Simultaneous and visual detection of infectious bronchitis virus and Newcastle disease virus by multiple LAMP and lateral flow dipstick

Abstract

Infectious bronchitis virus (IBV) andNewcastle disease virus (NDV) are both important viruses seriously affecting poultry industry worldwide. In this study, reverse-transcription LAMP (RT-LAMP) was combined with lateral flow dipstick (LFD) forming a novel detection tool which could simultaneously detect IBV and NDV visually. Primers targeted the 5 -untranslated region (5 -UTR) of IBV genome and the conserved region of NDV large polymerase gene (LP). The specificity and sensitivity of this multiple reverse transcription-LAMP-LFD (mRT-LAMP-LFD) assay were compared with those of conventional RT-PCR, nested RT-PCR (nRT-PCR), quantification RT-PCR (qRT-PCR), and RT-LAMP monitored by electrophoresis. No non-specific amplifications were observed when the assays were tested with unrelated viruses. According to the sensitivity study, when detecting IBV or NDV alone, the lowest detection limits of mRT-LAMP-LFD were 10 0.8 IBV RNA copies/reaction and 10 0.7 NDV RNA copies/reaction. Furthermore, when detecting IBV and NDV simultaneously, the lowest detection limit was the same as that of the single detection assays. In the clinical sample study, mRT-LAMP-LFD performed the best among these assays. When tested with IBV or NDV single infected samples, the mean detection rates were 98.65% and 97.25%, respectively. In the IBV and NDV co-infected sample study, the mean detection rates of IBV and NDV were both 95%. This study showed that mRT-LAMP-LFD was a promising qualitative detection tool suitable for field single or multiple IBV and NDV detection.

Development of a TaqMan loop-mediated isothermal amplification assay for the rapid detection of pigeon paramyxovirus type 1

“…However, these methods require specialized facilities and highly trained workers and are time consuming, which renders them unsuitable for detecting pathogens under field conditions. Several studies on the development of diagnostic methods for other pathogens have shown that LAMP assays exhibit equal or even higher sensitivity when compared with qPCR assays [ 8 , 27 ]. Probe based LAMP techniques are among the most specific and sensitive diagnostic methods available [ 20 ].…”

A closed-tube, single-step, real time, reverse transcription-loop-mediated isothermal amplification assay for infectious bronchitis virus detection in chickens

“…Given the above, it is not surprising that in recent years scientists have been developing RT-LAMP assays for IBV (Chen et al, 2010;Chandrasekar et al, 2015;Wu et al, 2019). These assays use naked eye, gel electrophoresis, and lateral flow strips for amplicon detection and are not amenable for quantification.…”

Loop-Mediated Isothermal Amplification (LAMP) as a Promising Point-of-Care Diagnostic Strategy in Avian Virus Research

“…While all the ALV field strains are oncogenic, the neoplastic lymphoid leukosis disease triggered by this cluster of RNA viruses on chicken flocks needs to be identified and addressed specifically according to the subgroup. Infectious bronchitis virus (IBV) is another major cause of respiratory disease in chicken farms that has been underlined with the urgency of rapid diagnosis via the optimized LAMP method [ 16 , 86 , 87 ]. With single-stranded RNA avian coronavirus as the causative agent, from the Coronaviridae family, IBV was first tested with RT-LAMP at the conserved regions of the nucleocapsid gene [ 16 ].…”

“…With single-stranded RNA avian coronavirus as the causative agent, from the Coronaviridae family, IBV was first tested with RT-LAMP at the conserved regions of the nucleocapsid gene [ 16 ]. Through an innovative mRT-LAMP-LFD, a multiple LAMP method was coupled with a lateral flow dipstick for simultaneous detection of IBV and the contagious Newcastle disease virus (NDV) [ 87 ]. In the diagnostic tests, the presence of IBV and NDV on clinical samples detected using the mRT-LAMP-LFD assay were at decent sensitivity rates of 98.65% and 97.25%, respectively.…”