摘要
根据Genbank中对虾白斑病由白斑综合征病毒（WSSV）和传染性皮下及造血器官坏死病毒（IHHNV）的基因序列，设计了两对能分别检测WSSV和IHHNV保守片段基因的特异性引物，而且这两对引物在同一反应体系中可以同时对WSSV和IHHNV的DNA模板进行多重PCR扩增，得到大小分别为110bp（WSSV）和356bp（IHHNV）的扩增条带。对影响PCR反应的主要因素Mg“浓度和退火温度进行了优化，证明当Mg^2＋浓度为2．0～4．0mmd，退火温度为57～59℃时可获得最佳的扩增和检测效果。特异性试验结果表明，这两对引物检测WSSV和IHHNV具有很好的特异性，对其它对虾常见病原的PCR扩增结果均为阴性。敏感性测定结果表明，该反应体系最低能检测100pg的WSSV和IHHNV的DNA模板。临床检测表明，所建立的双重PCR方法可以适用于WSSV和IHHNV的同时检测和鉴别。
Two pairs of specific primers were designed to detect respectively two pathogens including white spot syndrome virus （WSSV） and infectious hypodermal and hematopoietic necrosis virus （IHHNV） according to the conservative sequences of WSSV and IHHNV in Genebank. The DNA sample, which contained WSSV and IHHNV, could be amplified by the multiplex PCR u- sing these two sets of primers, yielding two sizes of specific bands 110 bp （WSSV） and 356 bp （ IHHNV）. The main factors of the different concentration of Mg^2＋ and the anneal temperature affecting the PCR reaction were tested, and it showed that the optimal reaction system was 2.0 to 4. 0 mM of Mg^2＋ and 57 to 59 ℃ for anneal temperature. The primers indicates high peculiarity in experiments, no specific bands were amplified from other panaeid shrimp pathogenic virus and bacteria. The minimum DNA of WSSV and IHHNV being able to cheek out was 100 pg. It suggested that this multiplex PCR could be used as a sensitive tool to detect WSSV and IHHNV simultaneously in clinical sample.
出处
《福建水产》
2008年第4期38-42,共5页
Journal of Fujian Fisheries
基金
国家质量监督检验检疫总局项目(2004IK028)资助
