开发并评估一种改良且特异的实时 PCR 检测方法，使用线粒体引物（Mit1C）检测农产品中的环孢子虫。

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.

机构信息

U.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, USA.

U.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, USA.

出版信息

J Food Prot. 2023 Feb;86(2):100037. doi: 10.1016/j.jfp.2022.100037. Epub 2023 Jan 4.

DOI:10.1016/j.jfp.2022.100037

PMID:36916572

Abstract

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.

摘要

用于检测食源原虫寄生虫环孢子虫的监管方法必须具有特异性和敏感性。为此，我们设计并评估了（在一次实验室验证中）一种用于实时 PCR 检测农产品中环孢子虫的新型改良引物/探针组合（Mit1C）。新开发的引物/探针组合针对环孢子虫线粒体基因组的保守区域，该区域在其他密切相关的生物体中存在差异。该引物/探针组合在计算机上和使用几种实时 PCR 试剂盒和聚合酶进行了评估，针对包含各种环孢子虫卵囊以及来自其他相关 Cyclospora 种和密切相关寄生虫的 DNA 的包容性/排他性面板进行了评估。新的引物/探针组合仅扩增环孢子虫，从而证明了特异性。通过用不同数量（200 和 5）的环孢子虫卵囊人工污染芫荽、覆盆子和生菜来评估敏感性。在污染的芫荽、覆盆子和生菜样本（分别为 75%、67.7%和 50%）中，均能检测到少至 5 个卵囊，所有未接种样本和无模板实时 PCR 对照均为阴性。改良的引物/探针组合有望成为在农产品中特异性检测环孢子虫的有效分析工具。