应用膜反向斑点杂交技术快速鉴定分枝杆菌菌种和耐药结核分枝杆菌基因型
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摘要
目的:建立一种简便、快速、灵敏、特异的分枝杆菌菌种鉴定方法,研究膜反向斑点杂交方法在快速鉴定分枝杆菌菌种方面的应用价值。
方法:通过16S rRNA聚合酶链反应(polymerase chain reaction,简称PCR)—单链构象多态性(single stranded conformation polymorphism,简称SSCP)分析253株分枝杆菌临床分离株;设计与合成用于鉴定分枝杆菌菌种的16S rRNA寡核苷酸探针,点于硝酸纤维素膜上。与待测的28种分枝杆菌标准菌株、9种非分枝杆菌和253株分枝杆菌临床分离株生物素标记的16S rRNA基因PCR产物进行反向斑点杂交。
结果:应用PCR—膜反向斑点杂交方法分析28种分枝杆菌标准菌株和9种非分枝杆菌菌株,结果显示寡核苷酸探针是特异的。253株分枝杆菌临床分离株中,经16S rRNA PCR-SSCP初步鉴定,198株为结核分枝杆菌复合群,经膜杂交,显示a1杂交阳性,两种鉴定方法结果一致:36株非结核分枝杆菌经膜杂交,11株鉴定为堪萨斯、瘰疬、胃、猿猴分枝杆菌,6株鉴定为胞内分枝杆菌,4株鉴定为耻垢分枝杆菌,3株鉴定为母牛分枝杆菌,3株鉴定为龟分枝杆菌,2株鉴定为戈登分枝杆菌,2株鉴定为偶发分枝杆菌,2株鉴定为结核分枝杆菌和胞内分枝杆菌复合感染株,1株鉴定为鸟胞内分枝杆菌复合感染株,1株鉴定为土分枝杆菌,1株鉴定为鸟分枝杆菌,另19株未出现分析探针阳性杂交。PCR-膜反向斑点杂交技术鉴定分枝杆菌菌种的灵敏度为92.49%,特异度为100%。
选用孔径为0.22μm的硝酸纤维素膜,选择各菌种特异性探针,探针终浓度为1.5pmol/μl,采用杂交温度42℃、杂交时间60分钟、45℃洗膜5分钟杂交结果较理想。
结论:影响杂交的主要因素是探针序列的组成、探针浓度、杂交和洗膜温度、洗膜时间等。PCR-膜反向斑点杂交技术可简便、快速、特异地鉴定分枝杆菌菌种,仅需2天时间。
Objective To establish a simple, rapid, sensitive and specific method for identification of mycobacterial species, and to evaluate the clinical value of polymerase chain reaction-membrane reverse dot blot hybridization technique in identifying rapidly mycobacterial species.Methods 253 clinical isolates of mycobacteria were identified by 16S rRNA polymerase chain reaction-single stranded conformation polymorphism(PCR-SSCP) analysis. The oligonucleotide probes of 16S rRNA used for identification of mycobacterial species were prepared and droped on nitrocellulose membrane. The target DNA fragments of 28 mycobacterial standard strains, 9 non-mycobacterial strains and 253 mycobacterial isolates were labeled with biotin by PCR amplification, and then hybridized with oligonucleotide probes on membrane.Results The standard strains of 28 mycobacteria and 9 nonmycobacteria were analyzed with membrane-reverse dot blot hybridization, the results showed that the oligonucleotide probe was specific. Of 253 clinical isolates of mycobacteria, 198 strains were identified as Mycobacterium tuberculosis complex with PCR-SSCP,, and were also positive hybridization with probe al with reverse dot blot hybridization; 36 strains were identified as non-tuberculous mycobacteria with PCR-SSCP, and their species were identified with specific probes as follow: 11 were M. kansasii, M. gastri, M. scrofulaceum, M. simiae with probe d1, 6 were M. inreacellulare with probe cl, 4 were M. smegmatis with probe ql, 3 were M. vaccae with probe yl, 3 were M. chelonae with probe p1, 2 were M. gordonae with probe k1, 2 were M. fortuitum with probe r1', 2 were M. tuberculosis and inreacellulare complex strains with probe al and c1, 1 was M. avium-inreacellulare with b1 and c1, 1 was M. terrae with probe i1', 1 was M. avium with probe bl. 19 strains were negative hybridization with all probes. The 16S rRNA PCR-membrane reverse dot blot hybridization technique showed that the sensitivity was 92.49%, and the specificity was 100%.The more ideal results could be obtained at the following experimental conditions: 0.22 μ. m nitrocellulose membrane with specific probes was used; the concentration of probe was 1.5pmol/μl; the temperature and time of hybridization and washing
方法:通过16S rRNA聚合酶链反应(polymerase chain reaction,简称PCR)—单链构象多态性(single stranded conformation polymorphism,简称SSCP)分析253株分枝杆菌临床分离株;设计与合成用于鉴定分枝杆菌菌种的16S rRNA寡核苷酸探针,点于硝酸纤维素膜上。与待测的28种分枝杆菌标准菌株、9种非分枝杆菌和253株分枝杆菌临床分离株生物素标记的16S rRNA基因PCR产物进行反向斑点杂交。
结果:应用PCR—膜反向斑点杂交方法分析28种分枝杆菌标准菌株和9种非分枝杆菌菌株,结果显示寡核苷酸探针是特异的。253株分枝杆菌临床分离株中,经16S rRNA PCR-SSCP初步鉴定,198株为结核分枝杆菌复合群,经膜杂交,显示a1杂交阳性,两种鉴定方法结果一致:36株非结核分枝杆菌经膜杂交,11株鉴定为堪萨斯、瘰疬、胃、猿猴分枝杆菌,6株鉴定为胞内分枝杆菌,4株鉴定为耻垢分枝杆菌,3株鉴定为母牛分枝杆菌,3株鉴定为龟分枝杆菌,2株鉴定为戈登分枝杆菌,2株鉴定为偶发分枝杆菌,2株鉴定为结核分枝杆菌和胞内分枝杆菌复合感染株,1株鉴定为鸟胞内分枝杆菌复合感染株,1株鉴定为土分枝杆菌,1株鉴定为鸟分枝杆菌,另19株未出现分析探针阳性杂交。PCR-膜反向斑点杂交技术鉴定分枝杆菌菌种的灵敏度为92.49%,特异度为100%。
