A Novel Quantitative PCR Mediated by High-Fidelity DNA Polymerase.

Traditional PCR methods can be used for viral diagnosis, however they are required to be highly sensitivity, specific and reproducible for this use. These criteria are largely dependent on the complementarity of the target nucleic acid to the primers and probe used in the PCR reaction. The primers and probe are designed to bind to a highly conserved genomic region and maximise the match. The presence of a mismatch reduces the amplification efficiency and sensitivity. Unfortunately, viruses have high genetic variability which makes it substantially more challenging to locate regions that are conserved in all subtypes of a particular virus.

A recent paper, published in Scientific Reports, investigated the development of a simpler qPCR method that is able to tolerate the presence of mismatches. The authors describe a method that is mediated by a high-fidelity DNA polymerase and uses a primer and a fluorescent primer (called a HFman probe).

The technique requires the mismatch to be located:

  • in the 3′-end of the HFman probe and the target sequence
  • or, in the 3′-end of the primer and the target sequence

This new technique will be particularly useful for viruses with a high mutation rate, such as HIV-1.

In order to demonstrate its flexibility, the paper details a test performed to compare the ability of the new and conventional methods to detect viral variants. To enable a direct comparison, the forward primer of the traditional technique was designed with an identical sequence to the HFman probe used in the new method. A further TaqMan probe was designed for use in the conventional method.

When using the wild template, the amplification curves from the two methods are close to each other, demonstrating that they are comparable. However, when using the mutant template, the amplification curve from the new method is substantially earlier than the conventional method (Cq values 17.98 and 26.35 respectfully). The new method also produced a stronger fluorescent signal and similar amplification curves for the wild and mutant templates.

To read the full paper, click here.