Preparation And Experimental Application Of DNA Probe Specific For Pneumocystis Carinii

As a basic technology of molecular biology, nucleic acid molecule hybridization has been widely used for basic and clinical medicine research. Being highly sensitive, specific and easy to operate, it has been widely used for detection of many pathogens.In this research, the rat model of Pneumocystis carinii pneumonia(PCP) was established by injecting and oral administration of acetate hydroprednisone to acquire necessary materials for experiments. After preparation of non-radioactive digoxingenin labeled probe by random priming, dot slot hybridization was used to detect Pneumocystis carinii(Pc) DNA from the lungs of rats model and clinical specimens of patients.1 .Establishment of the rat model with PCP, detection of PC and collection of samplesThe rat model with PCP was successfully induced by injecting acetate hydroprednisone subcutaneously to the groin of SD rat for two months.The lungs were taken out from the dead rats aseptically, and made imprints with the cross-section of ones.The imprints were stained by Giemsa's staining and improved Gomori's methenamine silver nitrate staining(GMS), and detected for PC microscopically.The cysts andtrophozoits of PC were isolated from the lungs to extract DNA for thetemplate of PCR. The PCR product was identified by agarose gelelectrophoresis and a about 350bp DNA band occurring in the ultravioletlight was thought to be positive.Results: The results of pathogen examination showed that the rat modelwith PCP was established successfully. The positive rate of the PCinfection found by Giemsa's staining, improved GMS and PCR was80%, 50 % and 95% respectively.The last was higher than the other both.The establishment of the rat model and the acquirement of the lungs laidbasis for the subsequent experiments.2.The acquirement of PC specific target DNA and sequencingRegular PCR was used to amplify the large subunit mitochondrial ribosomal RNA(LSU mt rRNA) gene of PC with the primers of PAZ102-E and PAZ102-H designed by Wakefield and his colleagues.The agarose gel comprising about 350bp DNA band was cut and recollected for the target DNA. After the purified DNA fragment being sequenced, software BLAST was used to align the acquired DNA sequence and those known sequences in the GeneBank.Results: The sequenced DNA fragment was 357bp long, and it shared 99% homology with the PC LSU mt rRNA gene announced by Sinclair and his colleagues. This result confirmed that the purified DNA fragment was specific for PC. The acquirement of the target DNA fragment was important and necessary for the preparation and detection of the probe, which was about to guarantee the specifity of the probe. 3.The preparation and experimental application of PC specific DNA probeDigoxingenin labeled non-radioactive probe was prepared by random priming, using the acquired targe DNA fragment specific for PC as the template. The density of the probe was confirmed through comparing with the density-known digoxingenin labeled control DNA, and thespecifity and sensitivity of the probe were identified by dot slot hybridization. Then the probe was used in dot slot hybridization to detect PC DNA from the lungs of rats model and clinical specimens of patients. Results: The density of the probe was 3 Ong/ul, and the lowest detectable amount of the probe was 2pg. The probe only reacted with the PC DNA, but did not react with the DNAs from Plasmodium berghei, P. falciparum, Toxoplasma gondii, human leukocytes and normal host lungs. So it showed that the probe was specific for PC. The dot slot hybridization could successfully detect PC DNA from 73.7% of the lungs of rats model, but not from the specimen (bronchoalveolar lavage, BAL ) of the kidney transplantation recipient with PCP. PCR combined with dot slot hybridization could detect PC from 94.7% of these samples, and PC DNA was detectable from the BAL of the patient.