The Application Analysis Of Multiplex Real-time PCR Assays For Detection Of Pathogenic Bacterium In Peritoneal Dialysis-associated Peritonitis

Objective Application of multiplex and monoplex real-time polymerase(RT-PCR)detection in patients with peritoneal dialysis-associated peritonitis(PDAP) through dialysis fluid specimens of common pathogenic bacteria distribution, and compared the results of the two methods. To estimate the clinical value of bacterial detection in PDAP by multiplex real-time polymerase chain reaction(RT-PCR).Methods Seventy peritoneal dialysate specimens were collected, conventional bacterial culture and SYBR Green RT-PCR detection of the bacterial universal primers were used respectively. According to references and the bacterial culture results of these 70 specimens, six common bacteria of PDAP were selected in this assay, multiplex and monoplex RT-PCR were used respectively to exam the positive specimens by SYBR Green RT-PCR detection. The results of SYBR Green RT-PCR and conventional bacterial culture were compared with each other, and multiplex RT-PCR compared with monoplex RT-PCR. We used SPSS16.0 statistical software to analysis those clinical data.Results 1.The positive rate of traditional culture among the 70 cases was 65.71%,gram-positive strains accounted for 71.74%(33/46), main species were: epidermis staphylococcus(9 cases), hemolysis staphylococcus(10 cases), escherichia coli(5 cases),and so on. 2. The results of clinical statistics:On admission, almost 70 patients were abdominal pain, tenderness or cloudy peritoneal fluid. three indicators of negative samples were significantly lower than positive ones(P<0.05),they were white blood cell count and multi-core percentage of diaiysis fluid, blood CRP. Also the blood albumin of the patients with negative culture test was obviously higher than the positive ones(P<0.05). And there was on statistical difference of other indicators between the two groups. 3. The SYBR Green RT-PCR detection results showed that 70 specimens total positive rate was 81.42%, among the 46 positive samples detected by conventional bacterial culture, 2 cases were not picked out by SYBR Green RT-PCR. With bacterial culture as the gold standard, the sensitivity of SYBR Green RT-PCR was 95.65%,specificity was 45.83%, there was statistical difference between the two methods(P<0.05).4. In the detection of these 6 common pathogenic bacteria, both of multiplex and monoplex RT-PCR assays found 38 cases of positive samples among the57 specimens detected by SYBR Green RT-PCR. The results of the two methods were completely identical. One of the positive samples examined by RT-PCR was different form classical bacterial culture, the remaining 19 cases failed to clear strains of pathogenic bacteria. 5. SYBR Green PCR for detecting pathogenic bacteria could show results in 4~5 hours, and in the experiments of the 6 common bacteria, monoplex RT-PCR could be finished in 7~9 hours, but multiplex RT-PCR just needed at most 3hours.Conclusion Compared with traditional culture method, all of the three RT-PCR assays are sensitive, specific, and more rapidly, but due to the multiplex RT-PCR can detect several kinds of bacteria simultaneously, it is more practical,convenient and economical then the monoplex RT-PCR.