CAJ | 학술논문

目的利用多重荧光PCR技术,建立一种快速、准确、特异的检测常见分枝杆菌的方法.方法以分枝杆菌16S rRNA作为检测靶基因,设计双启动寡核苷酸(DPO)引物和TaqMan探针,探针的5’端分别用FAM 、ROX、HEX和JOE进行荧光标记,3’端标记淬灭荧光.通过对4种分枝杆菌标准株基因组DNA的扩增,评价多重荧光PCR方法的特异性和灵敏度.采用建立的多重荧光PCR方法,采用该方法检测2010年6至12月杭州市红十字会医院68例结核科住院患者清晨痰液标本,结果与细菌培养和抗酸染色镜检进行比较.采用x2检验比较3种方法的阳性率.结果该方法可准确、特异地鉴定结核分枝杆菌和3种常见非结核分枝杆菌,检测灵敏度均为1 ×101 cfu/ml.对68份痰液标本进行检测,结果显示31份检测结果阳性,阳性率45.6％,明显高于涂片染色镜检(阳性率14.7％),差异具有统计学意义(x2=15.4,P ＜0.05),其中28例为结核分枝杆菌,1例为鸟分枝杆菌,2例为胞内分枝杆菌,与细菌培养鉴定方法一致.1例培养阳性而PCR检测结果阴性标本经16SrRNA扩增测序鉴定为龟分枝杆菌.结论本研究建立的多重荧光PCR方法敏感、特异、快速,能有效检测结核分枝杆菌和常见非结核分枝杆菌,可用于分枝杆菌感染者的实验室快速诊断.
목적 이용다중형광PCR기술,건립일충쾌속、준학、특이적검측상견분지간균적방법.방법 이분지간균16S rRNA작위검측파기인,설계쌍계동과핵감산(DPO)인물화TaqMan탐침,탐침적5’단분별용FAM 、ROX、HEX화JOE진행형광표기,3’단표기쉬멸형광.통과대4충분지간균표준주기인조DNA적확증,평개다중형광PCR방법적특이성화령민도.채용건립적다중형광PCR방법,채용해방법검측2010년6지12월항주시홍십자회의원68례결핵과주원환자청신담액표본,결과여세균배양화항산염색경검진행비교.채용x2검험비교3충방법적양성솔.결과 해방법가준학、특이지감정결핵분지간균화3충상견비결핵분지간균,검측령민도균위1 ×101 cfu/ml.대68빈담액표본진행검측,결과현시31빈검측결과양성,양성솔45.6％,명현고우도편염색경검(양성솔14.7％),차이구유통계학의의(x2=15.4,P ＜0.05),기중28례위결핵분지간균,1례위조분지간균,2례위포내분지간균,여세균배양감정방법일치.1례배양양성이PCR검측결과음성표본경16SrRNA확증측서감정위구분지간균.결론 본연구건립적다중형광PCR방법민감、특이、쾌속,능유효검측결핵분지간균화상견비결핵분지간균,가용우분지간균감염자적실험실쾌속진단.
Objective To establish a rapid,accurate and specific method to detect the common mycobacteria based on multiplex real-time PCR.Methods The dual priming oligonucleotide ( DPO)primers and TaqMan probes labeled with FAM,ROX,HEX or JOE fluoresceins at 5' end and eclipse at 3' end respectively were designed to detect the 16S rRNA of mycobacteria.Both specificity and sensitivity were estimated on multiplex real-time PCR detecting genome DNA from 4 mycobacterial model species.Sixty eight early morning sputum specimens collected from hospitalized patients in the Red Cross Hospital of Hangzhou were detected by multiplex real-time PCR,bacterial culture and smear microscopy simultaneously.The positive rates were analyzed by chi-square.Results Mycobacteria including Mycobacterium tuberculosis and three common non-tuberculosis mycobacteria spp.were identified by multiplex real-time PCR accurately and specifically,with the limited load at 101 cfu/ml.In 68 sputum specimens,31 were positive (positive rate 45.6％ ) by this method,which was significant higher than that by smear microscopy ( positive rate 14.7％,x2 =15.4,P ＜0.05 ).The positive cases were identified as 28 Mycobacterium tuberculosis,1 Mycobacterium avium and 2 Mycobacterium intracellulare in agreement with the culture results.One case,which is detected by culture,but not by PCR,was identified as Mycobacterium chelonae by sequencing.Conclusion The multiplex real-time PCR characterizing as sensitive,specific and time-saving for Mycobacterium tuberculosis and common non-tuberculosis mycobacteria could be chosen as the rapid laboratory test of mycobacterial infection.