Instituto Tecnológico de la Producción, Dirección de Investigación, Desarrollo, Innovación y Transferencia Tecnológica (DIDITT), Callao 07046, Peru.
Instituto Tecnológico de la Producción, Dirección de Investigación, Desarrollo, Innovación y Transferencia Tecnológica (DIDITT), Callao 07046, Peru.
J Virol Methods. 2018 Dec;262:20-25. doi: 10.1016/j.jviromet.2018.09.006. Epub 2018 Sep 17.
There are many problems associated with extracting viral genetic material from contaminated samples of bivalve molluscs, specifically because the hepatopancreas has many PCR inhibitors. For this reason, nucleic acid extraction methods must consider a process control virus (PCV) that may help to measure extraction efficiency. In the market, there are many commercial kits to extract nucleic acid from RNA viruses, as well as others to perform one-step real time RT-PCR, but most of them have not been evaluated for bivalve molluscs. For this reason, the aim of this study was to evaluate the extraction efficiency of the PCV (Mengovirus), it was performed using 3 different RNA extraction kits and 2 one-step real time RT-PCR kits. 10 μL of Mengovirus at a concentration of 1.6 × 10 viral particles/μL was added to 29 samples of hepatopancreas of Donax sp. Sample processing was performed according to the ISO/TS 15216-2: 2013 standard. RNA was extracted from each sample with the kits: (1) BioMerieux NucliSens®system (BioMérieux SA, France), (2) PureLink™ RNA Mini Kit (Ambion-Life Technologies™, USA) and (3) Hugh Pure RNA Tissue Kit (Roche SA, Germany). Once RNA was extracted, one-step real time RT-PCR was conducted by using the following kits: (A) Ultrasense One-step qRT-PCR Kit (Invitrogen, USA) according to ISO/TS 15216-2:2013, and (B) Mengovirus@ceeramTools™ Kit (Ceeram, France) according to the manufacturer's specifications. The extraction efficiency of PCV when using the extraction kits 1, 2 and 3 combined with real time RT-PCR kit A were: 10.82, 1.90 and 0.64, respectively; and when using real time RT-PCR kit B were: 7.34, 0.97 and 0.47, respectively. It is concluded that the BioMerieux NucliSens®system RNA extraction kit was the most efficient and that the Ultrasense One-step qRT-PCR Kit performed better than the Mengovirus@ceeramTools™ RT-PCR kit.
从受污染的双壳贝类样本中提取病毒遗传物质存在许多问题，特别是因为肝胰腺中有许多 PCR 抑制剂。出于这个原因，核酸提取方法必须考虑可能有助于衡量提取效率的过程控制病毒 (PCV)。市场上有许多用于提取 RNA 病毒的商用试剂盒，以及用于进行一步实时 RT-PCR 的试剂盒，但其中大多数尚未针对双壳贝类进行评估。出于这个原因，本研究的目的是评估 PCV（甲病毒）的提取效率，使用了 3 种不同的 RNA 提取试剂盒和 2 种一步实时 RT-PCR 试剂盒进行了评估。将浓度为 1.6×10 病毒粒子/μL 的 10μL 甲病毒添加到 29 个 Donax sp. 肝胰腺样本中。根据 ISO/TS 15216-2:2013 标准进行样本处理。使用试剂盒从每个样本中提取 RNA：(1)BioMerieux NucliSens®系统（法国 BioMérieux SA），(2)PureLink™ RNA Mini Kit（美国 Ambion-Life Technologies™）和 (3)Hugh Pure RNA Tissue Kit（瑞士 Roche SA）。提取 RNA 后，使用以下试剂盒进行一步实时 RT-PCR：(A)Ultrasense One-step qRT-PCR Kit（美国 Invitrogen），根据 ISO/TS 15216-2:2013，和 (B)Mengovirus@ceeramTools™ Kit（法国 Ceeram），根据制造商的规格。当使用提取试剂盒 1、2 和 3 结合实时 RT-PCR 试剂盒 A 时，PCV 的提取效率分别为 10.82、1.90 和 0.64；当使用实时 RT-PCR 试剂盒 B 时，提取效率分别为 7.34、0.97 和 0.47。研究得出结论，BioMerieux NucliSens®系统 RNA 提取试剂盒是最有效的，而 Ultrasense One-step qRT-PCR Kit 的性能优于 Mengovirus@ceeramTools™ RT-PCR 试剂盒。
