Terminology

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Overview

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Chapter 19b

Introduction

Cyclospora cayetanensis is a protozoan parasite that causes a human diarrheal disease called cyclosporiasis (1). Infected individuals shed spherical (8-10 μm in diameter) unsporulated oocysts into the environment with feces. Person-to-person transmission is considered unlikely because oocysts require a week or longer in the environment to sporulate and become infective. C. cayetanensis oocysts are not culturable, which makes the application of viability assays impractical. According to the latest surveillance data, 2,272 cases of cyclosporiasis were reported from 40 states in 2023 without travel history outside the US indicating that they were acquired domestically. Illnesses and outbreaks caused by C. cayetanensis are mainly associated with the consumption of fresh produce such as herbs, berries, and leafy greens (2).

The present FDA BAM Chapter 19b method for detection of C. cayetanensis in fresh produce was validated under the FDA Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods and Feeds, third Edition (4). The method includes three steps:

1. Produce wash procedure;
2. Isolation of DNA from produce wash, and
3. Detection by qPCR amplification based on the detection of the C. cayetanensis mitochondrial Cox3 gene (Mit1C qPCR).

The Mit1C target provides a region on the mitochondrial genome of C. cayetanensis that is specific to C. cayetanensis based on current multiple sequence alignment using BLAST searches against C. cayetanensis and other genera/species (e.g., Eimeria spp. and Isospora spp.) in the Apicomplexa phylum. The real-time PCR was optimized using Prime TimeTM Gene Expression master mix from Integral DNA technologies (IDT) and the method was developed and validated for use in the ABI 7500 Fast platform. The method was validated by a single validation study that included exclusivity, inclusivity, and Mit1C qPCR detection in C. cayetanensis inoculated food matrices in three commodities (raspberries, cilantro and Romaine lettuce) compared to the previous 18S qPCR method (4). Then, the new method was validated for Romaine lettuce in a multi-laboratory validation study (5) approved by the Microbiology Methods Validation Subcommittee in November 2024. A matrix extension was completed for detection of C. cayetanensis on fresh basil and parsley and approved by the Microbiology Methods Validation Subcommittee in March 2026.

The validation results (single laboratory validation and multi-laboratory validation) are available in the study publications (reference 4 and reference 5).

Molecular Detection of Cyclospora cayetanensis in Fresh Produce Using Real-Time PCR

A. Produce Washing and DNA Extraction Procedures

This analytical procedure provides steps for isolation of C. cayetanensis oocysts from produce by washing and subsequent DNA template preparation from produce washes.

A4. Wash Procedure for Fresh Produce Samples

The standard wash procedure described below is optimal for leafy greens and herbs or sturdy vegetables. It is important to take note of modifications which are described in the protocol and required for fragile matrices such as raspberries which release larger amounts of debris or pectin if not handled carefully.

Centrifugation of wash solution is performed as described below using a swinging bucket rotor with a brake setting of 6 (on a scale of 0-9) for deceleration.

A.

Weigh produce to be analyzed in a BagPage®+ filter bag (25 grams of fresh produce or 50 grams of fresh berries).

B.

Add 100 mL of 0.1% Alconox to the produce sample in the filter bag. Lay the bottom portion of the bag flat on the bench with the opening edge folded up against a vertical support (Figure 1a). Bags containing leafy greens or sturdy vegetables (but not those containing fragile matrices such as berries) should be massaged gently with fingertips up the length of the bag a few times to remove most of the air. Bags containing berries should be sealed without massaging and without removing air. Seal the bags with the bag clips.

C.

Lay sealed bags containing leafy greens flat in a tray on a rocker platform with the sealed opening edges propped up against the sides of the tray (Figure 1b) to control potential leaks. The bags are stacked on top of one another in order to accommodate all. Agitate for 30 minutes at 85 rpm (Stovall Belly Dancer set at 7.0 with maximum tilt) at room temperature, inverting the bags after 15 minutes. Bags containing berries are stood upright in the tray (Figure 1c) to achieve better coverage of matrix with wash solution and slowly rocked on a platform rocker for 30 minutes at low speed (e.g., 12 rocks per minute using a Hoefer Red Rocker set at 5.0).

D.

Open bags and transfer the supernatant from the filtrate side of each BagPage®+ filter bag into two labeled 50 mL conical centrifuge tubes using serological pipets.

E.

Isolate wash debris containing oocysts by centrifugation in a swinging bucket rotor for 20 minutes at 2,000 × g with a brake setting of 6 (on a scale of 0-9) for deceleration.

F.

During the centrifugation, add an additional 100 mL of 0.1% Alconox to the produce in each filter bag and tip the bag from side to side three to four times to rinse the food and bag surfaces. Lean the bags containing the produce and rinse solution against a vertical surface until needed in step A4H.

