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当前位置: 首页 > 文章 > 应用二温式PCR检测家蚕核型多角体病毒的方法 蚕业科学 2015,41 (3) 565-570
Position: Home > Articles > Method for Detection of Bombyx mori Nucleopolyhedrovirus by Two-temperature PCR Science of Sericulture 2015,41 (3) 565-570

应用二温式PCR检测家蚕核型多角体病毒的方法

作  者：
覃玥;陈保善
单  位：
河池学院化学与生命科学系;广西大学生命科学与技术学院
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
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。
译  名：
Method for Detection of Bombyx mori Nucleopolyhedrovirus by Two-temperature PCR
作  者：
Qin Yue;Chen Baoshan;College of Chemistry and Bioengineering,Hechi University;College of Life Science and Technology,Guangxi University;Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization;
关键词：
Bombyx mori nucleopolyhedrovirus(Bm NPV);;Polyhedrin gene(polh);;Two-temperature PCR;;Reaction condition
摘  要：
In order to develop a simple,quick and accurate method for detection of Bombyx mori nucleopolyhedrovirus( Bm NPV),polyhedrin gene( polh,Gen Bank accession No. JQ991011. 1) of Bm NPV 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 infection by Bm NPV isolated at Yizhou of Guangxi were used as template to detect Bm NPV by two-temperature PCR method.The optimal PCR amplification reaction system( 15 μL) was as follows: 10 × PCR buffer 1. 5 μL,5 mmol / L Mg Cl22 μL,2. 5 mmol / L d NTPs 0. 5 μL,10 μmol / L forward primer 1 μL,10 μmol / L reverse primer 1 μL,5 U / μL Taq DNA polymerase 0. 1 μL,18 ng / μL DNA template 1 μL. The optimal PCR amplification procedure was as follows: pre-denature at 95℃ for 3 min; 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 infectedsilkworm midgut tissue was 1. 8 pg / μL). This newly established method for detection of Bm NPV 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 nucleopolydhedrosis.

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