Objective To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray. Methods Four wild-type and 8 mutant probes were used to detect rifampin resistant strains. Target DN...Objective To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray. Methods Four wild-type and 8 mutant probes were used to detect rifampin resistant strains. Target DNA of M. tuberculosis was amplified by PCR, hybridized and scanned. Direct sequencing was performed to verify the results of oligonucleotide microarray Results Of the 102 rifampin-resistant strains 98 （96.1%） had mutations in the rpoB genes. Conclusion Oligonucleotide microarray with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of rifampin resistance in M. tuberculosis isolates.展开更多
Using PCR-denaturing gradient gel electrophoresis （DGGE） targeting the RNA polymerase beta subunit （rpoB） gene, a simultaneous detection method for Vibrio species was established, rpoB gene-based PCR-DGGE was carr...Using PCR-denaturing gradient gel electrophoresis （DGGE） targeting the RNA polymerase beta subunit （rpoB） gene, a simultaneous detection method for Vibrio species was established, rpoB gene-based PCR-DGGE was carried out with eight Vibrio reference strains （each from different species）, mixed sample （including these Vibrio reference strains）, two non Vibrio strains, four environmental Vibrio strains, and three unidentified environmental strains. For comparison, 16S rRNA gene-based PCR-DGGE of the eight Vibrio reference strains was performed with universal primers. In addition, three unidentified strains were identified by 16S rRNA and gyrB gene sequencing and API20E system in order to confirm the accuracy of rpoB gene-based PCR-DGGE detection. Results revealed that rpoB-based PCR-DGGE could well discriminate eight Vibrio reference strains and could not discriminate different strains within the same species. The bands derived from two non Vibrio strains could not match with any bands in reference marker. Meanwhile, 16S rRNA gene-based DGGE failed to distinguish these reference strains. Further-more, four out of eight Vibrio species exhibited heterogenous bands in 16S rRNA gene-based DGGE. Sequencing and API 20E identification of unidentified strains coincided with the detection by rpoB gene-based PCR-DGGE. The results demonstrated that rpoB-based PCR-DGGE provided a rapid and efficient method for simultaneous detection of muhiple Vibrio species, which can avoid the limitations inherent in 16S rRNA gene-based PCR-DGGE.展开更多
Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a...Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a result, after mycobacterium and non-mycobacterium standard specimens were duplicated by PCR, mycobacterium standard specimens reproduced 360bp DNA fragments; on the other hand, non-mycobacterium specimens did not reproduce any fragments except for hemolytic streptococcus and corynebacterium pseudodiphtheriticum which had the same results as mycobacterium standard specimens. Sensitive test is able to detect lpg tuberculosis mycobacterium DNA. The probe test showed that, among 21 oligonucleotide probes, probe-M. fortuitum and M. marinum were cross-hybrid; the other probes were specific. We used the new method to identify 126 mycobacterium clinical isolated specimens. The test results of this new method matched with conventional method. In conclusion, compared to the traditional method, the use of rpob gene chip technology to identify mycobacterium species will be faster, more accurate and higher value in application.展开更多
目的:了解我国利福平敏感结核杆菌rpoB基因利福平耐药决定区(rifampicin resistancedetermining region,RRDR)序列的多态性特征。方法:采用"rpoB AND Mycobacterium tuberculosis AND(China OR Hong Kong OR Macon OR Taiwan)"...目的:了解我国利福平敏感结核杆菌rpoB基因利福平耐药决定区(rifampicin resistancedetermining region,RRDR)序列的多态性特征。方法:采用"rpoB AND Mycobacterium tuberculosis AND(China OR Hong Kong OR Macon OR Taiwan)"检索策略在Pubmed数据库中检索文献,循证分析Pubmed数据库中截止至2013年4月30日有关我国结核杆菌rpoB基因突变分子特征的研究文献。结果:36篇文献纳入研究,5 136株利福平敏感结核杆菌rpoB基因RRDR序列信息得以分析,38株(0.74%)rpoB基因RRDR序列呈现基因突变,涉及8个位点,分别为511(11株)、533(7株)、531(6株)、526(5株)、516(4株)、518(2株)、519(2株)、521(2株)等,其中1株涉及511/526双位点。结论:我国结核杆菌rpoB基因RRDR序列存在沉默突变现象,并非所有RRDR序列改变均与结核杆菌利福平药物敏感性有关。展开更多
利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定。以分枝杆菌rpoB基因编码序列为靶基因,用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株。分枝杆菌与其它细菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bp...利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定。以分枝杆菌rpoB基因编码序列为靶基因,用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株。分枝杆菌与其它细菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bp DNA片段,在其它细菌中,除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外,其它细菌均未见扩增。21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交外,其余均为特异性杂交。对126株临床分离株进行鉴定,89株为结核分枝杆菌,占70.6%(89/126),非结核分枝杆菌(NTM)占9.2%(9/98)。应用rpoB基因芯片技术鉴定分枝杆菌菌种,是一种快速、准确的方法,具有较高的临床应用价值。展开更多
基金supported by the grant from the National Natural Science Foundation of China (No. 30400018)
文摘Objective To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray. Methods Four wild-type and 8 mutant probes were used to detect rifampin resistant strains. Target DNA of M. tuberculosis was amplified by PCR, hybridized and scanned. Direct sequencing was performed to verify the results of oligonucleotide microarray Results Of the 102 rifampin-resistant strains 98 （96.1%） had mutations in the rpoB genes. Conclusion Oligonucleotide microarray with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of rifampin resistance in M. tuberculosis isolates.
