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Genes 2017, 8(11), 332; doi:10.3390/genes8110332

Gene-Based Pathogen Detection: Can We Use qPCR to Predict the Outcome of Diagnostic Metagenomics?

National Food Institute, Technical University of Denmark, Kemitorvet, Building 204, DK-2800 Kgs. Lyngby, Denmark
Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark
Author to whom correspondence should be addressed.
Received: 29 September 2017 / Revised: 2 November 2017 / Accepted: 14 November 2017 / Published: 20 November 2017
(This article belongs to the Special Issue Genetics and Genomics of Foodborne Pathogens)
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In microbial food safety, molecular methods such as quantitative PCR (qPCR) and next-generation sequencing (NGS) of bacterial isolates can potentially be replaced by diagnostic shotgun metagenomics. However, the methods for pre-analytical sample preparation are often optimized for qPCR, and do not necessarily perform equally well for qPCR and sequencing. The present study investigates, through screening of methods, whether qPCR can be used as an indicator for the optimization of sample preparation for NGS-based shotgun metagenomics with a diagnostic focus. This was used on human fecal samples spiked with 103 or 106 colony-forming units (CFU)/g Campylobacter jejuni, as well as porcine fecal samples spiked with 103 or 106 CFU/g Salmonella typhimurium. DNA was extracted from the samples using variations of two widely used kits. The following quality parameters were measured: DNA concentration, qPCR, DNA fragmentation during library preparation, amount of DNA available for sequencing, amount of sequencing data, distribution of data between samples in a batch, and data insert size; none showed any correlation with the target ratio of the spiking organism detected in sequencing data. Surprisingly, diagnostic metagenomics can have better detection sensitivity than qPCR for samples spiked with 103 CFU/g C. jejuni. The study also showed that qPCR and sequencing results may be different due to inhibition in one of the methods. In conclusion, qPCR cannot uncritically be used as an indicator for the optimization of sample preparation for diagnostic metagenomics. View Full-Text
Keywords: zoonoses; testing; feces; DNA sequencing; bioinformatics zoonoses; testing; feces; DNA sequencing; bioinformatics
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Andersen, S.C.; Fachmann, M.S.R.; Kiil, K.; Møller Nielsen, E.; Hoorfar, J. Gene-Based Pathogen Detection: Can We Use qPCR to Predict the Outcome of Diagnostic Metagenomics? Genes 2017, 8, 332.

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