Abstract
The biggest challenge for accurate diagnosis of viral infectious disease is the high genetic variability of involved viruses, which affects amplification efficiency and results in low sensitivity and narrow spectrum. Here, we developed a new simple qPCR mediated by high-fidelity (HF) DNA polymerase. The new method utilizes an HFman probe and one primer. Fluorescent signal was generated from the 3′–5′ hydrolysis of HFman probe by HF DNA polymerase before elongation initiation. Mismatches between probe/primer and template have less influence on the amplification efficiency of the new method. The new qPCR exhibited higher sensitivity and better adaptability to sequence variable templates than the conventional TaqMan probe based-qPCR in quantification of HIV-1 viral load. Further comparison with COBAS TaqMan HIV-1 Test (v2.0) showed a good correlation coefficient (R2 = 0.79) between both methods in quantification of HIV-1 viral load among 21 clinical samples. The characteristics of tolerance to variable templates and one probe-one primer system imply that the probe/primer design for the new method will be easier and more flexible than the conventional method for highly heterogeneous viruses. Therefore, the HF DNA polymerase-mediated qPCR method is a simple, sensitive and promising approach for the development of diagnostics for viral infectious diseases.
Highlights
The biggest challenge for accurate diagnosis of viral infectious disease is the high genetic variability of involved viruses, which affects amplification efficiency and results in low sensitivity and narrow spectrum
We call the new method as HF DNA polymerase mediated real-time PCR and defined the probe as HFman probe or fluorescent primer (Fig. 1)
Various new methods were developed for the detection and diagnostics of infectious diseases in the last two decades[39]
Summary
The biggest challenge for accurate diagnosis of viral infectious disease is the high genetic variability of involved viruses, which affects amplification efficiency and results in low sensitivity and narrow spectrum. MGB (minor groove binder) modification had been demonstrated to increase the stability of duplexes formed by probe and target, and allows shorter probes[33], it is still a big challenge for the development of qPCR methods for high-efficient detection and quantification of all genotypes/subtypes of highly heterogenetic viruses such as HIV-1 and HCV. We developed a new simple qPCR method that is mediated by high-fidelity DNA polymerase and utilizes a primer and a fluorescent primer (called as HFman probe). The new method allows the presence of mismatches between the 3-ends of the HFman probe/primer and the target sequence, it has better performance in quantification of gene expression and viral load than the conventional TaqMan probe-based qPCR. Tolerance to mismatches between primer/probe and target and the one primer-one HFman probe system simplified the method, improved the sensitivity and accuracy for the quantification of gene expression, and diagnosis of various pathogens (especially for viruses with high heterogeneity)
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