| Objective: This study attempts to detect Mycoplasma pneumoniae from clinical specimens in children. By comparing the diagnosis of M.pneumoniae at home and abroad, research the early stage diagnosis of M.pneumoniae. In the mean while, investigating the mutation of resistance for macrolide-resistant gene 23S rRNA , L4 and L22 ribosomal protein of and genotyping of M.pneumoniae isolates using molecular biology technique, incluing PCR-RELP, nPCR and Rapid-Cycle PCR Comparison of the three typing methods of molecular biology characteristics and clinical value in rapid diagnosis and drug monitoring.Methods: Five hundred and sixty-six pediatric patients with signs of lower respiratory tract infections were selected for the study. Collecting swab and lavage fluid specimens and acute-phase serum from children with clinical symptoms about pneumonia or a cough and fever. Mp-IgM was determined by the most conventional serological method in the patiaonts who were doubtful of M.pneumoniae infection. Extracting DNA using TIANamp Bacteria DNA Kit. Then Mp-DNA was detected by PCR and Real-time PCR.Comparing the two PCR and serological methods on the detection situation of M.pneumoniae infection in children. In the mean time, 200 clinical cases of swab and lavage fluid specimens were cultivated in medium for rapid identification of M.pneumoniae and SP4 broth. Then, portions of macrolide-resistant genes encoding 23S rRNA and ribosomal proteins L4 and L22 were amplified by PCR. At last, Genotyping of M.pneumoniae isolates using molecular biology technique including PCR-RFLP, nPCR and Rapid-Cycle PCR and observing of the three methods in genotyping characteristics. Results: Among the 566 clinical cases, there were 566 serum samples from 566 patients which Mp-IgM positive 285(50.35%)cases. Beside there were also 566 swab and lavage fluid specimens from 566 children which include 508 swab and 58 lavage fluid specimens. 45 (7.95%) of patients with respiratory samples had positive PCR amplification of 16srRNA gene, which Mp-DNA positive and serum Mp-IgM negative in 20 cases(7.11%). By Real-Time PCR, the positivity rate of samples was 175(30.91%) and the copy number of Mp-DNA showed to be more than 1.00e+005copies/ml, which Mp-DNA positive and serum Mp-Ab negative in 67 cases(38.28%). Additionally, 35(31.53%) isolates obtained from 200 cases clinical samples by rapid culturation of M.pneumoniae. But there were just there positive cases (2.70%) by PCR and Real-Time PCR. Among the positive cases, 32(91.42%) isolates were false positive which fungi account for 93.75%(30/32) by colonies identification. 6(4.95%) isolates obtained from 200 cases clinical samples by SP4 broth sand agar culture which colonies identification, PCR and Real-Time PCR results were identical. There were 2 cases of throat swab and 4 cases of lavage fluid that culture ware positive. Of 24 M. pneumoniae strains, all showed the mutation of 23S rRNA gen, 14(58.33%) showed the mutation of L4 ribosomal protein, 6(25%) showed the mutation of L22 ribosomal protein. In 23S rRNA gen, 16 strains (66.66%) harbored an A-to-G transition mutation at position 2063 which was predominant mutation, 14(58.33%) harbored an missing at position 640, 10(41.66%) harbored an missing at position 2471, 8(33.33%) harbored an G-to-A transition at position 759. Beside, there were other mutation, base missing and insertion, including mutation of A649T, G2661C, A2635C and base missing of 1957 A, 1974A, 1966 C and Base insertion of 2783. In L4 ribosomal protein, what the most mutation were C162A and A430G. 4(16.66%) harbored an C-to-A transition at position 162 and an A-to-G transition at position 430. Beside, there was 1 habored an C-to-T transition at position 456 and 1 habored an A-to-T transition at position 209. In L22 ribosomal protein,What the most was base insertion which had 11(45.83%) at position 498 or 499. Beside, there were 3(12.5%) habored an T-to-C transition at position 279. In the genotyping of clinical samples with diagnosis of Mp-DNA positive,Rapid-Cycle PCR can be quickly and effectively type Mp-DNA .The results show that 21 isolates (46.66%) can be classified as typeâ… and 24 isolates (53.33%) as typeâ…¡.Conclusion:1) The result showed that Real-Time PCR might be a kind of rapid ,sensitive and apecific assessment in detecting the Mp-DNA, thereby, it has an important significance for establishing the earlydiagnosis of M.pneumoniae in clinical testing..2) Among clinical isolates, the predominant mutation were A2063G in domain V of 23S rRNA, C162A and A430G in L4 ribosomal protein3) Rapid-Cycle PCR can be quickly and effectively type Mp-DNA.Compared with nPCR and PCR-RFLP, Rapid-Cycle PCR is more rapid and effective method. |
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