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Validation of the TadpoleTM Campylobacter jejuni Real-Time PCR Identification Kit

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Background: The gene-based real-time PCR method for identification of Campylobacter jejuni is more simple, rapid and accurate than the traditional biochemical method. Objective: A performance validation of the TadpoleTM Campylobacter jejuni Real-Time PCR Identification Kit was performed. Method: The assay uses TaqMan Real-time PCR technology to amplify target genes from isolated colonies. Bacterial deoxyribonucleic acid (DNA) from inclusivity and exclusivity organisms cultured on Columbia Blood Agar, Campy-Cefex agar and modified Charcoal Cefoperazone Deoxycholate was extracted and analyzed on three instruments: Applied Biosystems (ABI) 7500 Fast, ABI StepOne Plus and Bio-Rad CFX96. Results: When 57 distinct strains of C. jejuni were tested for inclusivity, all 57 strains produced positive results on the three instruments. In exclusivity testing, all 35 strains of related organisms, including 7 non-target Campylobacter strains and other common species, produced negative results on the three instruments. The Independent Laboratory validation consisting of an inclusivity and exclusivity evaluation for 10 C. jejuni isolates and 10 nontarget Campylobacter isolates also showed 100% expected results on the three instruments. In addition, in robustness testing, small, deliberate changes to the assay parameters, including cell suspension turbidity, heat lysis time, and DNA template volume in the PCR reaction, did not affect the kit performance. Finally, the combined lot-to-lot and stability study on both the ABI 7500 Fast and the ABI StepOne Plus showed that the 11 C. jejuni strains and 5 nontarget Campylobacter strains can be correctly identified by the three independently manufactured, lots and it supported a shelf life of 9 months when stored at –20°C. Conclusions: The Tadpole method offers a rapid, accurate, and robust alternative for C. jejuni identification. Highlights: Rapid and accurate method to identify C. jejuni, which has a good robustness and high stability. It is flexible and offers the advantages of reduced labor and time saving.
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Document Type: Research Article

Affiliations: 1: Shanghai Customs, 13 Zhongshan East 1st Rd, Shanghai 200001, China 2: Shanghai Entry-Exit Inspection and Quarantine Bureau, Technical Center of Animal Plant and Food Inspection and Quarantine, 1208 Minsheng Rd, Shanghai 200135, China 3: Nanjing Agricultural University, College of Veterinary Medicine, 1 Weigang Rd, Nanjing 210095, China 4: Technology Center of Zhangjiagang Entry-Exit Inspection and Quarantine Bureau, 59 Renmin Middle Rd, Zhangjiagang 215600, China 5: Nanjing Xiaozhuang University, 41 Beiwei Rd, Nanjing 210017, China 6: Suzhou Tadpole Biotechnology Co., Ltd, 218 Xinghu St, Suzhou 215000, China 7: U.S. Food and Drug Administration, Center for Food Safety and Nutrition, 5001 Campus Dr, College Park, MD 20740 8: Brodsky Consultants, 73 Donnamora Crescent, Thornhill, ON L3T 4K6, Canada 9: Consultant, Association of Public Health Laboratories, 1488 Madison St, Denver, CO 80206

Publication date: May 1, 2019

This article was made available online on November 19, 2018 as a Fast Track article with title: "Validation of the TadpoleTM Campylobacter jejuni Real-Time PCR Identification Kit".

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  • The Journal of AOAC INTERNATIONAL publishes refereed papers and reviews in the fields of chemical, biological and toxicological analytical chemistry for the purpose of showcasing the most precise, accurate and sensitive methods for analysis of foods, food additives, supplements and contaminants, cosmetics, drugs, toxins, hazardous substances, pesticides, feeds, fertilizers and the environment available at that point in time. The scope of the Journal includes unpublished original research describing new analytical methods, techniques and applications; improved approaches to sampling, both in the field and the laboratory; better methods of preparing samples for analysis; collaborative studies substantiating the performance of a given method; statistical techniques for evaluating data. The Journal will also publish other articles of general interest to its audience, e.g., technical communications; cautionary notes; comments on techniques, apparatus, and reagents.
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