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.

中文翻译：

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错过比赛可能不会让您输掉比赛：在不同甲型肝炎病毒变体中研究的引物模板不匹配。

模板序列与逆转录 (RT) 或聚合酶链式反应 (PCR) 引物之间的不匹配可能会导致序列多样性病毒检测和定量过程中的低估或假阴性结果。我们对 ISO 15216-1 中描述的广泛使用的用于检测食品中甲型肝炎病毒 (HAV) 的 RT-PCR 测定法进行了计算机包容性分析。最常见的错配之一是位于反向引物区域末端 3' 端的单个 G（引物）与 U（模板）错配。这种错配存在于 GenBank 中可用的所有基因型 III 序列中。通过体外转录产生具有常见或潜在严重错配的部分 HAV 基因组，并使用 RT-ddPCR 和 RT-qPCR 进行分析。当使用标准条件进行 RT-qPCR 时，发现的错配导致模板浓度被低估 1.7-1.8 倍，并且检测限从 8.6 拷贝/反应增加到 19 拷贝/反应，95% 的检测限增加。使用全长病毒基因组验证了这种错配的影响。在这里，同样的不匹配导致模板浓度被低估 2.8 倍。对于部分基因组，额外错配的存在导致模板浓度被低估高达 232 倍。在分析反向引物区域内存在错配的 RNA 模板时，RT-ddPCR 和 RT-qPCR 的定量同样受到影响。然而，在分析具有相同错配的 DNA 模板时，我们发现由于终点检测技术，ddPCR 定量比 qPCR 受错配影响更小。