Establishment And Application Of The Gram Stain-specific-probe-based Real-time PCR Assay

Sepsis is a serious bacterial disease with high mortality in newborns,which is obscure in the representation and is not specific in the clinical symptom.A fast and correct diagnosis,followed by rapid treatment,plays an important role in the reduction of infant mortality resulting from sepsis.Currently,bacterial culture is required as a standard method for diagnosis of the presence of bacterial pathogens in clinic.However, this technique has some disadvantages with regard to the desired rapidity and sensitivity. In this study,we describe a Gram stain-specific-probe-based real-time PCR(GSPBRTPCR) system involving the 16S rRNAgene that allows simultaneous detection and discrimination of clinically relevant gram-positive and -negative bacteria directly from clinical specimens.The study was composed of two parts.Part one:Establishment of the Gram stain-specific-probe-based real-time PCR(GSPBRT-PCR);part two: Application of the GSPBRT-PCR technology for clinical specimens.Part One Establishment of the Gram stain-specific-probe-based real-time PCRMethods:A pair of universal primers and a set of probes(including Gram-positive probe and Gram-negative probe) were designed based on the bacterial highly conserved region of 16S rRNA gene.With the GSPBRT-PCR,53 clinically important strains representing 25 gram-positive and 28 gram-negative bacteria species were evaluated for their specificity.To determine the sensitivities and the linear relationship of GSPBRT-PCR, S.aureus as a representative of gram-positive bacteria and E.coli as a representative of gram-negative bacteria were used to establish the standard curve and to detect the limits of the assay,based on a series of 10-fold dilutions.Results:The limits of the gram-specific probes based real-time PCR assay to serial dilutions of the bacteria revealed that S.aureus could be detected at concentrations of 3 CFU per PCR and E.coli at concentrations as low as 1 CFU per PCR.All 25 gram-positive bacteria examined showed fluorescence signals,and the gram-negative bacteria showed no fluorescence with the gram-positive probe.When tested simultaneously by the gram-negative probe,DNAs from all of the gram-negative species were positive,and the gram-positive bacteria showed on fluorescence.No fluorescence was detected and no cross-reaction was showed to DNAs extracted from the CMV,EBV,HBV,fungi,human genome and the blank control group.Conclusions:This study suggests that the bacterial gram-specific probes based real-time PCR is very useful for the rapid and accurate diagnosis of bacterial infection and has an important impact on the current inappropriate and unnecessary use of antibiotics in the treatment of newborns.Part two Application of the GSPBRT-PCR technology for clinical specimensMethods:The GSPBRT-PCR assay was further evaluated on 1000 blood specimens from the patients with suspicious sepsis and 125 clinical CSF specimens suspected bacterial meningitis and compared to the results obtained from routine culture.The results were analyzed using SPSS software.Results:A total of 1000 blood samples were analyzed by both blood culture and GSPBRT-PCR.There were 64 positive results(64/1000,6.40%) with GSPBRT-PCR and 50 positive results(50/1000,5.00%) with blood culture.The positive rate of GSPBRT-PCR was significantly higher than that of blood culture(P＜0.01).Among 125 CSF specimens from children with suspected bacterial meningitis,17 samples were tested positive by GSPBRT-PCR,while only 6 specimens were positive results with the cerebrospinal fluid culture method.These two groups gave significant different results (P＜0.01).Compared with the total results of bacterial culture,the sensitivity and specificity were 91.07%and 97.36%respectively,and the Youden's Index was 0.8843, the value of kappa was 0.89.Conclusions:This GSPBRT-PCR technology,which is rapid,specific and sensitive,serves as an effective technique for simultaneous detection and Gram identification of clincallly relevant bacterial pathogens causing sepsis or bacterial meningitis directly from clinical samples,and has a condiderable value in erarly diagnosis of bacterial infection in clinic.