| This paper mainly includes establishment and optimization of RT-PCR and multiplex RT-PCR detection technique for grapevine viruses, RT-PCR and multiplex RT-PCR detection technique for Korla pear viruses, cDNA probe detection technique for Korla pear viruses, and in situ RT-PCR detection technique Korla pear viruses. 1.Using grapevine leaves and cortexes as materials, three methods for total RNA extraction were compared and the fittest method was screened out. PCR amplification conditions were optimized based on the effectual RT-PCR detection system of Grapevine leafroll virus Ⅲand Grapevine fanleaf virus. Concentration of primary ingredients in RT reaction and PCR reaction were optimized. Multiple RT-PCR detection system of GLRaV Ⅲand GFLV was established base on the optimized RT-PCR detection of two virus respectively. The concentrations of multiple PCR reaction ingredients, i.e. dNTPs, Taq E, primer, Mg2+ , were optimized. Studies indicated that the multiple PCR system was economical and timesaving. 2.Using Korla pear leaves and cortexes as materials, seven methods for total RNA extraction were compared and the fittest method was screened out. Because pear tissues are abundant in amylose,hydroxybenzene, we improved these methods in order to obtain high quality products. Based on the effectual RT-PCR detection system of two viruses, the concentrations of RT reaction ingredients, including of dNTPs,primer,AMV,template and RNasin, were optimized, and the concentrations of RCR reaction ingredients, including of dNTPs,primer,Mg2+;Taq E and cDNA, were optimized too. Results indicated that concentrations of dNTPs,primer,AMV,template are not lower than 0.1mmol·L-1,0.2μmol·L(-1),0.05U·μL(-1),0.01μg·μL(-1) in RT system respectively. RNasin restrained the action of RNase,which ensured the process of RT go on wheels. Denaturalization of dsRNA should be 95℃3-5 min. RT time is no less than 40 min. Concentrations of dNTPs and primer should be 0.2～0.3mmol·L-1 and0.3～0.7μmol·L-1 in PCR system, respectively. The proper concentration Mg2+ is more than 1.26mmol·L-1. 3.A NADH dehydrogenase subunit 5 (nad5) 181bp fragment cloned from mitochondrial DNA of apple was as internal control used in detection of three pear viruses by multiplex RT-PCR assays. The recovered special fragments were cloned, sequenced and identified. 4.Using Korla pear leaves and cortexes as materials, we extracted total RNA using two methods. The fittest method for Korla pear was screened out. Biotin labeled cDNA Probe of ASPV and ACLSV was yielded with RT-PCR. The main factors that affected on sensitivity of hybridization were studied, and the results indicated that the highest sensitivity was obtained at probe concentration 400ng/ml, 45%formamide and 42℃for 6 hours. The best result of hybridization was obtained on import nitrocellulose membrane. Blocking with Tween20 was better than blocking with Albumin Bovine. The result of comparing of detection of the total RNA of different tissues and extracted methods indicated that all of the total RNA of fresh leaf, old leaf, cortexes and freezing leaf extracted with two methods showed sign of hybridization. 5.Korla pear leaf with ACLSV was used to establish detection method of fruit virus using in situ PCR. Parameters, such as concentrations of dNTPs,RNasin and AMV reverse transcriptase, complement primer for cDNA, annealing temperature,concentration of Taq DNA polymerase,Mg2+ and primer in PCR, and cycle number were studied systematically. Result showed that: the strength of signal started appearing and increase when RNasin amount increased and its concentration reached 0.2U·μl-1, certain amount cDNA would be generated when concentration of dNTPs in reaction solution reached 1.0mol·L-1,reverse transcription can be carried out when the concentration of AMV was over 0.3 U·μl-1,the quantity of outcome grew with the concentration of AMV raising in the range from 0.3U·μl-1 to 0.5U·μl-1,the effective reverse transcription can be carried out only when the concentration of complement primer reached 0.9μmol·L-1,and the amount of cDNA would increase following to its increase.Fifty-six centigrade was a suitable annealing temperature for in situ...
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