Rapid Diagnosis Of Septicemia And Grouping Of The Bacteria By 16srRNA Gene PCR

Septicemia is a kind of general disease caused by invasion of bacteria into the blood stream and progresses rapidly with high mortality. Early diagnosis and effectively therapy at the onset of diseases is impotant for the recovery of the patients. At present, the gold standard for the diagnosis of septicemia is still blood culture. It is a time-consuming and low positive rate assay, so it can not provide the useful information to clinical doctors for early diagnosis. And the doctors were forced to select the broad-spectrum antibiotic, which could lead to increasing production of drug-resistance bacteria and heavy burden to patients; therefore, rapid diagnosis of septicemia and grouping the bacteria initially is a problem needed to be solved in medicine.16SrRNA gene is the DNA sequence encoding 16S rRNA, which is present in all bacteria chromosome but not among non-prokaryotic organism, such as virus and fungi. 16SrRNA gene not only contains regions well conserved in all bacteria, but also 9 to 10 species-specific variable regions that allow species identification, which alternate each other. This study explored the PCR method to amplify the conserved region and variable region respectively to detect septicemia and grouping the Gram bacteria, providing the etiology evidence for doctors earlier and more efficiently.1. Rapid diagnosis of septicemia1.1 The specificity of the universal primer based on the conserved region was evaluated on the positive group (S.aureus, S.epidermidis, E.coli, K.pneumoniae, S.saprophyticus and Enterococcus) and the negative controls (Candida albicans, Cryptococcus and Heptitis B virus), the results showed high specificity with positive bands appearing in the positive group and none in the control group.1.2 Optimized the PCR system and evaluated the sensitivity by detecting a series of different density bacteria suspensions. PCR assay can detect the minimum density of bacteria to 1.5×103CFU/ml,which is more significantly sensitive than the blood culture. 1.3 The blood samples from 60 septicemia patients were tested with 16srRNA gene PCR and blood culture respectively, and 20 health peoples were enrolled as the negative control. The positive rate was 86.6% and 38.3% respectively by PCR and blood culture. The positive rate by PCR assay was more significantly higher than that of blood culture.1.4 10 blood samples, which were positive by PCR method and negative by blood culture were selected randomly to sequence the 16SrRNA gene.The species of eight sequences were searched and identified by BLAST software.2. Grouping of the Gram bacteria2.1 Gram-positive primers could specifically amplify 16SrRNA gene of the gram-positive bacteria such as (S.aureus, S.epidermidis, S.pneumoniae, S.pyogenes, S.saprophyticus and Enterococcus) and the one of Gram-negative isolates (E.coli, K.pneumoniae, E.cloacae, P.mirabilis and P.aeruginosa) could be amplified with Gram-negative primers, the results showed high specificity in all isolates.2.2 The results of 20 samples with nested-PCR were consistent with that of the blood culture.In the all, the study indicated that we can make a rapid diagnosis of septicemia amplification of the conserved region of the 16SrRNA gene by PCR. The positive rate by PCR assay was significantly higher than that of blood culture because of its high sensitivety and not being influenced by antibiotics. And the study showed that we can group Gram-positive and -negative bacteria by nested-PCR, providing the etiology evidence for doctors earlier and more efficiently.