Appendices

Chapter 26

Authors
Jacquelina Williams-Woods
Rachel Rodriguez
Joey Marchant
Angela Gail Swinford
William Burkhardt III
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Appendices
Chapter 26
Last Updated
2026-06-01
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81 minute(s)
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16079 words

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Appendices

Appendix A: Validation Data for the Concentration, Extraction, and Detection of Norovirus Genogroup I, Norovirus Genogroup II, and Hepatitis A Virus from Green Onion and Leafy Greens

Green onions were artificially contaminated with three levels of HAV and the murine norovirus extraction control. In this method validation, 8 of the 10 participating FDA, CFSAN, or FERN laboratories produce acceptable data consistent with the FDA Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods, 1st Ed (2011) for a level 3 validation (Tables A1 and A2). The overall detection frequency of HAV from green onion spikes was 97% and 75% for the 50 pfu and 5 pfu/g test portions, respectively (Table A3).

For the leafy greens matrix, romaine lettuce was spiked with three levels of norovirus and HAV. This matrix extension produced acceptable data consistent with the FDA Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods and Feeds, 2nd Ed (2015). HAV and norovirus GII were detected in all replicates at all inoculum levels. In romaine lettuce, norovirus GI had a detection frequency of 80% and 60% in the 3 genome copies (g.c.)/g and 0.3 g.c./g inoculum levels (Table A4 A-B). In spinach, norovirus GI had a detection frequency of 47% and 60% at the 3 g.c./g and 0.3 g.c./g inoculum levels (Table A4 A-B).

Sample Preparation

Green onions were purchased from a local retail market. The test portions were cut in 2” and 5” segments and placed into Whirl-pak® bags. Samples were spiked and held at 4 °C for 3 days prior to shipment. Twenty test portions, in triplicate, were prepared and shipped in coolers with ice bricks to the participating laboratories by CFSAN’s Moffett Center Institute of Food Safety and Health. Sample analysis was begun within 24 hrs of receipt.

Virus Inoculum

HAV inoculum used for seeding was the vaccine strain (HAV175/18f) propagated in house utilizing FrHK cell line. Murine norovirus (MNV) used for seeding was murine norovirus-1 propagated in house using RAW 264.7 cell line. Norovirus GI and GII from chloroform extracted clinical specimen were used for seeding samples. Three inoculation levels were used for HAV spikes; Low (5 PFU/g of HAV), High (50 PFU/g of HAV) and uninoculated. The norovirus spikes were 330 genomic copies for the high, 33 for the medium, and 0.3-3 genomic copies for the low inoculum. MNV was inoculated in all test portions at 4 x 103 PFU.

Table A1. Percentage of samples with expected results for each participating laboratory

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Table A2. Laboratory data from detection of HAV in green onion

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Table A3. Detection Frequencies of HAV

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Table A4 (A-B). Matrix extension data for norovirus GI, GII, and HAV in leafy greens

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A. Norovirus GI and GII

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B. HAV

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Conclusion

The MLV of the HAV concentration, extraction, and detection method for green onion has demonstrated acceptable sensitivity and specificity for regulatory analysis of green onion samples. Data supporting the reliability of the real-time RT-qPCR detection assays for HAV and norovirus are provided in Appendix F and Appendix G, respectively. In addition, a matrix extension for the concentration and extraction of enteric viruses from leafy greens (romaine lettuce and spinach) has been completed using this method. The results show the method is sensitive, reproducible, and robust and has established the "Fitness of Purpose" for concentration, extraction, and detection of HAV, norovirus GI, and norovirus GII from leafy greens. The detection frequency of the concentration, extraction, and detection method was 97 100% for the medium and high inocula, and 47-100% for the low inocula, depending on the specific matrix/target combination. The internal amplification control demonstrated little to no inhibition (< 4 Cts) for all detection assays. The limit of detection for this validated concentration, extraction, and detection method is 1-5 PFU/g.

Our conclusion from the MLV data is that the concentration, extraction, and detection method can be used for determination of HAV in green onion samples. The subsequent matrix extension demonstrates the concentration, extraction, and detection method can be used for HAV, norovirus GI, and norovirus GII from green onion and leafy greens. These assays are ready to be incorporated into the Bacteriological Analytical Manual and ongoing Office of Regulatory Affairs Field Assignments.

Appendix B: Validation Data for the Concentration, Extraction, and Detection of Norovirus Genogroup I, Norovirus Genogroup II, and Hepatitis A Virus from Soft Fruit: Fresh and Frozen

See Attached

Appendix C: Validation Data for the Concentration, Extraction, and Detection of Norovirus Genogroup I, Norovirus Genogroup II, and Hepatitis A Virus from Molluscan Shellfish

See Attached

Appendix D: Validation Data for the Concentration, Extraction, and Detection of Norovirus Genogroup I, Norovirus Genogroup II, and Hepatitis A Virus from Scallops and Tuna

See Attached

Appendix E: Data Analysis and Supplemental Material for the Murine Norovirus Detection Assay

The murine norovirus (MNV) RT-qPCR detection assay is used to assess the recovery of murine norovirus from spiked samples and to determine if the extraction was performed correctly. Valid norovirus and/or HAV sample results are contingent upon the successful detection of the extraction control from the sample being tested. Figures E1 and E2 demonstrate typical linear and log amplification curves for the RT-qPCR assays. Figures E3 and E4 demonstrate false positives or non-linear amplification. The MNV assay utilizes IAC primers and probe that are multiplexed (simultaneously amplified) with MNV primers and probe for each RNA sample. All primers and probes should be hydrated to a concentration of 100 µM using primer TE prior to making primer and probe mixes.
Data Analysis for the Murine Norovirus Detection Assay
Figure E1. Linear amplification plot of positive virus sample. This is a typical representation of a positive sample.
30 Figure E2. Log amplification plot of positive virus sample. This is a typical representation of a positive sample. Figure E3. Linear amplification plot of false positive virus sample.
Figure E4. Log amplification plot of false positive virus sample.
31 Supplemental Material for the Murine Norovirus Detection Assay 32 Table E1. Master Mix components for RT-qPCR assays for AB 7500 Reagent Volume per 25 µl for 1 reaction Buffer Mix 15.55 µl Volume for 75 reactions Primer Mix 2-plex 1875 µl 2 µl Probe Mix
150 µl 1 µl Enzyme Mix 75 µl 1.25 µl FAM dye 93.75 µl 2 µl Internal Control RNA* 150 µl 0.2 µl 15 µl RNA 3 µl Amount varies with concentration of IAC RNA. The amount of IAC template needs to be adjusted based on the prepared stock concentration to report Cycle threshold (Ct) of 20-25 PCR cycles when no inhibition is present in the reaction.
Table E2. AB7500 HAV, MNV, and Norovirus Buffer Mix Setup Reagents Volume (50 mM MgCl) DNase/RNase free H2O 1760 µl Volume (25 mM MgCl) 5X Buffer 1610 µl 1000 µl MgCl 1000 µl 150 µl dNTPs 300 µl 200 µl 200 µl

Appendix F: Validation Data, Data Analysis, and Supplemental Material for the Hepatitis A Virus Detection Assay and Control Exclusion Assay

Single Laboratory Validation of HAV RT-qPCR Detection Assay The real time RT-qPCR assay for HAV was validated for use on the Cepheid® SmartCycler in multiple phases. For the SLV of the HAV detection assay, three HAV strains were used to establish inclusivity (Table F1). Eleven enteric viruses and five pathogenic enteric bacterial species were used to establish assay exclusivity (Table F2). HAV-positive and HAV-negative human sera samples from CDC were tested using the HAV detection assay and results demonstrated 100% accuracy in detection (Table F3). The amplification efficiency ranged between 97% and 103% (Table F4). There was no inhibition in the assay with the addition of the internal amplification control (IAC) and competitive RNA poliovirus (Table F5). The dynamic range of the assay was 7 logs (Figure F1) and the limit of quantification (LOQ) and limit of detection (LOD) were 0.11 and 0.001 PFU/reaction, respectively (Table F6). RNA Template Controls and Clinical Specimens Viral Nucleic Acid Templates (for inclusivity/exclusivity testing) Template RNA was isolated from stock suspensions and diluted for inclusive and exclusive, viruses (Tables F1 and F2) and stool samples using the QIAamp Viral RNA Mini Kit (Qiagen) protocol for cell culture. The RNA was eluted from the spin columns with 60 µl AVE elution buffer (provided in kit) and stored at -80 °C until used. RNA Template for Competitive RNA Testing Poliovirus RNA was isolated from stock suspensions, diluted, and added to the HAV RT-qPCR multiplex assay to determine if the presence of additional enteric viral RNA would be competitive with detection of HAV viral RNA. The poliovirus RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) protocol for cell culture. The RNA was eluted from the spin columns with 60 µl AVE elution buffer (provided in kit) and stored at -80 °C until used. Bacterial Templates (for exclusivity testing) DNA templates were prepared by transferring 1 ml of overnight Tryptic Soy Broth culture to a microcentrifuge tube and centrifuged at 12,000 × g for 3 min. The supernatant was removed and the pellet completely resuspend in 1 ml 0.85% NaCl. The tube was centrifuged 12,000 × g for 3 min. The supernatant was removed and the pellet was completely re-suspended in 1 ml sterile water. The tube was place in a water bath or heat block and maintained at 100 °C for 10 min. Following boiling the tube was then centrifuged 12,000 × g for 1 min, and the supernatant was removed and transferred to a new microcentrifuge tube. This bacterial extracted was frozen at -20 °C and served as the appropriate control. 35

Table F1. Inclusivity of the HAV detection assay

*Average Ct value of 6 replicates.

