[Application of nested PCR and sequencing technique to detect point mutations of the 23S rRNA gene of Mycoplasma pneumoniae].
OBJECTIVE
To establish a quick method to detect drug resistance of Mycoplasma pneumoniae (MP) and study the condition of drug resistance in MP infection.


METHODS
MP 23S rRNA target gene in throat swab specimens from 200 patients with suspected MP infection was detected by using nested PCR and DNA sequencing. The result of 23S rRNA gene detection was confirmed by MP isolation and drug susceptibility test in vitro for reliability.


RESULTS
Of the 200 clinical specimens, 64 were proved to be positive for MP through MP-IgM antibody, MP specific 16S rRNA nested PCR and MP isolation . The 23S rRNA gene was amplified and the gene sequence was compared with MP reference strain in Genbank, 26 were identical to the reference strain, 38 had a point mutation in 23S rRNA. Among them, 35 had A to G mutation at position 2063, 1 had A to C mutation at position 2063 and 2 had A to G mutation at position 2064, the percentage of drug resistance was 59.4%. The sensitivity of the gene detection method was 10(2) ccu/ml and it was confirmed to be reliable by MP isolation and drug susceptibility test.


CONCLUSIONS
The gene detection method could detect MP drug resistant gene directly from clinical specimen, which has the advantages of high specificity, high sensitivity and quickness. It is of great significance for diagnosis of MP infection because MP isolation is difficult and time-consuming.
Xin
De-li
DL
Department of Pediatrics, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100050, China.
Mi
Zu-huang
ZH
Han
Xu
X
Qin
Ling
L
Li
Jing
J
Liu
Xi-jie
XJ
Ma
Shao-jie
SJ
Hou
An-cun
AC
Li
Gui
G
D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D024881:Drug Resistance, Bacterial; D005260:Female; D020459:Genes, rRNA; D006801:Humans; D007223:Infant; D008297:Male; D008826:Microbial Sensitivity Tests; D009177:Mycoplasma pneumoniae / Q000235:genetics* / Q000302:isolation & purification; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics*
D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S