鹅星状病毒基因Ⅰ型和Ⅱ型双重荧光定量RT-PCR检测方法的建立与应用

doi:10.11843/j.issn.0366-6964.2023.04.025

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (4): 1616-1623.doi: 10.11843/j.issn.0366-6964.2023.04.025

鹅星状病毒基因Ⅰ型和Ⅱ型双重荧光定量RT-PCR检测方法的建立与应用

王宏宇^1,2, 朱寅初², 云涛², 张存^2*, 鲍恩东^1*

1. 南京农业大学动物医学院, 南京 210095;
2. 浙江省农业科学院畜牧兽医研究所, 杭州 310021

收稿日期:2022-07-07 出版日期:2023-04-23 发布日期:2023-04-27
通讯作者: 鲍恩东,主要从事畜禽热休克蛋白研究及畜禽疾病的病理研究,E-mail:b_endong@njau.edu.cn;张存,主要从事畜禽疾病的防控研究,E-mail:zhangcun@aliyun.com
作者简介:王宏宇(1992-),男,山东潍坊人,博士生,主要从事畜禽疾病的防控及病理研究,E-mail:wanghongyuwhy92@163.com
基金资助:
浙江省自然科学基金资助项目（LGN22C180005）；浙江省重点研发计划项目（2019C02052）；浙江省农业重大技术协同推广项目（2021XTTGXM04-02）；科技部外传项目（产业技术创新类，G20200010082）；江苏省农业科技自主创新资金项目（CX（22）2016）

Establishment and Application of Double Real-time PCR Detection Simultaneously for GAstV Ⅰ and GAstV Ⅱ

WANG Hongyu^1,2, ZHU Yinchu², YUN Tao², ZHANG Cun^2*, BAO Endong^1*

1. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;
2. Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2022-07-07 Online:2023-04-23 Published:2023-04-27

摘要/Abstract

摘要： 鹅星状病毒（goose astroviruses，GAstV）作为一种雏鹅新发病原，对养鹅业造成巨大经济损失，在尚无有效治疗手段情况下，加强疫病及时检测和防控十分重要。为建立快速、特异且灵敏的鉴别GAstV Ⅰ型和GAstV Ⅱ型的病毒检测方法，本研究根据GAstV Ⅰ和GAstV Ⅱ的保守区域RdRp基因序列，设计合成2对特异性引物和2种不同荧光基团标记的TaqMan探针，建立了一种双重荧光定量PCR检测方法，以达到在同一反应体系中同时定量检测GAstV Ⅰ和GAstV Ⅱ的目的。结果显示，GAstV Ⅰ和GAstV Ⅱ的最小检出量分别为43.3、6.49 copies·μL^-1；重复试验的变异系数不超过0.5%；该方法对DPV、GPV、GTMUV、GRV、AIV （H9N2）等病毒核酸无交叉反应。综上表明，本研究建立的可鉴别GAstV Ⅰ和GAstV Ⅱ的双重荧光定量PCR方法灵敏度高、特异性强、重复性好。可用于临床GAstV Ⅰ和GAstV Ⅱ的快速鉴别诊断，也可用于两种基因型GAstV的定量分析。

关键词: 鹅星状病毒Ⅰ型, 鹅星状病毒Ⅱ型, 双重荧光定量RT-PCR, TaqMan探针

Abstract: Goose astrovirus (GAstV), as an emerging pathogen causing visceral gout and high mortality in goslings and result in huge economic losses. Thus, it is very important to develop credible detection methods to prevent this diseases wide spread in the absence of effective treatment measure. In order to establish a rapid, specific and sensitive method for detection of genotype GAstV Ⅰ and GAstV Ⅱ viruses, a duplex TaqMan real-time quantitative reverse transcription PCR (RT-qPCR) assay was established. Two pairs of specific primers and two kinds of TaqMan probes labeled with different fluorescent groups according to the RdRp gene sequences were designed and synthesized. The results showed that the minimum detectable amounts of GAstV Ⅰ and GAstV Ⅱ were 43.3 and 6.49 DNA copies·μL^-1, respectively; the coefficient of variation of repeated tests was less than 0.5%; and the method had no cross reaction to viral nucleic acids such as DPV, GPV, GTMUV, GRV, AIV (H9N2), etc. In summary, the results above indicate that this duplex real-time PCR method can distinguish GAstV Ⅰ and GAstV Ⅱ with high sensitivity and specificity. It can be used for the clinical rapid differential diagnosis of GAstV Ⅰ and GAstV Ⅱ, and quantitative analysis of two genotypes of GAstV.

Key words: GAstV Ⅰ, GAstV Ⅱ, double real-time RT-PCR, TaqMan probe

中图分类号:

S852.659.6

王宏宇, 朱寅初, 云涛, 张存, 鲍恩东. 鹅星状病毒基因Ⅰ型和Ⅱ型双重荧光定量RT-PCR检测方法的建立与应用[J]. 畜牧兽医学报, 2023, 54(4): 1616-1623.

WANG Hongyu, ZHU Yinchu, YUN Tao, ZHANG Cun, BAO Endong. Establishment and Application of Double Real-time PCR Detection Simultaneously for GAstV Ⅰ and GAstV Ⅱ[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1616-1623.

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鹅星状病毒基因Ⅰ型和Ⅱ型双重荧光定量RT-PCR检测方法的建立与应用

Establishment and Application of Double Real-time PCR Detection Simultaneously for GAstV Ⅰ and GAstV Ⅱ

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