The Methodologic Establishment And Application To Detect OprI Gene In The Pseudomonas Aeruginosa With Fluorescence Quantitive Polymerase Chain Reaction

Pseudomonas aeruginosa is a common normal pathogenic bacteria in hospital infections, especially the in-hospital onset of nosocomial pneumonia, which is aggressive, and tend to drug resistance and high fatality rate. Traditional bacterial culture and related biochemical identification is called the "gold standard" to diagnose the bacteria infections. As the cultural is time consuming, susceptible to application of antibiotics, the inoculation duration and method, the false negative rate is so high that it could not meet the clinical need for early diagnosis. Fluorescence Quantitive Polymerase Chain Reaction (FQ-PCR) is not only characterized with high sensitivity and specificity, but also simple, rapid and less-pollution. It could meet the instant and accurate requirement clinically detection to bacteria for the capability of quantitatively analyzing trace pathogenic bacteria or the gene fragments in the blood at the same moment. Those characteristics are conducive to the early diagnosis and rational treatment in clinics. The oprI gene encoding the outer member proteins of the Pseudomonas aeruginosa is highly conserved, which could be utilized as the target gene of FQ-PCR detection. In this investigation we exert to build and test a fast and accurate method to diagnose the Pseudomonas aeruginosa infection by checking oprI gene with FQ-PCRChapter One Construct the methods to detect Pseudomonas aeruginosa with fluorescence real-time quantitative PCRObjective To construct the methods to detect the oprI gene of pseudomonas aeruginosa with fluorescence real-time quantitative PCR, and to apply it in quantifying the pseudomonas aeruginosa, and to compare the sensitivity and specificity of which with those of the traditional bacterial cultrue.Methods The primers and The probes were designed based on the major outer membrane lipoprotein I (oprI) gene which is specific for pseudomonas aeruginosa. Bacterial genome DNA was isolated from a typical pseudomonas aeruginosa, from which the oprI gene was got with PCR and was linked with pMD 18-T Vector to construct a recombined plasmids, then the constructed plasmid was transformed into competent E.coli cells. The true plasmids which were targeted in white colonies selected by Ampicillin, were linearrizated by HindIII and were identified their specificity by direct PCR, value of OD and DNA sequencing. The copy numbers were calculated by the value of OD and then diluted to serial concentrations as standards for standard curve of FQ-PCR by which the copy numbers were indicated.Results The target fragment of oprI gene of pseudomonas aeruginosa was successfully prepared, and the recombined plasmid contained the target fragment was constructed which was stable and its sequence integrity and specificity were kept. The correlation coefficient of the standard curve reached 0.994. The copy numbers of typical and cultivated strains of pseudomonas aeruginosa were calculated, while neither E.coli nor other control groups were detected. In the identification essays for every strains, all 31 strains of pure pseudomonas aeruginosa were all positive. The copy numbers of the typical strain ATCC27853 decreased by rank gradient after it was diluted by order concentration, and the minimal detectable clone number is 100 per ml. The FQ-PCR results of mixture of pseudomonas aeruginosa to other Gram staining negative bacteria, Gram staining positive bacteria or Blastomyces albicans are all positive. All Non-pseudomonas aeruginosa bacterium, Blastomyces albicans, hepatovirus B, cytomegalovirus and Mycoplasma pneumonia are negative checking with those primers. This method is not only for its good repeatability with the within-batch CV of 0.95, between -batch CV of 3.04%, but also for its simple and fast procedure that whole range of that is less than 4 hours. Conclusion The method to detect oprI gene in pseudomonas aeruginosa with FQ-PCR established successfully, which can detect the bacterial burden of pseudomonas aeruginosa fast and accuratelyChapter Two Applying FQ-PCR to check the rat model with Pseudomonas aeruginosa septicemiaObjective: To probe into the oprI gene in