建立并应用 TaqMan 一步法实时 RT-PCR 检测新型鹅源星状病毒。
Establishment and application of a TaqMan-based one-step real-time RT-PCR for the detection of novel goose-origin astrovirus.
机构信息
College of Animal Science and Technology, Shandong Agricultural University, 61 Daizong Street, Tai'an, Shandong Province, 271018, China; Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Tai'an, Shandong, 271018, China; Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Tai'an, Shandong, 271018, China.
College of Animal Science and Technology, Shandong Agricultural University, 61 Daizong Street, Tai'an, Shandong Province, 271018, China; Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Tai'an, Shandong, 271018, China; Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Tai'an, Shandong, 271018, China.
出版信息
J Virol Methods. 2020 Jan;275:113757. doi: 10.1016/j.jviromet.2019.113757. Epub 2019 Oct 24.
The outbreak of an infectious disease characterized by severe symptom of gout has set great threat to several major goose-producing regions in China since December 2016. The causative agent for the novel infection has been identified was a novel goose-origin astrovirus (GoAstV). Lack of effective detection methods indeed hinders further research, as well as prevention and control of GoAstV. Keep this in mind, a TaqMan-based one-step real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for rapid detection of GoAstV was developed. Primers and probe were targeting the capsid protein gene sequence (ORF2). The method is capable of detecting quite low number of targeting nucleic acid as low as 10 copies/μL. What's more, it is also of great specificity and repeatability for GoAstV detection. No cross-activity was found with other goose-origin viruses. The assay had excellent intra-assay and inter-assay repeatability with the coefficient of variation (CV) value from 0.48% to 0.99%. A total of 340 GoAstV specimens from different regions of China were used in this study to verify the feasibility and effectiveness of this method in clinical diagnosis. The results indicated that qRT-PCR is a highly sensitive, specific and repeatable method for quantitative detection of GoAstV, which can be used to detect this virus, thereby facilitating epidemiological investigations of gout in goslings.
自 2016 年 12 月以来，一种以严重痛风症状为特征的传染病在中国几个主要的鹅产区爆发，对其构成了严重威胁。新型感染的病原体已被确定为一种新型鹅源星状病毒（GoAstV）。缺乏有效的检测方法确实阻碍了进一步的研究，以及对 GoAstV 的预防和控制。鉴于此，开发了一种基于 TaqMan 的一步实时定量逆转录聚合酶链反应（qRT-PCR）检测方法，用于快速检测 GoAstV。引物和探针针对衣壳蛋白基因序列（ORF2）。该方法能够检测到低至 10 拷贝/μL 的低数量的靶向核酸。此外，它对 GoAstV 的检测具有很高的特异性和重复性。与其他鹅源病毒没有交叉活性。该检测方法在内部和外部检测中具有极好的重复性，变异系数（CV）值为 0.48%至 0.99%。本研究共使用了来自中国不同地区的 340 个 GoAstV 标本，以验证该方法在临床诊断中的可行性和有效性。结果表明，qRT-PCR 是一种高度敏感、特异和可重复的方法，用于定量检测 GoAstV，可以用来检测这种病毒，从而有利于鹅痛风的流行病学调查。
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