Missing the Match Might Not Cost You the Game: Primer-Template Mismatches Studied in Different Hepatitis A Virus Variants

Abstract

Mismatches between template sequences and reverse transcription (RT) or polymerase chain reaction (PCR) primers can lead to underestimation or false negative results during detection and quantification of sequence-diverse viruses. We performed an in silico inclusivity analysis of a widely used RT-PCR assay for detection of hepatitis A virus (HAV) in food, described in ISO 15216-1. One of the most common mismatches found was a single G (primer) to U (template) mismatch located at the terminal 3'-end of the reverse primer region. This mismatch was present in all genotype III sequences available in GenBank. Partial HAV genomes with common or potentially severe mismatches were produced by in vitro transcription and analysed using RT-ddPCR and RT-qPCR. When using standard conditions for RT-qPCR, the mismatch identified resulted in underestimation of the template concentration by a factor of 1.7-1.8 and an increase in 95% limit of detection from 8.6 to 19 copies/reaction. The effect of this mismatch was verified using full-length viral genomes. Here, the same mismatch resulted in underestimation of the template concentration by a factor of 2.8. For the partial genomes, the presence of additional mismatches resulted in underestimation of the template concentration by up to a factor of 232. Quantification by RT-ddPCR and RT-qPCR was equally affected during analysis of RNA templates with mismatches within the reverse primer region. However, on analysing DNA templates with the same mismatches, we found that ddPCR quantification was less affected by mismatches than qPCR due to the end-point detection technique.

Keywords: Digital PCR; Hepatitis A virus; Mismatch; Primer; Real-time PCR; Reverse transcription.

Fig. 1

Mismatches within the reverse primer region. a Effects in one-step RT-PCR (RNA templates) and b effects in PCR (DNA templates). Four (RT)-PCR runs were conducted for each template and method, and each dot displays the average from a single run. The horizontal bars represent averages of four runs. R: reverse primer region. For instance, R: C19U means that a C is substituted to a U in the template at the position where the 19th base of the reverse primer binds. The 19^th base corresponds to the terminal 3′-end of the primer

Fig. 2

Mismatches and the performance of ddPCR. Fluorescence amplification plots of DNA templates for strain A and C in ddPCR, amplified with MM and PM primers, in a run containing five replicate wells of each sample. NTC no template control

Fig. 3

The RNA template of strain A in RT-qPCR at low concentrations. Strain A has a single G (primer) to U (template) mismatch at the terminal 3′-end of the reverse primer. The figure shows estimated probability of detection for MM and PM primers versus expected concentration. The observed proportions of positive samples are shown as dots and the estimated 95% limits of detection are indicated as vertical lines. n =16 tested sample wells per dilution level and primer type

Fig. 4

Partial genome of strain A and full-length genomes with the same mismatch in RT-qPCR. Dots represent single RT-qPCR wells and lines represent mean values