Table F2. Exclusivity testing of the HAV detection assay

Organism Source Results Frequency of Result Poliovirus ATCC VR-193 Negative 6 of 6 Astrovirus HuAst1 Negative 6 of 6 San Miguel Sea Lion virus serogroup 17 Dr. Alvin Smith, Univ. OR, Corvallis Negative 6 of 6 Rotavirus ATCC VR 2018 Negative 6 of 6 Adenovirus ATCC VR-1083 Negative 6 of 6 Feline Calicivirus ATCC VR-2057 Negative 6 of 6 Human Paraechovirus ATCC VR-1063 Negative 6 of 6 Echovirus 1 ATCC VR-1038 Negative 6 of 6 Coxsackievirus ATCC VR-1007 Negative 6 of 6 Norwalk Virus GI Human Stool Negative 6 of 6 Norovirus GII Human Stool Negative 6 of 6 Escherichia coli ATCC 25922 Negative 6 of 6 Salmonella enterica ATCC 9700 Negative 6 of 6 Shigella sonnei ATCC 9290 Negative 6 of 6 Vibrio cholerae ATCC 14035 Negative 6 of 6 Listeria monocytogenes ATCC 7646 Negative 6 of 6

Strain Source ATCC HAV Average Ct* SD IAC Average Ct SD Frequency HM175/18f (sub-genotype 1B) VR-1402 29.08 0.208 20.88 0.22 6 of 6 PA21 (sub-genotype IIIA) VR-1357 23.24 0.261 20.85 0.33 6 of 6 PA21 (sub-genotype IIIA) VR-2281 20.07 0.352 21.221 1.08 6 of 6 36

Table F3. Specificity testing of HAV detection assay with human serum samples

Sample Number HAV Detection Ct Value* IAC Ct Value 17000 Positive 34.65 23.63 14000 Positive 32.23 23.16 13516 Positive 25.67 23.14 12010 Positive 39.86 22.74 12009 Positive 27.18 23.42 17500 Positive 37.49 23.53 12144 Positive 28.74 23.03 16000 Positive 36.25 23.33 12121 Positive 23.91 22.82 12113 Positive 28.66 23.65 12101 Positive 23.27 23.15 12112 Positive 29.21 23.58 13518 Positive 32.42 23.32 12319 Positive 29.73 23.61 12399 Positive 29.75 23.11 12320 Positive 28.25 23.18 12312 Positive 30.68 23.19 12323 Positive 28.58 23.33 12322 Positive 26.80 23.34 12305 Positive 25.12 22.87 12330 Positive 24.25 23.10 12385 Positive 28.26 23.19 12316 Positive 26.09 23.21 12346 Positive 33.88 23.18 12363 Positive 28.09 23.24 37

Sample Number HAV Detection Ct Value* IAC Ct Value 12325 Positive 22.38 23.22 12364 Positive 32.32 23.21 12359 Positive 31.70 23.12 12326 Positive 28.10 23.60 12302 Positive 29.08 23.58 12313 Positive 33.28 23.28 12352 Positive 33.74 23.40 12303 Positive 29.06 23.32 12304 Positive 26.83 23.55 12306 Positive 28.95 23.53 12329 Positive 29.44 22.75 12330 Positive 25.12 23.35 12307 Positive 31.42 23.29 12331 Positive 31.35 23.15 12345 Positive 28.54 23.12 12003 Negative** - 23.41 12013 Negative** - 23.34 19300 Negative** - 23.23 13517 Negative** - 23.28 *Serum samples tested in a single reaction due to limited amount of serum provided **Negative for HAV based on CDC’s data
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Table F4. HAV RT-qPCR Intra-Assay Variability: 3 - 100-fold dilutions of HAV RNA

*From 9 replicates Mean Efficiency: 100%; r2 = 0.997 Intra-Assay Reproducibility: Five samples of varying low (Ct 33-36) medium (Ct 27-28) and high (Ct 21-22) concentrations were tested in 9 reactions on the same run. The results show excellent reproducibility. Inter-Assay Reproducibility: The Ct value of the daily positive control was analyzed over a period of 3 days on two different, calibrated, Smart Cycler blocks. The results show consistent reproducibility over time on the same sample.

Table F5. Detection of HAV and IAC in the presence of competitive RNA
Average of 6 replicates.
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Ct High Med Low High Med Low High Med Low High Med Low High Med Low Mean 21.09 27.85 34.37 28.24 34.82 21.38 27.92 34.70 34.73 21.26 28.24 34.82 21.38 27.92 34.70 SD 0.182 0.320 0.243 0.250 0.599 0.249 0.338 0.523 0.347 0.350 0.250 0.599 0.249 0.338 0.523 SE 0.061 0.103 0.061 0.083 0.200 0.083 0.113 0.198 0.116 0.117 0.083 0.200 0.083 0.113 0.198 Amplification Efficiency 100% 103% 97% 97% 100% r2 0.998 0.998 0.997 0.995 0.996 Internal Control Impact No Significant Difference (p= 0.113) No Significant Difference (p= 0.415) No Significant Difference (p= 0.183) No Significant Difference (p= 0.311) No Significant Difference (p= 0.939) HAV Detection IAC Detection No Competitor With Polio at
4 x 104 pfu/rxn No Competitor With Polio at
4 x 104 pfu/rxn Average Ct* SD Average Ct SD Average Ct* SD Average Ct SD HAV 2 × 103 pfu/rxn 27.06 0.164 26.97 0.207 22.36 0.126 22.28 0.094 HAV 20 pfu/rxn 32.90 0.326 33.42 0.408 22.18 0.264 22.31 0.278 HAV 0.2 pfu/rxn 41.03 0.768 40.72 1.440 22.34 0.129 22.41 0.193 39 Figure F1. Dynamic Range of Assay: The assay has a dynamic range of 7 logs and a mean efficiency of 99.4% Figure F2. Standard Curve Table F6. Limit of Detection (LOD)/Limit of Quantitation (LOQ) using HAV HM175/18f Average Ct SD* 0.11 PFU 41.13 Pos/Total 0.55 0.01 PFU 42.44 10/10 0.82 0.001 PFU 43.48 7/10 1.04

Table F8. Stage #1 Results

Detection of HAV via RT-qPCR Organism/ Strain Lab #1 Lab #5 Lab #6 Lab #7 HAV HM- 175 3 of 3 6 of 6 6 of 6 6 of 6 HAV PA 21 3 of 3 6 of 6 6 of 6 6 of 6 HAV HAS15 3 of 3 6 of 6 6 of 6 6 of 6 Poliovirus 0 of 3 0 of 6 0 of 6 0 of 6 Astrovirus 0 of 3 0 of 6 0 of 6 0 of 6 NoV; SMSV-17 0 of 3 0 of 6 0 of 6 0 of 6 HuNoV GII 0 of 3 0 of 6 0 of 6 0 of 6 Salmonella 0 of 3 0 of 6 0 of 6 0 of 6 HAV: 63 of 63 reactions positive; 100% accuracy Non-HAV: 105 of 105 reactions negative; 100% accuracy

Table F9. Stage #2 Results

Detection of HAV via RT-qPCR Organism/ Strain Lab #1 Lab #2 Lab #3 Lab #4 Lab #5 HAV HM-175 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 HAV PA 21 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 HAV HAS15 5 of 6 6 of 6 6 of 6 6 of 6 6 of 6 Poliovirus 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 Astrovirus 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 NoV; SMSV-17 0 of 6 2 of 6 0 of 6 0 of 6 0 of 6 HuNoV GII 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 Salmonella 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 HAV: 89 of 90 reactions positive; 99% accuracy Non-HAV: 178 of 180 reactions negative; 99% accuracy

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Table F10. Stage #3 Results

Detection of HAV via RT-qPCR Organism/ Strain Lab #1 Lab #4 Lab #6 Lab #7 HAV HM- 175 6 of 6 6 of 6 6 of 6 6 of 6 HAV PA 21 6 of 6 6 of 6 6 of 6 6 of 6 HAV HAS15 6 of 6 6 of 6 6 of 6 6 of 6 Poliovirus 0 of 6 0 of 6 0 of 6 0 of 6 Astrovirus 1 of 6 0 of 6 0 of 6 0 of 6 NoV; SMSV-17 0 of 6 0 of 6 0 of 6 0 of 6 HuNoV GII 0 of 6 0 of 6 0 of 6 0 of 6 Salmonella 0 of 6 0 of 6 0 of 6 0 of 6 HAV: 72 of 72 reactions positive; 100% accuracy Non-HAV: 179 of 180 reactions negative; 99% accuracy

Table F11. Stage #4 Results

Detection of HAV via RT-qPCR Organism/Strain Lab #1 Lab #2 Lab #5 Lab #7 Lab #8 HAV HM- 175 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 HAV PA 21 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 HAV HAS15 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 Poliovirus 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 Astrovirus 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 NoV; SMSV-17 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 HuNov GII 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 Salmonella 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 HAV: 89 of 90 reactions positive; 99% accuracy Non-HAV: 149 of 150 reactions negative; 99% accuracy
Table F12. Inter-Laboratory Repeatability - Stage #1
43 HAV Strain Lab #1 Lab #2 Lab #3 Lab #4 Lab #5 Mean*
SD SE Mean
SD SE Mean
SD SE Mean
SD SE Mean
SD SE HM-175 25.1 0.21 0.09 27.9** 0.77 0.31 25.4 0.35 0.14 25.3 0.31 0.13 25.2 0.16 0.06 PA 21 24.5 0.20 0.08 27.7** 0.18 0.07 24.7 0.68 0.28 24.2 0.19 0.08 24.3 0.36 0.15 HAS-15 26.3 0.19 0.08 26.3 0.40 0.16 27.2 1.21 0.50 26.6 0.51 0.21 26.2 0.21 0.09 HAV HM-175: 1.3 × 103 pfu/rxn; HAV PA 21: 7.2 × 10 pfu/rxn; HAV HAS-15: 1.1 × 10 pfu/rxn

Results Table F18 (A-C). Intra- Assay Variability of the HAV Detection Assay. Three 10-fold dilutions of 9 replicates of HAV RNA were tested (10, 1, and 0.1 PFU/reaction) in 3 µl reactions. This experiment was repeated in three independent trials.

A. Trial 1 Smart Cycler AB 7500 Spike Level High Medium Low High Medium Low Mean Ct * 28.28 31.19 34.61 28.9 33.06 36.21 SD 0.16 0.44 0.37 0.18 0.12 0.25 SE 0.05 0.14 0.12 0.05 0.38 0.08 Amplification efficiency 107% 93% r2 0.99 0.99

B. Trial 2 Smart Cycler AB 7500 Spike Level High Medium Low High Medium Low Mean Ct * 28.24 31.04 34.88 25.83 29.49 33.03 SD 0.23 0.39 0.42 0.49 0.45 0.39 SE 0.07 0.12 0.13 0.15 0.143 0.12 Amplification efficiency 100% 90% r2 0.99 0.99

C. Trial 3 Smart Cycler AB 7500 Spike Level High Medium Low High Medium Low Mean Ct * 28.38 32.06 35.27 29.47 32.64 36.05 SD 0.84 0.37 0.22 0.15 0.30 0.31 SE 0.26 0.12 0.07 0.049 0.09 0.10 Amplification efficiency 95% 101% r2 0.99 0.99 *Cycle threshold averaged for 9 replicates of 3 µl reactions

E2 Mean Efficiency: Smart Cycler 101%; r2 = 0.99
AB7500 95%; r2 = 0.99

Intra-Assay Reproducibility (Table F18A-18C): Nine reactions of varying low (Ct 33-36) medium (Ct 29-32) and high (Ct 25-29) concentrations were tested in triplicate on the same run for the Smart Cycler and AB7500. The results show excellent reproducibility and amplification efficiencies that fall within the acceptable range (90-110 %).

