Study Of Real-time Quantitative Pcr On The Early Diagnosis Of Deep-seated Candida Infections

Study of Real-time Quantitative PCR on the Early Diagnosis of Deep-seated Candida InfectionsWith the wide use of antibiotics, corticosteroids and immunosuppressive agents, the morbidity and mortality of deep fungal infection were increasing year after year. Moreover, clinical datas and experiences showed curative effect and prognosis of deep fungal infection largely depend on early diagnosis and treatment. Particularly, the patients infected by Candida albicans accounted for 60% of the total inpatients that suffered from deep fungal diseases, of which pathogenicity or colonization state was associated with procreation. The positive rate of non-Candida increased as well as drug resistance.Therefore, we needed not only an early, rapid diagnosis way on deep fungal diseases, but also identified pathogens to select sensitive drugs for treatment as soon as possible.Currently, clinical diagnostic technology of fungal infection was faced with enormous difficulties and challenges. Some traditional methods still were applied. The process may be time-consuming, easily contaminated and hardly quantitative. These disadvantages greatly restricted the early diagnosis and treatment, even delayed the best chances of treatment.Given the current problem, the subject lay on the basis of other studies. The basic principle was Fluorescence inlay legitimate of Real-time quantitative PCR . The specific primer of Candida albicans was designed and the standard strains were applied to construct recombinant plasmids.The reaction conditions of Real-time quantitative PCR were constantly optimized.The standard curve of Candida albicans was constructed, which revealed the association between circle threshold (Ct) and log (Copy number).It was used as a standard in detection of other samples.Furthermore, in order to choose sensitive, reliable drugs, five kinds of Candida needed to be identified rapidly. We tried to build five different kinds of recombinant plasmid by means of fungal universal primers. The specific melting temperature was respectively obtained by Real-time quantitative PCR method. The melting temperature was related to the sequence of products and the temperature was constant for one kind of amplification products. Therefore, according to the different melting temperatures, five kinds of Candida could be quickly identified. PARTⅠReal-timequantitative PCR in detection of Candida albicansObjective To investigate a rapid and reliable new method by Real-time quantitative PCR in detection of Candida albicansMethods Firstly ,18SrRNA, 5.8SrRNA, 28SrRNA and ITSⅠ, ITSⅡgene sequences of different strains were searched in Genbank of NCBI. Secondly, specific gene sequences of Candida albicans were obtained following BLAST comparison.it made certain that this paragraph was highly conserved. According to the principle of primer design and Primer 5.0, the specific primers of Candida albicans were designed. The gene fragment was about 270bp. Candida albicans strains were carried out by PCR. After 1% agarose gel electrophoresis, Gel was cut under violet lamp and retrieved by Gel Kit. The recovered fragment was connected with pMD18-T vector so as to construct the recombinant plasmid of Candida albicans, the plasmid was transformed into DH5 cells. DH5 cells was cultured on LB solid medium with ammonia benzyl resistance overnight, recombinant plasmid clones was extracted. 10μl Calbicans recombinant plasmid was sent to the Mobil Corporation in Shang hai for sequencing. the rest were preserved at -20℃for Real-time quantitative PCR.After recombinant plasmids of Candida albicans were well purified, their molecular weights were calculated under UV Spectrophotometer according to the length of the base of the recombinant plasmid. The copy numbers were calculated on the basis of molecular weight. It was diluted into 5×107-5×103 concentration gradient. At the optimum reaction conditions, it was added into three reaction tube simultaneously. The computer automatically drew the standard curve after the reactions finished.Results After recombinant plasmid of Candida albicans were diluted, standard curve and amplification curves were obtained under the optimal reaction conditions.Moreover, the equation of circle number (Ct) and Copy number log (Copy number) was drew. The expression was y =–3.385x +42.80. The lowest 10 copy genes [equivalent to (1-5) CFU / ml] could be detected. Additionally, it was non-cross-positive with other fungi, bacteria and viruses etc. Conclusion The method of Real-time quantitative PCR was a quantitative way in detection of Candida albican, which showed high sensitivity and specificity. PARTⅡRapid identification of five kinds of common Candida Species by Real-time quantitative PCRObjective To expose a rapid method by real-time quantitative PCR in identification of five kinds of common CandidaMethods Candida albicans, Candida glabratawas, Candida parapsilosis, Candida krusei, Candida tropicalis, were separately carried out by PCR by means of fungal universal primer ITS4 and ITS86. Their amplified gene fragments separately were 300bp, 360bp, 260bp, 248bp, 214bp. After 1% agarose gel electrophoresis, Gel was cut under violet lamp and retrieved by Gel Kit. The recovered fragment was separately connected with pMD18-T ector so as to construct the five different kinds of recombinant plasmids, which was separately transformed into DH5 cells. After DH5 cells were cultured on LB solid medium with ammonia benzyl resistance overnight, recombinant plasmid clones was extracted. Five different kinds of recombinant plasmid were sent to the Mobil Corporation in Shanghai for sequencing. The rest of five different kinds of recombinant plasmid were preserved at -20℃for Real-time quantitative PCR.After recombinant plasmids of five different kinds of Candida were well purified, their molecular weights were calculated under UV Spectrophotometer. According to the length of the base of the recombinant plasmid, the copy numbers were calculated on the basis of molecular weight. They were diluted into 5×107-5×103 concentration gradient. At the optimum reaction conditions, they were added into three reaction tube simultaneously.The computer automatically drew melting temperature and melting temperature curves after the reactions finished.Results After five different kinds of recombinant plasmid were diluted, under the optimum reaction conditions, melting temperature and melting temperature curves were obtained by Real-time quantitative PCR. Moreover, the melting temperature is related to the sequence of products of PCR. It is fixed for one kind of amplification products. Therefore, the specificity was determined by the melting temperature and melting curve. Therefore, five different kinds of Candida have fixed melting temperature. Therefore, according to the different melting temperatures, five kinds of Candida could be quickly identified.Conclusion The method of Real-time quantitative PCR was a rapid, easy, reliable way in identification of five different kinds of Candida.