Establishment and application of reverse transcription loop-mediated isothermal amplification for the detection of three viruses infecting Rosa chinensis
[Objective]Chinese rose is an important ornamental flower in China.Prunus necrotic ringspot virus(PNRSV),ap-ple stem grooving virus(ASGV)and rose cryptic virus 1(RCV1)are important viruses infecting Chinese rose.When these three viruses co-infect roses alongside other viruses,they severely impact the growth and development of the plants.The pur-pose of this study was to establish a sensitive and convenient RT-LAMP method for detecting these three viruses in Chinese rose.[Methods]Specific primers targeting the viral CP gene sequence were designed to establish an RT-LAMP detection meth-od for PNRSV,ASGV,and RCV1 infections.The reaction system's Mg2+concentration,dNTP concentration,reaction tem-perature,and reaction time were optimized.Sensitivity and specificity were evaluated to ensure the reliability of the method.[Results]The optimized RT-LAMP method successfully detected the target viruses at a constant temperature of 61℃.The op-timal concentrations were 5.0 mmol/L for Mg2+and 1.2 mmol/L for dNTPs.This method exhibited high sensitivity,detect-ing RCV1 at a minimum concentration of 3.19×105 copies/µL,PNRSV at 6.44×104 copies/µL,and ASGV at 4.04×104 copies/µL.These sensitivities were 100 to 1 000 times higher than those achieved by RT-PCR.Furthermore,the detection system exhibited excellent specificity,with no cross-reactions observed with other rose-infecting viruses.The RT-LAMP method was applied to nineteen field samples,enabling accurate viruses identification through visual color changes without the need for agarose gel electrophoresis or specialized instruments.[Conclusion]The RT-LAMP detection method developed in this study is a rapid,convenient,and highly specific tool for diagnosing and monitoring PNRSV,ASGV,and RCV1 infections in roses.Its visualization-based approach,allowing direct dye addition during testing,is particularly suitable for field applica-tions and rapid diagnostics.
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维普