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Table F19. Limit of Detection (LOD)/Limit of Quantitation (LOQ)- HAV175/18f

PFU/rxnα Ave. Ct SD* Pos/Total 0.11 PFU 36.21 0.23 9/9 0.01 PFU 40.72 0.97 8/9 0.001 PFU 42.97 0.42 2/9

Table F20. No Template Control (PCR Grade Water)

Sample HAV Ct HAV IAC Ct Sample HAV Ct HAV IAC Ct Sample HAV Ct HAV IAC Ct Sample HAV Ct HAV IAC Ct NTC 0.00 24.23 NTC 0.00 24.20 NTC 0.00 24.20 NTC 0.00 24.18 NTC 0.00 24.18 NTC 0.00 24.11 NTC 0.00 24.15 NTC 0.00 24.15 NTC 0.00 24.12 NTC 0.00 24.13 NTC 0.00 24.08 NTC 0.00 24.10 NTC 0.00 24.05 NTC 0.00 24.05 NTC 0.00 24.03 NTC 0.00 24.07 NTC 0.00 24.09 NTC 0.00 24.01 NTC 0.00 23.95 NTC 0.00 24.18 NTC 0.00 24.05 NTC 0.00 23.95 NTC 0.00 24.00 NTC 0.00 24.06 NTC 0.00 24.02 NTC 0.00 24.00 NTC 0.00 24.00 NTC 0.00 24.02 NTC 0.00 24.05 NTC 0.00 23.97 NTC 0.00 23.99 NTC 0.00 24.02 NTC 0.00 24.09 NTC 0.00 24.04 NTC 0.00 24.03 NTC 0.00 24.11 NTC 0.00 24.16 NTC 0.00 24.12 NTC 0.00 24.11 NTC 0.00 24.20 NTC 0.00 24.24 NTC 0.00 24.17 NTC 0.00 24.19 NTC 0.00 24.19 NTC 0.00 24.36 NTC 0.00 24.37 NTC 0.00 24.20 NTC 0.00 24.19 NTC 0.00 24.16 NTC 0.00 24.24 NTC 0.00 24.20 NTC 0.00 24.27 NTC 0.00 24.16 NTC 0.00 24.11 NTC 0.00 24.13 NTC 0.00 24.16 NTC 0.00 24.10 NTC 0.00 24.05 NTC 0.00 24.01 NTC 0.00 24.16 NTC 0.00 24.07 NTC 0.00 24.00 NTC 0.00 24.02 NTC 0.00 24.05 NTC 0.00 24.00 NTC 0.00 23.97 NTC 0.00 24.00 NTC 0.00 24.01 NTC 0.00 24.00 NTC 0.00 23.92 NTC 0.00 24.00 NTC 0.00 24.07 NTC 0.00 23.96 NTC 0.00 23.95 NTC 0.00 23.98 NTC 0.00 24.01 NTC 0.00 24.00 NTC 0.00 23.97 NTC 0.00 23.98 NTC 0.00 24.01 NTC 0.00 24.04 NTC 0.00 23.99 NTC 0.00 24.06 NTC 0.00 24.12 NTC 0.00 24.05 NTC 0.00 24.05 NTC 0.00 24.11 NTC 0.00 24.16 NTC 0.00 24.15 NTC 0.00 24.09 NTC 0.00 24.15 NTC 0.00 24.18 NTC 0.00 24.26 NTC 0.00 24.20 NTC 0.00 24.28 POS 29.05 24.70

Table F21. Negative Finfish Matrix
49 Sample HAV Ct HAV IAC Ct Sample HAV Ct HAV IAC Ct Sample HAV Ct HAV IAC Ct Sample HAV Ct HAV IAC Ct Tuna 1 0.00 24.58 Tuna 2 0.00 24.42 Tuna 3 0.00 24.41 Tuna 4 0.00 24.50 Tuna 1 0.00 24.63 Tuna 2 0.00 24.38 Tuna 3 0.00 24.37 Tuna 4 0.00 24.50 Tuna 1 0.00 24.54 Tuna 2 0.00 24.44 Tuna 3 0.00 24.36 Tuna 4 0.00 24.46 Tuna 1 0.00 24.64 Tuna 2 0.00 24.40 Tuna 3 0.00 24.41 Tuna 4 0.00 24.46 Tuna 1 0.00 24.52 Tuna 2 0.00 24.47 Tuna 3 0.00 24.36 Tuna 4 0.00 24.48 Tuna 1 0.00 24.55 Tuna 2 0.00 24.44 Tuna 3 0.00 24.39 Tuna 4 0.00 24.52 Tuna 1 0.00 24.43 Tuna 2 0.00 24.46 Tuna 3 0.00 24.43 Tuna 4 0.00 24.55 Tuna 1 0.00 24.54 Tuna 2 0.00 24.44 Tuna 3 0.00 24.40 Tuna 4 0.00 24.54 Tuna 1 0.00 24.60 Tuna 2 0.00 24.38 Tuna 3 0.00 24.35 Tuna 4 0.00 24.64 Tuna 1 0.00 24.52 Tuna 2 0.00 24.34 Tuna 3 0.00 24.42 Tuna 4 0.00 24.57 Tuna 1 0.00 24.53 Tuna 2 0.00 24.38 Tuna 3 0.00 24.40 Tuna 4 0.00 24.51 Tuna 1 0.00 24.50 Tuna 2 0.00 24.31 Tuna 3 0.00 24.34 Tuna 4 0.00 24.57 Tuna 1 0.00 24.50 Tuna 2 0.00 24.38 Tuna 3 0.00 24.33 Tuna 4 0.00 24.62 Tuna 1 0.00 24.58 Tuna 2 0.00 24.41 Tuna 3 0.00 24.44 Tuna 4 0.00 24.49 Tuna 1 0.00 24.55 Tuna 2 0.00 24.42 Tuna 3 0.00 24.39 Tuna 4 0.00 24.54 Tuna 1 0.00 24.55 Tuna 2 0.00 24.46 Tuna 3 0.00 24.36 Tuna 4 0.00 24.51 Tuna 1 0.00 24.52 Tuna 2 0.00 24.29 Tuna 3 0.00 24.43 Tuna 4 0.00 24.58 Tuna 1 0.00 24.48 Tuna 2 0.00 24.29 Tuna 3 0.00 24.38 Tuna 4 0.00 24.65 Tuna 1 0.00 24.38 Tuna 2 0.00 24.24 Tuna 3 0.00 24.43 Tuna 4 0.00 24.77 Tuna 1 0.00 24.43 Tuna 2 0.00 24.34 Tuna 3 0.00 24.32 Tuna 4 0.00 24.52 Tuna 1 0.00 24.43 Tuna 2 0.00 24.37 Tuna 3 0.00 24.46 Tuna 4 0.00 24.55 Tuna 1 0.00 24.49 Tuna 2 0.00 24.30 Tuna 3 0.00 24.42 Tuna 4 0.00 24.61 Tuna 1 0.00 24.49 Tuna 2 0.00 24.48 Tuna 3 0.00 24.56 NTC 0.00 24.48 Tuna 1 0.00 24.54 Tuna 2 0.00 24.31 Tuna 3 0.00 24.52 POS 29.59 24.57 Table F22. Frozen Strawberry Matrix on Smart Cycler and AB 7500 Strawberry Matrix Medium 5 PFU/g Uninoculated 0 PFU/g Smart Cycler Low 0.5 PFU/g 3 of 3 1 of 3 Smart Cycler – Percent Positive 0 of 2 100 33 AB 7500 0 3 of 3 3 of 3 AB 7500 – Percent Positive 0 of 2 100 Conclusion 100 0 The results of the validations for the HAV RT-qPCR detection assay demonstrate the assay is sensitive, specific, reproducible and robust. Our conclusion is that this assay can be used for the detection of HAV in RNA preparations obtained from any food matrix. This assay is ready to be incorporated into the Bacteriological Analytical Manual and ongoing Office of Regulatory Affairs Field Assignments. Data Analysis for HAV Detection Assay and Control Exclusion Assay
50 The HAV multiplex RT-qPCR assay is used to detect HAV RNA in food matrices. The Cy5 and Texas Red channels correlating to the HAV and the IAC targets, respectively. Positive HAV detection occurs when the primary fluorescence curve crosses the threshold for HAV and the IAC is positive. For HAV samples where HAV is detected, the HAV laboratory strain has sequence identities that can be used to differentiate between the laboratory and majority of wild-type strains. The Control Exclusion Assay uses a RT-qPCR assay for the differentiation of the HAV laboratory control strain. All primers and probes should be hydrated to a concentration of 100 µM using primer TE prior to making primer and probe mixes.
Data Interpretation for HAV RT-qPCR Detection Assay For this HAV multiplex assay, Cy5 is the HAV probe fluorescent label and Texas Red (TxR) is the internal amplification control (IAC) probe fluorescent label. 1. 2. 3. Sample is “negative" if: a. b. c. d. e. RT-qPCR negative control is negative for HAV, RT-qPCR positive control is positive for HAV, Matrix control sample (if included) is negative for HAV, Unknown is negative for HAV, Internal amplification control (IAC) is positive. No further analysis is needed. Sample is “positive” if: a. b. c. RT-qPCR negative control is negative for HAV, RT-qPCR positive control is positive for HAV, Unknown sample is positive for the detection HAV. Samples are invalid if: a. b. The negative RT-qPCR control sample demonstrates positive results crossing the Cy5 or if the IAC is negative, repeat the RT-qPCR assay. The average of the IAC Ct values for the sample replicates are more than 4.0 Cts greater than the Negative Control IAC Ct value, repeat the RT-qPCR assay using remaining RNA or RNA from a newly extracted saved tube with a 1 µl RT-qPCR reaction in triplicate. If the 1 µl template reactions yield an average IAC Ct value greater than 4.0 Ct higher than the Negative Control IAC Ct value, repeat the sample analysis from the beginning using additional food sample. With the new sample, the concentrates will be split into 5 tubes and complete RT-qPCR with 1 µl reactions in triplicate. *Note: A positive sample is a result that demonstrates log amplification. Log amplification can be viewed as a graph on the AB 7500 platforms. If the sample does not exhibit log amplification and crosses the threshold, the RT-qPCR reaction should be repeated. Refer to figures in Appendix E for appropriate amplification curves.
Data Analysis for Control Exclusion Assay (CEA) 51 1. Any sample CEA RT-qPCR assay which is negative for Cy5 (0 Ct value) and all controls are satisfactory, the virus detected in the HAV RT-qPCR assay was not the laboratory strain.
Note: If there was inhibition in the HAV RT-qPCR, this will be reflected in the internal amplification control for this assay also.
2. 3. Any sample CEA RT-qPCR reaction which is positive for Cy5 and all controls are satisfactory will be considered a ‘cannot rule out’ and will require gel analysis with 3% agarose or genetic bioanalyzer for confirmation of the presence or absence of the wild-type strain.
If the RT-qPCR negative control demonstrates positive Ct results for HAV in Cy5, if the RT qPCR positive control is negative (no Ct from Cy5) for HAV, or if the IAC is negative (no Ct from TxRed) the results are invalid and repeat assay due to invalid results. Assessment of CEA positive samples The HAV CEA probe targets the HAV laboratory control strain and typically will not generate a positive RT-qPCR result unless wild-type HAV is present in titers ≥103, including positive controls from culture (genomic) RNA. All positive samples will need to be analyzed by 3% agarose gel or on a genetic bioanalyzer such as an Agilent Tapestation or equivalent genetic bioanalyzer. If a genetic bioanalyzer will be used, manufacturer’s instructions for analysis of product should be followed. Detailed instructions for the 3% agarose gel analysis are listed below. The HAV laboratory control strain will yield a product size of 180 bp while other strains (wild-type) will yield a product size of 169 bp (Figure F1). You will also observe the internal control band size of 148 bp. On rare occasions, amplification of wild-type strains may produce bands with sizes ranging between 180 bp and 160 bp but not 169 bp; these bands should be excised and sequenced to confirm HAV RNA. 3.0% Agarose Gel Analysis: 1. 2. 3. 4. 5. 6. 7. 8. 9. Prepare 1X TAE buffer. Rinse and dry the gel tray. Place gel tray in gel caster; adjust to fix tray and level. Add comb to the first slot of the tray, making sure the comb fits securely in the slots. Pour 200 ml of 1X TAE buffer into 250 ml glass bottle with magnetic stirrer. Weigh 6 g of agarose and add to 200 ml buffer.
Shake briefly and microwave for 1-3 min (until the agarose is completely dissolved and there is a nice momentary boil). Place on stirrer and let cool for 5-10 min. Add 4 µl of ethidium bromide, stir for additional 30 sec. Carefully pour the 200 mL agarose into the casting tray.
52 Note: The depth of the gel should be 5 mm so the volume of the gel may need to be adjusted depending on the size of the gel casting tray. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. Allow the gel to solidify (about 1 hr); then remove the comb. Add 1X TAE buffer to electrophoresis chamber; careful not to overfill. Insert the casting tray (with the gel on it) into the electrophoresis chamber closest to the negative (black) electrode. DNA is negatively charged. During electrophoresis, it will migrate from the negative (black) to the positive (red) electrode. If gel is not covered with enough 1X TAE, gradually add 1X TAE to electrophoresis chamber until the buffer just covers the top of the gel. Add 0.5 µl of loading dye to each sample. Skip first and last well and load 15 µl of each sample to individual wells, then add the 100 bp ladder to the first and last well, taking care not to puncture the well bottoms. Do not attempt to load a sample if there is an air bubble in your pipet tip. Also, if a well has an air bubble in it, push it out using a clean tip before loading a sample in it.
Attach lid; make sure cords are correctly plugged into the power supply (red to red, black to black). Plug in the power supply. Turn the power on and adjust to 100 volts.
Electrophorese (run) the gel for 2 hrs. Shut off the power supply, unplug the leads, and unplug the power supply. Lift the gel casting tray from the chamber. Place gel in gel imager and analyze according to manufacturer’s instructions.
Figure F1. 3% Low-melt temp agarose gel with positive wild-type and controls 53 Supplemental Material for HAV Detection Assay
54 Table F23. HAV, MNV, and Norovirus Buffer Mix Setup Reagents Volume (50 mM MgCl) DNase/RNase free H2O 1760 µl Volume (25 mM MgCl) 5X Buffer 1610 µl 1000 µl MgCl 1000 µl 150 µl dNTPs 300 µl 200 µl Made with components from Qiagen One-Step RTqPCR kit and PCR grade water
200 µl Table F24. Enzyme Mix Setup Component One-Step Qiagen enzyme Volume Superase-IN (10000U) ~200 µL 50 µL Table F25. HAV Primer Mix Setup Primers/H2O Volume GAR2F GAR1R 30 µl IC46F 30 µl IC194R 7.44 µl DNase/RNase water 7.44 µl *Made with 100 µM primers stocks and PCR grade water 725.12 µl Table F26. HAV Probe Mix Probe/H2O Volume HAVP ICP 30 µl DNase/RNase water 22.5 µl Made with 100 µM probe stocks and PCR grade water
547.10 µl Supplemental Material for Control Exclusion Assay
55 Table F27. CEA Buffer Mix Setup Reagents Volume (50 mM MgCl) DNase/RNase free H2O 1660 µl Volume (25 mM MgCl) 5X Buffer 1360 µl 1000 µl MgCl2 1000µl 300 µl dNTPs 600 µl 150 µl Made with components from Qiagen One-Step RT-qPCR kit and PCR grade water
150 µl Table F28. CEA Primer Mix Setup Primers/H2O Volume HAVCROF JWCROR 7.44 µl IC46F 7.44 µl IC194R 7.44 µl DNase/RNase water 7.44 µl *Made with 100 µM primers stocks and PCR grade water 770.24 µl Table F29. CEA Probe Mix Probe/H2O JWCROP Volume ICP 10 µl DNase/RNase water 15 µl *Made with 100 µM probe stocks and PCR grade water

