一种可降低PCR假阴性的槟榔潜隐病毒检测方法的建立  

Establishment of a Detection Method for Areca Palm Velarivirus 1 that can Reduce the False Negative of PCR

作　　者：李梦雨 尹慧祥[2] 羊彬彬 汪志伟[1] 王健华[2] Li Mengyu;Yin Huixiang;Yang Binbin;Wang Zhiwei;Wang Jianhua(Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province,School of Horticulture,Hainan University,Haikou,570228;Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Science,Haikou,571101)

机构地区：[1]海南大学园艺学院,海南省热带园艺作物品质调控重点实验室,海口570228 [2]中国热带农业科学院热带生物技术研究所,海口571101

出　　处：《分子植物育种》2024年第1期108-116,共9页Molecular Plant Breeding

基　　金：中国热带农业科学院基本科研业务费专项(1630052019016)资助。

摘　　要：为建立一种可降低PCR假阴性的槟榔隐症病毒(areca palm velarivirus 1,APV1)检测方法,参考已报告的槟榔引物保守基因,设计槟榔内标准物引物和APV1引物,槟榔内标准物引物合成3对,APV1引物合成2对,进行PCR扩增筛选槟榔内标准物引物和APV1引物。运用双重PCR扩增方法,优化反应参数。内标准物引物和APV1引物的最适引物浓度比为1:4,内标准物引物终浓度为0.2μmol/L,APV1引物终浓度为0.8μmol/L,最适退火温度为54℃,最适循环次数为30次。灵敏度结果表明,槟榔cDNA浓度稀释到10^(-6)时,仍能扩增出目的条带。特异性结果显示,只有槟榔能检测到内标准物和APV1扩增出的目的条带。利用建立的双重PCR检测方法,对海南省万宁市的40份槟榔样品进行了检测,发现有25份样品感染APV1。本研究结果可为降低槟榔潜隐病毒PCR假阴性提供理论依据。To establish a detection method for areca palm velarivirus 1(APV1) that can reduce the false negative of PCR.Referring to the reported conserved genes of Areca catechu primers,the Areca catechu internal standard primers and APV1 primers were designed,three pairs of Areca catechu internal standard primers and two pairs of APV1 primers were synthesized,and PCR amplification was performed to screen Areca catechu internal standard primers and APV1 primers.The duplex PCR amplification method was used to optimize the reaction parameters.The optimal primer concentration ratio of internal standard primer and APV1 primer is 1:4,the final concentration of internal standard primer is 0.2 μmol/L,the final concentration of APV1 primer is 0.8 μmol/L,and the optimal annealing temperature is 54 ℃,the optimum number of cycles is 30 times.The sensitivity results showed that the target band could still be amplified when the concentration of Areca catechu cDNA was diluted to 10^(-6).The specific results showed that only Areca catechu could detect the target band amplified by the internal standard and APV1.Using the established duplex PCR detection method,40 samples of Areca catechu were tested from Wanning City,Hainan Province,and 25 samples were found to be infected with APV1.The results of this study can provide a theoretical basis for reducing the false negative of areca palm velarivirus 1 PCR.

关 键 词：槟榔 PCR假阴性 内标准物 槟榔隐症病毒 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治]