Xing Jian-ming, Zhang Su, Zhang Hong-he, Shen Cui-fen, Bi Dan, Li Gang, Yao Li-hui
Department of Laboratory Medicine, Huzhou Maternity and Child Care Hospital, Huzhou 313000, China.
Zhonghua Liu Xing Bing Xue Za Zhi. 2008 Aug;29(8):815-8.
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.
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, Salmonella 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.
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 oligonucleotide microarray could reach 10(3) 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.
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.
以16S rDNA和23S rDNA基因作为靶序列，开发一种基于寡核苷酸微阵列的系统，用于检测七种常见临床致病细菌。
采用双重聚合酶链反应（PCR）扩增靶细菌的16S rDNA和23S rDNA基因片段。构建寡核苷酸微阵列，同时检测肠出血性大肠杆菌O157:H7、副溶血性弧菌、沙门氏菌属、霍乱弧菌、单核细胞增生李斯特菌、空肠弯曲菌和志贺氏菌属。检测微阵列在检测过程中的特异性、敏感性和可重复性。然后使用微阵列检测81例腹泻和呕吐患者粪便标本中的微生物。
双重PCR方法可同时扩增七种病原体的16S rDNA和23S rDNA基因的靶序列。所开发的寡核苷酸微阵列的敏感性可达10(3) cfu/ml，但非靶细菌未出现阳性结果。微阵列芯片同一批次或不同批次间的差异系数为3.89% - 5.81%。与常规细菌学检查结果相比，寡核苷酸微阵列对粪便标本的阳性检出率为39.5%（32/81），患者检测结果的符合率为96.3%（78/81），细菌属或种鉴定的符合率为96.8%（31/32）。
本研究建立的基于寡核苷酸微阵列检测七种致病细菌的方法具有方便、快速、准确、稳定、高通量等优点，适用于临床标本检查和流行病学现场调查。
