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当前位置: 首页 > 文章 > 梨组织中苹果褪绿叶斑病毒的原位RT-PCR检测 园艺学报 2007,34 (1) 53-58
Position: Home > Articles > Detection of Apple chlorotic leaf spot virus in Pear by in situ RT-PCR Acta Horticulturae Sinica 2007,34 (1) 53-58

梨组织中苹果褪绿叶斑病毒的原位RT-PCR检测

作  者：
牛建新;周民生;马兵钢;赵英;刘宏
单  位：
石河子大学农学院园艺系
关键词：
梨;库尔勒香梨;苹果褪绿叶斑病毒;原位PCR
摘  要：
为了建立梨树苹果褪绿叶斑病毒原位RT-PCR检测技术,用已知带有苹果褪绿叶斑病毒的库尔勒香梨和无病毒实生苗叶片为材料,利用DIG标记,研究了香梨病毒的叶片石蜡切片IS-RT-PCR检测技术。包括AMV逆转录酶、dNTPs、RNasin及互补引物浓度对cDNA合成的影响,退火温度、TaqDNA聚合酶、Mg2+、引物浓度及循环次数对原位PCR效果的影响。结果表明:RNasin的用量大于0.2U.μL-1时,信号强度随着RNasin量的加大而增强;只有当dNTPs浓度达1.0mmol.L-1时才能生成一定量的cDNA;AMV浓度在0.3~0.5U.μL-1均可进行正常的逆转录,而且在该范围内产物的量随AMV浓度提高而增多;引物浓度达到0.9μmol.L-1以上时才能进行有效的逆转录,并且生成的cDNA的量随引物浓度增大而增加。原位扩增ACLSV的cDNA适合退火温度为56℃。循环20~30次可出现较强的蓝色信号,引物浓度在0.8~1.2μmol.L-1时显色较好;Taq酶浓度为20U.mL-1以上,均显示较深的蓝色;Mg2+浓度为1.5mmol.L-1就可满足原位PCR所需。获得了苹果褪绿叶斑病毒的原位PCR优化检测体系,利用建立的优化程序对香梨样品进行了检测验证,取得了很好效果。
译  名：
Detection of Apple chlorotic leaf spot virus in Pear by in situ RT-PCR
作  者：
NIU Jian-xin1,2*,ZHOU Min-sheng1,MA Bing-gang1,2,ZHAO Ying1,and LIU Hong1(1Department of Horticulture,Agricultural College of Shihezi University,Shihezi,Xinjiang 832003,China;2Key Laboratory of Oasis Ecology Agriculture of Xinjiang Bingtuan,Shihezi,Xinjiang 832003,China)
关键词：
Pear;Korla pear;Apple chlorotic leaf spot virus;In situ PCR
摘  要：
The detection technique of Apple chlorotic leaf spot virus(ACLSV)in pear plant by IS-RT-PCR with Digoxigenin labels for leaf paraffin slice samples and shoot tips 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 primers,effect of AMV reverse transcriptase and complement 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 amount when its concentration reached over 0.2 U·μL-1.Certain amount of cDNA would not be generated until the concentration of dNTPs reached 1.0 mmol·L-1 in reaction solution.Reverse transcription could be carried out when the concentration of AMV reverse transcriptase was between 0.3 U·μL-1 and 0.5 U·μL-1,and the quantity of cDNA production improved with the increase of the concentration of AMV.The reverse transcription could not be carried out until the concentration of antisense primer reached 0.9 μmol·L-1,and the amount of cDNA would increase along with the increase of primers concentration.The suitable annealing temperature for in situ amplification of cDNA was 56℃.The minimum amplification cycle number was 20 times.The stain signal was weak when primer concentration was less than 0.8 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 when Mg2+ concentration was at 1.5 mmol·L-1.An optimized 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.

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