G.

After the centrifugation use a short glass Pasteur pipet or regular [non-filtered] pipets tips at the end of [non-filtered] serological pipettes connected with tubing to a filter flask and house vacuum to aspirate all but approximately 4 mL of the supernatant from each of the 50 mL tubes to waste without disturbing the wash debris pellets.

H.

Transfer the rinse from the filtrate side of each BagPage®+ filter bag to the corresponding two 50 mL conical tubes containing the first wash debris pellets from step A4G. Centrifuge for 20 minutes at 2,000 × g to pellet the combined wash and rinse debris. After the centrifugation aspirate all but approximately 4 ml of the supernatant from each of the 50 mL tubes to waste without disturbing the pellets.

I.

Pool each pair of wash debris pellets by resuspending with a 5 mL serological pipet in the residual wash liquid and transferring to a single 15 mL conical centrifuge tube. Rinse the pair of empty 50 mL tubes sequentially with 2 mL of dH2O and add to the contents of the 15 mL tube. Centrifuge for 20 minutes at 2,000 × g to pellet the debris. After the centrifugation aspirate all but approximately 1 mL of the supernatant from the 15 mL tube. Resuspend the debris pellet in the 15 ml tube in the residual supernatant and transfer to a single empty 2 mL FastPrep lysing tube (without beads). Rinse the empty 15 mL tube with 0.4 mL of dH2O and add to the contents of the 2 mL tube. If the total volume of the resuspended pellet and tube rinse exceeds the capacity of the 2 mL tube, centrifuge a portion in the 2 mL FastPrep tube at 14,000 × g for 4 minutes, aspirate the supernatant without disturbing the pellet, and then add the remaining resuspended pellet and tube rinse.

J.

Centrifuge the 2 mL FastPrep tubes containing wash debris from step A4I at 14,000 × g for 4 minutes. Aspirate all but approximately 100-200 µL of the supernatant without disturbing the pellet. Note: If a pooled debris pellet sample is greater than approximately 850 µL, the sample must be split into two 2 mL FastPrep lysing tubes.

K.

Store at 4°C overnight or proceed as described in Section A5 for isolation of DNA immediately.

Detection of Cyclospora cayetanensis in Produce

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A5. Isolation of DNA from Fresh Produce Wash Debris Pellets using the FastDNA® SPIN Kit for Soil

DNA is extracted from produce washes in a Biological Safety Cabinet using the FastDNA SPIN Kit for Soil following the modified instructions detailed below.

Prepare the following items for the DNA extraction procedure before beginning:

• Add 100 mL of 100% ethanol to SWES-M bottle of wash solution*
• Lysing Matrix E tubes (containing beads)*
• 2 mL microcentrifuge tubes
• 15 mL Falcon tubes containing 1 mL resuspended Binding Matrix*
• Spin Filters in catch tubes*
• Second set of catch tubes*

*Items provided in the FastDNA® SPIN Kit for Soil.

Modified FastDNA Spin Extraction Protocol

A.

Assemble the samples to be extracted from wash procedure step A4J and add an empty FastPrep tube as a DNA extraction control.

B.

Carefully transfer into each tube in step A5A the beads from a Lysing Matrix E tube (supplied with the FastDNA Spin Kit).

C.

Add 122 µL MT buffer (FastDNA protocol step 3).

D.

Add 978 µL (or less) Sodium Phosphate Buffer (FastDNA protocol step 2) to the maximum fill height; leave at least 1.0 cm of air space at the top of the tube to allow for efficient bead-beating (see Figure 2). Screw on cap securely. Figure 2

E.

Transfer the samples to a FastPrep-24 bead beater and homogenize at a setting of 6.5 m/s (approximately 4000 rpm) for 60 seconds. Immediately remove the sample holder containing the tubes from the instrument and place on ice for 3 minutes. Return the sample holder to the bead beater and repeat the bead beating and the incubation on ice as above.

F.

Remove the tubes from the sample holder and centrifuge at 14,000 × g for 15 minutes (FastDNA protocol step 5).

G.

Transfer the supernatant to a clean 2 mL tube. Add 250 µL PPS and mix by inverting by hand 10 times (FastDNA protocol step 6).

H.

Centrifuge at 14,000 × g for 5 minutes (FastDNA protocol step 7) then transfer supernatant to a clean 15 mL Falcon tube containing 1.0 mL of resuspended Binding Matrix.

I.

Place on a rotator or invert by hand for 2 minutes and then allow silica matrix to settle for 3 minutes (FastDNA protocol step 9). Centrifuge the 15 mL tubes briefly at 1000 × g for 1 minute in a swinging bucket rotor.

