Construction of Lentivirus-Based Reference Material for RT-PCR Detection of Middle East Respiratory Syndrome Coronavirus and Its Application in External Quality Assessment

Abstract

Nucleic acid amplification technologies (NAT) have been used most for rapid detection of Middle East Respiratory Syndrome coronavirus (MERS-CoV) since MERS-CoV was first found in 2012. It is important to develop stable and safe reference materials for assessing the quality of NAT kits and external quality assessment (EQA) of different labs. In this study, the MERS-CoV RNA fragments including upE, ORF1b and N were packed within human immunodeficiency virus type 1 (HIV-1) like particles. The lyophilized virus-like particles (VLPs) were found stable at 37˚C or below and safe to be used not only as the control material for PCR detection of MERS-CoV, but also as the reference material for EQA. In an EQA organized by Ningbo International Travel Healthcare Center in China, 49 participating institutes achieved 100% agreement for detecting MERS-CoV using various commercial diagnosis kits and different extraction methods. But the different Ct values reported by the different labs for the same sample implied that there needed to standardize the RNA extraction method and/or the PCR detection conditions between the labs.

A Novel Primer Probe Set for Detection of SARS-CoV-2 by Sensitive Droplet Digital PCR

“…First of all, nucleic acid detecting is the most direct way to demonstrate the presence of the virus. Secondly, ddPCR may be more proper in which nucleic acid detection is the most direct way to demonstrate the presence of the virus [24] , it could achieve absolute quantification of SARS-CoV-2 without the standard curve [24] . Thirdly, through detection within portioned droplets, ddPCR is much more sensitive [25] and accurate [26] in portioned droplets detection, which is might be a strong tool for the detection of very low viral load in mild symptomatic and asymptomatic infection, it is also suitable for monitoring the change of the viral load in the convalescent patients [27] .…”

Comparison of Seven Commercial Severe Acute Respiratory Syndrome Coronavirus 2 Nucleic Acid Detection Reagents with Pseudovirus as Quality Control Material

“…Therefore, construction of pseudoviruses similar to SARS-CoV-2 virus is necessary to evaluate the performance of NAT kits, as well as to control the quality of experimental procedure. Vectors of lentivirus and bacteriophage have been widely used to produce stable quality control materials [4][5][6][7] .…”