Establishment and Optimization of RT-LAMP Assay System for Tobacco Tomato Spotted Wilt Virus
To rapidly detect tobacco tomato spotted wilt virus(TSWV),5 sets of primers were designed based on the conserved sequence of TSWV nucleocapsid protein(NP)for screening,and parameters such as reaction temperature and time,volumetric molar concentrations of dNTPs,Mg2+,betaine,and internal and external primer ratios in the reaction system were optimized using the univariate method to establish the tobacco TSWV reverse transcription loop-mediated isothermal amplification(RT-LAMP),the specificity,sensitivity,and practicality of the optimized RT-LAMP were verified by parallel comparison tests using the revers transcription-polymerase chain reaction(RT-PCR)assay.The results showed that the best primer set for the tobacco TSWV RT-LAMP assay was TS-N-4,and the optimal addition of each component in the 25 µL reaction system were 2.5 µL of buffer,0.5 µL of MgSO4(100 mmol·L-1),0.5 µL of dNTPs(10 mmol·L-1),1.5 µL of FIP/BIP(10 mmol·L-1),0.5 µL of F3/B3(10 mmol·L-1),1.5 µL LF/LB(10 mmol·L-1),6 µL of Betaine(5 mmol·L-1),0.5 µL of Bst 2.0 WarmStar DNA Polymerase(8 000 U·mL-1),0.125 µL of M-MLV enzyme(10 000 U·mL-1),0.125 µL of RNA(≥64.7 fg),and added DEPC H2O to 25 µL.The optimum reaction temperature and time were 58℃and 60 min,respectively.The optimized RT-LAMP was 1 000 times more sensitive than RT-PCR,and the results of field samples were consistent with RT-PCR.The RT-LAMP method established in this study was specific,sensitive,and simple to operate,which was important for the detection and monitoring of TSWV in tobacco.
国家科技期刊平台
NSTL
万方数据
