细菌16S rRNA基因荧光定量PCR诊断新生儿败血症

目的探讨新生儿败血症的快速可靠诊断方法,以提高临床检测细菌的速度及准确性。方法以细菌16SrRNA基因为靶序列,设计通用引物及TaqMan探针,建立细菌16SrRNA基因实时荧光定量PCR反应体系;选择临床引起新生儿败血症的常见菌如金黄色葡萄球菌、表皮葡萄球菌和大肠埃希菌等进行浓度梯度实验;对临床疑为新生儿败血症的830例感染性疾病的新生儿抽取静脉血分别做细菌16SrRNA基因荧光定量PCR和血培养检测。结果临床常见分离株金黄色葡萄球菌、表皮葡萄球菌、大肠埃希菌荧光定量PCR检测结果均为阳性;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。细菌荧光定量PCR最低能检测到3个细菌,其荧光定量CT值为37．90;对临床疑为感染性疾病的830例患儿标本中,荧光定量PCR检测血标本阳性率5．18％（43／830）,血培养阳性率2．41％（20／830）,前者明显高于后者,差异具有统计学意义,P〈0．01,30例非感染性疾病患儿血标本荧光定量PCR检测及细菌培养均为阴性。若以血培养作为对照,荧光定量PCR方法的诊断敏感性为100％,特异性为97．16％,正确诊断指数为0．972。结论建立了细菌16SrRNA基因荧光定量PCR诊断新生儿败血症方法。其检测快速、简便,为新生儿败血症提供早期、敏感的病原学诊断依据。

A broad-range 16S rRNA gene real-time PCR assay for the diagnosis of neonatal septicemia

OBJECTIVE: To evaluate the usefulness of a broad-range real-time PCR assay aimed at the 16S rRNA gene of bacteria in a clinical setting in rapid and reliable diagnosis of neonatal septicemia for improving the speed and accuracy of bacterial detection. METHODS: The universal primer and TaqMan probe were designed based on the highly conserved sequences of the bacterial 16S rRNA gene. The chosen primers and probe did not show any likely cross hybridization with human, viral or fungal genome sequences. The TaqMan assay used the fluorescent signal on the probe, such as 6-carboxyfluorescin (6-FAM), and quenched by the standard 6-carboxytetramethylrhodamine (TAMRA) probes. The broad-range 16S rRNA gene real-time PCR array was established. Then, three common pathogenic microorganisms including Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli, which were prepared by a 10-fold dilution series respectively from 10(8) colony forming unit (CFU)/ml to 10(3) CFU/ml, as well as controls, were used for testing of both sensitivity and specificity of the real-time PCR assay. The blood samples from 830 cases of suspected septicemia, who were hospitalized in our neonatal ward and the neonatal intensive care unit (NICU) and developed clinical signs suggestive of infection, were tested with routine culture and bacterial 16S rRNA genes real-time PCR separately. In addition, 30 neonates without infection were enrolled as the negative control group. RESULTS: All the three common pathogenic bacterial species were positive on the 16S rRNA genes real-time PCR assay. There were no cross-reaction with cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B virus (HBV), fungi, human DNA and blank control, and the technique showed high specificity and sensitivity. The detection limit of the TaqMan assay was tested by amplifying serial dilutions of the three common pathogenic bacterial DNA. The minimal detection limit of the TaqMan system was equivalent to 3 CFU of bacteria, the threshold cycle (CT), which is inversely proportional to the log of the amount of target DNA initially present, was 37.90 by calculation. The real-time PCR assay was evaluated on 830 blood specimens for suspected neonatal septicemia, as compared to the results obtained from the routine bacterial cultures. The positive rate by the real-time PCR assay was 5.18% (43/830) in 830 samples, and was significantly higher than that of blood culture [2.41% (20/830) (P < 0.01)]. The real-time PCR was positive in all the 20 positive blood culture samples. Thirty non-infectious blood samples were negative by both the PCR assay and blood cultures. When blood culture was used as control, the sensitivity of the real-time PCR assay was 100%, the specificity was 97.16%, and the index of accurate diagnosis was 0.972. Moreover, three of the PCR positive amplicons were confirmed by sequencing to confirm the accuracy of the real-time PCR assay in testing clinical specimens. The sequencing showed that except for one sequence, all the others were demonstrated to be Staphylococcus aureus and Escherichia coli respectively, which was in accord with the results of the blood cultures. CONCLUSIONS: The bacterial 16S rRNA genes real-time PCR had been established to diagnose the neonatal septicemia. The sensitivity and specificity the real-time PCR assay were higher than those of blood culture. This technique can provide a rapid way for the etiological diagnosis of neonatal septicemia, and was a convenient and accurate method in etiologic diagnosis of neonatal septicemia.