摘要
建立扩增内标存在下同时检测副溶血弧菌tlh基因和trh基因的多重PCR方法。以细菌16SrDNA片段为扩增内标对照(internal amplification control,IAC),副溶血弧菌不耐热溶血毒素(thermolabile hemolysin,TLH)基因tlh和相对耐热直接溶血毒素(thermostable related hemolysin,TRH)基因trh为检测基因,设计引物,并优化多重PCR体系。特异性及灵敏度试验结果表明:该多重PCR体系检测致病性副溶血弧菌表现出极好的特异性。纯培养条件下,扩增内标存在时tlh基因和致病基因trh的检测灵敏度分别为1.3×102 cfu.mL-1和1.3×103 cfu.mL-1;人工污染牡蛎样品,不经富集培养,扩增内标存在时tlh基因和致病基因trh的检测灵敏度分别为2.6×103 cfu.mL-1和2.6×104 cfu.mL-1;经过6h的富集培养,tlh基因和trh基因的检测限均能达到2.6×102 cfu.mL-1。该检测体系特异性强、灵敏度高,并且扩增内标的存在可排除PCR检测致病性副溶血弧菌可能导致的假阴性结果,可提高检测的速度和精准度。
A multiplex PCR assay was developed to simultaneously detect tlh and trh genes of Vibrio parahaemolyticus in the presence of an internal amplification control（IAC）.Using 16SrDNA fragment of bacteria as IAC primers were designed to detect the genes of thermolabile hemolysin（tlh） and thermostable related hemolysin（trh） in vibrio parahaemolyticus,and the assay was optimized.The experiment of specificity and sensitivity showed that this multiplex PCR assay had a good specificity,and for pure culture,the detection limit of tlh and trh was 1.3×102 CFU·mL-1 and 1.3×103 CFU·mL-1 respectively in the presence of IAC.For samples of artificially contaminated oysters,however,the detection sensitivity of tlh and trh was 2.6×103 CFU·mL-1 and 2.6×104 CFU·mL-1 respectively in the presence of IAC without enrichment.After 6h enrichment as little as 2.6×102 CFU·mL-1 of tlh and trh could be detected by this multiplex PCR.This test system had a good specificity and sensitivity.The existence of IAC could successfully eliminate false-negative results using PCR technique to detect pathogenic Vibrio parahaemolyticus.This method could improve the detection speed and accuracy.
出处
《沈阳农业大学学报》
CAS
CSCD
北大核心
2010年第6期701-705,共5页
Journal of Shenyang Agricultural University
基金
教育部留学回国人员基金项目(2006331)
辽宁省科技厅资助项目(20062109)
沈阳市科技局资助项目(1063312-1-00
090009)
关键词
扩增内标对照
多重PCR
副溶血弧菌
特异性
灵敏度
internal amplification control
multiplex PCR
Vibrio parahaemolyticus
specificity
sensitivity
