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


Autoři: Guillaume Roux aff001;  Christophe Ravel aff002;  Emmanuelle Varlet-Marie aff002;  Rachel Jendrowiak aff002;  Patrick Bastien aff002;  Yvon Sterkers aff002
Působiště autorů: Univ. Montpellier, and Laboratoire de Microbiologie, Centre Hospitalier Universitaire (CHU) of Nîmes, Nîmes, France aff001;  Univ. Montpellier, Centre Hospitalier Universitaire (CHU) of Montpellier, Dept. of Parasitology-Mycology, Research Unit MiVEGEC, CNRS, IRD, Montpellier, France aff002
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0219276

Souhrn

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 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.

Klíčová slova:

Albumins – Cerebrospinal fluid – DNA extraction – Polymerase chain reaction – Toxoplasma – White blood cells – Pneumocystis – Amniotic fluid


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