Coudray-Meunier Coralie, Fraisse Audrey, Martin-Latil Sandra, Delannoy Sabine, Fach Patrick, Perelle Sylvie
Université Paris-Est, ANSES, Food Safety Laboratory, Enteric viruses Unit, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort Cedex, France.
Université Paris-Est, ANSES, Food Safety Laboratory, Identypath, 14 rue Pierre et Marie Curie, 94701 Maisons-Alfort Cedex, France.
PLoS One. 2016 Jan 29;11(1):e0147832. doi: 10.1371/journal.pone.0147832. eCollection 2016.
Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.
人类肠道病毒被认为是全球食源性和水源性疾病的主要病因。人类肠道病毒的灵敏定量检测通常通过定量逆转录聚合酶链反应（RT-qPCR）来实现。一种纳米流体实时PCR系统被用于开发新型高通量方法，用于定性分子检测（RT-qPCR阵列）以及通过数字RT-PCR（RT-dPCR）对人类致病病毒进行定量分析。研究了高通量PCR方法在检测19种人类致病病毒和食品病毒学中使用的两种主要过程对照物方面的性能。将传统实时PCR系统与RT-dPCR和RT-qPCR阵列进行了比较。基于通过分光光度法计算的基因组拷贝数，发现对于14种病毒，RT-qPCR的灵敏度比RT-dPCR略高，倍数范围为0.3至1.6个对数10。相反，对于7种病毒，RT-dPCR的灵敏度比RT-qPCR好，倍数范围为0.10至1.40个对数10。有趣的是，RT-dPCR确定的基因组拷贝数总是比RT-qPCR标准曲线计算的预期拷贝数低1至2个对数10。发现RT-qPCR和RT-qPCR阵列检测对于两种病毒的灵敏度相似，对于18种病毒，RT-qPCR的灵敏度比RT-qPCR阵列好，倍数范围为0.7至3.0个对数10。相反，对于诺如病毒GIV检测，RT-qPCR阵列的灵敏度仅比传统RT-qPCR检测高0.30个对数10。最后，RT-qPCR阵列和RT-dPCR检测成功地一起用于筛查临床样本和定量致病病毒。此外，该方法使得识别临床样本中的合并感染成为可能。总之，鉴于其快速性和检测大量病毒靶标的潜力，这种纳米流体RT-qPCR检测方法应该会对人类致病病毒监测和疫情调查产生重大影响，并且可能对公共卫生有益。
