多重RT-PCR方法对人肺炎链球菌菌种、血清型及耐药性的鉴别检测

Novel Multiplex Real-Time PCR Assays for the Detection,Serotyping and Drug resistance of S. Pneumoniae

首先,选择肺炎链球菌(Streptococcus pneumoniae,SP)基因LytA、肺炎支原体(Mycoplasma pneumoniae,MP)基因P1、肺炎衣原体(Chlamydia pneumoniae,CP)基因ompA作为目标检测靶点设计引物探针,建立鉴别肺炎链球菌菌种的多重RT-PCR方法.其次,针对肺炎链球菌cps基因座序列,利用MGB-TaqMan探针的多重RT-PCR方法对肺炎链球菌的血清型进行鉴别检测.最后,针对肺炎链球菌耐药基因ermB、mefA为靶标设计引物探针,采用多重RT-PCR方法对其临床阳性样本的耐药性进行鉴别检测.肺炎链球菌鉴别检测多重RT-PCR方法,线性范围为102~107 copies/mL,R2分别为0.999、0.996和0.995,灵敏度可达102copies/mL,与其他病原体无交叉反应.512例临床样本经多重RT-PCR方法鉴别检测,肺炎链球菌189例(189/304,62.17%),肺炎支原体83例(83/304,27.30%),肺炎衣原体32例(32/304,10.53%),与单重FQ-PCR及基因测序法比较无统计学差异(P>0.05).189例肺炎链球菌临床样本,经多重RT-PCR检测,血清分型为:19(19.58%,37/189)、23(15.87%,30/189)、6(13.23%,25/189)、14(6.3%,12/189),其他血清型(22.75%,43/189),其他血清型(22.22%,42/189).肺炎链球菌抗药菌株突变检测为:ermB突变115例(115/189,60.85%),mefA突变56例(56/189,29.63%),两基因共突变15例(15/189,7.94%).

First of all,the conserved genes LytA of SP,P1 of MP and ompA of CP were chosen as targets for design of primers and TaqMan probes. The Multiplex RT-PCR methods were developed for diagnosis ofthree pathogens. Secondly,according to sequence ofcp5 ,the TaqMan/MGB probes were designed and the Mul-tiplex RT-PCR methods were developed for serotyping of SP. Last,the erm B,m/A were chosen as targets fordesign of primers and probes for SP drug resistance, then it used for the detection of drug resistance in CAP clinical samples. The standard curve of multiplex RT-PCR showed great linear relationship. The sensitivity of methods for three pathogens were 10^2 copics/mL, while the detection range were 10^2- 10^7 copics/mLandno cross reaction with other microbial floras. 304 pathogen-positive clinical specimens was identified from 512 spu-tum, the positive of SP, MP and CP were 189 （ 62.17 %）, 83 （27. 30%） and 32（10. 53%） , respectively.T＇hcrc were good consistency between Multiplex RT＇-PCR and Simplex RT-PCR, and the resplex RT-PCR and Sequence methods were no significant difference. The serotype of SPin 189 positive samples were 19 （19.58%,37/189）,23 （15.87%,30/189）,6 （13.23%,25/189） ,11 （6.3% ,12/189）,other scro- types （22.75% , 43/189）,and unknown serotypes （22.22%, 42/189）. The gene mutations of SP resistance strainswere identified as follows： ermB （60.85%, 115/189）, mefA（29.63%, 56/189）, and both ermB and mfA （7.94% ,15/189）.