无乳链球菌和海豚链球菌早期预警分子检测

Molecular Detection and Early Warning of Streptococcus agalactiae and S.iniae

为提高罗非鱼链球菌病发前的早期预警,根据海豚链球菌的16S rRNA基因的部分序列和无乳链球菌的Sip基因特异性序列,分别合成特异性检测引物,经过对反应体系和反应条件的优化,建立检测较低含量的无乳链球菌和海豚链球菌的分子技术。试验结果显示,所建立的双重PCR可扩增出870 bp无乳链球菌和614 bp海豚链球菌的特异性片段,而迟钝爱德华氏菌、维氏气单胞菌、创伤弧菌、温和气单胞菌、溶藻弧菌、大肠杆菌均为阴性;无乳链球菌和海豚链球菌基因组DNA最低检测量分别为9.84×10^(-5) ng/μL和9.30×10^(-5) ng/μL,菌液最低检测量分别为2.76×10^(3) cfu/mL和2.51×10^(3) cfu/mL,远低于其半致死密度107 cfu/mL;对无乳链球菌和海豚链球菌混合感染的模拟临床样品的检出率为100%。研究结果表明,该分子技术的特异性较强、灵敏度较高、检出率较高,可用于较低含量的无乳链球菌和海豚链球菌的快速鉴别,为罗非鱼链球菌的早期预警分子检测提供技术支持。

Two pairs of specific primers designed according to the 16S rRNA partial gene sequence of Streptococcus agalactiae and the Sip gene sequence of S.iniae,respectively,were applied to establish a rapid and sensitive PCR detection system,aiming to detect trivial amounts of S.agalactiae and S.iniae simultaneously.Two distinct DNA fragments of 870 bp and 614 bp covering the 16S rRNA partial gene sequence and the Sip gene sequence were amplified from the genomic DNA of S.agalactiae and S.iniae,respectively.No cross-reactivity was observed between the two Streptococcus species or with other fish/nonfish pathogens,including Edwards ellatarda,Aeromonas veronii,Vibrio vulnificus,A.sobria,V.alginolyticus,and Escherichia coli.The two primer pairs were capable of detecting genomic DNA of S.agalactiae and S.iniae in concentrations as low as 9.84×10^(-5) ng/μL and 9.30×10^(-5) ng/μL,respectively,and of detecting the bacterial cells of S.agalactiae and S.iniae in amount as low as 2.76×10^(3) cfu/mL and 2.51×10^(3) cfu/mL.The duplex PCR designed in this study had strong specificity,high sensitivity,high detection rate,and was used for rapid identification of lower amount of S.agalactiae and S.iniae.The finding provides a valuable tool for early warning detection of the pathogens leading to streptococosis.