Appendix G: Validation Data, Data Analysis, and Supplemental Material for the Norovirus Genogroup I and Genogroup II Detection Assay

The RT-qPCR detection assay includes oligonucleotide primers and dual-labeled hydrolysis (TaqMan® style) probes for the in vitro detection of norovirus GI and GII. The norovirus primers target the ORF 1 and 2 junction of the norovirus genome. This region of the genome is highly variable, and these primers and probes have previously been shown to detect multiple strains of norovirus GI and GII (Kageyama et al., 2003). This assay also incorporates an RNA internal amplification control (IAC) to monitor any potential matrix-derived inhibitory effects. All primers and probes should be hydrated to a concentration of 100 µM using primer TE prior to making primer and probe mixes.
Single Laboratory Validation of Norovirus RT-qPCR Detection Assay The norovirus detection assay has been effectively optimized and has been used extensively in research and outbreak analysis for norovirus (DePaola et al., 2010; Woods et al., 2016). The single laboratory validation (SLV) followed the FDA Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods, 2nd Ed (2015). The SLV was conducted at GCSL in accordance with a level 2 validation on the Smart Cycler and AB 7500 platforms. Results are presented separately for each instrument platform. For the RT-qPCR assay for the SLV inclusivity, 17 different strains of GI or GII were used (Table G1). For the exclusivity assay, 19 different strains of enteric bacteria or viruses were used (Table G2). In addition, known positive clinical samples from state and international laboratories were tested with the norovirus multiplex assay yielding 100% detection on the Smart Cycler and AB 7500 platforms (Table G3 and G10). The amplification efficiencies ranged from 91% to 99% on the Smart Cycler and 95% to 99% for the AB 7500 (Tables G4 and G11). There was no inhibition in the assay with the addition of the internal amplification control (IAC) or competitive RNA with and without norovirus GI or GII (Table G5 and G12). On the Smart Cycler, the dynamic range of the assay was 7 logs (Figure G1) and the LOQ and LOD were 1 and 10 genomic copies/reaction, respectively (Table G7). For the AB 7500 platform, the dynamic range of the assay was 6 and 7 logs for GI and GII respectively (Figure G4) and the LOQ and LOD were 1 and 10 genomic copies/reaction, respectively (Table G13). Sample Preparation Viral Nucleic Acid Templates (for inclusivity/exclusivity and competitive RNA testing) RNA was isolated from stock suspensions and diluted for inclusive and exclusive viruses (Tables G1 and G2) and stool samples using the QIAamp Viral RNA Mini Kit (Qiagen) protocol for cell culture.
The RNA was eluted from the spin columns with 60 µl AVE elution buffer (provided in kit) and stored at -80 ºC until used.
Bacterial Templates (for exclusivity testing) DNA templates were prepared by transferring 1 ml of overnight Tryptic Soy Broth culture to a microcentrifuge tube and centrifuging at 12,000 x g for 3 min. The supernatant was removed and the pellet resuspend in 1 ml 0.85% NaCl. The tube was centrifuged at 12,000 x g for 3 min. The supernatant was removed and the pellet was completely resuspend in 1 ml sterile water. The tube was placed in a water bath or heat block maintained at 100 °C for 10 min. Following heating, the tube was centrifuge 57

12,000 x g for 1 min, and the supernatant was removed to a new microcentrifuge tube. This bacterial extract was frozen at -20 °C until used.

SLV Results for the Norovirus Detection Assay on the Smart Cycler

Table G1. Inclusivity of the norovirus detection assay

Genotype/Strain Source Norovirus Average Ct* SD IAC Average Ct SD Frequency of Result GII.4 Henry clinical 34.87 0.34 30.99 0.20 6 of 6 GII.4 Grimsby clinical 31.55 0.70 23.72 0.23 6 of 6 GII.4 Minerva clinical 27.12 0.66 25.21 0.62 6 of 6 GII.4 Hunter clinical 26.36 0.57 24.51 0.14 6 of 6 GII.4 Osaka clinical 25.38 0.37 23.82 0.37 6 of 6 GII.4 New Orleans clinical 24.14 0.66 23.72 0.84 6 of 6 GII.1 clinical 20.63 0.38 24.22 0.35 6 of 6 GII.6A clinical 25.78 0.33 25.15 0.32 6 of 6 GII.12 clinical 21.69 0.63 25.69 0.42 6 of 6 GII.16 clinical 35.62 0.34 24.41 0.47 6 of 6 GI.6A clinical 33.97 0.39 26.18 0.29 6 of 6 GI.1 clinical 33.41 0.55 27.17 0.15 6 of 6 GI.2 clinical 29.09 0.32 26.34 0.23 6 of 6 GI.2 Constellation clinical 27.36 0.18 26.48 0.14 6 of 6 GI.4 clinical 27.67 1.07 26.55 0.17 6 of 6 GI.3C clinical 26.72 1.08 26.18 0.13 6 of 6 GI.3B clinical 37.39 0.56 26.10 0.34 6 of 6 *Average Ct value of 6 replicates.

