| Oligonucleotides and their derivatives have extensive applications in many fields, such as polymerase chain reaction (PCR), biological DNA chips, gene diagnoses, analysis of molecular structure and treatment of illness. With the rapid development of biotechnology, the demand of Oligonucleotides is increasing. Therefore the products of oligonucleotides have more widespread applications and larger market share.Cucumber green mottle mosaic virus (CGMMV) is an important quarantine plant virus in our country. CGMMV, a member of the Tobamovirus genus, mainly infects the cucurbit species, resulting in the decrease of crop yield or no harvest. Formerly, the virus didn't existed in China. In recent years, it was detected from cucurbit plants imported to China which led to the death of thousand acres of watermelon and great economic losses for exporting agricultural products. It is significant to establish a rapid, accurate, and sensitive detective technique for prevention of CGMMV from invading and spreading in our country. In this thesis, the synthesis and purification methods of primer and Taqman probes were established and the methods of detecting the CGMMV by RT-PCR and RTF-PCR were set up. The details are as follows:A pair of specific primers had been synthesized by solid phase synthesis method on DNA synthesizer and been purified by OPC, PAGE and RP-HPLC method. The synthesis and purification method were established that we reported here. For the first time, the RT-PCR detection of CGMMV from pumpkin fruit was reported in China. The primers were used for the RT-PCR amplification of infected pumpkin fruit. The expected positive amplified fragments were obtained, sequenced and analyzed. The results showed that fragment contains the coat protein gene and its 3' noncoding region and is 660nts in size. Comparing with the sequences of other isolators of CGMMV which had been published, the result showed that nucleotides homology was 98-99%.The above data showed first time in China that CGMMV exists in pumpkin fruit, sequences of the primers synthesized and purified in our lab were correct, and they worked efficiently for PCR amplification.FAM/TAMRA, FAM/BHQ2 Taqman probes and a pair of specific primers were synthesized automatically and purified by RP-HPLC, and they were used for real-time-flurorecent PCR (RTF-PCR) detection of CGMMV. RTF-PCR detection of CGMMV in pumpkin fruit is first reported in the world. It is demonstrated that the sensitivity of RTF-PCR with the FAM/BHQ2 probe was higher than that of FAM/TAMRA probe due to its low fluorescence background. Up to date, the RTF-PCR is the fastest, the most sensitive and accurate molecular detection method. The qualities of primers and probes have important influences to the sensitivity and accuracy of detection of the method. The results showed that the synthesis and purification system of primers and probes was high efficient and laid the foundation of the high performance of RTF-PCR. |
