CAJ | 학술논문

目的建立TaqMan荧光定量PCR检测方法 ,用于流感嗜血杆菌和肺炎链球菌的检测和鉴别诊断.方法针对流感嗜血杆菌种属特异性基因bexA和肺炎链球菌种属特异性基因lytA,设计合成引物和TaqMan探针,研究不同引物和探针荧光定量PCR检测的特异性和灵敏度,确定标本检测中循环阈值(Ct)的临界值(cut-off值).将荧光定量PCR、乳胶凝集和细菌培养3种检测方法同时应用于278份细菌性脑膜炎患者脑脊髓液标本的检测.结果 bexA基因引物和探针能特异性检测流感嗜血杆菌a、b、c、d血清型的菌株,检测灵敏度为每个反应10个基因组DNA拷贝;lytA基因引物和探针能特异性检测肺炎链球菌常见致病的血清型肺炎链球菌菌株,检测灵敏度为每个反应90个基因组DNA拷贝.通过荧光定量PCR方法 ,278份脑脊髓液标本中共检测出 4份流感嗜血杆菌阳性和7份肺炎链球菌阳性,其中各有2份培养出相应的病原菌,另有1份流感嗜血杆菌和2份肺炎链球菌乳胶凝集结果阳性.结论 TaqMan荧光定苗PCR方法能特异地检测和鉴定流感嗜血杆菌和肺炎链球菌,具有较高的灵敏度和快速检测的特点,能提高临床流感嗜血杆菌和肺炎链球菌感染患者标本的阳性检出率.
목적 건립TaqMan형광정량PCR검측방법 ,용우류감기혈간균화폐염련구균적검측화감별진단.방법 침대류감기혈간균충속특이성기인bexA화폐염련구균충속특이성기인lytA,설계합성인물화TaqMan탐침,연구불동인물화탐침형광정량PCR검측적특이성화령민도,학정표본검측중순배역치(Ct)적림계치(cut-off치).장형광정량PCR、유효응집화세균배양3충검측방법 동시응용우278빈세균성뇌막염환자뇌척수액표본적검측.결과 bexA기인인물화탐침능특이성검측류감기혈간균a、b、c、d혈청형적균주,검측령민도위매개반응10개기인조DNA고패;lytA기인인물화탐침능특이성검측폐염련구균상견치병적혈청형폐염련구균균주,검측령민도위매개반응90개기인조DNA고패.통과형광정량PCR방법 ,278빈뇌척수액표본중공검측출 4빈류감기혈간균양성화7빈폐염련구균양성,기중각유2빈배양출상응적병원균,령유1빈류감기혈간균화2빈폐염련구균유효응집결과 양성.결론 TaqMan형광정묘PCR방법 능특이지검측화감정류감기혈간균화폐염련구균,구유교고적령민도화쾌속검측적특점,능제고림상류감기혈간균화폐염련구균감염환자표본적양성검출솔.
Objective To establish TaqMan real-time PCR method for detection and identification of Haemophilus influenzae and Streptococcus pneumonia. Methods Two sets of primers and FAM-labeled probes targeting different genes of Haemophilus influenzae and Streptococcus pneumoniae were designed and synthesized. The bexA gene was used for identification of Haemophilus influenzae and lytA for Streptococcus pneumoniae. The sensitivity and specificity of real-time PCR were assessed for different primers and probes. Cut-off values of cycle threshold (Ct) were determined. Two hundred and seventy-eight cerebrospinal fluid (CSF) specimens from suspected bacterial meningitis cases were detected by real-time PCR assay, latex agglutination test and bacteria culture simultaneously. Results Haemophilus influenzae isolates of serotype a to d could be detected and identified by bexA primers and probe. All Streptococcus pneumoniae isolates of different serotypes could be detected and identified by lytA primers and probe. The respective sensitivities for Haemophilus influenzae and Streptococcus pneumoniae were 10 and 90 genome DNA copies in each PCR reaction. Of the 278 CSF specimens, four were positive by Haemophilus influenzae and seven positive by Streptococcus pneumoniae when detected by real-time PCR. Of the four Haemophilus influenzae positive specimens, two were positive by culture and one positive hy latex. Of the seven Streptococcus pneumonia positive specimens, two were positive by culture and two positive by latex. Conclusions Real-time PCR could rapidly detect and identify Haemophilus influenzae of serotype a to d and Streptococcus pneumoniae of different serotypes with high sensitivity. TaqMan real-time PCR could be widely used for the diagnosis of invasive meningitis caused by Haemophilus influenzae and Streptococcus pneumoniae. It can improve the rate of positivity for diagnosis of suspicious bacterial meningitis cases.