Establishment And Application Of The 16SrRNA Gene Real-Time PCR And Microarrays

Neonatal sepsis is obscure in the representation and is not specific in the clinical symptom, quite a few survivals have sequelaes. So looking for the early, fast, sensitive, and differential diagnosis index of neonatal sepsis is important in supervising the clinical therapy, reducing the mortality rate of neonate and improving the prognosis. In this study, we analysed the bacterial 16S rRNA gene sequence, desigened primers and probes in the conserved regions, followed by the FQ-PCR and gene chip hybridization analysis, finally established the method of bacterial 16S rRNA gene FQ-PCR combined with gene chip hybridization to detect the DNA quickly, reported the results of a preliminary application that was performed to evaluate the feasibility of this technique of clinical specimens furtherly. The reseach was composed of two parts. Part one: Establishment of the 16SrRNA gene Real-Time PCR and Microarrays;part two: Application of the 16SrRNA gene Real-Time PCR and Microarrays for clinical specimens in children.Part OneEstablishment of the 16SrRNA gene Real-Time PCR and MicroarraysMethods: A pair of universal primer and a universal fluorescence probe were designed based on the bacterial 16S rRNA gene highly conserved district, then the standard strains and negative controls were detected and analysed by FQ-PCR. We also desigened primers and probes targeting the bacterial 16rRNA gene conserved district. These probes included the Gram-positive probes, Gram-negative probes, and other specific probes, and then the DNA microarrays format were made based on the standard microscopic glass slides. The standard strains and negative controls were hybridized, washed, scaned in these chips, Finally the slides were imaged and the data were analysed.Results:1. The UnProbe results of FQ-PCR in detecting 10 kinds of standard strains (35 plants) were positive, and their Ct between 14.24 and 25.08. CMV, EBV, HBV, Cryptococcus histolyticus, and Blastomyces albicans were detected to be negative by FQ-PCR. Therefore the 16S rRNA gene FQ-PCR did not cross-react human DNA, virus or fungi, and it was highly specific for bacteria. Serial dilutions of the plasmid were prepared and were detected by FQ-PCR within the range of 10~0~10~7copy/μl. The Ct numbers produced by the Rn vs Cycles curves of FQ-PCR were increased by degrees. 10 copies were detected by FQ-PCR at least, equaled to 2 bacteria nearly, which Ct was 38.2. The blank controls were negative, which Ct 40.2. The FQ-PCR amplified the products of 49 strains were tested by the gene chip hybridization, including 33 Gram-positve and 16 Gram-negative strains. Using the amplified DNA of the 33 Gram-positive bacteria as templets, there were positive findings for the two universal probes and the one Gram-positve probe. The Gram-negative probes and universal probes showed positive results when 16Gram-negative bacteria were used as templates. Each strain specifically hybridized with its own probe. These results showed that the gene chip hybridization not only distinguished Gram-positve and Gram-negative strains but also detected the special bacteria.Conclusions: FQ-PCR is sensitive, rapid in detection. It is not disturbed byantibacterial exist in the samples. The quantitative analysis is. accurate and is helpful to evaluate the curative effect, estimate the prognosis, and also solved the pollution problems of PCR. The sensitivity of DNA microarrays is high;it could detecte some bacteria which was unusual or difficult to culture. It was able to assay any asepsis body fluid, such as blood, CSF and pleural fluid. The gene chip was fast which could deteced within 4 h and was high flux which could do with many items once. The technique combinding the two methods aboved will provide a new way for identifying the bacteria infection early and fast.Part twoApplication of the 16SrRNA gene Real-Time PCR and Microarrays for clinical specimensMethods : 49 clinical CSF specimens suspected Purulent Meningitishospitalized children from our neonatal ward, the NICU and the 15th ward. Each chidren was drawn 2ml CSF, 1ml was routine cultured, and 1ml was done by the FQ-PCR and DNA microarrays. 