| Cucumber mosaic virus(CMV),lily mottle virus(LMoV)and lily symptomless virus(LSV)are the three major viruses that seriously harm lily at present.When lilies were infected by CMV and LMoV separately,they showed mosaic and petal mottle,and LSV was generally asymptomatic.However,lilies are often infected by LSV,LMoV and CMV,simultaneously.And it can cause seriously damage to the growth,yield and quality of the lilies,resulting in serious economic losses.Thus,it has become an urgent need to develop a method for detection of lilies viruses in lily industry.Published methods for detecting LSV,LMoV and CMV in infected lily include virus purification,enzyme-linked immunosorbent assay(ELISA),dot-blot hybridization,reverse transcription-polymerase chain reaction(RT-PCR)and immunocapture RT-PCR(IC-RT-PCR).However,these assays are relatively time-consuming,labour-intensive,and dependent on specialized equipment.Furthermore,ELISA can have limited detection sensitivity with very low virus concentration and PCR-based detection methods require skilled technicians.Therefore,it is very necessary to establish a simple,rapid and visual method for detection of LSV,LMoV and CMV.This study is designed to establish LSV,LMoV and CMV LAMP detection methods and develop the live visualization LAMP rapid qualitative detection kit for LSV,LMoV and CMV,optimize the reaction system and the parameters,and research the preparation process.The main contents and results are as follows:1.The establishment of the lily symptomless virus(LSV)LAMP visual rapid detection method and the developmentof the kitThe objective of this study was to develope effective diagnostic technique and diagnostic kit for lily symptomless virus(LSV),to establish a rapid,sensitive and specific detection of LSV.Loop-mediated isothermal amplification(LAMP)method,it provided an accurate and reliable tool for the diagnosis of LSV.According to the LSV CP sequence published in GenBank,LAMP primers were designed in its conserved sequence using Primer Explorer V4,by optimizing conditions such as the proportion of primer,the annealing temperature,the reaction time,the components of the PCR,etc,the LAMP detection method for LSV virus-specific amplification was established,the LAMP assay kit was developed,and the sensitivity,specificity,reproducibility.And shelf life properties of the kit were evaluated.The specific amplification of high efficiency for plasmid and cDNA of LSV was obtained at58.5 ?,59 min.The diluted plasmid samples were tested by this test kit,the detection limit for LSV by the LAMP detection was 10 copies/?L,whereas the detection limit by conventional PCR was 1000 copies/?L,the results indicated that the LAMP method was highly sensitive.The detection did not cross-react with other lily viruses.The stability test showed that this kit can survive at least 40 times post-repeated freezed and thawed.The results indicated that the establishment of this method and the test kit are specific,sensitive and stable.Moreover,reverse transcription LAMP(RT-LAMP)was applied to detect LSV RNA directly.And it is conducive to lily field and customs import testing.2.The establishment of the lily mottle virus(LMoV)LAMP visual rapid detection method and the development of the kitThe objective of this study was to develope effective diagnostic technique and diagnostic kit for lily mottle virus(LMoV),to establish a rapid,sensitive and specific detection of LMoV loop-mediated isothermal amplification(LAMP)method,it provide an accurate and reliable tool for thedetection of LMoV.According to the LMoV CP sequence published in GenBank,LAMP primers were designed in its conserved sequence using PrimerExplorer V4,by optimizing conditions such as the proportion of primer,the annealing temperature,the reaction time,the components of the PCR,etc,the LAMP detection method for LMoV virus-specific amplification was established,the LAMP assay kit was developed,and the sensitivity,specificity,reproducibility and shelf life properties of the kit were evaluated.The specific amplification of high efficiency for plasmid and cDNA of LMoV was obtained at58.5 ?,60 min.The diluted plasmid samples were tested by this test kit,the detection limit for LMoV by the LAMP detection was 10 copies/?L,whereas the detection limit by conventional PCR was 1000 copies/?L,the results indicated that the LAMP method was highly sensitive.The detection did not cross-react with other lily viruses.The stability test showed that this kit can survive at least 40 times post-repeated freezed and thawed.Moreover,reverse transcription LAMP(RT-LAMP)was applied to detect LMoV RNA directly.The results indicated that the establishment of this method and the test kit are specific,sensitive and stable and provide a simple,rapid and visual method for detection of LMoV.3.The establishment of the cucumber mosaic virus(CMV)LAMP visual rapid detection method and the development of the kitThe objective of this study was to develope effective diagnostic technique and diagnostic kit for cucumber mosaic virus(CMV),to establish a rapid,sensitive and specific detection of CMV loop-mediated isothermal amplification(LAMP)method,it provide an accurate and reliable tool for the detection of CMV.According to the CMV CP sequence published in GenBank,LAMP primers were designed in its conserved sequence using Primer Explorer V4,by optimizing conditions such as the proportion of primer,the annealing temperature,the reaction time,the components of the PCR,etc,the LAMP detection method for CMV virus-specific amplification was established,the LAMP assay kit was developed,and the sensitivity,specificity,reproducibility and shelf life properties of the kit were evaluated.The specific amplification of high efficiency for plasmid and cDNA of CMV was obtained at58.5 ?,60 min.The diluted plasmid samples were tested by this test kit,the detection limit for CMV by the LAMP detection was 10 copies/?L,whereas the detection limit by conventional PCR was 1000 copies/?L,the results indicated that the LAMP method was highly sensitive.The detection did not cross-react with other lily viruses.The stability test showed that this kit can survive at least 40 times post-repeated freezed and thawed.Moreover,reverse transcription LAMP(RT-LAMP)was applied to detect CMV RNA directly.The results indicated that the establishment of this method and the test kit are specific,sensitive and stable and provide a simple,rapid and visual method for detection of CMV. |
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