鹅肾型星状病毒SYBR GreenⅠ实时荧光定量PCR方法的建立  

Establishment of a SYBR GreenⅠ-based real-time PCR for goose nephritic astrovirus

作　　者：邱思语 徐蓉[1] 郭远奎 吕英军[1] QIU Si-yu;XU Rong;GUO Yuan-kui;LU Ying-jun(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Rongcheng Yinzi Animal Husbandry and Veterinary Station,Rongcheng 264301,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]荣成市荫子畜牧兽医站,山东荣成264301

出　　处：《中国兽医科学》2020年第3期300-306,共7页Chinese Veterinary Science

基　　金：江苏高校优势学科建设工程资助项目(PAPD)。

摘　　要：本研究旨在建立一种灵敏、快速检测鹅肾型星状病毒的SYBR GreenⅠ实时荧光定量PCR方法。根据鹅肾型星状病毒的ORF1b保守序列,设计出1对引物,建立了检测该病毒的SYBR GreenⅠ实时荧光定量PCR方法。结果显示,该方法的Ct值与标准质粒在2.42×10^4~2.42×10^9copies/μL范围内呈良好的线性关系;敏感性高,最低检测限为24.2 copies/μL;该方法特异性强,对禽流感H9N2、鹅副黏病毒、鹅细小病毒、鸭坦布苏病毒均无交叉反应;重复性好,批内和批间变异系数均小于2.5%。运用该方法对动物回归试验中攻毒的雏鹅的各脏器进行检测,结果显示,肾、脾和肝中病毒含量较高,其次是胰腺、肺及肠道,而心和脑中的病毒含量最低。上述结果表明,本研究成功建立了鹅肾型星状病毒的SYBR GreenⅠ实时荧光定量PCR方法,并根据此方法检测出该病毒对肾、脾及肝具有较强的亲嗜性,为该病原致病机制研究奠定了基础。The purpose of the study is to establish a sensitive and rapid real-time PCR method for the detection of goose nephritic astrovirus.A pair of primers was designed based on the ORF1b conserved sequence of goose nephritic astrovirus,and then,a SYBR GreenⅠ-based real-time PCR method for goose nephritc astrovirus was established.The results showed that the Ct value of the established SYBR GreenⅠ-based real-time PCR method had a good linear relationship with the standard plasmid in the range of 2.42×10^4-2.42×10^9 copies/μL.The method showed high sensitivity and the limit of detection concentration was 24.2 copies/μL.The method was specific for goose nephritic astrovirus and there was no cross-reaction with avian influenza(H9N2)virus,goose paramyxovirus,goose parvovirus and duck tembusu virus.It had a good repeatability,the intra-assay and inter-assay coefficients of variation were less than 2.5%.The virus load in different infected goose organs were detected using the method,the results showed that the kidney,spleen and liver had high virus load,followed by pancreas,lung and intestine,while the virus load in heart and brain was the lowest.In conclusion,a goose nephritic astrovirus SYBR GreenⅠ-based real-time PCR detection method was successfully established in the study,and a strong affinity of the virus to kidney,spleen and liver was observed according to the method,which supplied a foundation for the pathogenic mechanism investigation.

关 键 词：鹅肾型星状病毒 实时荧光定量PCR SYBR GreenⅠ 病毒分布 

分 类 号：S852.659.6[农业科学—基础兽医学]