基金the National Basic Research Programme of China(No.2006CB101803)the National Natural Science Foundation of China(No.30700016)
文摘Using PCR-denaturing gradient gel electrophoresis （DGGE） targeting the RNA polymerase beta subunit （rpoB） gene, a simultaneous detection method for Vibrio species was established, rpoB gene-based PCR-DGGE was carried out with eight Vibrio reference strains （each from different species）, mixed sample （including these Vibrio reference strains）, two non Vibrio strains, four environmental Vibrio strains, and three unidentified environmental strains. For comparison, 16S rRNA gene-based PCR-DGGE of the eight Vibrio reference strains was performed with universal primers. In addition, three unidentified strains were identified by 16S rRNA and gyrB gene sequencing and API20E system in order to confirm the accuracy of rpoB gene-based PCR-DGGE detection. Results revealed that rpoB-based PCR-DGGE could well discriminate eight Vibrio reference strains and could not discriminate different strains within the same species. The bands derived from two non Vibrio strains could not match with any bands in reference marker. Meanwhile, 16S rRNA gene-based DGGE failed to distinguish these reference strains. Further-more, four out of eight Vibrio species exhibited heterogenous bands in 16S rRNA gene-based DGGE. Sequencing and API 20E identification of unidentified strains coincided with the detection by rpoB gene-based PCR-DGGE. The results demonstrated that rpoB-based PCR-DGGE provided a rapid and efficient method for simultaneous detection of muhiple Vibrio species, which can avoid the limitations inherent in 16S rRNA gene-based PCR-DGGE.
文摘Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a result, after mycobacterium and non-mycobacterium standard specimens were duplicated by PCR, mycobacterium standard specimens reproduced 360bp DNA fragments; on the other hand, non-mycobacterium specimens did not reproduce any fragments except for hemolytic streptococcus and corynebacterium pseudodiphtheriticum which had the same results as mycobacterium standard specimens. Sensitive test is able to detect lpg tuberculosis mycobacterium DNA. The probe test showed that, among 21 oligonucleotide probes, probe-M. fortuitum and M. marinum were cross-hybrid; the other probes were specific. We used the new method to identify 126 mycobacterium clinical isolated specimens. The test results of this new method matched with conventional method. In conclusion, compared to the traditional method, the use of rpob gene chip technology to identify mycobacterium species will be faster, more accurate and higher value in application.
文摘目的:了解我国利福平敏感结核杆菌rpoB基因利福平耐药决定区(rifampicin resistancedetermining region,RRDR)序列的多态性特征。方法:采用"rpoB AND Mycobacterium tuberculosis AND(China OR Hong Kong OR Macon OR Taiwan)"检索策略在Pubmed数据库中检索文献,循证分析Pubmed数据库中截止至2013年4月30日有关我国结核杆菌rpoB基因突变分子特征的研究文献。结果:36篇文献纳入研究,5 136株利福平敏感结核杆菌rpoB基因RRDR序列信息得以分析,38株(0.74%)rpoB基因RRDR序列呈现基因突变,涉及8个位点,分别为511(11株)、533(7株)、531(6株)、526(5株)、516(4株)、518(2株)、519(2株)、521(2株)等,其中1株涉及511/526双位点。结论:我国结核杆菌rpoB基因RRDR序列存在沉默突变现象,并非所有RRDR序列改变均与结核杆菌利福平药物敏感性有关。
文摘利用rpoB基因芯片技术快速进行分枝杆菌菌种鉴定。以分枝杆菌rpoB基因编码序列为靶基因,用基因芯片技术检测21种分枝杆菌标准株;8种其它细菌标准株;126株临床分离株。分枝杆菌与其它细菌标准株经PCR扩增后,分枝杆菌标准株均扩增出360 bp DNA片段,在其它细菌中,除甲型溶血性链球菌和假白喉棒状杆菌出现同样片段外,其它细菌均未见扩增。21种寡核苷酸探针除海分枝杆菌与偶然分枝杆菌的探针有交叉杂交外,其余均为特异性杂交。对126株临床分离株进行鉴定,89株为结核分枝杆菌,占70.6%(89/126),非结核分枝杆菌(NTM)占9.2%(9/98)。应用rpoB基因芯片技术鉴定分枝杆菌菌种,是一种快速、准确的方法,具有较高的临床应用价值。
