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| Establishment and Application of Multiplex PCR Assay for Detection of Five Freshwater Bacterial Species |
| AI Kete,XU Pei,ZHOU Jingwen,WAN Jing,YANG Daiqin ,XU Qiaoqing |
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Abstract Detection of bacteria with traditional method of microscope examination leads to high inaccuracy .On the contrary , the PCR method is specific and accurate to identify bacterial species . A multiplex PCR method for accurate detection of conventional bacteria in aquatic animals was developed and used to amplify Hly A gene in Aeromonas hydrophila ,Khe gene in Klebsiella pneumoniae ,Sip gene in Streptococcus agalactiae ,Ser C gene ,Eip gene in Edwardsiella ictaluri ,and Muk F gene ,and Gad B gene in Edwardsiella tarda .The results showed that the amplification was found to be high specificity .A double PCR method for simultaneous detection of S . agalactiae and E .ictalur and a multiplex PCR method for simultaneous detection of A . hydrophila , K . pneumoniae , E . tarda and E .ictalur were developed .In order to verify the accuracy of the multiplex PCR method , the diseased ricefield eel (Monopterus albus)were collected for isolation and culture of bacteria ,as well as for DNA extraction from the cultured bacteria .K .pneumoniae and A .hydrophila were detected by multiplex PCR method .And the established multiplex PCR had important significance for rapid detection of aquatic bacterial conventional pathogenic bacterium and molecular epidemiological research .
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Received: 25 May 2015
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