分子信标探针技术检测沙门菌invA基因

南方医科大学学报 ›› 2004, Vol. 24 ›› Issue (11): 1257-1259.

分子信标探针技术检测沙门菌invA基因

万成松, 李俊艾, 罗军

第一军医大学热带军队卫生学系微生物学教研室, 广东, 广州, 510515

出版日期:2004-11-20 发布日期:2004-11-20
基金资助:
收稿日期:2004-6-7。
基金项目:广东省科技计划项目(2002B3100101)
作者简介:万成松(1965- ),男,1996年毕业于中国科学院武汉病毒研究所,硕士,副教授,电话:020-61640114-89123,E-mail:gzwcs@fimmu.com

Detection of the Salmonella invasion gene invA using molecular beacon probe

WAN Cheng-song, LI Jun-ai, LUO Jun

第一军医大学热带军队卫生学系微生物学教研室, 广东, 广州, 510515

Online:2004-11-20 Published:2004-11-20

摘要/Abstract

摘要： 目的研究沙门菌的分子信标基因检测方法。方法在PCR反应体系中加入分子信标探针,探针的5’端标记6-fluorescine(6-FAM),3’端标记4-(dimethylaminophenylazo)benzoic acid (DABCYL),对沙门菌invA基因PCR产物进行荧光检测。结果肠炎沙门菌、甲型副伤寒沙门菌、乙型副伤寒沙门菌、伤寒沙门菌“H”、伤寒沙门菌和肠侵袭型大肠杆菌的荧光值分别为161.6、104.5、85.9、83.1、94.8、46.1,Ax值(样本荧光值-空白对照荧光值)分别为121.3、64.2、45.6、42.8、54.5、5.8,沙门菌的Ax值均大于21,结果阳性,与琼脂糖电泳分析结果一致。结论分子信标探针技术可以准确、快速、简便进行沙门菌invA基因检测。

Abstract: Objective To detect Salmonella invasion gene invA.Methods The invA gene of Salmonella was amplified with PCR reaction system containing the molecular beacon probe labeled with 6-carboxy fluorescein (6-FAM) at its 5’ end and with (4-dimethylaminophenylazo)benzoic acid (DABCYL) at its 3’ end, and the fluorescence signal was read at the end of PCR.Results The fluorescence values of S.enteritidis, S. paratyphi A, S. paratyphi B, S.typhi antigen H, S.typhi, and enteroinvasive E.coli were 161.6, 104.5, 85.9, 83.1, 94.8 and 46.1 respectively. The results of PCR of Salmonella was consistent with that of agarose gel electrophoresis.Conclusion PCR with fluorogenic molecular beacon probe is rapid, specific, sensitive, and convenient for detecting Salmonella with invA gene.

中图分类号:

R378.2

万成松, 李俊艾, 罗军. 分子信标探针技术检测沙门菌invA基因[J]. 南方医科大学学报, 2004, 24(11): 1257-1259.

WAN Cheng-song, LI Jun-ai, LUO Jun. Detection of the Salmonella invasion gene invA using molecular beacon probe[J]. Journal of Southern Medical University, 2004, 24(11): 1257-1259.

参考文献

[1] Rahn K, De Grandis SA, Clarke RC, et al. Amplification of an invA gene sequences of Salmonella typhimurium polymerase chain reaction as a specific method of detection Salmonella [J]. Mol Cell Probes, 1992, 6(4):271-9.
[2] 程钢,何蕴韶,周新宇,等.结核分支杆菌荧光PCR试剂盒的研制及临床试验[J].第一军医大学学报,2002,22(6):533-5.Cheng G, He YS, Zhou XY, et al. Development and clinical trial of fluorescence PCR diagnostic kit to detect mycobacterium tuberculosis[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2002,22(6):533-5.
[3] 王艳,马文丽,毛向明,等.痘苗病毒寡核苷酸检测芯片的设计及研制[J].第一军医大学学报,2004,24(2):180-3.Wang Y, Ma WL, Mao XM, et al. Design and preparation of oligonucleotide microarray for vaccinia virus detection [J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2004, 24(2):180-3.
[4] Parkhill J, Dougan G, James KD, et al. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18[J]. Nature, 2001, 413 (6858):848-52.
[5] Deng W, Liou, SR, Plunkett Ⅲ G, et al. Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18 [J]. J Bacteriol, 2003, 185 (7):2330-7.
[6] McClelland M, Sanderson KE, Spieth J, et al. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2 [J]. Nature,2001, 413(6858):852-6.
[7] Hashimoto Y, Itho Y, Fujinaga Y, et al. Development of nested PCR based on the ViaB sequence to detect Salmonella[J]. J Clin Microbiol, 1995, 33(3):775-7.
[8] Song JH,Cho H, Park MY, et al. Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction[J]. J Clin Microbiol, 1993, 31(6):1439-43.
[9] Mahon J, Lax AJ. A quantitative polymerase chain reaction method for the detection in avian faeces of Salmonellas carrying the spvR gene[J]. Epidemiol Infect, 1993, 111(3):455-64.
[10] Cohen JH, Mechanda SM, Lin W. PCR amplification of the fimA gene sequence of Salmonella typhimurium,a specific method for detection of Salmonella spp [J]. Appl Environ Microbiol, 1996, 62(12):4303-8.
[11] Chen W, Martinez G, Mulchandani A. Molecular beacons:A realtime polymerase chain reaction assay for detecting Salmonella [J].Anal Biochem, 2000, 280:166-72.
[12] Tyagi S, Kramer FR. Molecular beacons:probes that fluoresce upon hybridization[J]. Natl Biotechnol, 1996, 14(3):303-8.