Borgmästars Emmy, Jazi Mehrdad Mousavi, Persson Sofia, Jansson Linda, Rådström Peter, Simonsson Magnus, Hedman Johannes, Eriksson Ronnie
Science Division, Biology Department, National Food Agency, Hamnesplanaden 5, 75319, Uppsala, Sweden.
Department of Medical Science, Uppsala University, Akademiska sjukhuset, 75185, Uppsala, Sweden.
Food Environ Virol. 2017 Dec;9(4):395-405. doi: 10.1007/s12560-017-9295-3. Epub 2017 Apr 11.
Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) detection of waterborne RNA viruses generally requires concentration of large water volumes due to low virus levels. A common approach is to use dead-end ultrafiltration followed by precipitation with polyethylene glycol. However, this procedure often leads to the co-concentration of PCR inhibitors that impairs the limit of detection and causes false-negative results. Here, we applied the concept of pre-PCR processing to optimize RT-qPCR detection of norovirus genogroup I (GI), genogroup II (GII), and hepatitis A virus (HAV) in challenging water matrices. The RT-qPCR assay was improved by screening for an inhibitor-tolerant master mix and modifying the primers with twisted intercalating nucleic acid molecules. Additionally, a modified protocol based on chaotropic lysis buffer and magnetic silica bead nucleic acid extraction was developed for complex water matrices. A validation of the modified extraction protocol on surface and drinking waters was performed. At least a 26-fold improvement was seen in the most complex surface water studied. The modified protocol resulted in average recoveries of 33, 13, 8, and 4% for mengovirus, norovirus GI, GII, and HAV, respectively. The modified protocol also improved the limit of detection for norovirus GI and HAV. RT-qPCR inhibition with C shifts of 1.6, 2.8, and 3.5 for norovirus GI, GII, and HAV, respectively, obtained for the standard nucleic acid extraction were completely eliminated by the modified protocol. The standard nucleic acid extraction method worked well on drinking water with no RT-qPCR inhibition observed and average recoveries of 80, 124, 89, and 32% for mengovirus, norovirus GI, GII, and HAV, respectively.
由于水传播RNA病毒水平较低，采用定量逆转录聚合酶链反应（RT-qPCR）检测通常需要对大量水样进行浓缩。一种常用方法是先进行死端超滤，然后用聚乙二醇沉淀。然而，该过程常导致PCR抑制剂共同浓缩，从而影响检测限并产生假阴性结果。在此，我们应用PCR前处理的概念，优化在具有挑战性的水基质中对诺如病毒基因组I（GI）、基因组II（GII）和甲型肝炎病毒（HAV）的RT-qPCR检测。通过筛选耐受抑制剂的预混液并用扭曲嵌入核酸分子修饰引物，改进了RT-qPCR检测方法。此外，还针对复杂水基质开发了一种基于离液剂裂解缓冲液和磁性硅胶珠核酸提取的改良方案。对地表水和饮用水的改良提取方案进行了验证。在所研究的最复杂地表水样本中，至少有26倍的改善。改良方案对脑心肌炎病毒、诺如病毒GI、GII和HAV的平均回收率分别为33%、13%、8%和4%。改良方案还提高了诺如病毒GI和HAV的检测限。标准核酸提取方法对诺如病毒GI、GII和HAV分别产生1.6、2.8和3.5的C 偏移导致的RT-qPCR抑制，改良方案可将其完全消除。标准核酸提取方法在饮用水中效果良好，未观察到RT-qPCR抑制，对脑心肌炎病毒、诺如病毒GI、GII和HAV的平均回收率分别为80%、124%、89%和32%。
