Efficiency evaluation of the process control virus “Mengovirus” in real time RT-PCR viral detection in the bivalve mollusc Donax sp

Abstract

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 × 104 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.