摘要
为建立一种可降低PCR假阴性的槟榔隐症病毒(arecapalmvelarivirus 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0.2 μmol/L,APV1引物终浓度为0.8 μmol/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.
Abstract
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 the-oretical basis for reducing the false negative of areca palm velarivirus 1 PCR.
基金项目
中国热带农业科学院基本科研业务费专项(1630052019016)
NETL
NSTL
维普
万方数据