选用孔径为0.22μm的硝酸纤维素膜,选择各菌种特异性探针,探针终浓度为1.5pmol/μl,采用杂交温度42℃、杂交时间60分钟、45℃洗膜5分钟杂交结果较理想。
结论:影响杂交的主要因素是探针序列的组成、探针浓度、杂交和洗膜温度、洗膜时间等。PCR-膜反向斑点杂交技术可简便、快速、特异地鉴定分枝杆菌菌种,仅需2天时间。
Objective To establish a simple, rapid, sensitive and specific method for identification of mycobacterial species, and to evaluate the clinical value of polymerase chain reaction-membrane reverse dot blot hybridization technique in identifying rapidly mycobacterial species.Methods 253 clinical isolates of mycobacteria were identified by 16S rRNA polymerase chain reaction-single stranded conformation polymorphism(PCR-SSCP) analysis. The oligonucleotide probes of 16S rRNA used for identification of mycobacterial species were prepared and droped on nitrocellulose membrane. The target DNA fragments of 28 mycobacterial standard strains, 9 non-mycobacterial strains and 253 mycobacterial isolates were labeled with biotin by PCR amplification, and then hybridized with oligonucleotide probes on membrane.Results The standard strains of 28 mycobacteria and 9 nonmycobacteria were analyzed with membrane-reverse dot blot hybridization, the results showed that the oligonucleotide probe was specific. Of 253 clinical isolates of mycobacteria, 198 strains were identified as Mycobacterium tuberculosis complex with PCR-SSCP,, and were also positive hybridization with probe al with reverse dot blot hybridization; 36 strains were identified as non-tuberculous mycobacteria with PCR-SSCP, and their species were identified with specific probes as follow: 11 were M. kansasii, M. gastri, M. scrofulaceum, M. simiae with probe d1, 6 were M. inreacellulare with probe cl, 4 were M. smegmatis with probe ql, 3 were M. vaccae with probe yl, 3 were M. chelonae with probe p1, 2 were M. gordonae with probe k1, 2 were M. fortuitum with probe r1', 2 were M. tuberculosis and inreacellulare complex strains with probe al and c1, 1 was M. avium-inreacellulare with b1 and c1, 1 was M. terrae with probe i1', 1 was M. avium with probe bl. 19 strains were negative hybridization with all probes. The 16S rRNA PCR-membrane reverse dot blot hybridization technique showed that the sensitivity was 92.49%, and the specificity was 100%.The more ideal results could be obtained at the following experimental conditions: 0.22 μ. m nitrocellulose membrane with specific probes was used; the concentration of probe was 1.5pmol/μl; the temperature and time of hybridization and washing
引文
1. Soini H, Botter E C, Viljanen M K.Identification of Mycobacteria by PCR-based sequence determination of the 32-kilodalton protein gene[J]. J Clin Microbiol, 1994,32(12):2944-2947.
2.吴雪琼,王晋洪,张俊仙,等.PCR-SSCP分枝杆菌菌种初步鉴定方法的建立及其应用[J].中国现代医学杂志,2000,10(7):25-26.
3.李洪敏,吴雪琼,张俊仙,等.应用PCR-SSCP快速鉴定结核分枝杆菌复合群[J].微生物学通报,2000,27(3):202-204.
4. Devallois A, Goh K S, Rastogi N.Rapid identification of Mycobacteria to species level by PCR-restriction fragment length polymorphism analysis of the hsp65 gene and proposition of analgorithm to differentiate 34 mycobacterial species. [J]. J Clin Microbiol, 1997,35(11):2969-2973.
2.吴雪琼,王晋洪,张俊仙,等.PCR-SSCP分枝杆菌菌种初步鉴定方法的建立及其应用[J].中国现代医学杂志,2000,10(7):25-26.
3.李洪敏,吴雪琼,张俊仙,等.应用PCR-SSCP快速鉴定结核分枝杆菌复合群[J].微生物学通报,2000,27(3):202-204.
4. Devallois A, Goh K S, Rastogi N.Rapid identification of Mycobacteria to species level by PCR-restriction fragment length polymorphism analysis of the hsp65 gene and proposition of analgorithm to differentiate 34 mycobacterial species. [J]. J Clin Microbiol, 1997,35(11):2969-2973.