恆溫環形核酸增幅法(LAMP)能快速增幅所需基因片段且具有較高靈敏度。在本研究中，我們利用恆溫環形核酸增幅檢測法用於檢測及鑑定可能引起幼蟲移行症之蛔蟲屬寄生蟲，包括犬蛔蟲及浣熊蛔蟲，並根據寄生蟲核糖體DNA中ITS-2的特異性遺傳標誌設計引子進行檢測。LAMP法在65度穩溫箱中反應60分鐘。增幅後的產物以電泳方式進行鑑定。LAMP法鑑定蛔蟲屬之寄生蟲具有高度特異性，與非標靶基因片段則無陽性反應。DNA的增幅產物檢出時間最快可達到30分鐘。以特定引子利用LAMP法檢測犬蛔蟲、貓蛔蟲及浣熊蛔蟲僅需10 fg、100 pg及1 fg的量即可有效檢出。傳統的PCR檢測在偵測犬蛔蟲及浣熊蛔蟲具有較低的靈敏度，分別需要 10 ng及1 pg的DNA，但是針對貓蛔蟲之靈敏度約為LAMP assay的10倍。本研究結果顯示，在動物或人體組織中所採集的蛔蟲屬幼蟲之種別鑑定方式中，相較於型態學的鑑定，分子鑑定技術提供更為快速且容易。該方法亦可運用於鑑定土壤或糞材中所含有的蛔蟲卵，將可有效防止人類因接觸污染源而遭受感染。Loop-mediated isothermal amplification (LAMP) is a novel technique which rapidly amplifies target DNA with high specificity and efficiency under isothermal conditions. In the present study, we developed a LAMP method for the detection and identification of the larva migrans causing ascaridoid nematodes, Toxocara canis, T. cati and Baylisascaris procyonis, using primer sets designed from species-specific genetic markers in the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA (rDNA) of the nematode species. LAMP reactions were carried out isothermally at 65C for 60 min in a simple incubator. Positive amplification products analyzed in agarose gel electrophoresis were also detected by the addition of SYBR Green I. The LAMP assay for each nematode species was specific and produced negative results when tested against non-target DNA. DNA amplifications were detected as early as 30 min. The LAMP primer sets for T. canis, T. cati and B. procyonis were capable of detecting down to 10 fg, 100 pg and 1 fg of genomic DNA, respectively. Conventional PCR on the other hand, had a comparatively lower sensitivity in detecting T. canis and B. procyonis DNA down to 10 ng and1 pg, respectively, except for T. cati which is 10-fold more sensitive compared to the LAMP assay. These results showed that the molecular diagnostic technique developed in this study would be potentially valuable in the rapid yet simple approach of differentiating and identifying ascaridoid larvae in tissues of humans and animals when morphological methods are difficult or inadequate to attain diagnosis. This would also be useful in the detection of ascaridoid eggs in soil and in feces of dogs and cats in the environment which could serve as source of infection for humans.TABLE OF CONTENTS
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ACKNOWLEDGMENTS …………………………………………………….. i
CHINESE ABSTRACT …….………………………………………………… ii
ENGLISH ABSTRACT ………………………………………………………. iii
TABLE OF CONTENTS …………………………………………………….. iv
LIST OF TABLES ……………………………………………………………. vi
LIST OF FIGURES ……………………………………………………………. vii
LIST OF APPENDICES ………………………………………………………. ix
INTRODUCTION ……………………………………………………………… 1
LITERATURE REVIEW ……………………………………………………… 3
1. Larva Migrans …………………………………………………………… 3
1.1. Categories of Larva Migrans …………………………………………….. 3
1.2. Helminths Causing Larva Migrans Syndromes ………………………….. 3
2. Toxocara spp. in Dogs and Cats …………………………………………. 4
2.1. Taxonomy and Morphology ……………………………………………... 4
2.2. Life Cycle ……………………………………………………………….. 5
2.3. Epidemiology ……………………………………………………………. 7
2.4. Clinical Disease in Animals ...……………………………………………. 8
2.5. Clinical Disease in Humans ……………………………………………… 8
2.6. Diagnosis ………………………………………………………………… 10
2.7. Treatment ………………………………………………………………… 11
3. Baylisascaris in Raccoons …………………………………………………. 12
3.1. Taxonomy and Morphology ……………………………………………... 12
3.2. Life Cycle ……………………………………………………………….. 13
3.3. Epidemiology ……………………………………………………………. 14
3.4. Clinical Disease in Animals ……………………………………………… 15
3.5. Clinical Disease in Humans ……………………………………………… 15
3.6. Diagnosis ………………………………………………………………… 16
3.7. Treatment ………………………………………………………………… 17
4. Molecular Diagnosis of Parasitic Diseases ……………………………… 18
5. Loop-mediated Isothermal Amplification (LAMP) Method ………….. 19
5.1. Primer Design …………………………………………………………… 20
5.2. Principle of LAMP Method …………………………………………….. 20
5.3. Using Loop Primers ……………………………………………………... 21
5.4. Principle of Visual Detection ……………………………………………. 22
MATERIALS AND METHODS ……………………………………………… 23
1. Parasites …………………………………………………………………… 23
2. Isolation of Genomic DNA ………………………………………………... 23
3. Oligonucleotide Primers …………………………………………………… 23
4. LAMP Reaction …………………………………………………………… 24
5. Detection of LAMP Products ……………………………………………... 24
6. Sensitivity and Specificity of LAMP ……………………………………... 24
7. PCR and Nested PCR …………………………………………………….. 25
RESULTS ……………………………………………………………………... 26
1. Specificities of the LAMP Assays ………………………………………… 26
2. Sensitivities of the LAMP Assays ………………………………………… 27
3. Earliest Time of Amplification …………………………………………… 28
DISCUSSION …………………………………………………………………. 29
CONCLUSION …………………………………………………………………. 34
REFERENCES …………………………………………………………………. 35
TABLES ………………………………………………………………………… 52
FIGURES ……………………………………………………………………….. 53
APPENDICES ………………………………………………………………….. 8