J.

Remove and discard a total of 1.4 mL of supernatant from each tube in two 700 µL aliquots.

K.

Resuspend the matrix in the remaining supernatant and transfer approximately 700 µL to a SPIN Filter in a catch tube. Centrifuge at 14,000 × g for 1 minute (FastDNA protocol step 11). Empty the catch tube and add any remaining resuspended mixture to the SPIN Filter and spin as before. Empty the catch tube again.

L.

Add 500 µL prepared SWES-M to each filter. Gently resuspend each by pipetting up and down (FastDNA protocol step 12).

M.

Centrifuge at 14,000 × g for 1 minute. Empty catch tube and replace (FastDNA protocol step 13).

N.

Centrifuge at 14,000 × g for 2 minutes to dry the matrix. Discard the catch tube and replace with a new catch tube (FastDNA protocol step 14).

O.

Air dry the filter for 5 minutes at room temperature (FastDNA protocol step 15).

P.

Add 75 µL DES to the matrix in the spin filter. Resuspend the Binding Matrix by gently stirring with a small pipet tip. Incubate for 5 minutes in a heat block at 55°C. (FastDNA protocol step 16).

Q.

Centrifuge at 14,000 × g for 1 minute to recover the eluted DNA and then discard the SPIN Filter (FastDNA protocol step 17).

R.

Store the DNA samples at 4°C for up to 2 days or at -20 or -80°C for longer term prior to performing the Real-Time PCR detection step described below.

B. Cyclospora cayetanensis Real-Time PCR Detection Method

The real-time PCR protocol described below offers several advantages including increased specificity. In addition, the use of a real-time PCR method minimizes laboratory environment contamination by amplicons commonly associated with conventional nested PCR. The method was developed for the Applied Biosystems 7500 Fast Real-Time PCR System for detection of C. cayetanensis in food samples and is based on a new target on the mitochondria of C. cayetanensis. The multi-copy nature of mitochondrial genomes in cells provides higher target sequence concentration than nuclear sequences for molecular methods, such as PCR and NGS. The real-time PCR assay is a duplex reaction which targets the C. cayetanensis multicopy mitochondrial gene and uses an internal amplification control to monitor for potential matrix derived inhibition of the reaction. The method also provides a synthetic positive control, allowing sequence verification to identify false positives stemming from inadvertent laboratory contamination.

B4. Reaction Setup and Execution

A primer/probe mix must be prepared for the C. cayetanensis target reaction and for the IAC target reaction. Briefly mix and centrifuge all reagents to resuspend and bring down contents before assembling mixes.

B4A. Primer/Probe Mixes: (store at -20°C in dark)

Table 5. 20X Mit1C Pr/Pro (12 µM each primer, 6 µM probe)

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Table 6. 20X synIAC Pr/Pro (5 µM each primer, 5 µM probe, 2E5 copies synIAC target)

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B4B. Real-Time PCR reaction mix for 20 µl volume reactions

All samples and all controls are always run in triplicate

Briefly mix and centrifuge all reagents to resuspend and bring down contents before assembling reaction mix. The master mix formula below is sufficient to run one (1) replicate of one sample. For each qPCR experimental run, prepare sufficient reaction mix to run the no template control (NTC), the positive control, and samples all in triplicate. Calculate the total number of replicates being run (N) in one experiment and prepare a volume of master mix between N+1 and N+3 to assure sufficient reagents for all replicates.

Table 7. Reaction mix for 20 microliters volume reactions

TODO

Aliquot 18 µL of reaction mix to each reaction well or tube.

Add 2.0 µL of sample or appropriate controls to each reaction plate well or tube (see Section C below).

B4C. Samples and Controls:

Table 8. Sample and Controls volume

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B4D.

Always briefly vortex and centrifuge controls and samples before adding to reaction wells or tubes.

ALL UNKNOWN SAMPLES ARE TO BE ANALYZED AT 1X AND A ¼ DILUTION IN THE SAME INITIAL EXPERIMENTAL RUN (Controls are not tested at a ¼ dilution). Prepare diluted samples following instructions below.

B4E. ¼ Sample Dilution Protocol:

Transfer 2.5 µL of sample to a clean microcentrifuge tube containing 7.5 µL of TE. Mix well and centrifuge briefly.

B4F.

_After the addition of samples and controls to reaction wells or tubes, seal the plate with the adhesive film or seal the tube strips with cap strips and centrifuge at 400 × g for 30 seconds.

Run the plate or tube strips in the ABI 7500 Fast Real-Time PCR Instrument using a pre-defined protocol template and run method as described in Sections I and J below.