830 blood samples were collected from neonates suspected septicemia, the collection of blood samples was done by cultured, 16SrRNA gene FQ-PCR and DNA microarrays respectively.Results: One—Application in Purulent Meningitis1.FQ-PCR was positive when the baseline was took as 0.02 and Ct<35. Of 49 specimens, 17 were positive by FQ-PCR, the positive rate was 34.7% (17/49), while 5 specimens (10.2%) were positive by CSF culture. The positive rate of FQ-PCR was significantly higher than that of CSF culture, x2= 10.481,P=0.005,P<0.01.2. The seventeen specimens with positive results by FQ-PCR were further hybridized on microarrays. All were positive by universal probes. Of those 17 specimens, seven were positive by E. coli probe, two by S. epidermidis, two by S. aureus, one by hemophilies influenzae, one by Streptococcus agalactiae, five by G-, one by G+ (17/49);While of the 5 positive CSF cultured specimens, four were positive by E. coli probe, one by Klebsiella Pneumoniae, the positive rate of CSF culture was 10.2% (5/49) .3. There were some difference among the bacterial DNA copy munbers and Ct numbers of different P.M status children. The Ct numbers of CSF FQ-PCR with severe children were usually lower than that of light sick children. The higher the Ct number, the lower the bacterial DNA copy numbers in CSF. They were in negative correlativity.4. Two PCR productions of positive FQ-PCR CSF DNA were sequenced. One sample was sequenced to E. coli with Ct 17.9, it supported the report of the FQ-PCR Ct number and accorded with the positive E. coli CSF culture. While another specimen was not achieved the sequence result, it showed that the specimen Ct 31.8 hinted the lower bacterial DNA copy number, matched the high Ct number and the result of CSF bacterial culture.Two— Application in Neonatal Septicemia1.Of 830 specimens, 43 were positive by 16SrRNA gene FQ-PCR, the positive rate was 5.18% (43/830) , while 20 specimens (2.41%) were positive by blood routine culture(20/830). The positive rate of FQ-PCR was significantly higher than that of blood culture, p<0.01.2. The 43 specimens with positive results by FQ-PCR were further hybridized on the microarrays. All were positive by universal probes. Of those 43 specimens, 32 were positive by G+ probes, including two were positive by Streptococcus pneumoniae probe, eight by S. epidermidis, eight by S. aureus, thirteen by CONS. While 11 specimens were positive by G- probes, including seven were positive by E. coli probe, one by hemophilies influenzae. Of the 20 positive blood culturedspecimens, fifteen were positive by G+ probes altogether, five by G- probes. They were able to hybridize to the corresponding special probes;the species determined by the microarray analysis corresponded to that determined by traditional blood culture basically. One specimen with positive E. coli both in blood routine culture and the blood microarray was showed both positive in E. coli probe of CSF gene chip and in the CSF culture.3. Three positive specimens in blood routine culture had not designed the corresponding special probe in the gene chip, but the blood microarrays were showed positive with the universal probe, the correspoing G- probe and G+ probe. Of one specimen with positive E. avium in blood routine culture, the hybridization showed both positive with universal probe and the G+ probe.Two with positive Sphingomonas paucimobilis and Burkholderia cepacia respectively in blood routine culture, the hybridization showed both positive with universal probe and G- probe accordingly. Of 23 specimens with negative in blood culture, all the corresponding FQ-PCR and hybridization showed both positive, 17 specimens were positive with G+ probes, 6 positive G-probes.Conclusion: The results of the specimens DNA microarrays with FQ-PCRpositive were identical to the results of the blood cultures basically, so these confirmed the combinative method possessed better especiality and exactness. The positive rate of FQ-PCR and DNA microarray was higher than that of blood routine culture, it demonstrated the combinative method had better sensitivity than the blood culture and decreased the lost diagnosis of septicemia. We can screen out primarily the pathogeny of neonatal septicemia by 16S rRNA gene FQ-PCR, reduce the lost assay of positive specimens, then ascertain the kinds of bacteria by the DNA microarray further. Finally this combinative method can diagnose the Septicemia and Purulent Meningitis earlier. It can be well-suited for the analysis of clinical specimens, and offer an effective method to instruct the using drug of clinical therapy.