58

Table G2. Exclusivity testing of the norovirus detection assay

Strain Source Results Frequency Murine norovirus David Kingsley – cell culture negative 6 of 6 San Miguel Sea Lion Virus serogroup 17 Dr. Alvin Smith, University of Oregon negative 6 of 6 Astrovirus clinical negative 6 of 6 HAS-15 ATCC VR2281 negative 6 of 6 Adenovirus 41 clinical negative 6 of 6 Adenovirus 40 clinical negative 6 of 6 Sabine poliovirus cell culture negative 6 of 6 Shigella sonnei ATCC 9290 negative 6 of 6 Paraechovirus clinical negative 6 of 6 Echovirus 30 clinical negative 6 of 6 Enterovirus 90 clinical negative 6 of 6 Vibrio parahaemolyticus Oyster extract negative 6 of 6 Vibrio spp. ATCC 14035 negative 6 of 6 Salmonella enterica ATCC 9700 negative 6 of 6 Rotavirus ATCC VR2018 negative 6 of 6 Listeria monocytogenes ATCC 7646 negative 6 of 6 HAV HM175f cell culture negative 6 of 6 Coxsackievirus A1 clinical negative 6 of 6 Norovirus GIV IDT RNA transcript negative 6 of 6

59

Table G3. Specificity testing of norovirus detection assay with clinical samples

GI IAC GII 1 42.40 29.10 25.57 2 0.00 28.70 32.89 3 42.19 30.43 34.94 4 0.00 33.62 40.76 5 47.22 30.08 25.48 6 0.00 32.05 31.86 7 46.05 27.76 28.53 8 0.00 29.30 28.01 9 0.00 29.09 26.45 10 0.00 28.94 42.54 11 42.71 28.27 0.00 12 26.57 28.46 0.00 13 0.00 27.86 0.00 14 33.34 27.27 0.00 15 34.87 28.27 0.00 16 39.88 27.39 0.00 17 37.63 27.05 0.00 18 28.19 27.96 0.00 19 34.41 27.31 0.00 20 37.92 28.42 0.00 21 0.00 25.13 0.00 22 0.00 28.05 0.00 23 0.00 30.84 0.00 24 0.00 27.74 0.00 Positive 29.30 27.38 28.73 Negative 0.00 27.38 0.00

60

Table G4. Norovirus RT-qPCR Intra-assay Variability: three 100-fold dilutions of GI and GII RNA, 9 replicates per dilution

Trial 1 Trial 2 Ct* High Med Low High Med Low Template GI GII GI GII GI GII GI GII GI GII GI GII Mean 17.69 22.47 24.19 29.04 31.79 37.30 17.77 22.27 24.10 28.96 31.87 36.62 SD 0.26 0.41 0.52 0.48 0.29 1.19 1.02 1.18 0.40 0.32 1.28 1.16 SE 0.08 0.13 0.17 0.16 0.09 0.45 0.34 0.39 0.13 0.10 0.42 0.44 Mean Amplification Efficiency 95% 99% r2 0.997 0.998

Trial 3 Trial 4 Ct* High Med Low High Med Low Template GI GII GI GII GI GII GI GII GI GII GI GII Mean 17.46 22.31 23.72 28.55 31.35 35.80 17.74 21.99 24.25 28.54 32.07 36.01 SD 0.37 0.60 0.47 0.40 0.62 0.39 0.104 0.16 0.171 0.26 0.377 0.63 SE 0.12 0.29 0.16 0.13 0.21 0.13 0.035 0.05 0.057 0.09 0.142 0.21 Mean Amplification Efficiency 91% 98% r2 0.997 0.997

Trial 5 Ct* High Med Low Template GI GII GI GII GI GII Mean 17.82 21.79 23.97 28.24 31.81 35.62 SD 0.32 0.17 0.29 0.25 0.32 1.01 SE 0.11 0.06 0.10 0.08 0.11 0.33 Mean Amplification Efficiency 95% r2 0.996

*From 9 replicates Mean Efficiency: 96%; r2 = 0.997

61

Intra-assay reproducibility: Five trials with the multiplex assay of low (Ct 31-37) medium (Ct 23-28) and high (Ct 17-21) concentrations were tested in 9 replicates. The results show excellent reproducibility.

Inter-assay reproducibility: The Ct value of the daily positive control was analyzed over a period of 5 days on 6 different, calibrated, Smart Cycler blocks. The results show consistent reproducibility over the period of time on the same sample with mean amplification efficiencies and r2 values of 96% and 0.997, respectively.

Table G5. Detection of norovirus GI in the presence of IAC and GII

Without IAC With IAC GI Average Ct GI SD IAC Average Ct IAC SD GI Average Ct GI SD With GII GI -2 Dilution 18.29 0.19 30.32 0.60 18.06 0.54 GI -3 Dilution 21.77 0.35 28.69 0.21 21.18 0.15 GI -4 Dilution 24.83 0.49 28.14 0.20 24.29 0.26 Without GII GI -2 Dilution 18.64 0.92 34.39 0.55 18.13 0.30 GI -3 Dilution 21.07 0.16 31.46 0.42 20.80 0.29 GI -4 Dilution 24.08 0.26 29.03 0.32 23.54 0.17

Table G6. Detection of norovirus GII in the presence of IAC and GI

Without IAC With IAC GII Average Ct GII SD IAC Average Ct IAC SD GII Average Ct GII SD With GI GII -2 Dilution 22.03 0.28 28.98 0.20 21.68 0.52 GII -3 Dilution 25.60 0.20 28.44 0.34 25.35 0.46 GII -4 Dilution 29.21 0.24 28.62 0.21 29.29 0.26 Without GI GII -2 Dilution 22.00 0.52 28.77 0.42 22.29 0.54 GII -3 Dilution 25.57 0.24 28.38 0.34 25.35 0.48 GII -4 Dilution 28.93 0.16 28.14 0.18 28.78 0.29

Figure G1 (A-B). Dynamic range of the norovirus GI and GII detection assay
62 GIA. GI ..
GIB. GII 63 Figure G2 (A-B). Standard curves 2
G2A. GI reaction efficiency of 91% G2B. GII efficiency of 90% 64

Table G7. LOD / LOQ detection assay using GI.1 and GII.4

Norovirus GI Norovirus GII Genomic copies/rxn Average Ct SD* Pos/Total Average Ct SD* Pos/Total 100 30.64 0.12 6 of 6 31.08 1.08 6 of 6 10 35.58 1.68 6 of 6 34.80 0.81 6 of 6 1 41.66 2.05 2 of 6 42.78 3.60 3 of 6 *standard deviation of positive samples only

LOQ = 10 genomic copies
LOD = 1 genomic copies

SLV Results for the Norovirus Detection Assay on the AB 7500

Table G8. Inclusivity of the norovirus detection assay

Genotype/Strain Source Norovirus Average Ct* SD IAC Average Ct SD Frequency GII.4 Henry clinical 33.88 0.34 28.66 0.20 6 of 6 GII.4 Grimsby clinical 30.26 0.70 23.72 0.23 6 of 6 GII.4 Minerva clinical 26.34 0.66 25.21 0.62 6 of 6 GII.4 Hunter clinical 25.21 0.57 24.51 0.14 6 of 6 GII.4 Osaka clinical 24.59 0.37 23.82 0.37 6 of 6 GII.4 New Orleans clinical 23.31 0.14 23.72 0.35 6 of 6 GII.1 clinical 36.35 0.38 24.22 0.32 6 of 6 GII.6A clinical 25.08 0.33 25.15 0.42 6 of 6 GII.12 clinical 20.18 0.63 25.69 0.47 6 of 6 GII.16 clinical 34.04 0.34 24.41 0.29 6 of 6 GI.6A clinical 33.97 0.39 26.18 0.15 6 of 6 GI.1 clinical 33.41 0.55 27.17 0.23 6 of 6 GI.2 clinical 29.09 0.32 26.34 0.14 6 of 6 GI.2 Constellation clinical 27.36 0.18 26.48 0.17 6 of 6 GI.4 clinical 27.67 1.07 26.55 0.13 6 of 6 GI.3C clinical 26.72 1.08 26.18 0.34 6 of 6 GI.3B clinical 37.39 0.56 26.10 0.31 6 of 6 *Average of 6 replicates

65

Table G9. Exclusivity testing of the norovirus detection assay

Strain Source Results Frequency Murine norovirus David Kingsley – cell culture negative 6 of 6 San Miguel Sea Lion Virus serogroup 17 Dr. Alvin Smith, University of Oregon negative 6 of 6 Astrovirus clinical negative 6 of 6 HAS-15 ATCC VR2281 negative 6 of 6 Adenovirus 41 clinical negative 6 of 6 Adenovirus 40 clinical negative 6 of 6 Sabine poliovirus cell culture negative 6 of 6 Shigella sonnei ATCC 9290 negative 6 of 6 Paraechovirus clinical negative 6 of 6 Echovirus 30 clinical negative 6 of 6 Enterovirus 90 clinical negative 6 of 6 Vibrio parahaemolyticus Oyster extract negative 6 of 6 Vibrio spp. ATCC 14035 negative 6 of 6 Salmonella enterica ATCC 9700 negative 6 of 6 Rotavirus ATCC VR2018 negative 6 of 6 Listeria monocytogenes ATCC 7646 negative 6 of 6 HAV HM175f cell culture negative 6 of 6 Coxsackievirus A1 clinical negative 6 of 6 Norovirus GIV IDT RNA transcript negative 6 of 6

66

Table G10. Specificity testing of norovirus detection assay with clinical samples

GI IAC GII 1 0.00 26.63 23.67 2 0.00 26.81 31.32 3 0.00 26.37 33.27 4 0.00 28.15 34.71 5 0.00 27.15 23.27 6 42.87 27.45 28.93 7 0.00 26.25 26.83 8 43.79 27.23 26.26 9 0.00 26.94 24.73 10 0.00 26.73 40.08 11 41.90 26.51 0.00 12 26.16 26.97 0.00 13 0.00 26.26 0.00 14 32.26 26.28 0.00 15 32.90 26.00 0.00 16 38.52 26.90 40.61 17 35.59 26.43 0.00 18 27.72 26.41 0.00 19 34.03 26.52 0.00 20 37.86 26.38 0.00 21 0.00 26.84 0.00 22 0.00 26.73 0.00 23 0.00 26.65 0.00 24 0.00 26.30 0.00 Positive 28.12 26.29 27.19 Negative 0.00 26.41 0.00

67

Table G11. Norovirus RT-qPCR Intra Assay Variability: three 100-fold dilutions of GI and GII RNA

Trial 1 Trial 2 Ct* High Med Low High Med Low Template GI GII GI GII GI GII GI GII GI GII GI GII Mean 18.05 21.90 24.62 28.59 32.20 36.24 17.63 21.37 24.30 28.18 31.91 35.47 SD 0.17 0.17 0.10 0.10 0.14 0.38 0.07 0.14 0.09 0.24 0.12 0.81 SE 0.05 0.06 0.03 0.03 0.02 0.13 0.02 0.04 0.12 0.08 0.04 0.27 Mean Amplification Efficiency 97% 95% r2 0.996 0.995

Trial 3 Trial 4 Ct* High Med Low High Med Low Template GI GII GI GII GI GII GI GII GI GII GI GII Mean 17.48 21.24 24.24 28.09 31.84 35.44 17.46 21.39 24.04 27.96 31.48 35.21 SD 0.15 0.09 0.13 0.26 0.27 0.40 0.09 0.12 0.10 0.13 0.28 0.44 SE 0.05 0.31 0.27 0.40 0.09 0.13 0.03 0.04 0.03 0.03 0.09 0.14 Mean Amplification Efficiency 98% 98% r2 0.998 0.997

Trial 5 Ct* High Med Low Template GI GII GI GII GI GII Mean 17.37 21.31 23.96 27.95 31.73 35.01 SD 0.17 0.17 0.19 0.23 0.45 0.31 SE 0.05 0.06 0.06 0.08 0.14 0.10 Mean Amplification Efficiency 99% r2 0.996

*From 9 replicates Mean Efficiency: 97%; r2 = 0.997

68

Intra-Assay Reproducibility: Five trials with the multiplex assay of varying low (Ct 31-36) medium (Ct 23-28) and high (Ct 17-21) concentrations were tested in 9 replicate reactions in the same run. The results show excellent reproducibility.

