Preliminary Studies On The Rapid Detection Of Clinical Common Bacteria And Fungi

Bacterial and fungal infections are clinical common infectious diseases with high morbidity and mortality, which threaten lives of patients without early diagnosis and treatment. The traditional methods including microbial culture, biochemical tests and immunoassay were lack of sensitivity and specificity and did not diagnose pathogenic infection rapidly and accurately. So it is very important to establish a quick diagnosis method. In this study, molecular biology methods were established for rapid diagnosis of common bacteria and fungi in clinical infection samples. The aims are to provide potential guides to rational antibiotic administration in clinical practice.Part1Establishment of real-time PCR for detection of bacteria and fungi simultaneouslyObjective:TaqMan probe-based real-time PCR was established for detection and differentiation of gram-positive bacteria, gram-negative bacteria and fungi infections simultaneous in one test. It would make it possible to diagnose pathogens at an early stage.Methods:Twenty bacterial16S rRNA gene and seven fungal18S rRNA sequences were obtained from GenBank and aligned with sequences to identify respective conserved sequences using the program ClustalX. According to general principle of design, bacterial universal primers and gram-stain typing probes were designed based on bacterial conserved sequences, and fungal universal primers and universal probe were designed based on conserved regions by the Primer Express2.0and Oligo6.0software. The gram-positive probe and fungal probe were labeled with6-carboxyfluorescein (FAM) reporter dye at the5’-terminal, gram-negative probe was labeled with HEX. The probes were labeled with fluorescent quencher TAMRA at the3’-terminal. A229bp fragment was amplified by bacterial primers and276bp amplified by fungal primers. After optimization of reaction system, the bacterial gram-positive and gram-negative probes were placed in a reaction tube, and fungal universal probe in another tube, thus PCR amplification of bacterial and fungal target DNA are two parallel reactions. Each assay included negative, positive and blank controls. PCR reactions were performed using an ABI7500real-time florescence quantification PCR machine. Standard and clinical strains isolated including eleven gram-positive bacteria, nine gram-negative bacteria and seven fungi were detected. At the same time, HBV DNA and human genomic DNA were detected in order to observe the specificity. The genomic DNA of Staphylococcus aureus, Escherichia coli and Candida albicans were amplified by PCR primers and their recombinant plasmids were also constructed. Ten-fold serial dilutions starting from108copies/μl-100copies/μl of plasmids were prepared. Each dilution was run in triplicate within an experiment by real-time PCR to evaluate the sensitivity and linear relationship. The plasmids of Candida albicans with three different concentrations were used as templates in the real-time PCR reactions to evaluate the reproducibility. Each concentration was repeated three times on three consecutive days, the repeatability and stability were evaluated by calculating CV of CT values.Results: A total of twenty bacteria were detected by designed primers and probes. Eleven gram-positive bacteria examined showed fluorescence signals with the gram-positive probe, but no fluorescence appeared with the gram-negative bacteria and fungi pobes. In nine gram-negtive bacteria, the fluorescence signal was only found in combining with gram-negtive bacteria probes, no fluorescence signal appeared with gram-positive and fungi probes. Bacterial DNA, viral DNA and human genomic DNA were not detected by fungal universal primers and probe. Fungi only be detected by fungi probe and results showed that CT values were between15.6and26.0. These showed designed primers and probes were specific. Ten-fold dilutions plasmids were prepared, CT values to be with108copies-101copies showed the remarkable inverse correlation and linear relationship. As CT value<35was a positive standard, the detection limit of the real-time PCR was defined as10copies of plasimd DNA when only two bacterial probes were placed in one reaction system. The detection limit was102copies of plasimd DNA if three probes were placed in one reaction system. The between-run CV values for the fungal plasmid DNA in the concentrations of107copies,105copies and103copies tested by real-time PCR were2.47%,1.89%and2.28%respectively, all are less than5%, it showed that this method was stable and fine repeatability.Conclusions:Multiple real-time PCR approach established which allows the detection of bacteria and fungi in a single sample with excellent analytical sensitivity and specificity. Gram-positive bacteria, gram-negative bacteria and fungi could be distinguished. It is applied to clinical laboratory diagnosis and will provide an important basis for early diagnosis of infectious diseases. Part2Application of real-time PCR for simultaneous detection of bacteria and