[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.
Wu
Yi-dong
YD
Department of Central Laboratory, Children's Hospital Affiliated to the Medical College, Zhejiang University, Hangzhou 310003, China.
Shang
Shi-qiang
SQ
Li
Jian-ping
JP
Yang
Zu-qin
ZQ
Zheng
Zhi-bei
ZB
Du
Li-zhong
LZ
Zhao
Zheng-yan
ZY
D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D020459:Genes, rRNA / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007231:Infant, Newborn; D057230:Limit of Detection; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D012235:Rhodamines; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000175:diagnosis* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000235:genetics; D013212:Staphylococcus epidermidis / Q000235:genetics
C106141:6-carboxytetramethylrhodamine; D017931:DNA Primers; D012336:RNA, Ribosomal, 16S; D012235:Rhodamines; D004247:DNA