摘要
针对鰤鱼诺卡氏菌16S—23SrRNA基因内转录间隔序列,设计了四条LAMP引物,在外内引物浓度比1:8、dNTP浓度0.8—1.2mmol/L、Mg2+浓度10—14mmol/L、反应温度60—65℃、反应时间60min的优化条件下,扩增产物经电泳后呈现特异性的LAMP梯形条带,建立的LAMP法具有高灵敏度,达到10-7μg/μlDNA浓度,比常规PCR方法高100倍。将该方法应用于取自养殖场的乌鳢、大黄鱼、黄姑鱼组织样品检测,结果在16份组织样品中有7份检出自然感染的鰤鱼诺卡氏菌,与同步取样品鱼组织的细菌分离、培养检查结果相一致,并显示既能检测发病鱼,又能检出未发病且已感染的病鱼。分析表明,这是一种能快速、简易、特异、敏感的检测鱼类致病鰤鱼诺卡氏菌的诊断方法。
To develop a loop-mediated isothermal amplification （LAMP） assay for detection of fish pathogenic Nocardia seriolae. Four primers were designed based on the sequence of the 16S--23S ribosomal RNA internal transcribed spacer region of N. seriolae for the use of LAMP assay. The assay was optimized to the following conditions： 1：8 outer primer concentration ratio, 0.8--1.2mmol/L of dNTP, 10--14mmol/L of Mg2＋, at 60--65℃ for 60min. LAMP amplification products had ladder-like bands when electrophoresed on an agarose gel. The assay could amplify up to 10^-7μg/μl of DNA. Hence, the sensitivity of LAMP assay was 100-fold higher than the standard PCR protocol. The assay was applied to detect the presence of Nocardia seriolae in different tissue samples from farm Ophiocephalus argus, Pseudnoseiaena crotea, Niber albiflora. Seven naturally infected 17. seriolae were detected in 16 tissue samples, and the results from the bacterial culture test were consistent with fish tissue samples taken simultaneously. This study showed LAMP assay could be used not only to diagnose the infected fishes, but also to recognize the carrier ofN. seriolae as well. LAMP assay is a rapid, simple, specific and sensitive test to diagnose for fish pathogenic N. seriolae.
出处
《海洋与湖沼》
CAS
CSCD
北大核心
2011年第1期27-31,共5页
Oceanologia Et Limnologia Sinica
基金
长江学者和创新团队发展计划资助
IRT0734号
农业部公益性行业科研专项
200903029号
浙江省自然科学基金项目
Y3080244号
