CAJ | 학술논문

目的以细菌16S rDNA和23S rDNA基因为靶序列建立可检测临床七种常见病原菌寡核苷酸芯片系统.方法采用双重PCR扩增标本中靶细菌16S和23S rDNA基因片段.构建能同时检测肠出血性大肠埃希菌O157:H7、副溶血性弧菌、沙门菌、霍乱弧菌、单核细胞增生李斯特菌、空肠弯曲菌和志贺菌的寡核苷酸芯片,并考核芯片的检测特异性、灵敏度和重复性.采用所建立的寡核苷酸芯片检测81例腹泻患者粪便样本.结果双重PCR可同时扩增上述七种病原菌的16S和23SrDNA基因靶序列.所研制的寡核苷酸芯片检测灵敏度可达103 cfu/ml,非靶细菌无阳性结果 ,不同批间和批内芯片的变异系数为3.89%～5.81%.寡核苷酸芯片检测粪便样本的阳性率为39.5%(32/81),与常规细菌学检查法检测结果的符合率达到96.3%(78/81),菌种鉴定结果符合率为96.8%(31132).结论研究建立的寡核苷酸芯片法在检测七种病原菌时具有简便、快速、准确、高通量等优点,适合于临床样本检测及流行病学现场调查.
목적 이세균16S rDNA화23S rDNA기인위파서렬건립가검측림상칠충상견병원균과핵감산심편계통.방법 채용쌍중PCR확증표본중파세균16S화23S rDNA기인편단.구건능동시검측장출혈성대장애희균O157:H7、부용혈성호균、사문균、곽란호균、단핵세포증생리사특균、공장만곡균화지하균적과핵감산심편,병고핵심편적검측특이성、령민도화중복성.채용소건립적과핵감산심편검측81례복사환자분편양본.결과 쌍중PCR가동시확증상술칠충병원균적16S화23SrDNA기인파서렬.소연제적과핵감산심편검측령민도가체103 cfu/ml,비파세균무양성결과 ,불동비간화비내심편적변이계수위3.89%～5.81%.과핵감산심편검측분편양본적양성솔위39.5%(32/81),여상규세균학검사법검측결과 적부합솔체도96.3%(78/81),균충감정결과 부합솔위96.8%(31132).결론 연구건립적과핵감산심편법재검측칠충병원균시구유간편、쾌속、준학、고통량등우점,괄합우림상양본검측급류행병학현장조사.
Objective Using 16S rDNA and 23S rDNA genes as the target sequences to develop a system based on oligonucleotide microarray and to detect the seven clinical pathogenic bacteria, commonly seen. Methods Double polymerase chain reaction(PCR) was applied to amplify the segments of 16S rDNA and 23S rDNA genes of the target bacteria. An oligonucleotide microarray was constructed to simultaneously detect EHEC O157:H7, Vibrio parahaemolyticus , Saimonella sp., Vibrio cholerae ,Listeria monocytogenes, Campylobacter jejuni and Shigella sp. Specificity, sensitivity and reproducibility of the microarray during detection were checked. And then microarray was used to detect the microbes in stool specimens of 81 patients with diarrhea and vomiting. Results The double PCR method could simultaneously amplify the target sequences of 16S rDNA and 23S rDNA genes of the seven pathogens. The sensitivity of the developed oligonueleotide microarray could reach 103 cfu/ml but no positive results were presented for non-targeted bacteria. The coefficients of differentiation in one lot or among different lots of the microarray slices were 3.89%-5.81%. The positive detection rate of the stool specimens by oligonucleotide microarray was 39.5 % (32/81), with a coincidence of 96.3 % (78/81) for the patients and another coincidence of 96.8% (31/32) for bacterial genus or species identification, when comparing to the results by routine bacteriological examinations. Conclusion The established assay in this study based on oligonucleotide microarray to detect the seven pathogenic bacteria has many advantages such as convenient,rapid, accurate, stable and high flux, which is suitable for clinical specimen examination and epidemiological field investigation.