Development and application of RT-TaqMan-LAMP method for detecting Newcastle disease virus in chicken

Abstract:

[Background] Newcastle disease (ND), one of the Class II infectious diseases induced by Newcastle disease virus (NDV), causes huge economic loss to the poultry industry. Thus, early and accurate screening of NDV is crucial for the prevention of ND outbreak. [Objective] To develop a rapid method for the detection of Newcastle disease virus (NDV) based on the reverse transcription loop-mediated isothermal amplification combined with a TaqMan probe (RT-TaqMan-LAMP). [Methods] The specific primer pairs and TaqMan probes were designed according to the NDV F gene sequence, and the reaction conditions were optimized with the recombinant plasmid pMD-NDV-F as a positive standard to verify the specificity, sensitivity, and reproducibility of the method. With 70 samples, the method was compared with the RT-qPCR method recommended in the national standard (GB/T 16550—2020). [Results] The optimal reaction conditions are as follows: 61 ℃, 60 min, 1.6 μmol/L (FIP/BIP), 0.2μmol/L (F3/B3), 0.8 μmol/L (LF/LB), and 0.2 μmol/L (GTP). The lowest detection limit was 1.651×10² copies/μL, and the sensitivity was 100 folds that of LAMP. No non-specific amplification was observed, and no cross-reactivity with Avian influenza virus (AIV), Mycoplasma gallisepticum (MG), Mycoplasma bursalis (MS), infectious bursal disease virus (IBDV), and herpes simplex virus (HSV) was detected. The intra- and inter-batch coefficients of variation (CV) were＜3%. The method detected one more positive sample than RT-qPCR in 70 clinical samples and the coincidence rate was 98.57% after two retests. [Conclusion] The NDV RT-TaqMan-LAMP method is highly specific and sensitive, with high repeatability, which can effectively avoid non-specific amplification and can be used for accurate detection of NDV and epidemic prevention.

Key words:Newcastle disease virus;LAMP;TaqMan probe;rapid detection