关键词: 梨, 库尔勒香梨, 苹果褪绿叶斑病毒, 原位PCR

Abstract: The detection technique of Apple chlorotic leaf spot virus (ACLSV) in pear plant by IS-RT-
PCR with Digoxigenin labels for leaf paraffin slice samp les and shoot tip s frost slice samples was established
in this paper. Korla pear leaves infected by ACLSV and virus-free seedling leaves of Pyrus betulaefolia Bge
as control were used as leaves materials in the tests. The IS-PCR parameters including concentration of
dNTPs, RNasin, Taq DNA polymerase, Mg²⁺ and p rimers, effect ofAMV reverse transcrip tase and comple
ment primer on cDNA synthesis, annealing temperature and cycle number were studied systematically. The
results showed that the stain signal strength increased accompanied with an increase of RNasin amountwhen
its concentration reached over 0.2 U·μL^-1. Certain amount of cDNA would not be generated until the con
centration of dNTPs reached 1.0 mmol·L ^-1 in reaction solution. Reverse transcrip tion could be carried out
when the concentration of AMV reverse transcrip tase was between 0.3 U·μL^-1 and 0.5 U·μL^-1, and the
quantity of cDNA p roduction imp roved with the increase of the concentration ofAMV. The reverse transcrip
tion could not be carried out until the concentration of antisense p rimer reached 0.9μmol·L ^-1 , and the a
mount of cDNA would increase alongwith the increase of primers concentration. The suitable annealing tem
perature for in situ amp lification of cDNA was 56℃. The minimum amp lification cycle number was 20 times. The stain signal was weak when primer concentration was less than 018 μmol·L - 1 and the signal
color was heavy blue as the concentration of Taq DNA polymerase was over 20 U·mL ^-1; In situ PCR could
be performed whenMg ²⁺ concentration was at 1.5 mmol·L^-1. An op timized detection system of ACLSV
by IS-RT-PCR has been established and used to detect virus in Korla pear orchard, and the stable detection
results have been obtained.

Key words: Pear, Korla pear, Apple chlorotic leaf spot virus, In situ PCR