Development And Application Of Molecular Detection Kit In Korla Aroma Pear's Main Viruses In Xinjiang

Virus disease is one of the main diseases in fruit tree. With the enlargement of the fruit tree area and the extension of the cultivated time, the harm of viruses is more and more obvious and attach important to scientist and technician. Viruses free seedling production is main measure to prevent and cure virus disease. Viruses free production of fruit crops demand a virus detection method mat is accutate, sensitive, brief and rapid. The traditional woody indicator is reliable, but it requires a long detection period. ELISA tests have a difficult antiserum preparation and the price is high. For the demand of fruit tree seedling quarantine and viruses free production, it is necessary to find a nicety, sensitive, brief and rapid detection method. Molecule detection technique provides a new effective tool for fruit crops virus disease detection lately. Korla aroma pear in Xinjiang is liked by a most customer because of its special flavor quality, and is one of the most export productions. Recently, Korla pear and grapevine in Xinjiang are found to have viruses at large, and it gives a enormous hidden trouble to the development of Korla pear in Xinjiang. So development of Korla aroma pear viruses' molecular detection in Xinjiang has a very important theoretical and practical meaning.The main content of this paper includes establishment and optimization of the main viruses RT-PCR detection method, cDNA probe detection method in Korla pear, assembly kit of RT-PCR, NASH and kits used to detection of samples.1. This paper acquired the total RNA extraction method suitable for different part of pear. Experiment results shows the RNA can be used to reverse transcription polymerase chain reation (RT-PCR) and Nucleic spot hybridization (NASH) in year-round.2. Two pairs specific primer was designed according to their genomic sequences of ASPV (apple stem pitting virus) and ACLSV (Apple chlorotic leafspot virus) in Genbank., their specific fragments of the conservative extent (683bp,361bp) are amplified. These fragments are used in RT-PCR, NASH detection. Sequenced, and alignment results show their nucleotides comparability difference in 83.7% and 79.5%.3. RT-PCR detection method of Korla pear viruses was optimized. Concentrations of dNTPs, primer, AMV and RNasin are studied in RT. When RNasin is 1.00 U/ μL, primer is 0.500umol/L,dNTPs is 1.0mmol/L,AMV is 1U / μL,and concentration of dNTPs, primer, Mg2+ and Taq E were also studied in PCR. MgC12 is 1.5 mmol/L, dNTPs is 0.20mmoVL, primer is 1.0μmol/L, Taq E is 0.1 U / μL, the result is best.4. With two kinds of plasmid, biotin labeled cDNA probes was synthesized by PCR method. The main factors of hybridization sensitivity were assayed, the results indicate that under the 400ng/ml probe concentration and 45% formamide in 42℃ for 6 hours conditions the detection result is highest sensitive. In hybridization, import nitrocellulose membrance is superior to nylon. Blocking affection of Tween20 is better than Albumin Bovine. Aimed at the disadvantages about long process and complex operation in routine NASH, NASH program was obviously improved. The detection period was cut down to 5 hours from 22 hours, and detection efficiency was enhanced.5. RT-PCR detection kit and NASH kit are assembled according to systems of RT-PCR and biotin labeled cDNA probe hybridization optimization. Detection of sample results shows these kits are efficient, rapid, accurate and credible.