库尔勒香梨主要病毒多重RT-PCR检测技术研究

 利用nad5基因作为内标系统,研究建立了库尔勒香梨上苹果茎痘病毒(ASPV)、苹果褪绿叶斑病毒(ACLSV)和苹果茎沟病毒(ASGV)等的RT-PCR检测技术,在此基础上研究建立了库尔勒香梨多重RT-PCR内标检测系统。并对回收的特异片段进行了克隆、鉴定和测序。测序结果表明:库尔勒香梨上的ACLSV与GenBank中D14996序列(日本苹果上的ACLSV分离物)中的6860~7536bp片段有116个碱基的差异,序列相似性为83.75%;库尔勒香梨上的ASPV与GenBank中D21828序列(德国梨脉黄病毒相关分离物)中的8869~9238bp片段有72个碱基的差异,序列相似性为79.5%;库尔勒香梨上的ASGV与GenBank中AB004063序列(日本ASGV分离物)中的6039~6311bp片段有21个碱基的差异,序列相似性为92.3%。

Multiple RT-PCR detection of viruses in pear

A 181 bp fragment of NADH dehydrogenase subunit 5 (nad5) gene cloned from mitochondrial DNA of apple was used as internal control in detection of three pear viruses by multiplex RT-PCR assays. The recovered fragments were cloned, sequenced and identified. Sequencing results indicate that the ACLSV on Kuala fragrant pear reveals 116 base difference between 6860 and 7536 bp and sequence identity is 83. 75% compared with sequence D14996 (isolates of Japan ACLSV); the ASPV on Kuala fragrant pear shows 72 base difference from 8869 to 9238 bp and sequence identity is 79. 5% compared with sequence D21828 (isolates of German pear vein yellows virus); and the ASGV on Kuala fragrant pear has only 21 base difference from nucleotide 6039 to 6311 bp and sequence identities attain 92. 3% highly compared with AB004063 in GenBank (isolates of Japan ASGV).