Aim: The clinical diagnosis of sepsis is difficult, particularly in neonates. It is necessary to develop a rapid and reliable method for detecting bacteria in blood and cerebrospinal fluid (CSF) Polymerase chain reaction (PCR) and reverse hybridization of the 16S rRNA gene would permit fast and sensitive determination of the presence of bacteria and differentiate gram‐positive bacteria from gram‐negative ones in clinical specimens. Methods: We developed a pair of primers according to the gene encoding 16SrRNA found in all bacteria. DNA fragments from different bacterial species and from clinical samples were detected with PCR, and with reverse hybridization using a universal bacterial probe, a gram‐positive probe and a gram‐negative probe. Results: A 371 bp DNA fragment was amplified from 20 different bacterial species. No signal was observed when human DNA and viruses were used as templates. The sensitivity could be improved to 10T‐12 g. All 26 culture‐positive clinical samples (22 blood samples and 4 CSF samples) were positive with PCR. The gram‐negative and gram‐positive probes hybridized to clinical samples and to known bacterial controls, as predicted by Gram's stain characteristics. Conclusions: Our results suggest that the method of PCR and reverse hybridization is rapid, sensitive and specific in detecting bacterial infections. This finding may be significant in the clinical diagnosis of sepsis in neonates.

The aim of the study was to develop a multiplex PCR-based DNA microarray technology for simultaneous detection and species identification of seven human herpes viruses, namely herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus 6 (HHV-6A, HHV-6B), and to apply this technology to accurate diagnosis of herpesvirus-associated diseases. Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase gene in human herpes viruses. DNA microarrays were made by printing the oligonucleotide probes onto special glass slides. After amplification and labeling with CY5, the PCR products were hybridized with the DNA microarrays and species identified. Sixty-one cerebrospinal fluid (CSF) and 132 blood specimens were analyzed by this technique, and the results were compared with those of TaqMan PCR. Several specimens were sequenced further after cloning. The PCR products of the seven human herpes viruses ranged from 224 to 252 bp, and could be species identified with DNA microarrays. The detection limits were 10(1) copies/microl for each virus. And the test showed no cross-reaction to DNA extracted from S. aureus, E. coli, hepatitis B virus, Cryptococcus neoformans, Candida albicans and human genome. Among 132 blood and 61 CSF specimens, 55 were tested positive for human herpes virus DNA. Compared with the results of TaqMan PCR, the sensitivity and specificity of the DNA microarray technology was 96.2% and 99.3%, respectively. This multiplex PCR-based DNA microarray technology, which is rapid, specific and sensitive, serves as an effective technique for simultaneous detection and species identification of seven human herpes viruses.

Aim: To detect and differentiate six major human herpesviruses in the cerebrospinal fluid (CSF) and blood of children by polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP). Methods: We synthesized two pairs of primers in the well‐conserved regions of the DNA polymerase gene in human herpesviruses. One pair was designed to amplify cytomegalovirus (CMV), Epstein‐Barr virus (EBV), herpes simplex virus type 1 (HSV‐1) and type 2 (HSV‐2), and the other pair to amplify varicella‐zoster virus (VZV) and human herpesvirus 6 (HHV‐6) by PCR. Virus species identification was achieved by restriction enzyme digestion with BamHI and BstUI. Ninety‐eight CSF and 75 blood specimens were analysed by this technique. At the same time, all blood specimens were also examined by enzyme‐linked immunosorbent assay (ELISA). Results: Thirteen (13.3%) of 98 CSF specimens and 26 (34.7%) of 75 blood specimens were positive for herpesvirus DNA in this PCR assay. Only 10 (13.3%) of the blood specimens were positive in ELISA for virus‐IgM antibody. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of PCR in detecting herpesvirus infections compared with ELISA were 100% (10/10), 75.4% (49/65), 38.5% (10/26) and 100% (49/49), respectively. These results indicate that the positive rate of PCR was significantly higher than that of ELISA (p<0.05). The herpesvirus type of these positive specimens was rapidly detected using restriction enzyme digestion with BamHI and BstUI. Conclusions: PCR‐RFLP is a specific, sensitive and accurate technique for the identification of herpesvirus infections in the CSF and blood of children.

PCR-RFLP is a specific, sensitive and accurate technique for the identification of herpesvirus infections in the CSF and blood of children.