Inter-assay reproducibility: The Ct value of the daily positive control was analyzed over a period of 5 days on a calibrated ABI 7500. The results show consistent reproducibility over the period of time on the same sample with mean amplification efficiencies and r2 values of 97% and 0.997, respectively.

Table G12. Detection of norovirus GI in the presence of IAC and GII

Without IAC With IAC GI
Average Ct GI SD IAC
Average Ct IAC SD GI
Average Ct GI SD With GII GI -2 Dilution 18.04 0.09 28.66 0.34 18.37 0.34 GI -3 Dilution 21.57 0.18 28.79 0.08 21.79 0.15 GI -4 Dilution 25.19 0.15 25.64 0.14 28.73 0.11 Without GII GI -2 Dilution 18.06 0.06 28.62 0.31 18.89 0.21 GI -3 Dilution 21.50 0.09 28.54 0.40 21.73 0.12 GI -4 Dilution 24.84 0.14 28.63 0.13 24.94 0.08

Table G13. Detection of norovirus GII in the presence of IAC and GI

Without IAC With IAC GII Average Ct GII SD IAC Average Ct IAC SD GII Average Ct GII SD With GI GII -2 Dilution 22.34 0.25 28.55 0.28 22.46 0.28 GII -3 Dilution 26.30 0.18 28.92 0.23 26.43 0.24 GII -4 Dilution 29.44 0.39 29.04 0.11 29.83 0.56 Without GI GII -2 Dilution 22.28 0.13 28.76 0.14 22.80 0.28 GII -3 Dilution 26.05 0.26 28.95 0.06 26.31 0.22 GII -4 Dilution 29.71 0.53 29.10 0.43 29.41 1.60 69

Figure G3 (A-B). Dynamic range of the assay. G3 A. The assay has a dynamic range of 7 logs for GI

G3 B. The assay has a dynamic range of 6 logs for GII

70

Figure G4A-B. Standard curves
G4 A. GI reaction efficiency of 90%

G4 B. GII reaction efficiency of 107%

y = -3.6025x + 30.4 R² = 0.9464 0 5 10 15 20 25 30 35 0 1 2 3 4 5 Ct value log10 Conc GI Ct Value Linear (Ct Value) y = -3.165x + 35.25 R² = 0.9523 0 5 10 15 20 25 30 35 40 0 2 4 6 Ct Values log 10 conc GII Ct Value Linear (Ct Value) Table G13. LOD/LOQ using GI.1 and GII.4
71 Norovirus GI Norovirus GII Genomic copies/rxn Average Ct SD* Pos/Total Average Ct 100 37.55 0.64 SD* Pos/Total 6/6 10 42.79 0.91 31.68 0.62 6/6 6/6 1 47.25 2.37 35.06 0.37 6/6 2/6 standard deviation of positive samples only. LOQ= 10 genomic copies
LOD= 1 genomic copies 41.30 4.13 4/6 Multi Laboratory Validation of Norovirus RT-qPCR Detection Assay The norovirus RT-qPCR detection assay was multi-laboratory validated yielding acceptable results for a Collaborative Validation Study based on FDA Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods, 2nd Ed (2015). There were 24 enteric organisms included in the MLV for the norovirus multiplex detection assay, with some of the inclusivity samples containing multiple genogroups and strains. There was 100% accuracy and specificity for the detection of norovirus GI while norovirus GII had 99.7% accuracy and 100% specificity-based data from 13 labs (Tables G14 and G15). As indicated in Table G17, there was no inhibition observed in the detection assay.
72 Table G14. Multi-laboratory validation data for norovirus multiplex detection assay in 3 µl triplicate reaction of inclusivity (norovirus GI and/or GII) or exclusivity (bacteria and other non-human noroviruses) microorganisms# Strain Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 Lab 7S Lab 8 Lab 9 Lab 7A Lab 11 GII.4 Minerva 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 Lab 12 Lab 13 Lab 14 1 2 3 2 of 3b 3 of 3 6 of 3 3 of 3 GII.12 4 of 3a 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 GI.3B/GII.4 Grim 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 GII.12 4 5 of 3a 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 5 GI.3C/GII.16 4 of 3a 4 of 3a 6 of 3 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 GI.4/GII.4∞ 5 of 6 6 of 6 6 of 6 6 of 6 6 of 6 5 of 6 6 of 6 5 of 6 3 of 6 6 of 6 6 of 6 6 of 6 6 of 6 3 of 6 Minerva GII.2 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 7 8 9 GI.6A/GII.4 Osaka 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 6 of 6 GII.4 Henry 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 2 of 2 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 10 GII.1 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 11 GII.4 New Orleans 3 of 3 3 of 3 4 of 3a 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 0 of 3 GII.6A 12 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 3 of 3 0 of 3 13 Murine norovirus 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 14 SMSV-17 15 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 Adenovirus 41 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 Sabine poliovirus 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 Shigella sonnei 16 17 18 19 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 Enterovirus 90 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 Vibrio parahaemolyticus 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 20 Vibrio spp. 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 21 Salmonella enterica 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 22 Rotavirus 23 Neg Control 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 HAV HM175f 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 24 Coxsackievirus A1 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 0 of 6 25 neg neg neg neg neg neg neg neg neg neg neg neg neg neg 26 GI/GII Pos Control^ pos pos pos pos pos pos pos pos pos pos pos pos pos pos Inclusivity GI: 100% accuracy and 100% specificity of 156 reactions Inclusivity GII: 99.7% accuracy and 100% specificity of 468 reactions Exclusivity GI and GII: 100% accuracy ∞For strain GII.4 Minerva – levels were found to be at limit of detection for norovirus GII, therefore detection produced sporadic positive or negatives.

Consists of up to 4,000 Ct values for GI, GII, or IAC

*Excluded data set – analysis performed on a non-calibrated instrument. a Clinical samples 3 and 4 were originally characterized as GII only; however, testing of multiple replicates demonstrated low levels of GI. Reported results of greater than 3 of 3 positive indicates GII was detected in all 3 replicates and GI was detected in additional replicate(s).
b False negative GII ^GI.1 and GII.4 RNA transcripts were used as positive control 73

Table G15. Summary of detection: inclusivity and exclusivity from 13 labs

Organism/Strain Accuracy False Negatives False Positives GII.4 Minerva 99.7% 0.3% - GI.3B 100% 0% - GII.4 Grimsby 100% 0% - GII.12* 100% 0% - GII.12* 100% 0% - GI.3C 100% 0% - GII.16 100% 0% - GI.4 100% 0% - GII.4 Minerva 100% 0% - GII.2 100% 0% - GI.6A 100% 0% - GII.4 Osaka 100% 0% - GII.4 Henry 100% 0% - GII.1 100% 0%
GII.4 New Orleans* 100% 0% - GII.6A 100% 0% - Murine norovirus 100% - 0% SMSV-17 100% - 0% Adenovirus 41 100% - 0% Sabine poliovirus 100% - 0% Shigella sonnei 100% - 0% Enterovirus 90 100% - 0% Vibrio parahaemolyticus 100% - 0% Vibrio spp. 100% - 0% Salmonella enterica 100% - 0% Rotavirus 100% - 0%

Table G16. Inter-laboratory repeatability for norovirus RT-qPCR from 13 labs

Norovirus Strain MEAN* SD SE GII.4 Minerva 27.20 0.59 0.34 GI.3B 34.05 0.28 0.16 GII.4 Grimsby 37.24 0.57 0.33 GII.12 18.45 0.83 0.48 GII.12 25.99 1.13 0.65 GI.3C 25.34 0.54 0.31 GII.16 26.25 0.51 0.29 GI.4 27.78 0.65 0.38 GII.4 Minerva 39.80 1.13 0.73 GII.2 26.33 0.47 0.27 GI.6A 36.82 0.38 0.22 GII.4 Osaka 38.50 0.56 0.32 GII.4 Henry 27.18 0.37 0.21 GII.1 19.73 1.29 0.74 GII.4 New Orleans 30.89 1.14 0.66 GII.6A 21.55 0.46 0.27

Table G17. IAC Inter-laboratory repeatability for IAC from 13 labs

Norovirus Strain MEAN* SD SE GII.4 Minerva 23.24 1.03 0.15 GI.3B 23.64 0.96 0.14 GII.4 Grimsby 23.64 0.96 0.14 GII.12 24.28 1.17 017 GII.12 25.18 1.93 0.29 GI.3C 23.70 1.16 0.16 GII.16 23.70 1.16 0.16 GI.4 24.10 1.40 0.21 GII.4 Minerva 24.10 1.40 0.21 GII.2 26.16 1.98 0.30 GI.6A 23.07 0.85 0.13 GII.4 Osaka 23.07 0.85 0.13 GII.4 Henry 23.08 0.92 0.13 GII.1 23.43 0.99 0.15 GII.4 New Orleans 23.38 1.01 0.14 GII.6A 24.14 1.43 0.22

Sample is “negative" if:
a. b. c. d. e. RT-qPCR negative control is negative for GI and GII,
RT-qPCR positive control is positive for GI and GII,
Matrix control sample (if included) is negative for GI and GII,
Unknown is negative for GI and GII,
Internal amplification control (IAC) is positive. No further analysis is needed. Sample is “positive” if: a. b. c. RT-qPCR negative control is negative for GI and GII,
RT-qPCR positive control is positive for GI and GII,
Unknown sample is positive for GI and/or GII. Samples are invalid if: a. b. c. The negative RT-qPCR control sample demonstrates positive results crossing the Cy5 or Cy3 threshold or if the IAC is negative, repeat the RT-qPCR assay, The RT-qPCR positive control is negative for GI and/or GII, The average of the IAC Ct values for the sample replicates are more than 4.0 Cts greater than the Negative Control IAC Ct value, repeat the RT-qPCR assay using remaining RNA or RNA from a newly extracted saved tube with a 1 µl RT-qPCR reaction in triplicate. If the 1 µl template reactions yield an average IAC Ct value greater than 4.0 Ct higher than the Negative Control IAC Ct value, repeat the sample analysis from the beginning using additional food sample. With the new sample, the concentrates will be split into 5 tubes and complete RT-qPCR with 1 µl reactions in triplicate. Note: A positive sample is a result that demonstrates log amplification. Log amplification can be viewed as a graph on the Smart Cycler and AB 7500 platforms. If the sample does not exhibit 76 log amplification and crosses the threshold, the RT-qPCR reaction should be repeated. Refer to figures in Appendix E for appropriate amplification curves. Supplemental Material for Norovirus GI and GII Detection Assay Table G6. AB7500 HAV, MNV, and Norovirus Buffer Mix Setup Reagents Volume (50 mM MgCl) DNase/Rnase free H2O 1760 µl Volume (25 mM MgCl) 5X Buffer 1610 µl 1000 µl MgCl 1000 µl 150 µl dNTPs 30 µl 200 µl

