| ObjectiveThis study aimed to understand the Characteristics and genotyping of Mycoplasma pneumoniae(MP) among children hospitalized with pneumonia in Tianjin. To explore a rapid diagnostic method for the early stage diagnosis of MP in children. At the same time, to know characterization and molecular mechanisms of Macrolide-resistant MP. A theory for rational choosing antibiotics can be provided according to this record.Method1. A total220bronchoalveolar lavage Fluid(BALF) specimens were collected from children with pneumonia hospitalized in Tianjin children’s hospital from October2010to November2011.The day of admissing to hospital or next day morning, venous blood were collected,then to be tested by ELISA for MP specific IgM antibody. Extract the220BALF specimens DNA, and then the genomic fragments of the strains were amplified by using polymerase chain reaction(PCR) method with primers. In addition to this,75specimens were chosed in random and using real-time PCR to judge the accuracy of the results.2. In base on the result of PCR, the positive strains were digested respectively with Hae Ⅱ and HaeⅢ. At the same time, the standard strain M129should be used to judge the genotyping of them. At last, part of positive strains were selected for sequencing to confirm the results of genotyping.3.50positive strains were selected randomly to be amplified with nest PCR method. Electrophoresising and purifying the amplifiable production, the23SrRNA gene sequences were sent to sequencing. The results of sequencing were compared with the sequence of MP M129(GenBank accession no.X68422) to detect wethere having point mutations or not.Results1. Among220venous blood specimens, the rate is41.8%. For220BALF,121samples produced specific brands after PCR. The rate is55%. The positive rates of male and female samples were50%and61.22%respectively. There was no statistical significance (x2=2.77, P＞0.05).The preschool-age and school-age children’s incidence of MP is higher than infant. The difference in the positive rate is highly statistical significance(x2=16.827, P＜0.05).The higher MP infection season is spring and autumn and the lower is summer and winte. There was no statistical significance (x2=2.28, P＞0.05)2.75strains which chose randomly, the positive rate is58.67%by real-time PCR,40%by ELISA and52%by PCR.The detection of ELISA is lower than real-time PCR and PCR. The difference in the positive rate is highly statistical significance (x2=4.76, P＜0.05和x2=7.68, P＜0.05),but the difference between real-time PCR and PCR have no statistical significance (x2=1.78, P＞0.05)3. By PFLP analysis,60clinical samples showed the characteristic band pattern for P1type1identical to the standard strain M129. Then the results of sequencing confirmed the fact that they are actually P1type1.4. Of the50strains that were selected to sequence,4strains and the standard strains were completely same. The other46strains all showed A-to-G transition at position2063. Different point mutations have not been found. The Macrolide-Resistant percentage was92%.Conclusion1. MP is a important pathogens for children with pneumonia hospitalized in Tianjin. There was no difference in different sex and seasons. But age was an important factor for MP antigen infection.2. PCR is a rapid diagnostic method for the early stage diagnosis of MP.3. The dominating type of MP in Tianjin is P1type1.4. Macrolide-resistant Mycoplasma pneumoniae is serious at present in Tianjin. The major molecular mechanisms is A-to-G transition at position2063of the23SrRNA domain V. |
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