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目的建立16S rRNA基因加基因芯片检测新生儿败血症的诊断技术,以提高临床检测细菌的速度及准确性。方法对125例拟诊为败血症的新生儿血标本及部分脑脊液标本进行细菌16S rRNA基因及基因芯片检测,包括DNA提取、设计引物和探针、聚合酶链反应(PCR)扩增、基因芯片的制备、杂交、激光扫描与读片。结果125例中PCR检测血标本阳性64例,占51.2%;血培养阳性32例,占25.6%;非特异性指标41例,占32.8%,差异有统计学意义(P<0.01)。若以血培养阳性和/或非特异指标至少两项阳性为诊断标准,PCR灵敏度为90.3%(56/62),特异度为87.3%(55/63),正确诊断指数为0.776。3例脑脊液标本中PCR阳性2例,培养阳性仅1例。对64例PCR阳性血标本进一步作基因芯片检测,结果通用探针均阳性。其中G+探针阳性60份,G-探针阳性4份。30份PCR和血培养均阳性的标本中其探针菌株与血培养细菌阳性结果相符。2例脑脊液标本PCR阳性,基因芯片检测结果与培养结果相符。结论16S rRNA基因PCR加基因芯片杂交可为新生儿败血症提供早期、敏感的病原学诊断依据。
Objective To establish a 16S rRNA gene plus gene chip for the diagnosis of neonatal sepsis in order to improve the speed and accuracy of clinical detection of bacteria. Methods A total of 125 cases of newborn blood samples and some cerebrospinal fluid samples diagnosed as sepsis were tested for bacterial 16S rRNA gene and microarray, including DNA extraction, design of primers and probes, polymerase chain reaction (PCR) amplification, Preparation, hybridization, laser scanning and reading. Results Of the 125 cases, 64 were positive for blood samples detected by PCR, accounting for 51.2%; 32 were positive for blood culture, accounting for 25.6%; 41 were nonspecific, accounting for 32.8%. The difference was statistically significant (P <0.01). If the blood culture positive and / or non-specific indicators of at least two positive diagnostic criteria, the PCR sensitivity was 90.3% (56/62), the specificity was 87.3% (55/63), the correct diagnostic index was 0.776.3 cases of cerebrospinal fluid PCR positive specimens in 2 cases, only 1 positive culture. 64 cases of PCR-positive blood samples for further gene chip detection, the results of the common probe were positive. Among them, 60 were positive for G + probe and 4 were positive for G-probe. Thirty PCR-positive and blood culture-positive specimens were found to be consistent with positive results from blood culture bacteria. 2 cases of cerebrospinal fluid specimens PCR positive, gene chip test results and culture results. Conclusion 16S rRNA gene PCR plus gene chip hybridization can provide early, sensitive etiological diagnosis of neonatal sepsis.