鸡新城疫病毒RT-TaqMan-LAMP检测方法的建立及应用

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

摘要:

【背景】新城疫病毒(Newcastle disease virus, NDV)的传染可能会引发作为二类传染病之一的新城疫(Newcastle disease, ND)，给养禽业带来巨大的经济损失，因而早期、精准的NDV筛查是防治ND暴发的关键。【目的】针对新城疫病毒(NDV)建立结合TaqMan探针的反转录环介导等温扩增技术(RT-TaqMan-LAMP)快速检测方法。【方法】根据NDV F基因序列设计特异性引物组和TaqMan探针，以重组质粒pMD-NDV-F为阳性标准品优化反应条件，验证该方法的特异性、灵敏性和重复性，同时与国家标准(GB/T 16550—2020)中推荐的RT-qPCR方法比较，对70份实际样本进行验证。【结果】最佳反应条件为61 ℃ 60 min。引物和探针最优浓度：1.6 μmol/L (FIP/BIP)、0.2 μmol/L (F3/B3)、0.8 μmol/L (LF/LB)、0.2 μmol/L (GTP)。最低检测限为1.651×10² copies/μL，灵敏性是LAMP方法的100倍。无非特异性扩增，与禽流感病毒(avian influenza virus, AIV)、鸡毒支原体(Mycoplasma gallisepticum, MG)、鸡滑液囊支原体(Mycoplasma synoviae, MS)、鸡传染性囊病病毒(infectious bursal disease virus, IBDV)及疱疹病毒(herpes virus, HSV)均无交叉反应，批次内和批次间变异系数(coefficient of variation, CV)均小于3%。在70份临床样品的检测中本方法比RT-qPCR方法多检测出1份阳性样本，经2次复测二者的符合率为98.57%。【结论】本研究建立的NDV RT-TaqMan-LAMP检测方法特异性强、灵敏度高、重复性好，并能有效避免非特异性扩增，可用于NDV的精准检测和流行病预防。

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.