Detection And Molecular Identification Of Arabis Mosaic Nepo Virus From The Infected Lily Hybridus

The synonyms of Arabis mosaic virus (ArMV) are Raspberry yellow dwarf virus, Rhabarber mosaic virus or Rhubarb mosaic virus, which is one of Nepovirus family that belongs to Comoviridae. ArMV is China's second-class quarantine plant viruse. Lily has been reported to be a new host of the virus which spread fast in recent years. Therefore, establishment of a detection method which is high sensitivity, specificity, speediness, safety and reliability is essential for entry-exit quarantine inspection and control of ArMV.Molecular biology methods were applied to clone and sequence the coat protein gene fragment of ArMV from Lily which is exported from abroad or cultured in Kunming, Yunna, in this study. The enzyme linked immunosorbent assay (ELISA), RT-PCR, nested PCR and fluorescent real-time quantitative RT-PCR (TaqMan RT-PCR) were established for the detection of ArMV at the same time. The sensitivity, specificity, safety and the cost of these four detection methods were compared and the main results are summarized as follows:The survey on different species of ArMV of lily indicated that Lily does not show symptoms after infected by the virus.The distribution of the virus in different parts of Lily was investigated by using DAS-ELISA method.The results showed that the highest concentration of the virus was in the bulb,lower leaves and middle leaves, the second was in the top leaves, and the lower concentration of the virus was in the shoot and petals. Therefore, spring is the best time to detect the virus. Bulb, lower and middle leaves are the best materials for the detection. In addition, DAS-ELISA detection method are high specifical, high sensitive, low cost,simple, and rapid, and has extensive application value.A 993bp specific band and a 276bp specific band were amplified via routine RT-PCR and nested RT-PCR, respectively, using specific primers. The routine RT-PCR and nested RT-PCR methods were showed to be stable, efficient and rapid to detect the existence of ArMV.The specific and sensitive test showed that the routine RT-PCR and nested RT-PCR coulde detect the presence of the virus in positive sample with 1.83×104 and 0.91×103 copies of ArMV RNA, respectively, after it was diluted by 10 series, indicating that the nested RT-PCR method has higher sensitivity and specificity than routine RT-PCR.According to the highly conservative sequence in ArMV genome, the specific primers and TaqMan probe was designed and synthesized. A real-time fluorescence quantitative PCR method was developed by optimizing the reaction conditions. The tests for the specificity, sensitivity and repeatability of this method showed real-time fluorescent TaqMan RT-PCR could detect positive samples with 102 copies of the virus RNA. The cycle number had a very good linear relationship with the copy number of the virus RNA in the standard curve and the correlation coefficient was 0.993. Comparison with conventional RT-PCR and nested RT-PCR, this method has higher specificity, sensitivity, speediness and a better repeatability. It could detect a large number of samples simultaneously. Moreover, real-time fluorescent TaqMan RT-PCR as a good method was able to accurately detect trace amount of ArMV virus.Taken together, the present study had successfully cloned the CP gene fragment of ArMV and established serological ELISA, conventional RT-PCR, nested RT-PCR and real-time fluorescence quantitative RT-PCR methods for detection of the virus. These detection systems has an important practical significance for the prevention and control of ArMV and provide a solid technical support for plant quarantine in China.