Prapon WilairatSakol PanyimPiengchan Rajkulchai2024-06-272024-06-27199219922024Thesis (M.Sc. (Biochemistry))--Mahidol University, 1992https://repository.li.mahidol.ac.th/handle/20.500.14594/99085Biochemistry (Mahidol University 1992)Approximately 45X of malaria cases in Thailand are caused by Plasmodium vivax. Unlike P.falciparum, in vitro culture of P.vivax has not been successful. Previous molecular cloning of P.vivax specific DNA probe had failed due to human DNA contamination In this study, DNA isolated from P.vivax-enriched patients' blood- was used to construct a genomic library. DNA was digested with Sau3A and ligated to Bluescribe M13+ vector at BamH I site. Approximately 4,000 recombinant clones were screened for repetitive DNA using P.vivax DNA as the probe. From 23 clones containing repetitive DNA, 7 clones did not hybridize with human DNA. When these DNA clones were further hybridized with P.falciparum DNA followed by P.vivax DNA, 3 clones gave positive signal with P.vivax DNA probe. One of these clones. Designated B2 contained 2 vectors and 2 inserted DNA. The 1.1 Kb inserted DNA fragment of B2 could detect P.vivax DNA to the level of 6.25 ng and did not hybridize to human, P.falciparum, P.chabaudi, P.berghei, P.cynomolgi and mosquito DNA. This P.vivax specific fragment was subcloned into Bluescribe M13+ vector an named pMU-PV2. The nucleotide sequence of pMU-PV2 was determined It contained 1,161 bp with 47.5% G+C content, with several short internal repeating sequences, runs of 5-7 consecutive A and T and n significant peptide and splice site sequences. Five primer pairs for amplification of P. vivax infected blood were designed based upon the above P.vivax specific sequence The amplification condition was optimized for sensitivity of the detection. One of these primer pairs which allowed amplification c 183 bp sequence of P.vivax DNA exhibited P.vivax specificity Moreover, the sensitivity of 183 bp detection in P.vivax-infected blood was at 10-6% parasitemia, which is equivalent to 1 parasite in 20 ul blood. For the field validity, 233 field samples were tested and evaluated. The specificity and sensitivity of PCR method comparing to microscopic examination were 70.8 and 75.6%, respectively, wit 25.3% disagreement.xv, 124 leaves : ill.application/pdfengผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้าDNA probesPlasmodium vivaxPolymerase Chain ReactionMalaria, Vivax -- etiologyMaster ThesisMahidol University