Reverse dot-blot hybridization method for identification of nine potyviruses
| Autor: | Tze-Jung Yeh, 葉慈容 |
|---|---|
| Rok vydání: | 2004 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 92 The genus Potyvirus in the family Potyviridae is the largest genus of plant viruses and can infect a wide range of crop plants. Because there are about 200 species in the genus, it is important to develop a rapid identification system for potyviruses to support the tasks of plant quarantine and inspection. Biochip has become an increasing popular tool of biotechnology industry at 21 century, because it can deal with hundreds to thousands information at the same time. The aim of this thesis is to develop a potyvirus identification chip based on the traits of potyviruses and biochip. Two kinds of probes, cDNA and oligonucleotide probes were prepared, and their sequences derived from the 3’end of the NIb gene and the 5’end of the CP gene. The DIG-labeled targets were prepared from viral cDNA clones or total RNA of infected tissues by means of PCR or RT-PCR with potyvirus degenerate primers. After cDNA probes immobilized onto the nylon membrane, the targets were tested by reverse dot-blot hybridization. The results indicated that our cDNA probes had high specificity to the targets. When using our cDNA chip to test the targets derived from plant total RNAs, it could successfully identify eight potyviruses including BaRMV, PRSV, PVA, PVY, TuMV, ZaMV and ZYMV. Moreover, the cDNA chip could also identify mix infection plants with two or three kinds of potyviruses. In order to define the optimal length of oligonucleotide probe, probes with different lengths of ZaMV and ZYMV sequence were designed, and the effectiveness of probes were compared. The results revealed that the 50-mer probe of specific virus sequence gave constant positive results and thus was used for further experimental design. The specificity of 50-mer oligonucleotide probes of potyvirus tested by reverse dot-blot hybridization was satisfying. When using our oligonucleotide chip to test the targets derived from plant total RNAs, it could successfully identify the aforementioned eight potyviruses and also mix-infection samples. Comparing the results of two kinds of potyvirus chips, cDNA probes showed better sensitivity than oligonucleotide probes, but oligonucleotide probes had the advantage of preparation without viral cDNA clones. If new viruses appear or the existing viruses produce quite a few mutations, we only need to add new oligonucleotide probes to maintain the completeness of the identification chip. Therefore, oligonucleotide chip is better choice than cDNA chip and will be a novel way of rapid identification for potyviruses in the future. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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