Rapid identification, pathotyping and quantification of infectious bursal disease virus by high-resolution melting curve quantitative reverse transcription PCR analysis: An innovative technology well-suited for real-time large-scale epidemiological surveillance

文献类型： 外文期刊

第一作者： Wang, Weiwei

作者： Wang, Weiwei;Zhang, Yan;Qiao, Yuanzheng;Shi, Jun;Huang, Jianni;Huang, Teng;Wei, Tianchao;Mo, Meilan;Wei, Ping;Wang, Weiwei;Zuo, Wenbo;He, Xiumiao;Zhang, Yan

作者机构：

关键词： Infectious bursal disease virus (IBDV); High-resolution melting curve (HRM); Quantitative reverse transcription PCR (qRT-; PCR); Pathotyping; Epidemiological surveillance

期刊名称：POULTRY SCIENCE （ 影响因子：4.2； 五年影响因子：4.5 ）

ISSN： 0032-5791

年卷期： 2024 年 103 卷 12 期

页码：

收录情况： SCI

摘要： 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 genomescreening 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/mu L, 61.1 copies/mu L and 67.5 copies/ mu L for vvIBDV, nvIBDV and attIBDV, respectively. The method exhibited excellent specificity, with no intergenotypes 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.

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