DNA sequence analysis of a specific trypanosoma evansi DNA probe and probe and its use for a highly sensitive detection of T. evansi in blood
8
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2024
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1993
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eng
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application/pdf
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xv, 135 leaves : ill.
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open access
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ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
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Mahidol University
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Thesis (M.Sc. (Biochemistry))--Mahidol University, 1993
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Nipa Chokesajjawatee DNA sequence analysis of a specific trypanosoma evansi DNA probe and probe and its use for a highly sensitive detection of T. evansi in blood. Thesis (M.Sc. (Biochemistry))--Mahidol University, 1993. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/100787
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DNA sequence analysis of a specific trypanosoma evansi DNA probe and probe and its use for a highly sensitive detection of T. evansi in blood
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การวิเคราะห์หาลำดับเบสไนดีเอ็นเอ (DNA sequence) ของ DNA probe สำหรับ Trypanosoma evansri และการนำไปใช้ในการตรวจเชื้อ T.evansi ในเลือดให้มีความไวสูง
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Trypanosoma evansi is a haemoflagellated parasitic protozoa which is mechanically transmitted by blood sucking insects, especially by Tabanid flies. It causes a syndrome refered to as "surra", an emaciating and sometimes fatal disease, targeting a wide range of livestock. Since T.evansi poses an important problem for the animal health and production development program, especially in the endemic areas inside and outside Thailand,.a practical method for a highly sensitive and reliable detection is needed. To this end, a method for direct detection of the parasites DNA by polymerase chain reaction (PCR) was developed. A previously developed T.evansi genomic DNA probe, pMUTec6, was subcloned into Bluescribe M13-. The clone pMUTec6.258 was selected for its signal intensity after hybridization with the T.evansi genome and for its size, suitable for PCR amplification. pMUTec6.258 showed an overall 50% GC content in its 258 bp sequence. From this sequence, a set of primers for PCR was designed allowing amplification of a 227 bp fragment from the original sequence. The amplification conditions were optimized for high sensitivity down to the detection of a single parasite in 10 ul blood. In an experimentally infected cow, the parasites were detected as early as 2 days post-infection and the signal totally disappeared 12 hr after successful treatment of the cow. The 227 bp amplification was specific for the detection of trypanosomes of the brucei group, T.brucei and T.equiperdum. No amplification product was seen from host blood in bovine, swine, equine, ovine, and caprine species, or from the following other blood parasites: Anaplasma sp., Theileria sp., Babesia bovis, and Babesia bigemina DNA. For easier manipulation during the sample-collections, the blood processing method was simplified by boiling the crude blood for at least 5 min prior the amplification. Either clotted- or EDTA anticoagulated blood, both from tube-collected and slide-collected could be used as starting materials for the developed PCR method. Seventy-one field specimens were tested for evaluation, the sensitivity and specificity of the PCR detection when compared with the mouse inoculation method was 100% and 88% respectively.
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Biochemistry (Mahidol University 1993)
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Master of Science
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Master's degree
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Faculty of Science
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Biochemistry
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Mahidol University