同时检测两种对虾病毒和4种弧菌的同步PCR方法的建立

Development of synchronous PCR for simultaneous detection of two viruses and four Vibrios in penaeid shrimp

通过检索、多重比对、分析和筛选GenBank中对虾白斑综合征病毒(WSSV)、传染性皮下和造血器官坏死病毒(IHHNV)、副溶血弧菌、创伤弧菌、哈维氏弧菌和溶藻胶弧菌的基因序列,设计了10对特异性引物,以已知毒株和菌株的DNA为模板进行PCR,均能扩增出与实验设计相符合的DNA片段,对PCR扩增条件进行优化,建立了可同时检测鉴别WSSV、IHHNV、副溶血弧菌、创伤弧菌、哈维氏弧菌和溶藻胶弧菌,并且能同时区分WSSV不同地理毒株的同步PCR方法。研究结果表明,该方法检测特异性好,检测通量大,适合于对虾多种病原的同时检测。

The conditions of a synchronous polymerase chain reaction （PCR） method was optimized to simultaneously detect six pathogens, including white spot syndrome virus （WSSV） of different geological stains, infectious hypodermal and hematopoietic necrosis virus （IHHNV）, Vibrio parahaemolyticus, V. algnolyticus, V. harveyi, V. vulnificus from penaeid shrimp. Ten pairs of specific primers were designed based on search, multiple comparison, analysis and selection of the sequences of WSSV, IHHNV,V. parahaernolyticus toxR gene,V. vulnificus toxR gene, V. harveyi toxR gene,V, algnolyticus hsp60 gene in GenBank. The PCR results showed that all samples contained pathogenic DNA could be amplified into specific bands that matched the design. This method was proved to be suitable for simultaneous detection of various pathogens with high specificity and throughput.