𫚕诺卡菌特异性PCR快速检测方法的建立

Establishment of a specific PCR assay to detect Nocardia seriolae

  • 摘要: 诺卡菌(Nocardia seriolae)是危害中国南方大口黑鲈(Micropterus salmoides)养殖业的主要病原之一,由于该菌在培养基上生长缓慢,对其分离鉴定造成诸多不便。文章根据诺卡菌16S~23S转录间隔区(ITS)序列设计特异性引物建立了诺卡菌特异性PCR快速检测方法。试验结果表明,利用设计的特异性PCR引物只能扩增出诺卡菌特异性片段,检出限为5 pg模板DNA。在此基础上研制了PCR快速检测试剂盒并对诺卡菌人工感染的大口黑鲈组织进行了检测,结果显示该试剂盒能从未出现明显发病症状的大口黑鲈组织中检出阳性片段,阳性率为100%,比传统细菌分离鉴定方法更加灵敏、快速且高效,具有较好的应用前景。

     

    Abstract: Nocardia seriolae, one of the main pathogenic bacterium of cultured largemouth bass Micropterus salmoides in South China, grows very slowly in culture medium, so it is difficult for its isolation and identification. In present study, two specific primers were designed according to genomic sequence of 16S~23S intergenic spacers (ITS) to establish a rapid specific PCR assay to detect N.seriolae. Results indicate that the 2 primers are very specific to detect N.seriolae and show positive to very little of DNA amount at 5 pg. In addition, a rapid PCR detection kit was developed which was then applied to detect the N.seriolae-infected tissues of M.salmoides. The PCR kit can detect positive fragments from M.salmoides without any obvious disease symptoms, so it is superior to traditional method in terms of sensitivity and efficiency, having good potential in clinical diagnose in aquaculture.

     

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