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Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification
Guillaume Roux1, Christophe Ravel2, Emmanuelle Varlet-Marie2
1Univ. Montpellier, and Laboratoire de Microbiologie, Centre Hospitalier Universitaire (CHU) of Nîmes, Nîmes, France.
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 (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.
Insights
Pathogen-specific amplification is more reliable than albumin gene amplification for detecting PCR inhibition in medical microbiology. Albumin qPCR frequently flags samples as inhibited, leading to potential misinterpretation of results.
Area of Science:
- Medical Microbiology
- Molecular Diagnostics
Background:
- PCR inhibition is a common issue in clinical microbiology, potentially causing false-negative results.
- Current methods for detecting PCR inhibition, such as pathogen-specific and human gene amplifications, lack standardization.
Purpose of the Study:
- To compare the effectiveness of pathogen-specific amplification versus albumin gene amplification for detecting PCR inhibition in microbiological quantitative PCR (qPCR) assays.
- To evaluate the reliability of albumin qPCR as an internal control for PCR inhibition in diverse sample matrices.
Main Methods:
- Quantitative PCR (qPCR) assays were performed on 3152 samples across various matrices.
- Analyzed cycle quantification (Cp) shifts (ΔCp) for both pathogen-specific targets and the human albumin gene.
- Assessed the agreement between inhibition detection using the two methods and evaluated the utility of Youden's index for threshold determination.
Main Results:
- Albumin qPCR identified 13.0% of samples as inhibited, while pathogen-specific amplification identified only 2.0%.
- Only 16 samples (0.5%) were flagged as inhibited by both methods, indicating poor concordance.
- Adequate Cp thresholds for albumin qPCR could not be established, even when accounting for sample matrix variations.
Conclusions:
- Albumin gene amplification is not a suitable method for detecting PCR inhibition in microbiological qPCR assays.
- The use of heterologous targets like the albumin gene to assess PCR inhibition in clinical microbiology should be discouraged.
- Pathogen-specific amplification offers a more reliable approach for monitoring PCR inhibition in diagnostic settings.
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