To optimize dual molecular beacon detection system for rapid identification of Mycobacterium tuberculosis / 国际检验医学杂志

Objective To optimize the experimental system of dual molecular beacon to rapidly detect My-cobacterium tuberculosis and its resistant strains.Methods Fluorescence quantitative PCR was carried out by selecting different magnesium ion concentration,annealing temperature and primer concentration respectively. Finally,the optimum reaction conditions were obtained.Results In order to ensure the efficiency of amplifica-tion and no non-specific amplification,the final selection of the best conditions were as follows,the concentra-tion of Mg2+was 3.0 mmol/L,annealing temperature was 60 ℃,and the concentration of primers was 0.3 mmol/L.Conclusion The optimal condition of dual molecular beacon experiment was established,which en-sured that the detection of Mycobacterium tuberculosis by molecular beacon quantitative PCR had the advanta-ges,such as simple operation,rapid speed,high sensitivity(the minimum detection limit was 1 CFU/mL)and specificity(only Mycobacterium tuberculosis complex including drug-resistant strains could be detected),good reproducibility(coefficient of variation was < 5%)and other advantages.The study provides the necessary conditions for the dual molecular beacon detection of Mycobacterium tuberculosis.

Evaluation for clinical application effect of gene chip for detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis / 中国人兽共患病学报

We evaluated clinical application effect of gene chip for detection of rifampin (RFP) and isoniazid (INH) resistance in Mycobacterium tuberculosis (MTB).Rifampin and isoniazid drug-resistance gene loci were detected by gene chip with sputum specimens from smear-positive tuberculosis patients and clinical strains,comparing the results of detection.BACTEC MGIT 960 drug susceptibility test results were used as control to evaluate the detection performance of gene chip.The sequences of the polymerase chain reaction products of the rpoB,katG and inhA genes from 999 strains identified as Mycobacterium tuberculosis were determined to confirm the mutations by DNA sequencing.Results showed that 100 cases were identified as nontuberculous mycobacteria by gene chip in the 1 108 cases of smear-positive samples.Among the rest 1 008 samples,there were only 9 cases of microarray results different from BACTEC MGIT960 culture-positive strains,achieving the coincidences of 99.1％.Compared with BACTEC MGIT 960 drug susceptibility test results,the gene chip method displayed a concordance of 98.1 ％ and 94.5 ％ for RFP and INH respectively in the 999 strains.Compared with the DNA sequencing method,the accuracy of gene chip method was 99.6％ for rifampin resistance and 99.8％ for isoniazid resistance.It's concluded that the gene chip technology can quickly and accurately detect rifampin and isoniazid resistance in MTB and can be used directly for the detection of sputum samples.

Distribution of nontuberculous mycobacteria of clinical mycobacterium isolates f rom Fujian Province,China / 中国人兽共患病学报

ABSTRACT:To understand the species distribution of nontuberculous mycobacteria (NTM ) in Fujian Province of China , we collected clinical Mycobacterium isolates in the Fuzhou Pulmonary Hospital from 2009 to 2012 .A total of 6 362 clinical My‐cobacteria isolates were identified as 5 713 (89 .8% ) M .tuberculosis complex and 649 (10 .2% ) NTM strains by conventional identification method .Then ,by means of hsp65‐and rpoB‐PCR‐RFLP methods ,649 NTM strains were identified as 24 spe‐cies or complex of NTM ,in which the top three species or complex with the highest occurrence frequency were M .intracellular , M .avium and M .abscessus ,accounting for 48 .5% ,21 .3% and 12 .5% respectively .The prevalence rate of NTM was 10 .2%among Mycobacterium culture‐positive patients .There are lots of NTM species infecting human being ,and the most prevalence NTM species was M .avium complex accounting for 67 .8% in Fujian Province .

Rapid identification of bacteriological negative pulmonary TB and lung disease by proteomic fingerprinting technology / 中国人兽共患病学报

To explore the application of protein fingerprint technique and differential diagnosis in bacteriological negative pulmonary tuberculosis and pneumonia ,60 patients with bacteriological negative pulmonary tuberculosis ,60 patients with pneumonia ,and 60 healthy volunteers were selected from known clinical cases .Surface strengthening laser desorption ioniza-tion time of flight mass spectrometry (SELDI ToF Ms) and protein chip technology were applied to detect serum proteins ,and analyze their protein peaks by Ciphergen protein chip 3 .1 .1 software .Comparison of the serum protein fingerprinting data from the pool of 180 patients and healthy volunteers showed significant difference in 5 protein peaks (1 028 .49 ,4 796 .56 ,7 564 .77 , 8 048 .02 ,and 11 526 .75 m/z) identified between pulmonary tuberculosis and pneumonia (P<0 .01) .The total effective rate of the 5 protein peaks as a diagnosis model for differential diagnosis of bacteriological negative pulmonary tuberculosis and pneumonia was 84 .2% (101/120) ,the specificity was 82 .5% (52/63) ,the sensitivity was 85 .9% (49/57) ,the positive pre-dictive value was 86 .7% (52/60) ,and the negative predictive value was 81 .7% (49/60) .The total effective rate of the diagno-sis model for differential diagnosis of bacteriological negative pulmonary tuberculosis ,pneumonia and healthy volunteers was 89 .4% (161/180) .The specificity was 100% (60/60) ,the sensitivity was 84 .2% (101/120) ,the positive predictive value was 100% (101/101) ,and the negative predictive value was 75 .9% (60/79) .Protein fingerprinting technology is advanta-geous of being a simple method ,quick detection ,and requires less amount of sample .It is an effective means to screening the tuberculosis specific markers .We found the good diagnosis model through the detection of serum protein by protein fingerprint-ing technology .

Rapid identification and differentiation of the species of the Mycobacterium chelonae /abscessus complex by hsp65 and rpoB PCR-RFLP / 中国人兽共患病学报

The Mycobacterium chelonae/abscessus (M.chelonae/abscessus) complex belongs to the rapidly growing genus Mycobacterium (RGM).It is one of the most important pathogenic members of Mycobacterium leading to nosocomial infections and outbreaks.It includes members of M.chelonae,M.immnunogenum,M.abscessus,M.massiliense,and M.bolletii.In order to investigate the epidemiological characteristics of the M.chelonae/abscessus complex in China and to conduct the molecular methods for species identification of M.chelonae/abscessus,we collected clinical M.chelonae/abscessus complex strains identified by phenotypic tests.Members were verified by sequencing of 16S rRNA,Species and subspecies were identified by hsp65 and rpoB PCR RFLP methods.In total,27 clinical specimens were identified as Mycobacterium chelonae/abscessus complex by phenotypic tests.16s rRNA gene sequence analysis of all 27 clinical samples shared over 99.7％ similarity with M.chelonae and M.abscessus.Species identification with hsp65 PCR-RFLP and rpoB PCR-RFLP revealed that 18 specimens were M.abscessus and 4 were M.absecces.The remaining 5 samples displayed a pattern that failed to match any previously reported pattern.Thus,this might represent a novel species that is part of the Mycobacterium chelonae/abscessus complex.We identified that a majority of the chronic lung infection in China is caused by the M.chelonae/abscessus complex.Specifically,the M.abscessus species might be the most infectious,while other species in the complex can still cause infection.Interestingly,there may be a novel or previously unidentified species that is a part of the complex.Finally,we show that species identification can be carried out more accurately by combined use of hsp65 and rpoB PCR-RFLP.