rat model with Pseudomonas aeruginosa septicemia with FQ-PCR, and to compare the sensitivity and specificity between FQ-PCR and traditional germiculture, and also to check the change of oprI gene before and after the antibiotic therapy as to rapidly judge its sensitivity.Methods: (1) The standard Pseudomonas aeruginosa with five different concentration of 1×10~9CFU/ml,1×10~8CFU/ml,1×10~7CFU/ml,1×10~6CFU/ml and 1×10~5CFU/ml were prepared, the drug-sensitive test to find the antibiotics sensitive (amikacin) and un-sensitive (oxacillin) to those were carried on. (2) 120 SD rats were divided into five groups randomly (each group with 24 rats), and five different concentration of Pseudomonas aeruginosa were injected into the rats with the volume of 0.5ml/100g, six rats of each group were picked up for taking 2ml blood for culture at the time points of 0h, 12h, 24h, and 48h after narcotization. Finally, the oprI gene of each blood samples were checked with FQ-PCR. (3)The 72 rats were divided into three groups, therapeutic group, treated group and untreated group randomly, each group with 24 rats. Pseudomonas aeruginosa with the concentration of 1×10~9CFU/ml 0.5ml/100g were injected into those rats. Sensitive antibiotics, un-sensitive antibiotics and 0.9%NaCl were given to the therapeutic, treated and untreated group rats respectively. Six rats of each group were picked up for taking 2ml blood for culture at the time point of 0h, 12h, 24h, and 48h after narcotized. Finally, the oprI gene of each blood sample were checked with FQ-PCR method.Results: (1) The blood culture were positive in each period of the concentrations 1×10~9CFU/ml and 1×10~8CFU/ml. Results of FQ-PCR showed that the copies number decreased with time going; the Ct value at 0h were 23.23±1.52 to 26.75±1.72, with 10~5-10~4 copies/μl, the Ct value at 48h were 33.15±1.17 to 37.75±0.97, with 10~2-10~1copies/μl, all of which were positive. (2) The blood culture were positive at the time points of 0h and 12h with the concentrations of 1×10~7CFU/ml and 1×10~6CFU/ml, were positive with concentration of 10~7CFU/ml at the time point of 24h, but negative at the time point of 48h, but were negative at the time points of 24h and 48h with the concentration of 1×10~6CFU/ml. Results of FQ-PCR showed that the Ct values increased with the time going, but copies number decreased with the time extend; the Ct value at the time point of Oh were 30.49±1.34 to 33.45±0.85, with 10~3-10~2copies/μl, the Ct value at the time of 24h was 39.77±1.47, with 10~0 copies/μl. But the Ct value at 48h was above 45, which was negative. Both the ct values at the time points of 24h and 48h were above 45, which were negative. (3)The blood culture were negative in each period of the concentration of 1×10~5CFU/ml. Results of FQ-PCR showed that the Ct value at the time point of Oh were above 45, which was negative. (4) The blood culture were positive in each period of both the treated and untreated group, but was negative in each period of therapeutic group. Results of FQ-PCR showed that the ct values at the time of Oh in the treated, untreated and therapeutic group were 23.30±1.49, 23.71±1.31, 23.69±1.54 respectively, there were no significant differences between these three groups. The Ct values at 12h were 27.57±1.35, 28.05±0.97, 30.56±1.74,at24h were 30.21±1.33, 30.87±1.42, 36.35±1.51,and at 48h were 33.43±1.28, 33.29±1.74, 39.73±1.09 in the treated, untreated and therapeutic group respectively. There were no significant differences between treated and untreated group at 12h,24h,48h, but had significant differences between therapeutic group with treated and untreated group, also before and after therapy at these tome points of 12h, 24h, 48h.Conclusion: (1) The coincidence rate between the method of FQ-PCR to detect the oprI gene for diagnose Pseudomonas aeruginosa septicemia and traditional germiculture were 100 % . Though the sensitivity of FQ-PCR was not increased, the time for diagnosis was shorter. (2)After treated it with effective antibiotic, the sensitivity of FQ-PCR to diagnosis Pseudomonas aeruginosa septicemia was higher than that of traditional germiculture, and the experiment time was shorter. (3)Detected the changes of the oprI gene copies number may be helpful to estimate the sensitivity of antibiotic.