基于核酸的检测,如聚合酶链反应(PCR),扩增和检测生物体特异性基因组序列是传染病监测的标准方法。然而,由于其遗传多样性,病毒监测面临挑战。这里,我们在计算机上计算了10种人类病毒物种基因组中核苷酸的变异性,发现地方性病毒表现出很高的可变核苷酸百分比(例如,诺如病毒基因组II的51.4%)。这种遗传多样性导致PCR测定的可变检测概率(包含测定的靶序列的病毒序列的比例除以病毒序列的总数)。然后,我们通过实验证实,靶序列检测的概率指示PCR测定和诺如病毒基因组之间的错配数量。接下来,我们开发了一种简并PCR检测方法,该方法在8个临床样本中检测到97%的已知诺如病毒基因群II基因组序列和识别的诺如病毒.相比之下,先前开发的检测概率为31%和16%的检测方法平均有1.1和2.5个错配,分别,这对RNA定量产生了负面影响。此外,检测概率较低的两种PCR检测也导致了废水流行病学的假阴性.我们的发现表明,检测概率可作为评估基于核酸的检测方法以进行遗传多样性病毒监测的简单指标。重要性基于核酸的检测,如聚合酶链反应(PCR),扩增和检测生物体特异性基因组序列被广泛用作传染病监测的标准方法。然而,由于病毒的快速进化和遗传变异,病毒监测面临挑战。该研究使用多种PCR测定法分析了临床和废水样品,发现PCR测定法在遗传多样性诺如病毒监测中的显着性能差异。这一发现表明,一些PCR检测可能会错过某些病毒株的检测,导致检测灵敏度的折衷。为了解决这个问题,我们提出了一个叫做检测概率的度量,可以使用本研究开发的代码简单地计算,评估用于遗传多样性病毒监测的基于核酸的检测方法。这种新方法可以帮助提高病毒检测的灵敏度和准确性,这对于有效的传染病监测和控制至关重要。
Nucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of their genetic diversity. Here, we calculated the variability of nucleotides within the genomes of 10 human viral species in silico and found that endemic viruses exhibit a high percentage of variable nucleotides (e.g., 51.4% for norovirus genogroup II). This genetic diversity led to the variable probability of detection of PCR assays (the proportion of viral sequences that contain the assay\'s target sequences divided by the total number of viral sequences). We then experimentally confirmed that the probability of the target sequence detection is indicative of the number of mismatches between PCR assays and norovirus genomes. Next, we developed a degenerate PCR assay that detects 97% of known norovirus genogroup II genome sequences and recognized norovirus in eight clinical samples. By contrast, previously developed assays with 31% and 16% probability of detection had 1.1 and 2.5 mismatches on average, respectively, which negatively impacted RNA quantification. In addition, the two PCR assays with a lower probability of detection also resulted in false negatives for wastewater-based epidemiology. Our findings suggest that the probability of detection serves as a simple metric for evaluating nucleic acid-based assays for genetically diverse virus surveillance.IMPORTANCENucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are employed widely as a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of the rapid evolution and genetic variation of viruses. The study analyzed clinical and wastewater samples using multiple PCR assays and found significant performance variation among the PCR assays for genetically diverse norovirus surveillance. This finding suggests that some PCR assays may miss detecting certain virus strains, leading to a compromise in detection sensitivity. To address this issue, we propose a metric called the probability of detection, which can be simply calculated in silico using a code developed in this study, to evaluate nucleic acid-based assays for genetically diverse virus surveillance. This new approach can help improve the sensitivity and accuracy of virus detection, which is crucial for effective infectious disease surveillance and control.