应用二温式PCR检测家蚕核型多角体病毒的方法  

Method for Detection of Bombyx mori Nucleopolyhedrovirus by Two-temperature PCR

作　　者：覃玥[1,2] 陈保善[2,3] 

机构地区：[1]河池学院化学与生物工程学院,广西宜州546300 [2]广西大学生命科学与技术学院,南宁530004 [3]广西亚热带生物资源保护利用重点实验室,南宁530004

出　　处：《蚕业科学》2015年第3期565-570,共6页ACTA SERICOLOGICA SINICA

基　　金：广西科技厅重大项目(No.桂科攻0718004-2B);广西亚热带生物资源保护利用重点实验室(省部共建国家重点实验室)开放课题基金项目(No.SB0710);广西壮族自治区教育厅科研项目(No.201010LX480)

摘　　要：为了简便、快速、准确地检测家蚕核型多角体病毒(Bm NPV),以Bm NPV广西分离株的多角体蛋白基因(polh)序列(Gen Bank登录号:JQ991011.1)设计引物,以感染Bm NPV(广西宜州分离株)2 h的5龄家蚕幼虫中肠组织提取的DNA为模板,建立二温式PCR检测Bm NPV的方法,最终确定的最佳反应体系为:10×PCR buffer 1.5μL,5 mmol/L Mg Cl22μL,2.5mmol/L d NTP 0.5μL,10μmol/L上、下游引物各1μL,5 U/μL Taq DNA聚合酶0.1μL,18 ng/μL模板DNA 1μL,加水至总体积15μL。确定的最佳反应条件为:94℃预变性3 min;95℃15 s,延伸和退火温度合并为62℃30 s,共25个循环。按上述条件PCR扩增得到大小约309 bp的特异性片段,测序结果与已知polh基因序列的相似度为100%。与普通PCR检测方法比较,采用二温式PCR方法对样本的检测时间节省1.5 h,反应特异性强,灵敏度高(能被检测的最小感病组织基因组DNA的质量浓度为1.8 pg/μL),并且选择最初2 h感病的幼虫中肠组织为检测材料,因而可用于家蚕血液型脓病的早期诊断。In order to develop a simple, quick and accurate method for detection of Bombyx mori nucleopolyhedrovirus （BmNPV）, polyhedrin gene （po/h, GenBank accession No. JQ991011.1 ） of BmNPV Guangxi isolate was used to design primers, and genomic DNAs extracted from the midgut tissue of the 5th instar silkworm larvae at 2 hours post in- fection by BmNPV isolated at Yizhou of Guangxi were used as template to detect BmNPV by two-temperature PCR meth- od. The optimal PCR amplification reaction system （15 μL） was as follows： 10xPCR buffer 1.5 μL, 5 mmol/L MgCI2 2 μL, 2.5 mmol/L dNTPs 0.5 μL, 10 μmol/L forward primer 1μL, 10 μmol/L reverse primer 1 FL, 5 U/FL Taq DNA pol- ymerase 0. 1μL, 18 ng/μL DNA template 1μL. The optimal PCR amplification procedure was as follows, pre-denature at 95 ℃ for 3 mini then 94 ℃ 15 s, elongate and anneal at 62 ℃ for 30 s, totally 25 cycles, Under these optimal conditions, a specific fragment of about 309 bp was obtained by PCR amplification. The sequence similarity of sequencing result and known polh gene was 100%. Compared with the common PCR method, two-temperature PCR method can save 1.5 hours and had the characteristics of highly specific and high sensitivity （the minimum detectable amount of genomic DNAs from infected silk-worm midgut tissue was 1.8 pg/μL）. This newly established method for detection of BmNPV by two-temperature PCR took the midgut tissue at 2 h after infection as materials, suggesting that it ould be used for early diagnosis of nucle- opolydhedrosis.

关 键 词：家蚕核型多角体病毒 多角体蛋白基因 二温式PCR 反应条件 

分 类 号：S884.51[农业科学—特种经济动物饲养] Q789[农业科学—畜牧兽医]