Identification of Anopheles dirus isomorphic species by chemiluminescent DNA probes
Issued Date
2024
Copyright Date
1992
Resource Type
Language
eng
File Type
application/pdf
No. of Pages/File Size
ix, 131 leaves : ill.
Access Rights
open access
Rights
ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (M.Sc. (Biochemistry))--Mahidol University, 1992
Suggested Citation
Mongkon Audtho Identification of Anopheles dirus isomorphic species by chemiluminescent DNA probes. Thesis (M.Sc. (Biochemistry))--Mahidol University, 1992. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/99930
Title
Identification of Anopheles dirus isomorphic species by chemiluminescent DNA probes
Alternative Title(s)
การจำแนกยุงที่มีสัณฐานวิทยาเหมือนกันชนิด Anopheles dirus โดยใช้ตัวติดตามดีเอ็นเอที่ติดฉลากด้วยสารที่ให้ปฎิกิริยาเรืองแสง
Author(s)
Advisor(s)
Abstract
Anopheles dirus is a major vector of malaria in Thailand and Southeast Asia. It was shown by cytogenetic and morphological studies to be a complex of at least six isomorphic species, provisionally designated species A, B, C, D, E and F on the Southeast Asian mainland. The five species found in Thailand (A-D, and F) exhibit distinct geographic distributions, seasonal variation in relative abundance and different nocturnal biting cycles. These biological characteristics of this group of mosquitoes may have implication for understanding the epidemiology of malaria in Southeast Asia. Identification of wild-caught female depends on rearing families from them and examination of larval chromosomes which is complicated, slow and laborious. Another identification method is using isozyme analysis which requires fresh or frozen stored specimens that is impractical for epidemiological studies. Species specific fragments, pMU-A40.1#5, pMU-B5, pMU-C19.2 and pMU-D9 which had been constructed since 1986, could be used as DNA probes for identification of species A, B, C and D respectively. But the disadvantages of the commonly used radio-isotopic DNA detection technique leads to its impractical use in the field conditions. Chemiluminescent DNA detection technique is another option for identification of vector sibling species by nonradioactive means. In this study, DNA probes were amplified by PCR, and then directly labeled with hoseradish peroxidase (HRP). The signal generating technique was done by adding detection reagent, luminol, enhancer and H2O2 which are substrates for HRP. However, the normally squash-blot technique could not be used for this detection system because substances in mosquito tissue generated strong chemiluminescent signal with detection reagent The signal from tissue could be eliminated by a phenolchloroform extraction step before hybridization process. In addition to direct labeling with HRP, the mosquito probes were labeled with fluorescein using both Taq DNA polymerase in PCR and terminal deoxynucleotidyl transferase. The fluorescein moieties were then detected by incubation with anti-fluorescein antibody-HRP conjugates Both fluorescein labeling techniques have sensitivity comparable to direct labeling techniques; 1-5 ng purified DNA of An. dirus could be detected after an exposure time of 30 minutes. Field-caught specimens were collected from 4 localities in Thailand and identified. The results of identification of 182 mosquitoes were corresponding to cytogenetic and isozyme analysis studies. Moreover, 75 mosquitoes which were not identified by non DNA methods, were clearly detected by this chemiluminescent technique operation, closed wound with drain, operated in rainy and summer season had the risks of wound infection 10.15, 5.06, 4.01, 3.07, 2.77, 2.16, 2.04, 1.88, 1.88, 3.85 and 0.67 more than surgical patients with clean wound , operated at most one time , serum albumin more than 3.5 mg/dl , female, operated by instructors, operated during the day time , operated less than 2 hours , no emergency in operation , closed wound with no drain and operated in winter respectively . The multiple logistic regression analysis, under 95% confidence, depicted that the factors associated with surgical wound infection were type of wound, serum albumin, closed wound with drain, season, number of operations in this admission, duration of operation, sex and surgeon. Surgical patients with clean-contaminated wound, serum albumin less than 3.5 mg/dl , closed wound with drain, operated in rainy and summer season, operated more than once, operated more than 2 hours, male and operated by extern / resident had the risks of wound infection 12.71 , 3.62 , 2.68 , 5.44. and 1.15 , 4. 30 , 3.06 , 2.18 and 1. 89 more than those patients with clean wound , serum albumin more than 3.5 mg/dl , closed wound with no drain, operated in winter, operated at most once, operated less than 2 hours , female and operated by instructors respectively. 107. 5 out of 63 transformants were characterized as recombinant clones which were designated as MEX-Taq 3, 18, 25, 30 and 48. The Taq Pol I product of clone MEX-Taq 30 provided the highest enzyme activity which was 16960 U/L of cell cultures. Analysis of the total intracellular proteins of E. coli (MEX-Taq 30) in SDS-PAGE showed an intense protein band of 94 kDa. This 94 kDa Taq Pol I protein represents about 3.4% of the total solubilized protein content in the E. col i cells. Taq Pol I was purified from E. coli using a simple purification scheme in two steps. First, since Taq Pol I protein was thermostable, any other E. coli proteins was inactivated and denatured by heating at 75C for 20 min and then was removed by centrifugation. Second, the E. coli nucleic acids were removed using ion-exchange chromatography, DEAE-sephacel. The Taq Pol I protein was eluted from DEAE-sephacel with 450 mM KCl TMK buffer. This partial purified Taq Pol I had polymeras activity of 16700 U/ ll cultures. From the characterization of Taq Pol I, the enzyme exhibited the optimum activity at the temperature in the range of 70-75C, pH in the range of 8.5-9, concentration of monovalent cations either KCl or NaCl at 50 mM while at 160 mM showed the inhibitory effects. The absolute requirement for Mg++ as the divalent cation cofactor at the optimum concentration was 2 mM whereas Mn++ displayed the inhibitory effect or ton lesser extent at I mM. The Taq Pol I enzyme lacks of exonuclease or endonuclease More than 10% glycerol in the total PCR reaction decreased the activity of Taq Pol I. Without adding stabilizers, Taq Pol I could be stored at room temperature (25-30C) for a week with the loss of approximately a half of activity.
Description
Biochemistry (Mahidol University 1992)
Degree Name
Master of Science
Degree Level
Master's degree
Degree Department
Faculty of Science
Degree Discipline
Biochemistry
Degree Grantor(s)
Mahidol University