Molecular Detection Of Main Korla Pear Viruses In Xinjiang

Most of the main fruit tree cultivars take viruses in our country. Along with the continual enlargement of the fruit trees cultivated area and the extension of their cultivated time, the harm of viruses is more and more obvious and attach importance by science and technology personnel. Viruses free seedling production is main method to prevent and cure virus disease. Viruses free production of fruit crops demand a virus detection method which is nicety, sensitive, brief and rapid. The traditional woody indicators detection method is credibility, but it requires long detection period. ELISA test has a difficulty antiserum preparation and costliness. 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 techniques provide a new effectively tool for fruit crops viruses detection recently, and it improves the fruit crops viruses' detection to a new level. Korla pear in Xinjiang is liked by most of customers because of its special flavor quality, and is one of mostly export productions. Recently, Korla pear in Xinjiang is found take viruses at large, and this gives an enormous hidden trouble to development Korla pear in Xinjiang. So, molecular detection of Korla pear viruses in Xinjiang has very important theory and practice meaning.This paper main includes establishment and optimization of RT-PCR and multiplex RT-PCR detection methods, cDNA probe detection methods and in situ RT-PCR detection methods in Korla pear main viruses.1. This paper acquires the total RNA, double-stranded RNA (dsRNA) and total nucleic acids extraction methods suitable for leaves and 1-2-year-old branches which can be taken all year round. Experiment results show this total RNA can bebetter used into reverse transcription polymerase chain reaction (RT-PCR) and this total nucleic acid can be better used into nucleic acid spot hybridization (NASH) detection kit and RT-PCR detection kit all year round.2. Three pair viruses specific primers of Apple chlorotic leaf spot (Trichovirus} virus (ACLSV), Apple stem grooving (Capillovirus) virus (ASGV) and Apple stem pitting (Foveavims) virus (ASPV) are designed according as their genomic sequences D14996, AB004063 and D21828 in Genbank. Using these primers pair, three viruses' specific fragments of the expected sizes (683bp, 273bp and 361bp) are amplified. These special fragments are used in RT-PCR, NASH and in situ RT-PCR (ISRT-PCR) detection. These three fragments are cloned into E. coli. XL1 blue and sequenced, and alignment results show their nucleotides comparability difference in 83.7%, 92.3% and 79.5%. The same annealing temperature in these primer pairs makes the multiplex RT-PCR assays to do better.3. RT-PCR detection methods of Korla pear viruses including concentrations of RT reaction ingredients dNTPs, primer, AMV and RNasin are optimized, and concentrations of RCR reaction ingredients dNTPs, primer, Mg2+ and Taq DNA polymerase are optimized too. Influences of denaturing temperature and reverse transcription time on PCR are studied. The results indicate that the concentration of dNTPs, primer and AMV is not lower than 0.175mmol/L, 0.35mol/L, 0.5U/uLin reverse transcription system, and RNasin control the action of RNase and insure the process of reverse transcription go on wheels. Denaturing temperature and time of dsRNA should different be 95 in 3-5 minutes. RT time is no less than 40 minutes; Concentration of dNTPs and primer should be 0.2~0.3mmol/L, 0.2~1.0mol/L in PCR system, respectively. The proper concentration of Mg2+ is more than 1.2mmol/L.4. A NADH dehydrogenase subunit 5 (nad5) 181 bp fragment is cloned which is a part of the mitochondrial DNA from apple, and this nad5 gene containing the exons a and b, separated by an 848 bp intron. The sense primer (nad5-s) of the internal control is designed in this manner, that the last four nucleotides at the 3'-end are homologous to the first four nucleotides of exon b and the comple...