| PREFACEMycoplasma pneumoniae is a common pathogen of human respiratory tract. Mycoplasma pneumonia always takes place in autumn and winter, especially in children and young adults. These cases tend to be relatively mild; however, this pathogen can lead to severe , even fatal, cases of pneumoniae. Therefore,the development of rapid, sensitive, and specific diagnostic techniques is necessary. Clinically, M. pneumoniae pneumonia cannot be differentiated from pneumoniae caused by other bacteria or viruses. The principal aim of this study was to compare three different approaches to the detection of M. pneumoniae pneumonia in pediatric patients. For this purpose, we used culture, culture - enhanced nested polymerase chain reaction and mi-cropartical agglutination.METHODSPatients. Clinical specimens were routinely obtained from patients admitted with acute respiratory complaints to the first affliated hospital of China Medical University. A total of 67 samples obtained in 2ml of broth as the transport medium were examined. Male,39,Female,28. Age,3month ~ 14years.Cultures. From each specimen, throat samples were collected on cotton - tipped swabs, which subsequently were agitated in 2ml oftransport medium and incubated. 0.4ml of sample was extracted after 24 hours for nPCR. The other was cultured as before. It was observed every day after 1 ~ 2 weeks. The sample cultured for 21 days but without color - turning would be looked as negative.DNA extraction. A 200 l sample of the transport medium in which the throat swab had been agitated was centrifuged at 12000g for 30min. The remaining 1. 8ml was stored at -20 . Pellets were resus-pended in 100 l of lysis buffer (50mM KCL, 10mM Tris - HCL, PH8.3 NP40 0. 45% , Tween 20 0. 45% , Proteinase K 10mg/ml) and incubated at 50 for 2h. Subsequently, samples were heated at 95 for 15min to inactivate Proteinase K . 10 l of the lysed sample were used as template in the first reaction of the nested PCR.nPCR. A nested PCR for amplification of M. pneumoniae DNA was designed, based on the P1 cytadhesin gene PCR of Ursi et al. The primers p1 -1 and p1 - 6 , which amplify a 272 - bp fragment of the p1 gene ,were used in the first PCR. The nested primers p1 -2 and p1 -3 used in the second PCR amplify a 133bp fragment. Amplifications were performed in a final volume of 50 l containing lOmM Tris - Hcl ( PH8.3 ) ,50mM Kcl, 1. 5mM MgCl2,200mM of each deoxynucleoside triphosphate, 20pmol of each primers, 2U Taq DNA polymerase (TAKARA biology company) and 10 l sample. Amplification was performed in a Thermocycler with a programme of 5min at 951 ,lmin at 65 ,1. 5min at 72 and a final step of 10min at 72 . 2 l of the first PCR product was added to the reaction mixture of the second PCR. The second PCR was performed under the same conditions as the first PCR. The 10 l. of the reaction products were analysed on Metaphor agarose 2% gels containing ethidium bromide 0.5 g/ml for visualisation of amplicons by UV transillumination.Serology. Determination of M. Pneumoniae - specific antibodies was performed by using micropartical agguliation ( Rebio , Tokyo, Japan ) according to the manufacturer's recommendation.RESULTSThe MAG on the first serum sample was positive for 6 patients (9. 0% ) and 14 patients for the second serum(20. 9% ). Mycoplasma culture was positive for 4 patients (6. 0% ) . nPCR for M. pneumoniae was positive for 17 patients(25. 4% ).The positive rate of detection was significantly higher with culture - enhanced nPCR than with mycoplasma culture ( P < 0.001). There was no significant difference between the positive rate of detection with culture - enhanced nPCR and that with the second serum sample (P > 0.05) . There was significant difference between the positive rate of detection with culture - enhanced nPCR and that with the first serum sample(P<0.05).DISCUSSIONThe detection of Mycoplasma pneumonisae by culture is time -consuming and of low sensitivity. The laboratory diagnosis of M. Pneumoniae infections presently relies upon conventional... |
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