Studies On IS-RT-PCR Detection Technology Of ACLSV In Korla Pear's Stem Tip

In situ reverse transcipte polymerase chain reaction(In situ RT-PCR) is a rapid and sensitive method for molecule biology and cytology,which was a combination of PCR and in situ hybridization,could examin low copy nucleic acids in tissue,yet could still identify the celluar origin and location of signals。Doing the study has great theoretical and pratical significances,as it would not only offer a kind of new channel for the virus detection of fruit tree,but also would give help in studies of virus'generation,stribution and virus transfer channel。In the experiment, using Kuala pear stem with pear ring pattern mosaic virus and as material, using digoxigenin -duTP as labele to cDNA。And studies on detection of viral disease in kuala pear using in situ PCR and situ hybridization.The paper includes major contents as follow,1.Studied systemically effect of making section manner on quality of in situ PCR.The result shown:it is suitable for using leaves as materials to do In situ PCR that the tissue is fixed in 4% PFA over 1h,dehydrated by 50%,70%,85%,95%,100% ethanol 1h respectively,transparented 1h by xylene,immersed paraffine twice each 2h,embed tissue in parafin cut sections at 7-8μm,and strech sections on ethanol-water.2.Using ACLSV's special primer paies,Dig labelled cDNA probes are synthesized.3.One-step ISRT-PCR assay is carried out by using Thermus thermophilus DNA polymerase.4. Have determined ACLSV some distribution in stem。In experiment many negative controls were made。The result shows:there are not blue-purple signals in negative control tissue,however,some cell expressions of the mesophyll of positive material tissues make obvious blue purple colour ,which explain ACLSV may distribute in peripheral vascular and cerebral cortex。Moreover,alkaline phosphatase system's reliablity is studied。The tissues on slide don't display blue-purple if the mix uninclude digoxigennin labled nucleotide only when the section is amplified,but be done other same treat as the positive。This demonstrate the result can't be affected by endogenous alkaline phosphatase,and the assay has strong speciality。...