Detection of streptomycin-resistance associated rpsL and rrs gene mutations in Mycobacterium tuberculosis by PCR-single-strand-conformational polymorphism / 中华临床感染病杂志

ObjectiveTo establish a novel rapid detection method based on PCR-single-strand-conformational polymorphism (PCR-SSCP) to determine mutation of streptomycin-resistance associated rpsL and rrs genes in isolates of Mycobacterium tuberculosis (MTB).MethodsStreptomycin-resistance of 112 MTB isolates was detected using the routine drug susceptibility test,and a special PCR-SSCP assay was established.The mutations of rpsL and rrs genes in streptomycin-resistant MTB isolates were detected by PCR-SSCP and PCR direct sequencing (PCR-DS) ; the results from two techniques were compared.Results All isolates had both rpsL and rrs genes.Fifty-two isolates (46.4％) were streptomycin susceptible,in which only 1 isolate showed abnormal PCR-SSCP fragments from rrs gene,and the specificity of PCR-SSCP was 98.1％ (51/52).Sixty isolates (53.6％) were streptomycin-resistant,in which 46 (76.6％) and 11 ( 18.3％ ) isolates presented the abnormal PCR-SSCP fragments of rpsL and rrs gene,respectively.One streptomycin-resistant isolate showed abnormal PCR-SSCP fragments from both rpsL and rrs genes.The sensitivity of PCR-SSCP was 93.3％ (56/60).ConclusionThe PCR-SSCP that established in this study is a specific and sensitive method for rapid detection of the streptomycin-resistance associated mutations in rpsL and rrs genes of MTB.

Expression of T and B cells combined epitopes protein of Leptospira interrogans and the immunity analysis / 中华微生物学和免疫学杂志

Objective To construct the T-cells and B-cells combined epitope peptide gene based on the LipL32,OmpL1 and LipL21 protein from Leptospira interrogans and E.coli expression system,and better understanding of the immunological activity of the recombinant protein. MethodsThe immunodomaint T- and B-cells combined epitopes of LipL32,OmpL1 and LipL21 were identified and used to synthetic a new gene and then construct its prokaryotic expression system.The expression of recombinant protein was determined by SDS-PAGE; MAT was used to determine the titer of the antiserum to L. interrogans standard strains of China ; Western blot and ELISA were used to identify the immunity activity of the recombinant protein.Results The synthetic gene was effectively expressed in E.coli BL21 ( DE3 ) strain and mainly presented in dissoluble protein.Western blot result showed that the expression protein react well with the antibodies from immunized rabbit by Leptospira or recombinant protein.ELISA and MAT results showed that the multiepitope protein could cross-react with different serogroup or serovar of Leptospira.Conclusion In this study,we successfully constructed the recombinant T- and B-cells combine epitope gene of leptospires and expressed it in E.coli.The recombinant protein had a good immune activity,and could cross-reacted with antibodies from different serogroups Leptospira infected patients.

The relevance of Mycobacterium tuberculosis isoniazid-resistance and mutations in two different regions of the katG gene / 中华检验医学杂志

Objective To analyze and compare the mutations in two different regions of the katG gene and study the relevance of Mycobacterium tuberculosis isoniazid-resistance and mutations in two different regions of the katG gene. Methods Fifty-three INH-resistant Mycobacterium tuberculosis strains isolated in cultures of sputum samples obtained from Zhejiang province were analyzed. PCR was used to amplify two regions of the katG gene (GenBank accession no. U06258) region 1 (from codon 1 to codon 150) and region 2 ( from codon 227 to codon 470) which were then sequenced in order to identify mutations. Results Three strains resistant to INH did not contain mutations in either region. Fourteen strains carried mutations in region 1. Among them 5 strains barbered deletions, and showed high-level resistance to isoniazid. Five strains had mutations only in region 1. Region 2 carried multiple point mutations, especially at codon 315, and there were S315 N ( AGC→AAC ) substitution in 18 of those cases. The frequency of mutations in the katG S315 of high-level INH-resistance isolates ( 84. 4%, 27/32) was significantly higher than those of low-level INH-resistance isolates( 15.6%, 5/32 ), there was statistically significant difference (x2 = 30. 25, P ＜ 0. 01 ).katG S315 mutations in high-level INH-resistance frequency (84. 4%, 27/32) was significantly higher than the other mutations of katG gene of high-level INH-resistance frequency (27. 7%, 5/18 ), there was significant difference (x2 = 16.02, P ＜ 0. 01 ). The analysis of region 2 allowed INH resistance to be diagnosed in 84. 9% of the strains. Five strains had mutations only in region 1 ,which allowed the proportion of INH-resistant strains identified to be increased to 94. 3%. Conclusions The number of mutations at codon 315 was high. Mutation type and location closely related with drug resistance and the analysis of region 1 resulted in a 9. 4% increase in the rate at which mutations were identified.