ABSTRACT
Objective To evaluate the efficiency of using nested PCR in restriction fragment length polymorphism analysis (P1-RFLP) for genotyping Mycoplasma pneumonia (M.pneumonia) in clinical specimens.Methods Based on the gene sequence of RepMp4 and RepMp2/3 in P1 gene of reference strains M129 (type 1) and FH (type 2),two sets of inner primers were designed with a HaeⅢ restriction enzyme site (GGCC).The nested PCR was set up to detect the target DNA in clinical specimens.The amplification products were mixed and digested with Hae Ⅲ enzyme.The genotypes were analyzed by comparing with various restriction maps and the results were verified by sequencing analysis.The concentration of DNA extracted from standard and clinical strains were detected by ten-fold dilution to evaluate the sensitivity of nested PCR-P1-RFLP and P1-RFLP.M.pneumonia-positive specimens isolated from Beijing in 2012 were analyzed by the nested PCR-P1-RFLP and the results were compared with those by P1-RFLP analysis.Results The nested PCR-P1-RFLP could effectively genotype M.pneumonia in clinical specimens and the results were consistent with those by sequencing analysis.The sensitivity of new assay was 103 times higher than that of the original P1-RFLP.Of the 115 M.pneumoniae positive clinical specimens,97.4％ (112/115) were type 1 and the rest were type 2.Conclusion The nested PCR-P1-RFLP shows high efficiency for genotyping of M.pneumonia in clinical specimens.It might be useful for the surveillance of M.pneumoniae infection.
ABSTRACT
Objective To inspect the source of an outbreak with Mycoplasma pneumoniae (Mp).Methods We carried out real-time PCR to analyze specimens collected from pediatric patients in Beijing during January 2010 to May 2012,diagnosed as pneumonia or a respiratory infection according to clinical symptoms.These positive samples were analyzed by the M-P typing system(M:multiple-locus variable-number tandem-repeat analysis,MLVA; P:P1-restriction fragment length polymorphism analysis,P1-RFLP).Results Sixty-nine specimens were tested positive to Mp by the real-time PCR in 446 specimens from pediatric patients.The infection rate was 11.69％,15.56％ and 20.00％ respectively in 2010,2011 and the first half of 2012.According to the M-P system,11 distinct genotypes were identified from 69 positive specimens,M43562P1 and M53562P1 were the two main genotypes that showed an increasing trend from 2010 to 2011,and M33562P1 and M63562P1 showed an increasing trend from 2011 to 2012 in China.Conclusion During this international Mp epidemic,the infection rate of Mp was also increase in Beijing in 2011,and M43562P1 and M53562P1 were the two main genotypes.Among them,M43562 were consistent with pop genotypes in Europe,and M53562 were consistent with pop genotype in Israel.The M-P system would be valuable to monitor the epidemic of Mp in different countries in the world.