fungiObjective: The clinical application of simultaneous real-time PCR detection was evaluated with clinical samples.Methods:A part of each sample was centrifuged at3000rpm for10min. The supernatant were cultured on broth medium, sediment of each sample was inoculated onto a blood agar medium, a chocolate agar medium and sabouraud agar plate. These plates were then incubated overnight at37℃in an atmosphere of5%CO2. The CSF India ink staining was initiated from patients with suspicion of meningitis, and meantime CSF routine analysis was also done. A total of137cerebrospinal fluid samples were detected by the real-time PCR established, and then compared with culture and India ink stain. The results were analyzed by SPSS13.0with McNemar test.Results:Among137clinical CSF samples, twenty-three samples were found to be positive by PCR while CT value<35as a positive standard, including seven positive bacteria and sixteen positive Cryptococcus, its total positive rate was16.8%. Becuase fastidious or slow-growing microorganisms may be difficult to be detected by traditional culture method, especially after continuous antibiotics treatment, only four CSF samples were cultured positive. India ink stain needs higher professional skills and usually with low sensitivity, ten samples were positive by India ink stain. Culture and ink stain methods were defined as control methods; the positive rate of PCR was higher than that of traditional methods. The results of two assays were significantly different (x2=5.82, P=0.012). The sensitivity and specificity were92.86%and91.87%respectively, the Youden’s Index was0.8473.Conclusions:Real-time PCR method was established for detection of clinical micro-organisms simultaneously and easy to operate. It may not only apply to diagnosis of cerebrospinal fluid samples, but also apply to diagnose other body fluids, such as blood, pectoral ascites and urine. It may be a potential guide for rational antibotics administration in clinical practice.Part3Establishment of real-time PCR for detection of Cryptococcus neoformans CAP10geneObjective:Cryptococcus neoformans CAP10gene detection was established using real-time PCR assay in order to provide an important basis for early diagnosis and efficacy judgment of Cryptococcus neoformans infection.Methods:Capsule associated protein gene(CAP10) sequence of Cryptococcus neoformans was obtained from GenBank database. A pair of primer was designed by Primer Premier5.0software. The RNA of strains were extracted and treated for reverse transcription. A191bp fragment of cDNA was amplified by PCR primers. PCR products amplified were cloned into pMD19-T vector and transformed into DH5a Escherichia coli competent cells, recombinant plasmids were constructed. Ten-fold serial dilutions plasmids were prepared. Real-time PCR was established to analyze amplication plot and melting curve, and the sensitivity, specificity and repeatability were evaluated.Results:The sensitivity of real-time PCR for detection of CAP10gene was6.8×101copies. The specificity of methods was high, and no positive results were found among Neisseria meningitidis, Candida albicans, Candida tropicalis, Aspergillus flavus, Aspergillus niger and Escherichia coli. With the detection of same plasmid, the CV of inter-assay and intra-assay were0.65%and2.45%. It showed that an excellent reproducibility. Conclusions:Real-time PCR for detection of CAP10gene of Cryptococcus neoformans was established successfully with excellent reproducibility, sensitivity and specificity. It may not only apply to early diagnosis of Cryptococcus neoformans, but also evalutate the prognosis and therapeutic effect.Part4Development and preliminary application of loop-mediated isothermal amplification method on Helicobacter Pylori detectionObjective:A loop-mediated isothermal amplification (LAMP) method was developed for detecting Helicobacter pylori rapidly and simply to provide an important basis for exploring diagnosis methods of clinical common bacteria and fungi infections.Methods:Helicobacter pylori16S rRNA gene sequence was obtained from GenBank database. Four specific LAMP primers(two inner primers and two outer primers) were designed by online software Primer Explorer4.0. PCR primers were designed by Primer Premier5.0software. After optimization of reaction system, LAMP and PCR methods were established. The sensitivity and specificity for detection of Helicobacter pylori were evaluated. Then the sensitivity was evaluated by detecting simulated feces samples.Results:The LAMP method could be finished within2h, and positive results were green and had a ladder-like pattern on the gel. The sensitivity of detecting Helicobacter pylori genomic DNA was12pg, and was10times as much as PCR. The detection limit of feces simulated samples was102CFU/ml. The results of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Enterococcus facealis were negative using two methods. Conclusions:LAMP assay which is high sensitivity and specificity, simple to operate and no need special equipments, is a promising testing method for Hp detection.