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.
Highlights
Hepatitis A virus (HAV) is one of the leading causes of acute viral hepatitis worldwide (Murray et al 2015)
Sub-genotype-wise multiple sequence alignments with the hepatitis A virus (HAV)-specific primers and probes recommended by ISO 15216-1 (ISO 2017) revealed that mismatches and/or insertions within the probe region were most common among genotype I, whereas mismatches at the 3′-end of the reverse primer region were most common among genotype III
Four sequences with common or potentially severe mismatches were analysed by reverse transcription (RT)-Droplet digital PCR (ddPCR) and reverse transcription quantitative real-time PCR (RT-qPCR) (Table 1)
Summary
Hepatitis A virus (HAV) is one of the leading causes of acute viral hepatitis worldwide (Murray et al 2015). The human HAV strains are divided into three genotypes (I, II, III) and seven sub-genotypes (IA, IB, IC, IIA, IIB, IIIA and IIIB) (Pintó et al 2012). Strains of sub-genotype IIIA seem to emerge rapidly in other parts of the world (Mukomolov et al 2012; Bosch et al 2016; Miyamura et al 2012) and the globalised food chain can be an important contributor to the spread of different HAV variants (Jacobsen 2018). HAV infection do not cause any symptoms in individuals below 6 years of age, but strains of sub-genotype IIIA are more frequently associated with symptomatic
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