Establishment and application of a quadruple real-time RT-PCR for detecting avian metapneumovirus

Abstract

In order to develop an appropriate method for high-throughput detection of avian metapneumovirus, a quadruple real-time reverse-transcription polymerase chain reaction assay was established with four pairs of specific primers and four specific probes based on the G or M gene of aMPV-A, aMPV-B, aMPV-C and aMPV-D. Its specificity and sensitivity were evaluated, and clinical samples were tested by the method. The results showed that all the four subgroups of avian metapneumovirus can be detected in the quadruple real-time RT-PCR assay simultaneously, with a detection limit of 100–1000 cRNA copies/reaction. The other common poultry viruses were negative. In the avian clinical sample detection, 39 out of 1920 clinical samples collected from 8 provinces were positive. Compared with published RT-PCR assays, the κ value of the quadruple real-time RT-PCR assay in 1920 avian clinical samples was 1.000 (P < 0.001). The established method could be used for the rapid detection of the four subgroups of avian metapneumovirus with high specificity and high sensitivity.

Establishment and Application Prospect of Reverse Transcriptase Recombinase-Aided Amplification Assay for Subgroup C Avian Metapneumovirus

“…Compared with traditional PCR detection, RT-RAA does not require a gel electrophoresis test and can be completed within 30 min, which saves more time. The lowest detection standard for aMPV-C by RT-qPCR was 10 3 copies/μL [ 24 ]. In our study, we yielded it as 10 1 copies/μL, which is 100 times more sensitive in contrast to RT-qPCR, and the amplification reaction can be carried out at a constant temperature of 41 °C without complicated temperature change procedures and equipment.…”

“…Current methods for aMPV detection can be divided into molecular detection and serological detection, including conventional PCR, real-time fluorescence quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and virus neutralization (VN) [ 7 ]. Traditional molecular detection methods encompassing conventional PCR and qPCR have high specificity and sensitivity, but require expensive and complex machinery and experienced laboratory personnel [ 23 , 24 ]. Compared with molecular detection, serological detection is conducive to the large-scale screening and monitoring of livestock and poultry.…”

An affordable detection system based on RT-LAMP and DNA-nanoprobes for avian metapneumovirus

“…Currently, RT-PCR and qRT-PCR tests have become the established gold standard methods in reference laboratories for diagnosing active infections. Most of the RT-PCR epidemiological studies on aMPV are based on M, N, and G genes (Ferreira et al 2009 ; Kariithi et al 2022 ; Wang et al 2022 ). In this study, we targeted its F gene based on its high conservation degree in the reference strains of aMPV subtypes A and B.…”

Development of a Fast and Affordable Diagnostic System for RNA Viruses Using Loop-Mediated Isothermal Amplification and DNA-Nanoprobe Detection

“…Currently, RT-PCR and qRT-PCR tests have become the established gold standard methods in reference laboratories for diagnosing active infections. Indeed, primer sequences for the RT-PCR have been designed for specific detection of the F, M, N and G genes (Ferreira et al, 2009;Kariithi et al, 2022;Wang et al, 2022). For instance, since the gene G shows the highest variability between subtypes it is the region most widely used at aMPV genotyping.…”