Establishment And Application Of Real-time Quantitative PCR And Isothermal Nucleic Acid Amplification For Detection Of Lotus Latent Virus

Lotus（Nelumbo nucifera Gaertn.）is a perennial aquatic herbaceous plant which belongs to Nymphaeaceae family,Nelumbo genus.Lotus is an important characteristic aquatic vegetable in China.It is popular with domestic and foreign consumers,because of its abundant nutrition.Some lotus roots in main producing areas of Jiangsu Province,are thin,small and stiff,with black-brown spots or streaks on the surface.This phenomenon is called "stiff lotus".Previous studies have shown that the "stiff lotus" is caused by virus infection.Lotus latent virus（tentative name）,Cucumber mosaic virus,Lotus streak virus,Dasheen mosaic virus and Apple stem grooving virus are the only reported pathogens in lotus.Preliminary studies of these viruses have found that LLV is the most prevalent and widely distributed.However,the stable detection technology of LLV has not yet been established.Therefore,the detection systems based on real-time quantification and isothermal nucleic acid amplification were established for LLV,and then the distribution of LLV in lotus root was explored by using this detection technology.The results are as follows.1.Three LLV detection systems,real-time quantitative PCR,LAMP and RPA,were established in this study.The sensitivity of RT-qPCR system is the highest,which is 100 times higher than that of RT-PCR.The minimum detection limit of this system is 7×10¹ copies ·μL^-1.It is suitable for the detection and identification of virus-free lotus root seedlings.LAMP system can be used for field diagnosis through directly observed by naked eyes.The sensitivity of LAMP detection was consistent with that of RT-PCR.RPA system has the shortest reaction time,thus,it can be used as an effective method for the detection of a large number of lotus root samples.The minimum detection limit of this system is 7×10³ copies·μL^-1.2.We used RT-qPCR system to detect LLV in different parts of "stiff lotus" samples,because of its high sensitivity.The results showed that the concentration of LLV in the first underground stem after the apical bud of "stiff lotus root" was the highest,and decreased gradually in the later underground stem.The inner part of the underground stem is divided into four parts,such as epidermis,basic tissue,vascular bundle and parenchymal cells.RT-qPCR was used to detect the concentration of LLV in these four parts.The results showed that the concentration of LLV in the basic tissue and vascular bundle was significantly higher than that in the epidermis and parenchymal cells.