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Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification.

ABSTRACT: PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (?Cp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0%), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden's index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.

SUBMITTER: Roux G

PROVIDER: S-EPMC6764677 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification.

Roux Guillaume G Ravel Christophe C Varlet-Marie Emmanuelle E Jendrowiak Rachel R Bastien Patrick P Sterkers Yvon Y

PloS one 20190927 9

PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qP ...[more]

PMID: 31560697

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Project description:IntroductionSchistosoma haematobium infection afflicts about 150 million people in 53 countries in Africa and the Middle East. In many endemic areas, S. haematobium is sympatric with Schistosoma bovis, Schistosoma mattheei, Schistosoma curassoni, Schistosoma intercalatum and Schistosoma magrebowiei, its closely related species. In addition, they also develop in the same intermediate snail hosts. Since these schistosome species often infect snails inhabiting the same bodies of water, examining cercariae or infected snails for estimating transmission of S. haematobium is always confounded by the need to differentially identify S. haematobium from these other species. Recently, differentiating S. haematobium by polymerase chain reaction (PCR) from S. bovis, S. mattheei, S. curassoni and S. intercalatum, but not from S. magrebowiei was reported. However, to be able to evaluate residual S. haematobium transmission after control interventions in areas where S. haematobium may be sympatric with S. magrebowiei, a differential tool for accurate monitoring of infected snails is needed.Materials and methodsThus in this study, we developed a new PCR assay using a pair of primers, ShND-1/ShND-2, to amplify a target sequence of 1117 bp (GenBank accession number KF834975) from S. haematobium mitochondrion complete genome (GenBank accession number DQ157222). Sensitivity of the assay was determined by PCR amplification of different concentrations of S. haematobium gDNA serially diluted from 10ng to 0.1pg. For assay specificity, different concentrations of gDNA from S. haematobium and the other schistosome species, 20 positive urine samples and five controls as well as 20 infected snails were subjected to PCR amplification, while some of the PCR products were sequenced.ResultsThe assay detected up to 1pg of S. haematobium gDNA, while a differential identification of S. haematobium DNA content from other closely related species was achieved when applied to urine and naturally infected snails. When a protein-protein blast search was carried out using Blastp, the amplified sequence was found to encode a protein that shows a 100% similarity with S. haematobium nicotinamide adenine dinucleotide dehydrogenase subunit 3 (GenBank accession number YP_626524.1).ConclusionThe PCR assay was sensitive, specific and was able to successfully differentiate S. haematobium from S. magrebowiei, in addition to its other closely related animal infective schistosome species.

Dataset Information

Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification.

Publications

Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification.

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