枣瘿蚊图尔病毒(DjTV-2a)PCR检测方法的建立

Establishment of PCR Method for Detection of Dasineura jujubifolia Toursvirus 2 (DjTV-2a)

为了建立枣瘿蚊图尔病毒(DjTV-2a)的PCR检测方法,通过提取枣瘿蚊总基因组DNA和设计DiTV-2a特异性引物,以DNA模板(80,100,120 ng),引物浓度(0.7,0.8,0.9μmol·L^(-1)),dNTP(0.2,0.3,0.4 mmol·L^(-1)),Taq酶(1.25,1.35,1.45 U)为变量,采用正交设计筛选最佳PCR反应体系,建立与优化DjTV-2a的PCR检测方法。结果表明:正交设计的9组PCR反应体系均能够扩增和检测该病毒,且DNA模板100 ng,引物0.7μmol·L^(-1),dNTPs 0.4 mmol·L^(-1)和Taq酶1.35U反应体系扩增效果最佳;对最佳PCR反应体系进行退火温度(46,48,50,52,54,56℃)优化,发现52℃为引物最佳退火温度。综上分析,在50μL反应体系下,引物退火温度为52℃,模板DNA量100 ng,引物0.7μmol·L^(-1),dNTPs 0.4 mmol·L^(-1),Taq酶1.35 U是DiTV-2a的最佳PCR扩增体系,可用于该病毒的检测。

To establish a PCR detection method for Dasineura jujubifolia toursvirus 2(DjTV-2a),the experiment extracted the total genomic DNA of Dasineura jujubifolia,designed DiTV-2a specific primers,the DNA template(80,100,120 ng)and the primer concentration(0.7,0.8,0.9μmol·L-1),dNTP(0.2,0.3,0.4 mmol·L-1)and Taq enzyme(1.25,1.35,1.45 U)were as variables,screening the optimum PCR reaction system to establish and optimize the PCR detection method of DjTV-2a by the orthogonal design.The results showed that all the nine group of PCR reaction systems from the orthogonal design could amplify and detect the virus,but the reaction system of 100 ng DNA template,0.7μmol·L-1 primers,0.4 mmol·L-1 dNTPs and 1.35U Taq enzyme had the best amplification effect.For the best PCR reaction system,the optimum annealing temperature of the primers were 52℃in the six treatment including 46,48,50,52,54,56℃.In summary,in a 50μL reaction system,52℃primer annealing temperature,100 ng DNA template,0.7μmol·L-1 primer,0.4 mmol·L-1 dNTPs and 1.35 U Taq enzyme is the optimum PCR amplification system for DiTV-2a which can be used for the detection of the virus.