Wang Weiwei, Zhang Yan, Zuo Wenbo, Qiao Yuanzheng, Shi Jun, Huang Jianni, Huang Teng, Wei Tianchao, Mo Meilan, He Xiumiao, Wei Ping
Institute for Poultry Science and Health, Guangxi University, Nanning 530004, China; Institute of Animal Husbandry and Veterinary Medicine/Fujian Industry Technology Innovation Research Academy of Livestock and Poultry Diseases Prevention and Control, Fujian Academy of Agricultural Sciences/Fujian Key Laboratory for Control and Prevention of Avian Diseases, Fuzhou 350013, China.
Institute for Poultry Science and Health, Guangxi University, Nanning 530004, China; Tianjin Wildlife Rescue and Domestication Breeding Center, Tianjin 301600, China.
Poult Sci. 2024 Dec;103(12):104440. doi: 10.1016/j.psj.2024.104440. Epub 2024 Oct 21.
With the virus continuing to evolve, very virulent IBDV (vvIBDV) and novel variant IBDV (nvIBDV) have become the predominant epidemic strains in China, exacerbated by the widespread use of attenuated vaccine strains (attIBDV), making a complex infection situation of IBDV in the field. Therefore, developing a rapid and accurate high-resolution melting curve quantitative reverse transcription PCR (HRM-qRT-PCR) for the identification and pathotyping of IBDV is crucial for clinical monitoring and disease control. Extensive data analysis and genome-screening of the three dominant IBDV pathotypes identified a specific region (nucleotides 2450-2603 in segment A) with distinct GC content as the detection target. Experimental testing of HRM-qRT-PCR revealed distinct melting curves and high sensitivity, with the detection limits of 61.2 copies/μL, 61.1 copies/μL and 67.5 copies/μL for vvIBDV, nvIBDV and attIBDV, respectively. The method exhibited excellent specificity, with no inter-genotypes cross-reactivity among the three pathotypes and no reactivity to other common avian pathogens. Applied to samples with double and triple co-infections of different IBDV pathotypes, the method displayed specific melting peaks corresponding to the viruses present in the samples, with an accuracy rate of 100 %. This method precisely identifies and differentiates all the single or co-infected samples, generating distinct peaks corresponding to the Tm values of each virus pathotype in traditional melting curve plots. Furthermore, the method overcomes the limitations of traditional pathotyping methods, requiring only one reaction to achieve rapid viral pathotyping and facilitating quantitative analysis of viruses within the samples. This study introduces an innovative HRM-qRT-PCR method, offering new technology to rapid and accurate identification, pathotyping and quantification of vvIBDV, nvIBDV, and attIBDV. With strong discriminatory power, user-friendliness and a short processing time, this method is highly attractive for the rapid IBDV pathotyping in real-time large-scale epidemiological surveillance during outbreaks.
随着病毒不断进化，高致病性传染性法氏囊病病毒（vvIBDV）和新型变异传染性法氏囊病病毒（nvIBDV）已成为中国的主要流行毒株，而减毒疫苗株（attIBDV）的广泛使用加剧了这种情况，导致了IBDV在田间复杂的感染状况。因此，开发一种用于IBDV鉴定和分型的快速、准确的高分辨率熔解曲线定量逆转录PCR（HRM-qRT-PCR）对于临床监测和疾病控制至关重要。对三种主要IBDV基因型的广泛数据分析和基因组筛选确定了一个具有独特GC含量的特定区域（A节段中的核苷酸2450-2603）作为检测目标。HRM-qRT-PCR的实验测试显示出明显不同的熔解曲线和高灵敏度，vvIBDV、nvIBDV和attIBDV的检测限分别为61.2拷贝/μL、61.1拷贝/μL和67.5拷贝/μL。该方法具有出色的特异性，三种基因型之间无种间交叉反应，对其他常见禽病原体也无反应。应用于不同IBDV基因型双重和三重共感染的样本时，该方法显示出与样本中存在的病毒相对应的特定熔解峰，准确率为100%。该方法能够精确鉴定和区分所有单一或共感染样本，在传统熔解曲线图中生成与每种病毒基因型的Tm值相对应的独特峰。此外，该方法克服了传统分型方法的局限性，只需要一次反应即可实现快速病毒分型，并便于对样本中的病毒进行定量分析。本研究介绍了一种创新的HRM-qRT-PCR方法，为快速、准确地鉴定、分型和定量vvIBDV、nvIBDV和attIBDV提供了新技术。该方法具有强大的鉴别力、用户友好性和较短的处理时间，对于疫情爆发期间实时大规模流行病学监测中的快速IBDV分型极具吸引力。
