摘要
目的建立快速、敏感、特异和精确的方法检测致病性副溶血弧菌（ＶＰ）。方法在耐热 溶血毒素（ＴＤＨ）结构基因序列的基础上，自行设计探针和引物，应用克隆杂交方法检测３０株不同 来源的ＶＰ，同时对１０株不同ＫＰ反应的ＶＰ ＤＮＡ行ＰＣＲ－ＴＤＨ检测。结果３０例实验菌株，２６ 例ＫＰ阳性株克隆杂交出现阳性信号，其余４例杂交阴性（２例ＫＰ弱阳性、２例ＫＰ阴性）。用ＰＣＲ 检测的１０株ＶＰ中，６例ＫＰ阳性株ＴＤＨ基因均阳性，但有１株ＫＰ弱阳性和１株ＫＰ阴性株也呈 ＴＤＨ基因阳性。ＰＣＲ阳性结果均用Ｓｏｕｔｈｅｒｎ ｂｌｏｔ法证实。结论应用克隆杂交技术检测ＴＤＨ基 因在合适的条件下能有效地鉴别致病性与非致病性ＶＰ。 ＰＣＲ检测ＴＤＨ基因为研究ＶＰ的致病机 理奠定基础，在临床上可作为一种检测致病性ＶＰ的快速敏感的初筛试验。
Objective To establish fast, sensitive and precise methods for detecting Kanagawa phenomenon (KP)-positive vibrio parahaemolyticus(VP). Methods Synthetic oligodeoxyri-bonu- cleotid was used in the colony hybridization test to examine thermostable direct hemolysim (TDH) gene , and polymerase chain reaction(PCR) was also used to detect TDH gene. Results 30 strains VP including different KP reactions and serotypes were detected with colony hybridization, 26 strains were positive, the Others were negative. All the positive strains were in accordence with the traditional KP test, but 2 strains which were weakpositive in KP test were negative. 6 KP- positive strains, 2 KP-negative strains and 2 KP-weak-positive strains weretested with PCR for the TDH gene. All the KP-positive strains were TDH gene positive. At the same time , I KP weak-positive strain and 1 KP-negative strain were also gene positive. These PCR positive results were confirmed with Southern blot. Conclusions Under optimal conditions, the probe was capable of detecting KP-positive VP and distinguishing pathogenic VP from inpathogenic VP. Detecting TDH gene by PCR was helpful in understanding the pathogenic mechanism of VP and benefical in quickly screenning pathogenicity VP in clinic, it also increases the sensitivity and specificity.
出处
《中华疾病控制杂志》
CAS
1999年第1期16-18,共3页
Chinese Journal of Disease Control & Prevention
关键词
副溶血弧菌
克隆杂交
聚合酶链反应
vibrio parahaemolyticus (VP)
colony hybridization
polymerase chain reaction(PCR )
