细菌革兰双检荧光定量PCR方法检测新生儿败血症

Gram-specific probes based real-time PCR assay for simultaneous detection of Gram-positive and -negative bacterial neonatal sepsis

目的建立细菌革兰阴、阳性菌双重实时荧光定量检测体系，探讨其检测败血症的临床应用价值。方法分析细菌16SrRNA基因序列，在高度保守区自行设计通用引物和革兰阴性和阳性分型探针，选取临床较常见的35株菌株进行细菌革兰双检实时荧光定量PCR方法检测；对临床疑为败血症的512例新生患儿分别做细菌革兰双检荧光定量PCR和血培养检测。结果细菌革兰双检荧光定量PCR具有较好的敏感性和特异性，能稳定检测到10CFU左右细菌数。35株菌株进行细菌革兰双检荧光定量PCR检测，均为阳性，且革兰阴、阳性菌能正确分型和定量。巨细胞病毒、EB病毒、乙肝病毒、新型隐球菌及白色念珠菌、人基因组DNA及空白对照均为阴性。对临床疑为败血症的512份新生患儿标本中，革兰双检荧光定量PCR检测血标本阳性率8．20％（42／512），血培养阳性率6．25％（32／512），前者明显高于后者，差异具有统计学意义（χ^2=8．10，P〈0．01），30例非感染性疾病同期患儿血标本革兰双检荧光定量PCR及细菌培养均为阴性。若以血培养作为对照，细菌革兰双检荧光定量PCR方法的诊断敏感度为100％，特异度为97．92％，准确性98．05％。结论建立了用通用引物和分型双荧光探针的细菌革兰双检荧光定量PCR方法。其检测快速、准确，具有很大的临床推广价值。

Objective To develop a method of simultaneous detection and Gram classification for pathogens causing sepsis with gram-specific probes based real-time PCR. Methods A pair of universal primers and a set of probes including Gram-positive probe and Gram-negative probe were designed based on the bacterial highly conserved region of 16SrRNA gene. With the gram-specific probes based real-time PCR, 35 clinical frequently-isolated strains including 17 gram-positive and 18 gram-negative bacteria were identified correctly with the corresponding gram probe. The blood samples from 512 cases of suspected septicemia, who were hospitalized in our neonatal ward and the NICU and developed clinical signs suggestive of infection, were tested with routine culture and bacterial gram-specific probes based real-time PCR separately. Results The detection limit of the gram-specific probes based real-time PCR assay was 10 CFU of the bacteria. The 35 isolates could be detected and classified correctly by gram-specific probes based realtime PCR. The PCR results were all negative for Cytomegalo virus, EB virus, hepatitis B virus, Cryptococcus neoformans, candida albican, human genomic DNA and negative control. The gram-specific probes based real-time PCR appeared to be quite specific. For 512 blood specimens from the patients with suspicious neonatal sepsis, the positive rate of the gram-specific probes based real-time PCR array was 8. 20% （42/512,）, which is significantly higher than that of blood culture （32/512, 6. 25% ） （χ^2 = 8.10, P 〈0. 01）. When blood culture was used as a standard, the sensitivity of the gram-specific probes based real-time PCR was 100%. The specificity was 97. 92% and the accuracy was 98. 05%. Conclusions Gram-specific probes based real-time PCR with universal primers and gram-specific probes are developed. This study suggests that the bacterial gram-specific probes based real-time PCR are very useful for the rapid and accurate diagnosis of bacterial infection.