Application of nested PCR in P1-RFLP for genotyping Mycoplasma pneumoniae strains
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. 
 
Key words: 
Mycoplasma pneumoniae;  Genotyping;  Nested-PCR;  P1-RFLP