Appendix H: Data Analysis and Supplemental Material for the Mengovirus Detection Assay

The method detection assay described here was developed by CFSAN’s Gulf Coast Seafood Laboratory for use as an alternative extraction control (to murine norovirus). This is an RT-qPCR assay for the detection of Mengovirus with the inclusion of an internal amplification control (IAC). All primers and probes should be hydrated to a concentration of 100 µM using primer TE prior to making primer and probe mixes.
Data Analysis for the Menogovirus Detection Assay For this Mengovirus multiplex assay, Cy5 is the Mengovirus probe fluorescent label and Texas Red (TxR) is the internal amplification control (IAC) probe fluorescent label. 1. 2. Repeat all invalid samples, a sample is “invalid” if: a. b. c. d. The negative RT-qPCR control sample demonstrates positive results crossing the Cy5 or if the IAC is negative, The RT-qPCR positive control is negative for Mengovirus, The Mengovirus RT-qPCR is negative in any sample,
The average of the IAC Ct values for the sample replicates are more than 4.0 Cts greater than the Negative Control IAC Ct value, repeat the RT-qPCR assay using remaining RNA or RNA from a newly extracted tube with a 1 µl template in the RT-qPCR reaction in triplicate. If the 1 µl template reactions yield an average IAC Ct value greater than 4.0 Cts higher than the Negative Control IAC Ct value, repeat the sample analysis from the beginning using additional food sample. With the new sample, the concentrates will be split into 5 tubes (refer to Work Instructions) and complete RT-qPCR with 1 µl reactions in triplicate. Sample is “valid” and can be reported if: a. b. c. d. RT-qPCR negative control is negative for Mengovirus, RT-qPCR positive control is positive for Mengovirus, RT-qPCR is positive for Mengovirus in all spike matrices, Internal amplification control (IAC) is positive in all reactions and average of the IAC Ct values for sample is within 4.0 Ct of the Negative Control IAC Ct Value. Note: For Mengovirus, if the average of the IAC Ct values for the sample replicates is more than 4.0 Cts greater than the Negative Control IAC Ct value AND the corresponding sample is positive for norovirus and/or hepatitis A virus, the Mengovirus RT-qPCR does not have to be repeated. If norovirus or hepatitis A virus is detected in a sample that has inhibition present in the RT qPCR reaction and has log amplification, this sample does not need to be repeated for norovirus or hepatitis A virus RT-qPCR and would be considered positive. Repeating RT 79 qPCR reactions due to inhibition is to ensure that you do not have false negatives. Refer to figures in Appendix E for appropriate amplification curves. Supplemental Material for the Mengovirus Detection Assay Table H1. Mengovirus Buffer Mix Setup Reagents Volume (50 mM MgCl) DNase/RNase free H2O 1760 µl Volume (25 mM MgCl) 5X Buffer 1610 µl 1000 µl MgCl2 1000 µl 150 µl dNTPs 300 µl 200 µl

Appendix I: Murine Norovirus, Hepatitis A Virus, Norovirus Genogroup I, and Norovirus Genogroup II Detection Assays on the Smart Cycler

*Note: Always wear gloves and never wear the same gloves when going between master mix and samples. Assembly of master mix should be done in a master mix PCR hood or BSC hood that has been decontaminated with 10% Bleach solution or Hype-Wipes followed by 70% Ethanol, or similar product and UV irradiated for 20 min. Change gloves often and when exiting and/or reentering the hood. Always use aerosol resistant pipette tips for PCR. RT-qPCR Detection of Murine Norovirus on Smart Cycler Platforms RT-qPCR Assays
Outlined Murine Norovirus RT-qPCR for detection of murine norovirus on Smart Cycler. Primers, probes, and master mix preparation are found in Tables I1 and I2.
Murine Norovirus Protocol
Reverse transcription: 50 °C for 3000 sec Activation: 95 °C for 900 sec
45 cycles of: 95 °C for 15 sec, 55 °C for 20 sec, 62 °C for 60 sec with optics on Murine Norovirus Reaction Set-Up Smart Cycler
*Note: Always use aerosol resistant pipette tips for PCR. 1. 2. 3. Thaw primer solutions, probe solutions, and buffer mix and place them in 4 °C bench top cool block or on ice in master mix set up hood. Vortex reagents for 2-3 sec at setting 7-10, and then briefly centrifuge for 3-5 sec in a mini-centrifuge to settle the liquid to the bottom of the tube. Place in ice or bench top cooler. Keep enzyme mix in cooling block or on ice at all times, these enzymes should not be defrosted.
Prepare master mix for all sample and control reactions as listed in table 2. Keep all thawed components, reagents, controls, and master mixes in cooling block or on ice. *Note: Viral RNA templates should be added to reaction tubes in a designated area separate from location where master mixes are prepared. A negative and positive control should be added to each reaction set-up. 4. Proceed to hood/area or room where the template is added and thaw IAC RNA and sample RNA in the designated hood where the template is added. Briefly centrifuge the tubes 3-5 sec in microcentrifuge to settle the liquid at the bottom of the tube. Add appropriate volume of IAC, (0.2 µl/rxn) to master mix (keep cold); Vortex briefly & Pulse spin. 5. Add 22 µl master mix to each designated reaction tube or sample wells. 82 6. 7. Add 3 µl of sample template to three designated reaction tubes or sample wells.
Close reaction tubes or seal sample plate once sample and appropriate controls have been added, briefly spin to mix bring down reagents. Instrument Set-Up Smart Cycler 1. 2. Place reactions tubes in the Smart Cycler and create run. Make sure the appropriate dye set (FCTC25) and protocols are selected for each site.
Start run; the entire reaction time for this assay is approximately 3 hrs. Data Analysis Smart Cycler 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. For results analysis, default instrument settings will be used, except the threshold is set at 10 for all channels utilized.
On the Smart Cycler Instrument, set the following Analysis Settings for TxRed and Cy5 channels. Update analysis settings if they are changed before recording results.
Usage: Assay
Curve Analysis: Primary
Threshold Setting: Manual
Manual Threshold Fluorescence Units: 10.0
Auto Min Cycle: 5
Auto Max Cycle: 10
Valid Min Cycle: 3
Valid Max. Cycle: 60
Background subtraction: ON
Boxcar Avg. Cycles: 0
Background Min. Cycle: 5
Background Max. Cycle: 40 Max Cycles: 45 Any sample which crosses the threshold in the Cy5 (Ch. 4) channel will be demonstrate detection of MNV.
17. The IAC will report in Channel 3 (TxRed).
83 Murine Norovirus Data Analysis 1. 2. Repeat any “invalid samples”. Sample is “invalid” if: a. b. c. d. The RT-qPCR negative control demonstrates positive Ct results for MNV in Cy5 or if the IAC is negative (no Ct from TxRed), The RT-qPCR positive control is negative (no Ct from Cy5) for MNV,
The MNV RT-qPCR is negative (no Ct from Cy5) for any sample, The average of the IAC Ct values for the sample replicates are more than 4.0 Cts greater than the negative control IAC Ct value, repeat the RT- qPCR assay using remaining RNA or RNA from a newly extracted tube with a 1 µl template in the RT-qPCR reaction in triplicate. If the 1 µl template reactions yield an average IAC Ct values greater than 4.0 Cts higher than the Negative Control IAC Ct value, repeat the sample analysis from the beginning using additional food sample. With the new sample, the concentrates will be split into 5 tubes (refer to Work Instructions) and complete RT-qPCR with 1 µl reactions in triplicate. Sample is “valid” and can be reported if: a. b. c. d. RT-qPCR negative control is negative for MNV, RT-qPCR positive control is positive for MNV, RT-qPCR is positive for MNV in all spiked matrices, Internal amplification control (IAC) is positive in all reactions and average of the IAC Ct values for sample is within 4.0 Cts of the negative control IAC Ct value. *Note: For MNV, if the average of the IAC Ct values for the sample replicates are more than 4.0 Cts greater than the Negative Control IAC Ct value AND the corresponding sample is positive for norovirus and/or hepatitis A virus, the MNV RT-qPCR does not have to be repeated. If norovirus or hepatitis A virus is detected in a sample that has inhibition present in the RT-qPCR reaction and has log amplification, this sample does not need to be repeated for norovirus or hepatitis A virus RT-qPCR and would be considered positive. Repeating RT-qPCR reactions due to inhibition is to ensure that you do not have false negatives.
84

Table I1. Primer and Probe Sequences for MNV and Internal Amplification Control

Identification Primers Location# MNVR@ 5’ CAC AGA GGC CAA TTG GTA AA 3’ 6645-6626 MNVF 5’- TGC AAG CTC TAC AAC GAA GG -3’ 6520-6539 IC46Fa 5’- GAC ATC GAT ATG GGT GCC G-3’ N/A IC194Ra 5’- AAT ATT CGC GAG ACG ATG CAG -3’ N/A

MNVP Cy5- 5’ CCT TCC CGA CCG ATG GCA TC3’-IB-RQ* 6578-6594 IACP TxR – 5’ TCT CAT GCG TCT CCC TGG TGA ATG TG -IB RQ 3’ * N/A @ Hewitt, Rivera-Aban, Greening 2009
a Depaola, Jones, Woods et. al. 2010 Internal Amplification Control (IAC) primers and probes are covered by U.S. Patent Application 0060166232.

