Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce
Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-02-01
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| Series: | Journal of Food Protection |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0362028X22119526 |
| _version_ | 1827908235295719424 |
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| author | Kannan V. Balan Mark Mammel David Lipman Uma Babu Lisa M. Harrison Sonia Almeria Mauricio Durigan Susan R. Leonard Hyein Jang Solomon Gebru John Grocholl Socrates Trujillo Kelli L. Hiett Steve Musser |
| author_facet | Kannan V. Balan Mark Mammel David Lipman Uma Babu Lisa M. Harrison Sonia Almeria Mauricio Durigan Susan R. Leonard Hyein Jang Solomon Gebru John Grocholl Socrates Trujillo Kelli L. Hiett Steve Musser |
| author_sort | Kannan V. Balan |
| collection | DOAJ |
| description | Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce. |
| first_indexed | 2024-03-13T01:19:05Z |
| format | Article |
| id | doaj.art-09a873c5f9fe4f329cbedf6111d0a4a4 |
| institution | Directory Open Access Journal |
| issn | 0362-028X |
| language | English |
| last_indexed | 2024-03-13T01:19:05Z |
| publishDate | 2023-02-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Food Protection |
| spelling | doaj.art-09a873c5f9fe4f329cbedf6111d0a4a42023-07-05T05:13:53ZengElsevierJournal of Food Protection0362-028X2023-02-01862100037Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in ProduceKannan V. Balan0Mark Mammel1David Lipman2Uma Babu3Lisa M. Harrison4Sonia Almeria5Mauricio Durigan6Susan R. Leonard7Hyein Jang8Solomon Gebru9John Grocholl10Socrates Trujillo11Kelli L. Hiett12Steve Musser13U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Virulence Assessment, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Molecular Biology, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of the Center Director, College Park, MD 20740, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Virulence Assessment, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Virulence Assessment, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Virulence Assessment, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Virulence Assessment, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Molecular Biology, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Virulence Assessment, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Virulence Assessment, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Virulence Assessment, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Division of Virulence Assessment, Laurel, MD 20708, USAU.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, Office of the Director, Laurel, MD 20708, USA; Corresponding author.U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of the Center Director, College Park, MD 20740, USARegulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.http://www.sciencedirect.com/science/article/pii/S0362028X22119526Cyclospora cayetanensisFoodborneLeafy greensMitochondriaParasiteReal-time PCR |
| spellingShingle | Kannan V. Balan Mark Mammel David Lipman Uma Babu Lisa M. Harrison Sonia Almeria Mauricio Durigan Susan R. Leonard Hyein Jang Solomon Gebru John Grocholl Socrates Trujillo Kelli L. Hiett Steve Musser Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce Journal of Food Protection Cyclospora cayetanensis Foodborne Leafy greens Mitochondria Parasite Real-time PCR |
| title | Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce |
| title_full | Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce |
| title_fullStr | Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce |
| title_full_unstemmed | Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce |
| title_short | Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce |
| title_sort | development and single laboratory evaluation of a refined and specific real time pcr detection method using mitochondrial primers mit1c for the detection of cyclospora cayetanensis in produce |
| topic | Cyclospora cayetanensis Foodborne Leafy greens Mitochondria Parasite Real-time PCR |
| url | http://www.sciencedirect.com/science/article/pii/S0362028X22119526 |
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