Assessment of commercial DNA clean-up kits for elimination of qPCR inhibitors in the detection of Cyclospora cayetanensis in cilantro.
Inhibited reactions have been occasionally observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using 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 clean-up treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA clean up kits [QIAquick® Purification kit from Qiagen (#1), One step™ PCR inhibitor removal by Zymo Research (#2), Nucleospin® Genomic DNA clean up XS from Macherey-Nagel (#3), DNA IQ™ system by Promega (#4) and DNeasy® Power Plant® Pro kit from Qiagen (#5)] to minimize qPCR inhibition using the US FDA validated BAM Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA clean up kits evaluated was able to reduce the qPCR internal amplification control (IAC) C T values to those considered to be normal for non-inhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both high oocysts level (200) and at low oocysts levels (10). Of the five kits compared, kits #1, #2 and #3 did not show significant differences in the detection of C. cayetanensis , while significant higher C T 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 clean-up commercial kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using BAM Chapter 19b method.
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