用聚合酶链反应加反相杂交扩增细菌16SrRNA基因快速诊断新生儿败血症

Rapid Diagnosis of Neonatal Sepsis by PCR Amplification and Reverse Hybridization of Bacterial 16SrRNA Genes

探讨新生儿败血症的快速诊断方法。方法采用聚合酶链反应(PCR)加反相杂交技术对131例拟诊败血症的新生儿血清中16 SrRNA基因进行检测,同时以血细菌培养及5项非特异性指标(包括C反应蛋白、微量血沉、外周血白细胞计数、血小板计数和未成熟粒细胞与中性粒细胞总数之比值)作为对比。结果 PCR检测阳性率为41.9%,显著高于血培养阳性率(17.6%)和非特异性指标的阳性率(P<0.05)。对55份PCR阳性标本进一步进行反相杂交,显示G^+菌27份,G^-菌28份,其结果与血培养结果相符。若以血培养加非特异性指标为诊断标准,PCR法的灵敏度为88.9%,特异性为90.9%,正确诊断指数达到0.798。结论 PCR加反相杂交技术检测血清中细菌16 SrRNA基因能快速诊断新生儿败血症并鉴别细菌属G^+抑或G^-菌,较血培养和非特异性指标等方法具有更好的敏感性与特异性,有较大的推广及应用价值。

Objective To explore a way of rapid diagnosis of sepsis in newborn infant. Methods Blood bacterial 16SrRNA genes PCR amplification were done in 131 newborn infants suspected of sepsis. The positive rate was compared with those of blood culture and 5 non-specific tests (including CRP, mESR WBC, PLT,and 1/T ratio, ). Samples with PCR positive were further analyzed by reverse hybridization to identify G^+ or G^- bacteria infection. Results The positive rate of PCR (41.9%) was significantly higher than those of blood culture (17.6%) and non-specific teste (33.6%), P<0.005 and P<0.05, respectively. Among 55 cases with positive PCR, 27 were Grampositive and 28 were Gram-negative species by reverse hybridization. It coincided with the blood cultures. The sensitivity, specificity and Youden's index of PCR were 88. 9%, 90.9% and 0.798 respectively when blood culture and five non-specific tests were used as a reference method. Conclusion Detection of the becterial 16SrRNA genes in blood by PCR amplification and reverse hybridization can diagnose neonatal sepsis apidly and distinguish bacteria between the Gram positive and Gram negative. This method may be valuable and practical in early diagnosis of neonatal sepsis with relative higher sensitivity and specificity.