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

Abstract

Pigeon paramyxovirus-1 (PPMV-1) is a strain of Newcastle disease virus (NDV) that has adapted to infect pigeons and poses a constant threat to the commercial poultry industry. Early detection via rapid and sensitive methods, along with timely preventative and mitigating actions, is important for reducing the spread of PPMV-1. Here, we report the development of a TaqMan loop-mediated isothermal amplification assay (TaqMan-LAMP) for rapid and specific detection of PPMV-1 based on the F gene. This system makes use of six novel primers and a TaqMan probe that targets nine distinct regions of the F gene that are highly conserved among PPMV-1 isolates. The results showed that the limit of detection was 10 copies μL-1 for PPMV-1 cDNA and 0.1 ng for PPMV-1 RNA. The reaction was completed within 25 min and was thus faster than conventional RT-PCR. Moreover, no cross-reactions with similar viruses or with peste des petits ruminants virus (PPRV) or NDV LaSota vaccine strains were observed under the same conditions. To evaluate the applicability of the assay, the TaqMan-LAMP assay and a commercial RT-PCR assay were compared using 108 clinical samples, and the concordance rate between two methods was found to be 96.3%. The newly developed PPMV-1 TaqMan-LAMP assay can therefore be used for simple, efficient, rapid, specific, and sensitive diagnosis of PPMV-1 infections.

Review detection of Newcastle disease virus

“…mRT-LAMP-LFD does not require special instrumentation, it is a promising qualitative detection tool and is suitable for some resource-poor areas ( 82 ). Recently, Liang et al ( 4 ) developed a TaqMan loop-mediated isothermal amplification assay for the rapid detection of pigeon paramyxovirus type 1. This method targeted nine distinct regions of the F gene and designed six novel primers and a TaqMan probe.…”

“…In addition, this detection method provides good specificity for NDV with no observed cross-reactions with other viruses. To evaluate the applicability of the assay, 108 clinical samples were used to compare the TaqMan-LAMP assay and commercial RT-PCR assay, and the concordance rate between the two methods was found to be 96.3% ( 4 ).…”

“…With the development of the global livestock and poultry industry and the increase in world trade, the prevention of ND is particularly important. Early detection via rapid, sensitive, and accurate methods is very important for reducing the spread of ND ( 4 ). This review introduces the latest research on ND, including historical overview, structural biology, and infection mechanism.…”

Loop-Mediated Isothermal Amplification: From Theory to Practice

“…The absence of 5'→3' exonuclease activity in Bst LF makes it difficult to use TaqMan probes, as well as molecular beacons [ 107 , 108 ] in the LAMP method for real-time detection of amplification. The TaqMan technology works in LAMP with some background amplification in no template controls [ 109 ] while molecular beacons are characterized by extremely low specificity [ 110 ]. Therefore, labeling inner primers with a fluorophore seems to be the optimal solution to the problem of direct detection [ 37 ].…”

Occurrence, Genetic Variability of Tomato Yellow Ring Orthotospovirus Population and the Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Its Rapid Detection

“…LAMP amplifies target DNA at isothermal conditions (usually 60–65 °C) and obviates the need for thermal cyclers and postamplification procedures for signal detection. The significant advantages of LAMP assay make it very popular and widely used by many researchers to detect plant and animal viruses [ 37 , 38 , 39 , 40 , 41 , 42 ].…”