Abstract
Objective To investigate and evaluate the PCR-RFLP method for identification of Mycobacterium abscessus (M.abscessus) group.Methods 46 clinical acid-fast bacilli (AFB) isolates from Pulmonary Hospital of Fujian Province,Center for Disease Control and Prevention of Zhaoyang District in Beijing and Beijing People Liberation Army General Hospital were collected in 2009 -2010 and identified by traditional bacteriological characteristics according to clinical laboratory handbook of mycobacteria (2004).The PCR-RFLP method was used for species identification of M.abscessus group using hsp65 (441 bp) or rpoB (380 bp) gene fragment as specific target,while the direct DNA sequencing was performed as a control method.Results Of 46 AFB isolates,30 strains were identified as Mycobacterium tuberculosis by its traditional bacteriological characteristics and 16 strains were identified as non-tuberculosis mycobacterium (NTM).10 strains of the NTM strains had identical bacteriological characteristics with the reference strain M.abscessus ATCC 19977.Identified by hsp65 PCR-RFLP,9 of these 10 strains got the same pattern of 235bp and 200 bp(BstE Ⅱ )/145 bp,70 bp,60 bp,55 bp,50 bp and 40 bp(Hae Ⅲ ),and 1 got pattern of 235 bp and 200 bp(BstE Ⅱ )/200 bp,70 bp,60 bp and 50 bp(Hae Ⅲ ).While identified by rpoB PCRRFLP,all 10 strains got the same pattern of 105 bp,95 bp and 80 bp( Msp Ⅰ )/130 bp,100 bp and 90 bp (Hae Ⅲ ).By analysis of the DNA sequence,hsp65 and rpoB sequence of these 9 strains showed 100％similarity with those of M.abscessus,while the hsp65 and rpoB sequence of the other one strain showed 100％similarity with those of M.massiliense.Conclusion PCR-RFLP is a rapid and effective method for species identification of M.abscessus group,and hsp65 PCR-RFLP is more effective than rpoB PCR-PFLP in the species identification of M.abscessus group. Key words: Mycobacterium; Bacterial proteins; Polymerase chain reaction; Polymorphism,restriction fragment length; Bacterial typing techniques
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