B4G.

_Real-Time PCR Cycling Protocol Templates for the ABI 7500 Fast Instrument:

Prior to initiating a run each laboratory should define a protocol template as described in Appendix 5 for ABI Fast instruments running v2.0 or 2.3 software or Appendix 6 for instruments running v1.4 software.

B4H.

_Run Methods on the ABI 7500 Fast Instrument:

Follow the run method detailed in Appendix 5 for ABI Fast instruments running v2.0 or 2.3 software or Appendix 6 for instruments running v1.4 (or any v1.x) software. The following software analysis settings are applied to data when instructions for protocol templates and run methods in Appendices 5 or 6 are followed:

• Manual Threshold = 0.05 (C. cayetanensis target; Mit1C)
• Manual Threshold = 0.05 (IAC target)
• Auto Baseline

Document the experimental run by saving the run file and an exported results data file according to the instructions in Appendices 5 or 6. Print the exported results data file and include with analytical worksheet packet.

B4I. Interpretation of Results:

Positive Samples

Samples are only considered Positive for the presence of C. cayetanensis if, on initial testing or re-testing, one (1) or more sample replicates produces a smooth exponential/sigmoidal amplification signal with Ct ≤ 38.0 for the C. cayetanensis mitochondrial target (Mit1C) reaction and the IAC target reaction is either Negative or Positive.

ONLY ONE REPLICATE OF AN UNKNOWN SAMPLE OR ¼ DILUTION OF THAT SAMPLE NEEDS TO BE POSITIVE FOR THE C. cayetanensis Mit1C TARGET IN ORDER TO CONSIDER A SAMPLE POSITIVE.

Samples for Further Analysis

Any sample producing a smooth exponential/sigmoidal amplification signal in one (1) or more replicates for the C. cayetanensis Mit1C target reaction crossing the threshold with Ct(s) > 38.0 and the IAC target reaction is either Negative or Positive. Questions about the protocol should be directed to the Chapter POCs.

Negative Samples

If a sample C. cayetanensis Mit1C target reaction produces all replicates with undetermined Ct or no replicate with Ct ≤ 38.0 and sample IAC target reaction produces an average Ct value which is not more than 3 cycles higher compared to the NTC:

• Sample is NEGATIVE, no further action.

Invalid Results

If one (1) or more replicates of the NTC sample or the DNA extraction control sample Mit1C target reactions produces a positive result crossing the threshold, the experimental run is invalid and must be repeated.

If after repeating an invalid experimental run, the DNA extraction control repeatedly produces a positive result and the NTC sample is negative, the DNA extraction procedure was likely contaminated. The DNA extraction procedure must be repeated for the entire set of samples using additional washed food samples if available.

If one (1) or more replicates of the positive control sample for C. cayetanensis Mit1C is undetermined, the experimental run is invalid and must be repeated.

Inconclusive Results

If on initial test (or after re-test if required), a sample produces no replicate with Ct ≤ 38.0 (for C. cayetanensis mitochondrial Mit1C target), and sample IAC target is undetermined or produces an average Ct value more than 3 cycles higher compared to the NTC:

• Sample is INCONCLUSIVE, contact the Chapter POCs.

Flowchart Interpretation of results in produce samples after analysis by real-time PCR for Cyclospora cayetanensis detection.

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Appendix 1. Alconox® Produce Wash Solution Recipe

Prepare 1.0% Alconox® stock solution

Dissolve 10 grams Alconox® in 1-liter distilled water.

Prepare and keep 1.0% Alconox for up to two weeks per manufacturer’s recommendations.

Prepare 0.1% Alconox® for wash solution

Mix 200 mL 1.0% Alconox® stock with 1800 mL distilled water.

Prepare 0.1% Alconox fresh daily when needed.

Appendix 2. Tris EDTA (TE) pH 7.5 buffer (10mM Tris, 0.1mM EDTA)

Table 1: Tris EDTA components

Appendix 3. Preparation of the Internal Amplification Control (IAC) Target Working Solution.

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Appendix 4. Preparation of the Positive Control Target Working Solution.

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Appendix 5. ABI 7500 Fast v2.0 or 2.3 Method

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(A) Define a Run Template Using Software v2.0 or 2.3 on the ABI 7500 Fast Instrument

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(B) Run Method Using Software v2.0 or 2.3 on the ABI 7500 Fast Instrument

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(C) Analysis Using Software v2.0 or 2.3 on the ABI 7500 Fast Instrument

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Appendix 6. ABI 7500 Fast v1.4 Method.

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(A) Define Protocol as a Template

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(B) Create Detectors: Mit1C & IAC (internal amplification control)

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(C) Start a new Run

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(D) Results

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