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研究生:何佳慧
研究生(外文):Joan Dee Parel
論文名稱:嚙齒動物蟯蟲分子生物鑑別法之研究
論文名稱(外文):Molecular Studies on the Differentiation of Rodent Pinworms
指導教授:黃鴻堅
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:39
中文關鍵詞:分子生物嚙齒動物蟯蟲
外文關鍵詞:molecularRodent pinworms
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實驗動物常見之鼠蟯蟲-Syphacia obvelata及S. muris 在形態學上十分相似,為能順利鑑別兩者,我們設計具有種別特異性的引子組來增幅核糖體DNA(rDNA)之ITS-1,5.8S及ITS-2基因組,再將PCR產物 進行定序。實驗結果顯示可由rDNA的定序結果鑑別出S. obvelata, S. muris及 Aspiculuris tetraptera。本研究是首次發表前述三種鼠蟯蟲 rDNA上ITS-1,5.8S及ITS-2基因組。使用限制酶AluI或是RsaI將能更明確鑑別前述三種蟯蟲。我們也設計對三種蟯蟲基因組之ITS-2具有專一性的引子組。本研究之檢驗方法可在同時加入異源DNA檢體時檢出特定蟯蟲,其他蟯蟲則無PCR產物出現。實驗所得的PCR產物進定時也證實為同一種蟯蟲。ITS-2的特定引子組可成為囓齒動物如大鼠、小鼠及野生鼠類等蟯蟲PCR-RFLP分析的有效檢測工具。此外,定序囓 齒動物蟯蟲的5S spacer可發現,不同種別之蟯蟲其5Sspacer 的長度及內容各有不同,亦可作為種別鑑定的另一項依據。
To differentiate the morphologically similar pinworms of the common laboratory rodents, such as Syphacia obvelata and S. muris, we amplified and sequenced the region spanning the internal transcribed spacer 1 (ITS-1), 5.8S gene, and ITS-2 of the ribosomal DNA followed by designing of species-specific primers for future use in the identification of the worms. It was observed that Syphacia obvelata, S. muris and Aspiculuris tetraptera can be differentiated from each other based on their rDNA sequences. This is the first report of the ITS-1, 5.8S, and ITS-2 of the rDNA of the three aforementioned rodent pinworm species. The use of restriction endonucleases, AluI or RsaI, further allowed the delineation of the three species. We also constructed species-specific primers that were designed to unique regions of the ITS-2 of the three species. This approach allowed their specific identification with no amplicons being amplified from heterogenous DNA samples, and sequencing confirmed the identity of the sequences amplified. Thus, the use of these ITS-2 specific primers along with PCR-RFLP can serve as useful tools for the identification of pinworms in rats, mice, and wild rodents. Aside from the ITS-2, the 5S rRNA intergenic spacer could also be utilized for pinworm identification since its length and composition varies between species. Furthermore, the finding of the putative 22 nucleotide- spliced leader motif and the Sm binding site sequences within the 5S spacer of Syphacia muris and S. obvelata provide evidence of the existence of the SL1 RNA gene in this spacer region.
ACKNOWLEDGEMENTS ………………………………………………………… …..i
CHINESE ABSTRACT ……………………………………………………………….ii
ENGLISH ABSTRACT ……………………………………………………………….iii
TABLE OF CONTENTS ………………………………………………………………iv
LIST OF FIGURES …………………………………………………………………….vii
LIST OF TABLES …………………………………………………………………….viii
INTRODUCTION ...……………………………………………………………………1
LITERATURE REVIEW ……………………………………………………………….3
1. Rodent pinworms ………………………………………………………………….3
2. Morphology of rodent pinworms .…………………………………………………4
a. Syphacia spp ………………………………………………………………..4
b. Aspiculuris tetraptera ……………………………………………………...4
3. Life cycle of Syphacia spp. ………………………………………………………5
4. Life cycle of A. tetraptera ………………………………………………………….5
5. Prevalence and distribution of rodent pinworms …………………………………6
6. Host factors associated with pinworm infection in mice and rats …………………6
7. Immune response to pinworm infection ……………………………………………7
8. Pathogenecity and clinical signs …………………………………………………8
9. Effects of pinworms on research …………………………………………………8
10. Diagnosis of pinworm infection ………………………………………………….8
11. Molecular tools for parasite identification ………………………………………9
12. Restriction fragment length polymorphism (RFLP) ……………………………..10
13. Polymerase chain reaction (PCR) ………………………………………………..10
14. Conventional PCR assays for the specific identification of parasites and diagnosis of infections ………………………………………………………….11
15. PCR-linked RFLP ……………………………………………………………….11
16. Real- time PCR ………………………………………………………………….12
17. Nuclear ribosomal RNA genes ………………………………………………….13
18. Use of the nuclear rDNA region in the identification of parasites ………………14
19. 5S rRNA intergenic spacer ……………………………………………………..14
20. Use of 5S intergenic spacer region in the identification of parasites ……………14
21. Presence of the SL1 RNA in the 5S spacer of nematodes ………………………15
22. Spliced leaders, SL RNA and trans- splicing ……………………………………16
MATERIALS AND METHODS ……………………………………………………..19
A. Detection of pinworm eggs (Syphacia spp.) by cellophane tape test ……………19
B. Characterization of the nuclear ribosomal DNA (rDNA) sequences and development of PCR- based methods for identification of rodent pinworms …..19
1. Collection of parasites …………………………………………………………19
2. DNA extraction and amplification of nuclear ribosomal DNA by PCR ………19
3. Sequencing and alignment of NC5- NC2 PCR products ………………………20
4. Characterization of the ribosomal DNA of pinworms …………………………21
5. Nuclear ribosomal DNA PCR-RFLP ………………………………………….21
6. Species-specific primer design ……………………………………………….21
7. Multiplex PCR ………………………………………………………………..22
C. Characterization of the 5S rRNA intergenic spacer of rodent pinworms, presence of the SL1 RNA gene, and 5S spacer PCR- RFLP ……………………22
1. Collection of parasites …………………………………………………………22
2. DNA extraction and PCR of 5S rRNA spacer ……………………………….23
3. Sequencing and SL1 RNA identification …………………………………….23
4. 5S rRNA spacer PCR-RFLP ………………………………………………….24
RESULTS …………………………………………………………………………….25
A. Rodent pinworms collected from mice and rats …………………………………24
B. Characterization of the nuclear ribosomal DNA (rDNA) sequences of rodent pinworms and development of PCR- based methods for identification of rodent pinworms ………………………………………………………………….24
1. PCR of ribosomal DNA (rDNA) ………………………………………………25
2. Characterization of ITS-1, 5.8S and ITS-2 rDNA regions …………………….25
3. Alignment and pairwise p-distances of the rDNA sequences …………………26
4. rDNA PCR-RFLP ……………………………………………………………..26
5. Species- specific primers ……………………………………………………...27
6. Multiplex PCR ………………………………………………………………..27
C. Characterization of 5S rRNA intergenic spacer, presence of the SL1 RNA gene and 5S rRNA spacer PCR-RFLP ………………………………………………..28
1. 5S rRNA intergenic spacer PCR …………………………………………….28
2. Characterization and alignment of 5S rRNA spacer sequences ………………28
3. Presence and orientation of the SL1 RNA ……………………………………28
4. 5S rRNA spacer PCR-RFLP ………………………………………………….29
DISCUSSION …………………………………………………………………………30
CONCLUSION ……………………………………………………………………….33
REFERENCES ………………………………………………………………………..34
FIGURES ……………………………………………………………………………..41
TABLES ……………………………………………………………………………..60
APPENDICES ……………………………………………………………………….67
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