猪流感病毒SYBR GreenⅠ定量RT-PCR检测方法的建立及应用  

Establishment of SYBR GreenⅠreal time RT-PCR for detecting swine influenza virus

作　　者：张鹏超[1,2] 师小潇[1,3] 哈卓[1,3] 刘永杰[2] 刘惠莉[1,3] 

机构地区：[1]上海市农业科学院畜牧兽医研究所,上海201106 [2]南京农业大学动物医学院,江苏南京210095 [3]上海市农业遗传育种重点实验室,上海201106

出　　处：《中国预防兽医学报》2010年第10期768-772,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：上海市农委重点攻关项目(沪农科攻字(2004)第12-2号、沪农科攻字(2009)第5-2号)

摘　　要：为研究猪流感病毒(SIV)感染后SIV在体内的分布规律,本研究设计针对SIV NP基因保守区引物,建立了SIV SYBR GreenⅠ荧光定量PCR方法。该方法对SIV核酸检测灵敏度为30TCID50,对健康猪肺组织cDNA、猪瘟病毒(CSFV)、猪蓝耳病病毒(PRRSV)核酸呈阴性反应。采用建立的荧光定量RT-PCR方法对人工感染A/swine/Shanghai/1/2007/H1N2(Sw/SH/1/2007)猪进行检测,结果显示:病毒在猪鼻腔粘膜持续存在至感染后第8d;肛门拭子在感染后2d～8d可持续检测到病毒核酸;喉头、气管在感染后第3d可检到病毒核酸,并持续至第10d;肺部淋巴结、脾脏在感染后5d～7d检测到病毒核酸阳性;其它脏器均未检测到病毒核酸,从而确定呼吸道系统及脾脏是SIV定殖的主要场所,SIV感染猪后向外排毒周期约为1周。The distribution of swine influenza virus（SIV） in pigs was investigated after artificially infected 40-day-old pigs with A/Sw/SH/1/2007 SIV strain.The infected pigs showed clinical symptom of cough and sneezing,and recovered within two weeks.The nasal swabs,anal swabs and tissue samples were taken at selected time points post inoculation（DPI） and detected for SIV by fluorescent quantitative PCR.Nucleic acid of the virus could be detected in nasal swabs immediately after inoculation and persistent for 8 days;The virus was detected positive in respiratory mucus from 3 DPI to 10 DPI,in the anal swabs from 2 DPI to 8 DPI,and in the lung lymph notes and spleen was 5 DPI to 7 DPI.It was concluded that the A/Sw/SH/1/2007 virus was mainly distributed in respiratory system and spleen,and the respiratory mucous is the main port of swine influenza virus colonization.

关 键 词：猪流感病毒 SYBR GreenI荧光定量PCR方法 组织分布 

分 类 号：S852.65[农业科学—基础兽医学]