Screening of rpsL mutations in streptomycin resistance gene of 104 strains of Yersinia pestis strains in south area of Qinghai Province by TaqMan-MGB fluorescent probe / 中国热带医学

@#Abstract: Objective To investigate the current status of streptomycin resistance of Yersinia pestis caused by point mutations of rpsL gene in Qinghai, so as to provide theoretical basis for precise clinical medication and prevention of drug resistance of human plague outbreak in South area of Qinghai Province in the future. Methods A total of 104 representative strains of Yersinia pestis collected from plague patients, vector insects and intermediate hosts in South area of Qinghai Province from 1957 to 2009 were screened, isolated and cultured by Hiss agar plates. The DNA of representative Yersinia pestis was extracted by sodium dodecyl sulfate lysis and phenol-chloroform method. The primers forward primer and reverse primer and TaqMan-MGB probes probe1 [FAM] and probe2 [VIC] were designed for the rpsL gene of streptomycin resistance gene in China. Real-time PCR with TaqMan-MGB fluorescent probe was used to detect the mutations of rpsL gene in streptomycin resistance locus of 104 strains of Yersinia pestis in South area of Qinghai Province. Results The FAM test results of 104 strains in South area of Qinghai Province were positive, corresponding to the detection of rpsL (128 : A ), RFU peak >1 000,negative <200. VIC test results of all tested strains were negative, corresponding to the detection of rpsL (128:G), RFU peak <200, positive >1 000. That is, no strains with rpsL gene mutation related to streptomycin resistance were found in the 104 strains of Yersinia pestis in Qingnan Province. Conclusion This study provides basic data on the distribution of streptomycin resistance of Yersinia pestis in South area of Qinghai Province, and lays a foundation for preventing the occurrence of drug resistance and clinical treatment of Yersinia pestis in South area of Qinghai Province.

Mutations in Streptomycin Resistance Genes and Their Relationship to Streptomycin Resistance and Lineage of Mycobacterium tuberculosis Thai Isolates / 결핵및호흡기질환

BACKGROUND: Streptomycin (SM) is recommended by the World Health Organization (WHO) as a part of standard regimens for retreating multidrug-resistant tuberculosis (MDR-TB) cases. The incidence of MDR-TB in retreatment cases was 19% in Thailand. To date, information on SM resistance (SMR) gene mutations correlated to the SMR of Mycobacterium tuberculosis Thai isolates is limited. In this study, the mutations in rpsL, rrs, gidB, and whiB7 were investigated and their association to SMR and the lineage of M. tuberculosis were explored. METHODS: The lineages of 287 M. tuberculosis collected from 2007 to 2011 were identified by spoligotyping. Drug susceptibility profiles were evaluated by the absolute concentration method. Mutations in SMR genes of 46 SM-resistant and 55 SM-susceptible isolates were examined by DNA sequencing. RESULTS: Three rpsL (Lys43Arg, Lys88Arg, and Lys88Thr) and two gidB (Trp45Ter and Gly69Asp) mutations were present exclusively in the SM resistant M. tuberculosis. Lys43Arg rpsL was the most predominant SMR mutations (69.6%) and prevailed among Beijing isolates (pC, 615A>G, and 330G>T, as lineage signatures for Beijing and EAI were underscored. This study identified 423G>A gidB as a novel sub-lineage marker for EAI6-BGD1. CONCLUSION: Our study suggested that the majority of SMR in M. tuberculosis Thai isolates were responsible by rpsL and gidB polymorphisms constantly providing the novel lineage specific makers.

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.

Drug susceptibility test for 16 Mycobacteria tuberculosis isolates from patients with cutaneous tuberculosis / 中华皮肤科杂志

Objective To test the drug susceptibility of 16 Mycobactena tuberculosis isolates from patients with cutaneous tuberculosis,and to provide a basis for the treatment of this entity.Methods Sixteen strains of mycobacterium were isolated from patients with cutaneous tuberculosis.and they were consistently identified as M.tuberculosis by biochemistry and molecular biology.Absolute concentration method was used to test the susceptibility of these isolates to isoniazid.streptomycin.rifampicin and ethambutol.For two strains resistant to streptomycin,PCR and sequencing were performed to analyse the mutation of rpsL gene.Results Out ofthe 16 strains of M tuberculosis.2 were resistant to streptomycin but sensitive to isoniazid,rifampicin and ethambutol,and 14 were sensitive to isoniazid,streptomycin,rifampicin and ethambutol.Sequencing of the rpsL gene revealed a mutation of AAG to AGG at codon 43 in one streptomycinresistant strain and a substitution of CGC bv CAC at codon 54 in another resistant strain.Conclusions In past five years,the resistance ratio of M tuberculosis was low in patients with cutaneous tuberculosis,and streptomycin resistance predominated in these strains.

Detecting 43Codon of High Mutation Site in rpsL of STR Resistant MTB by Molecular Beacons Technology:An Experimental Study / 中华医院感染学杂志

OBJECTIVE To design molecular beacon probe of 43 Codon of high mutation site in rpsL of STR resistant MTB and its amplification system, meanwhile, to find the way of detecting the fluorescent light and making a qualitative judgment by use of fluorescence microscope and image analysis software. METHODS The software, Beacon designer, was used to design the 43 Codon molecular beacon probe and the amplification system, and the fluorescence microscope and image analysis software were used to detect the fluorescent light and make a qualitative judgment. RESULTS The strap of amplification product was shown clearly. The difference between PCR products from standard strain and STR resistant one was obvious in detecting the fluorescent light by use of fluorescence microscope. The rate of resistant STR detected was about 80% (P

Detection of rpsL drug-resistant gene in L-form of Mycobacterium tuberculosis by PCR-SSCP / 临床检验杂志

Objective To study the relationship between mutation of rpsL drug-resistant gene in L-forms of Mycobacterium tuberculosis and drug-resistance to streptomycin in pneumoconiosis patients.Methods A total of 52 clinical isolated strains of Mycobacterium tuberculosis L-forms were collected from 97 pneumoconiosis patients.The mutation of rpsL gene was detected by PCR-SSCP,and the drug-resistance to streptomycin was performed by routine antimicrobial susceptibility test(AST).Results The results of drug susceptibility test showed that 26 in the 52 clinical isolated strains were drug-resistant to streptomycin.The streptomycin-resistant rate was 50.00%(26/52).The gene mutation rate of rpsL detected by PCR-SSCP was 40.38%(21/52).The coincidence rate of two experimental results was 80.77%(21/26).Conclusion High detectable rate of streptomycin-resistant strains in Mycobacterium tuberculosis L-forms was found by PCR-SSCP.The application of PCR-SSCP may possess important value for guiding clinical medication of pneumoconiosis patients complicated with tuberculosis among coal workers