Based on accession no. JF320650

Table I2. Smart Cycler Amplification Reaction Components and Master Mix Volume for MNV

Reagent Initial Concentration Volume per 25 µl reaction Final Concentration RNase Free H20 11.8 µl - 5X OneStep RT-PCR Buffer 5X 5.0 µl 1X MgCl2 ~ 50 mM 0.75 µl 1.5 mM dNTP Mix 10 mM 1 µl 0.4 mM
MNVF 10 µM 0.50 µl 0.2 µM MNVR 10 µM 0.50 µl 0.2 µM IC 46F 10 µM 0.1875 µl 0.075 µM IC 194R 10 µM 0.1875 µl 0.075 µM MNVP 10 µM 0.25 µl 0.1 µM IACP 10 µM 0.375 µl 0.15 µM OneStep RT-PCR Enzyme Mix 1.00 µl
Superase·in 20 Units/µl 0.25 µl 5 Units Internal Amplification Control RNA *0.2 µl -

RNA 3 µl

RT-qPCR Detection of Hepatitis A Virus Smart Cycler Platform 85 RT-qPCR Assay Outlined Hepatitis A Virus RT-qPCR for detection of hepatitis A on Smart Cycler. Primers, probes, and master mix preparation are found in Tables I3-4. Hepatitis A Virus Protocol
Reverse transcription: 50 °C for 3000 sec Activation: 95 °C for 900 sec
50 cycles of: 95 °C for 10 sec, 53 °C for 25 sec, 64 °C for 70 sec with optics on Hepatitis A Virus Reaction Set-Up Smart Cycler *Note: Always use aerosol resistant pipette tips for PCR. 1. 2. 3. Thaw primer solutions, probe solutions, and buffer mix and place them in 4 °C bench top cool block or on ice in master mix set up hood. Vortex reagents for 2-3 sec at setting 7-10, and then briefly centrifuge for 3-5 sec in a mini-centrifuge to settle the liquid to the bottom of the tube. Place in ice or bench top cooler. Keep enzyme mix in cooling block or on ice at all times, these enzymes should not be defrosted. Prepare master mix for all sample and control reactions as listed in table 6. Keep all thawed components, reagents, controls, and master mixes in cooling block or on ice. *Note: Viral RNA templates should be added to reaction tubes in a designated area separate from location where master mixes are prepared. A negative and positive control should be added to each reaction set-up.
4. 5. 6. 7. Proceed to hood/area or room where the template is added and thaw IAC RNA and sample RNA in the designated hood where the template is added. Briefly centrifuge the tubes 3-5 sec in microcentrifuge to settle the liquid at the bottom of the tube. Add appropriate volume of IAC, (0.2 µl/rxn) to master mix (keep cold); Vortex briefly & Pulse spin. Add 22 µl master mix to each designated reaction tube or sample wells. Add 3 µl of sample template to three designated reaction tubes or sample wells.
Close reaction tubes once sample and appropriate controls have been added, briefly spin to mix bring down reagents. Instrument Set-Up Smart Cycler 86 1. 2. Place reactions tubes in the Smart Cycler and create run. Make sure the appropriate dye set (FCTC25) and protocols (see creating protocol) are selected for each site. Name the run with the assay, sample number, and analysts initials. Start run; the entire reaction time for this assay is approximately 3 hrs. Data Analysis Smart Cycler 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. For results analysis, default instrument settings will be used, except the threshold is set at 10 for all channels utilized.
On the Smart Cycler Instrument, set the following Analysis Settings for TxRed and Cy5 channels. Update analysis settings if they are changed before recording results.
Usage: Assay
Curve Analysis: Primary
Threshold Setting: Manual
Manual Threshold Fluorescence Units: 10.0
Auto Min Cycle: 5
Auto Max Cycle: 10
Valid Min Cycle: 3
Valid Max. Cycle: 60
Background subtraction: ON
Boxcar Avg. Cycles: 0
Background Min. Cycle: 5
Background Max. Cycle: 40 Max Cycles: 50 Any sample which crosses the threshold in the Cy5 (Ch. 4) channel will be demonstrate detection of HAV.
The IAC will report in Channel 3 (TxRed).
87

Table I3. Primer and Probe Sequences for HAV and Internal Amplification Control RNA

Identification Primers Locationc GAR2F 5’ ATA GGG TAA CAG CGG CGG ATA T 3’ 448-469 GAR1R 5’-CTC AAT GCA TCC ACT GGA TGA G-3’ 517-537 IC46Fa.b 5’GAC ATC GAT ATG GGT GCC G-3’ N/A IC194Ra,b 5’-AAT ATT CGC GAG ACG ATG CAG-3’ N/A Probes
GARP Cy5- 5’ AGA CAA AAA CCA TTC AAC GCC GGA GG 3’ -IB-RQ* 483-508 IACPa,b TxR –TCT CAT GCG TCT CCC TGG TGA ATG TG -IB RQ* N/A a Internal Amplification Control (IAC) primers and probes are covered by U.S. Patent Application 0060166232 b Depaola, Jones, Woods, et al. 2010. c Based on GenBank accession # M14707

Table I4. Smart Cycler Amplification Reaction Components and Master Mix Volume for HAV

Reagent Initial Concentration Volume per 25 µl reaction Final Concentration RNase Free H20 11.05 µl - 5X OneStep RT-PCR Buffer 5X 5.0 µl 1X MgCl2a 50 mM 0.75 µl 1.5 mM dNTP Mix 10 mM 1 µl 0.4 mM GAR2F 10 µM 0.75 µl 0.3 µM GAR1R 10 µM 0.75 µl 0.3 µM IC 46F 10 µM 0.1875 µl 0.075 µM IC 194R 10 µM 0.1875 µl 0.075 µM GARP 10 µM 0.5 µl 0.2 µM IACP 10 µM 0.375 µl 0.15 µM OneStep RT-PCR Enzyme Mix 1.00 µl
Superase·in 20 Units/µl 0.25 µl 5 Units Internal Amplification Control RNA b0.2 µl -

RNA 3 µl
aWith the addition of 1.5 mM MgCl, the final concentration per reaction is 4.0 mM MgCl. bAmount varies with concentration of IAC RNA. The amount of IAC template needs to be adjusted based on the prepared stock concentration to report Cycle threshold (Ct) of 20-25 when no inhibition is present in the reaction.

RT-qPCR Detection of Norovirus GI and GII on Smart Cycler Platforms 88 RT-qPCR Assay Outlined norovirus RT-qPCR for detection norovirus GI and GII on Smart Cycler. Primers, probes, and master mix preparation are found in Tables I5-6.
Norovirus Virus Protocol
Reverse transcription: 50 °C for 3000 sec Activation: 95 °C for 900 sec
50 cycles of: 95 °C for 10 sec, 53 °C for 25 sec, 62 °C for 70 sec with optics on Reaction Set-Up Smart Cycler Note: Always use aerosol resistant pipette tips for PCR. 1. 2. 3. Thaw primer solutions, probe solutions, and buffer mix and place them in 4 °C bench top cool block or on ice in master mix set up hood. Vortex reagents for 2-3 sec at setting 7-10, and then briefly centrifuge for 3-5 sec in a mini-centrifuge to settle the liquid to the bottom of the tube. Place in ice or bench top cooler. Keep enzyme mix in cooling block or on ice at all times, these enzymes should not be defrosted. Prepare master mix for all sample and control reactions as in Appendix F. Keep all thawed components, reagents, controls, and master mixes in cooling block. Note: Viral RNA templates should be added to reaction tubes in a designated area separate from location where master mixes are prepared. A negative and positive control should be added to each reaction set-up.
4. 8. 9. 10. Proceed to hood/area or room where the template is added and thaw IAC RNA and sample RNA in the designated hood where the template is added. Briefly centrifuge the tubes 3-5 sec in microcentrifuge to settle the liquid at the bottom of the tube. Add appropriate volume of IAC, (0.2 µl/rxn) to master mix (keep cold); Vortex briefly & Pulse spin. Add 22 µl master mix to each designated reaction tube or sample wells. Add 3 µl of sample template to three designated reaction tubes or sample wells. Close reaction tubes once sample and appropriate controls have been added, briefly spin to mix and bring down reagents. Instrument Set-Up Smart Cycler 89 1. 2. Place reactions tubes in the Smart Cycler and create run. Make sure the appropriate dye set (FCTC25) and protocols (see creating protocol) are selected for each site. Name the run with the assay, sample number, and analysts initials. Start run; the entire reaction time for this assay is approximately 3 hrs. Data Analysis Smart Cycler 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. For results analysis, default instrument settings will be used, except the threshold is set at 10 for all channels utilized.
On the Smart Cycler Instrument, set the following Analysis Settings for TxRed and Cy5 channels. Update analysis settings if they are changed before recording results.
Usage: Assay
Curve Analysis: Primary
Threshold Setting: Manual
Manual Threshold Fluorescence Units: 10.0
Auto Min Cycle: 5
Auto Max Cycle: 10
Valid Min Cycle: 3
Valid Max. Cycle: 60
Background subtraction: ON
Boxcar Avg. Cycles: 0
Background Min. Cycle: 5
Background Max. Cycle: 40 Max Cycles: 50 Any sample which crosses the threshold in the Cy5 (Ch. 4) channel will be demonstrate detection of norovirus GI, any sample which crosses the threshold in the Cy3 (Ch. 2) will demonstrate detection of norovirus GII.
The IAC will report in Channel 3 (TxRed).
Table I5. Primer and Probe Sequences for Norovirus and Internal Amplification Control RNA
90 Identification Primers Location COG1Ra,d 5’ CTT AGA CGC CAT CAT CAT TYA C 3’
COG2Ra,e 5350-5371 5’ TCG ACG CCA TCT TCA TTC ACA 3’ COG1Fa,d 5080-5100 5’ CGY TGG ATG CGN TTY CAT GA 3’
COG2Fa,e 5287-5306 5’ CAR GAR BCN ATG TTY AGR TGG ATG AG 3’ IC46Fb,c 5003-5028 5’GAC ATC GAT ATG GGT GCC G-3’ IC194Rb,c N/A 5’-AAT ATT CGC GAG ACG ATG CAG-3’ N/A Probes
COGPa,d Cy5- 5’ (TAO) AGA TYG CGA TCY CCT GTC CA 3’ -IB-RQ COGP1ba,d 5317-5336 Cy5- 5’ (TAO) AGA TCG CGG TCT CCT GTC CA 3’ -IB-RQ* COG2Pa,e 5317-5336 Cy3- 5’ TGG GAG GGC GAT CGC AAT CT 3’ -IB-RQ* IACPb,c 5048-5067 TxR –TCT CAT GCG TCT CCC TGG TGA ATG TG -IB RQ* a Kageyama et al., 2003, b Internal Amplification Control (IAC) primers and probes are covered by U.S. Patent Application 0060166232 c Depaola, Jones, Woods, et al. 2010. d Based on GenBank accession # KF039728 e Based on GenBank accession # EF684915 N/A

Table I6. Smart Cycler Amplification Reaction Components for Norovirus

Reagent Initial Concentration Volume per 25 µl reaction Final Concentration RNase Free H20 9.3 µl - 5X OneStep RT-PCR Buffer 5X 5.0 µl 1X MgCl2a 50 mM 0.75 µl 1.5 mM dNTP Mix 10 mM 1 µl 0.4 mM
COG1F 10 µM 0.75 µl 0.3 µM COG1R 10 µM 0.75 µl 0.3 µM COG2F 10 µM 0.75 µl 0.3 µM COG2R 10 µM 0.75 µl 0.3 µM IC 46F 10 µM 0.1875 µl 0.075 µM IC 194R 10 µM 0.1875 µl 0.075 µM COG1P 10 µM 0.25 µl 0.1 µM COG1Pb 10 µM 0.25 µl 0.1 µM COG2P 10 µM 0.25 µl 0.1 µM IACP 10 µM 0.375 µl 0.15 µM OneStep RT-PCR Enzyme Mix 1.00 µl
Superase·in 20 Units/µl 0.25 µl 5 Units Internal Amplification Control RNA b0.2 µl -

RNA 3 µl
a With the addition of 1.5 mM MgCl2, the final concentration per reaction is 4.0 mM MgCl2 b Amount varies with concentration of IAC RNA. The amount of IAC template needs to be adjusted based on the prepared stock concentration to report Cycle threshold (Ct) of 20-25 PCR cycles when no inhibition is present in the reaction. The required concentration was provided to each laboratory participating in the validation study.