16S rRNA基因芯片诊断新生儿败血症

目的建立16SrRNA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16SrRNA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168 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。

Using of 16S rRNA gene chip hybridization in the diagnosis of neonatal sepsis

Objective To improve the speed and accuracy of bacteria detection, and develop the test of 16S rRNA genes PCR amplification plus gene chip hybridization to diagnose neonatal sepsis. Methods Bacterial 16S rRNA genes were detected in blood and CSF samples of 125 suspected neonatal sepsis, and the results were compared with blood culture, CSF culture, and non-specific diagnostic parameters (WBC, PLT, CRP). 30 non-infectious neonates were regarded as the negative control group. Gene chip test were performed by extraction of DNA, primers and probes design, PCR amplification, preparation of gene chip, hybridization, laser scan and reading of the results. 18 specific probes, including universal 1, universal 2, Gram positive bacterial probe, Gram negative bacterial probe 1, Gram negative bacterial probe 2, Staphylococcus aureus, Staphylococcus epidermidis, CoNS (Coagulase Negative Staphylococcus), Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Bacteroides fragilis, Bacillus, Meningococcus, Corynebacterium, Propionibacterium, were used in the test. Results The positive rate of PCR test was 51.2% in 125 blood samples, and was significantly higher than the positive blood culture (25.6%), or the indicator of two abnormal non-specific parameters (32.8%) (P<0.001). The PCR results were negative in 30 negative control cases. With the blood culture and non-specific parameters being the diagnostic standard, the sensitivity of PCR amplification is 90.3%, specificity is 87.3%, the accurate diagnostic index is 0.776. 2 of 3 CSF samples were positive by PCR amplification, but one culture positive. Moreover, 64 PCR positive products were tested by universal probes and all showed positive results. Among them, there were 31 Staphylococcus epidermidis, 17 CoNS, 9 Staphylococcus aureus, 2 Bacillus, 1 Listeria monocytogenes, 4 Escherichia coli. The bacteria detected by gene chip were coincident with the results of blood culture in 30 cases with both PCR and blood culture positive. 2 PCR positive CSF results, Staphylococcus epidermidis and Escherichia coli respectively, were in accord with the culture results. Conclusions 16S rRNA gene amplification plus gene chip hybridization is an early and sensitive method in pathogenic diagnosis of neonatal sepsis.