Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland, College Park, Maryland 20740.
U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Applied Research and Safety Assessment, 8301 Muirkirk Road, Laurel, Maryland 20805, USA.
J Food Prot. 2020 Nov 1;83(11):1863-1870. doi: 10.4315/JFP-20-139.
Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration-validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method.
在使用实时定量 PCR（qPCR）检测环孢子虫时，偶尔会观察到芫荽样本的抑制反应。PCR 反应（包括 qPCR）可能会部分或完全受到抑制，从而导致灵敏度降低或出现假阴性结果。如果发生抑制，这意味着需要对提取的 DNA 进行额外的纯化或清洗处理，以去除分子检测前的抑制剂。我们的目标是评估五种商业 DNA 清洗试剂盒（Qiagen 的 QIAquick 纯化试剂盒[试剂盒 1]、Zymo Research 的 OneStep PCR 抑制剂去除试剂盒[试剂盒 2]、Macherey-Nagel 的 NucleoSpin 基因组 DNA 清洗 XS 试剂盒[试剂盒 3]、Promega 的 DNA IQ 系统试剂盒[试剂盒 4]和 Qiagen 的 DNeasy PowerPlant pro 试剂盒[试剂盒 5]）的性能，以使用美国食品和药物管理局验证的细菌分析手册（BAM）第 19b 章方法最小化 qPCR 抑制，用于检测芫荽样本中的环孢子虫，样本中含有土壤。评估的五种商业 DNA 清洗试剂盒均能够将 qPCR 内部扩增对照的循环阈值值降低到被认为是非抑制样本的正常水平，从而可以在芫荽样本中清晰地解释高卵囊水平（200 个卵囊）和低卵囊水平（10 个卵囊）的结果。在比较的五个试剂盒中，试剂盒 1、2 和 3 在检测环孢子虫方面没有显著差异，而在接种 200 和 10 个卵囊的样本中，试剂盒 4 和/或 5 的循环阈值值显著升高，表明目标 DNA 的回收较低（P < 0.05）。这项比较研究提供了使用商业清洗试剂盒的建议，当使用 BAM 第 19b 章方法检测芫荽样本中的环孢子虫时，如果观察到抑制，可以采用这些建议。
