| 邢建明,张霆,张红河,沈翠芬,毕丹,李刚,姚丽惠.基于16S和23S rDNA基因芯片检测和鉴定七种临床常见病原菌[J].Chinese journal of Epidemiology,2008,29(8):815-818 |
| 基于16S和23S rDNA基因芯片检测和鉴定七种临床常见病原菌 |
| Detection and identification of seven clinical common pathogenic bacteria by oligonucleotide microarray |
| Received:December 13, 2007 Revised:August 15, 2012 |
| DOI： |
| KeyWord: 寡核苷酸芯片 病原菌 检测 |
| English Key Word: Oligonucleotide microarray Pathogenic bacteria Detection |
| FundProject:国家“十五”科技攻关计划(2001BA705B10);香港大学合作研究基金资助项目(10202653-05680-20400-323-01) |
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| Abstract: |
| 目的<\b> 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<\b>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0157：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昔酸芯片，并考核芯片的检测特异性、灵敏度和重复性。采用所建它的寡核苷酸芯片检测8l例腹泻患者粪便样本。结果<\b> 双重PCR可同时扩增卜述七种病原菌的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103cfu/ml，非靶细菌无阳性结果<\b>，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<\b>的符合率达到96．3％(78/81)，菌种鉴定结果<\b>符合率为96．8％(31/32)。结论<\b>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。 |
| English Abstract: |
| 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. |
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