How do polymerase inhibitors work

Samples without Cp alb value due to a flat amplification curve were excluded from the graph and the correlation. The Dept. These laboratory-developed qPCR assays may therefore be considered as robust and finely optimized. LightCycler real-time PCR amplification curves in a serial dilution assay. During the year , samples were referred to the laboratory as part of the routine activity Fig 2 : samples were analysed by Toxoplasma -qPCR, by Pneumocystis -qPCR, and 37 samples were analysed by both methods S1 Table.

Two samples analysed by Toxoplasma -qPCR were excluded from this study, one due to cancellation of the analysis and another due to identity problem S1 and S2 Figs. The percentage of positive samples for the Toxoplasma -qPCR was 1. For Pneumocystis -qPCR, the percentage of positive sample was 7. Of the analysed samples, 62 2. However, this percentage varied from 1. The remaining sample was a cord blood tested for the presence of Toxoplasma for which PCR remained inhibited despite the dilution, showing flat amplification curves for both the target- and the albumin-specific inhibition controls before and after tenfold dilution.

No additional dilution was applied to this sample due to the risk of reduced sensitivity. Interestingly, blood samples, which are reported elsewhere [ 26 ] to be prone to PCR inhibition did not exhibit the highest inhibition rate in our study.

Samples like sputum or placenta were also prone to PCR inhibition, probably because it is difficult to optimize a DNA extraction protocol for heterogeneous matrixes with highly different structures and properties from one sample to another. These discrepant results illustrate that PCR inhibition measurement is a problem of variable importance in routine practices, probably depending on the ability of extraction methods to remove inhibitors, on the susceptibility of PCR methods to inhibitors and on the performance of PCR inhibition detection.

Thus, i the albumin-qPCR identified many more samples as containing inhibitors than the pathogen-specific controls, and this was so in both qPCR assays, i.

In addition, ii it is noteworthy that only 13 and 3 samples were detected as inhibited using both inhibitor-detection methods, for the Toxoplasma and Pneumocystis assays, respectively Fig 2. We therefore conclude that i the human gene-based qPCR detected inhibitors much more often than the pathogen-specific qPCR and ii it most often did not detect them correctly. Considering this poor correlation between pathogen-specific controls and albumin qPCR to detect PCR inhibitors, we wished to determine Cp alb thresholds more adapted to our assays.

We therefore concluded that no Cp alb cut-off values can be determined to efficiently detect pathogen-specific inhibitions for both pathogen-specific PCR assays. To check the influence of the cellularity of clinical samples on Cp alb values, we compared white blood cell counts to albumin qPCR results for blood samples.

The lack of sensitivity and specificity of albumin PCR to detect target specific inhibitions may be explained by a differential susceptibility of each qPCR assay to inhibitors and by the variable quantity of human DNA in clinical samples. These results prevent using human gene-based PCR, e. We based our demonstration on two models, i.

Toxoplasma and Pneumocystis but these results should be expanded to the detection of other pathogens in human samples. A and C. Red curves: sensitivity; blue curves: specificity. The maximum value was 0. B Graphical analysis of sensitivity and specificity curves in function of cut-off values.

Both curves intersect at a Cp alb value of At this Cp alb cut-off value sensitivity is D Graphical profiles of sensitivity and curves in function of cut-off values. Semi-log scale graph where leucocyte count x-axis was plotted against Cp alb orange and green dots corresponding to amplification control inhibited and not inhibited samples respectively. Differential susceptibility to inhibitors and efficiency discrepancies between PCR assays should also prove problematic in this approach.

Indeed, the size and GC rate of the amplicons of the foreign DNA used will have an impact on the detection of the inhibition [ 13 ].

So, implementation of one of these controls should be avoided until their performances have been assessed in routine practice. In conclusion, pathogen-specific amplification controls appear to be a method of choice for screening the presence of inhibitors in a PCR assay for infectious diseases as compared to the use of a human gene-based qPCR. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.

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. Methods We retrospectively analysed Toxoplasma and Pneumocystis qPCR tests for all the clinical samples analysed in in the Department of Parasitology-Mycology at the academic hospital of Montpellier Montpellier, France.

PCR controls and definition of inhibition Each PCR plate contained one well for negative control sterile water and one for positive control calibrated positive sample. Determination of the PCR efficiencies PCR efficiencies of the techniques were determined using a standard curve generated by performing a logarithmic serial dilution of DNA extracts. Statistics Data were analyzed using R 3. Results and discussion General description The Dept. Download: PPT.

Table 1. Inhibition rates in clinical samples as assessed from the pathogen-specific amplification control Of the analysed samples, 62 2. Fig 3. Fig 4. Correlation between leucocyte counts and Cp alb values in blood samples. Supporting information. S1 Table.

Spreadsheet of raw data. S1 Fig. Flow chart of data cleaning and tidying of Toxoplasma PCR results. S2 Fig. Flow chart of data cleaning and tidying of Pneumocystis PCR results. S3 Fig. S4 Fig. Acknowledgments We are grateful to S. Douzou, B.

Sanichanh and G. Serres for their technical help. References 1. Hedman J, Radstrom P. Overcoming inhibition in real-time diagnostic PCR. Methods Mol Biol. Quality in the molecular microbiology laboratory. PCR inhibitors—occurrence, properties and removal. J Appl Microbiol. Inhibition controls for qualitative real-time PCR assays: are they necessary for all specimen matrices?

J Clin Microbiol. A key goal of this program is to identify non-rifamycin, small molecule inhibitors of RNAP that can maximally inhibit the enzyme at clinically attainable doses and therefore shorten therapy. New drugs that function outside the rifamycin binding site on RNAP will be prioritized as these will likely be effective against rifamycin-resistant M. Likewise, potent small molecule inhibitors may offer improvements in TB dosing regimens improved pharmacokinetic profile relative to rifamycin , and delivery opportunities for fixed dose combinations with other TB agents.

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