Detection Of The Viral Pathogens Of Fujian Narcissus Disease And Establishment Of Treble RT-PCR System

Narcissus virus disease brings serious harm to the yield and quality of narcissus,causing huge losses,and widely distributed in the world of narcissus cultivation area.According to the reports,There are about 30 species of virus can be naturally infected narcissus,the reported 20 virus isolation mainly distributed in Nepovirus,Potyvirus and Carlavirus.The main infected virus were Narcissus yellow stripe virus and Narcissus mosaic virus,the symptoms were leaves and yellow flowers,respectively.At present,the main method to prevent the narcissus virus disease is cultivation of virus-free plantlets,and the accurate virus detection technology is the key to obtain excellent virus-free plants.In this study,through field survey,we found that the type of symptoms in the narcissus of Fujian Zhangzhou and Pingtan were various,the main symptoms were leaf mottle,color yellow,chlorotic stripe spot etc..Severe symptoms of narcissus plants,showed leaves massive lesions,perforation or cracks,flower color clutter and breaking.In the electron microscope,we can observe linear virus particles,the length was about 550-850nm.Combined with the biological method,specific primers designed by narcissus virus were used in RT-PCR detection,the detection result of the Zhangzhou and Pingtan narcissus virus mainly include NMV,NYSV,NLSYV,NDV and NLV.To achieve the purpose of accurate identification of narcissus diseases,require sensitive and fast assays as guarantee.In this study,we based the conservative gene sequences of NMV,NDV and NYSV,designed 3 pairs of virus specific primers.After extraction of total RNA,used the total RNA as template,using the 3 pairs of primers in the amplification of RT-PCR,respectively,then,obtained 3 fragments of DNA.We got the clip connected,transformed,then cloned and sequenced,the results of sequencing showed that the nucleotide homology reached more than 98%,indicated that the obtained DNA fragment were gene fragments of virus.Through single RT-PCR,the detection technology of conventional RT-PCR of NMV,NDV,NYSV were set up.Based on single RT-PCR detection,we explored and optimized the treble RT-PCR mothod of detection NDV,NYSV and NMV simultaneously.To the reaction system,we optimized the concentration of Mg2+,template cDNA and Tag polymerase;To the reaction conditions,the annealing temperature and cycle times were optimized.We got bands of 751bp,623bp and 483bp successfully according with experimental design.The reliability of the results has been verified by sequence homology analysis.Thus,we successfully established a treble RT-PCR system of detection NYSV,NDV and NMV simultaneously.To prove the pathogen species of narcissus virus disease in Fujian Zhangzhou and Pingtan,in this study,we used RT-PCR detection method to detect 35 narcissus samples from Fujian Zhangzhou and Pingtan,which showed typical narcissus virus disease symptoms.There were 5 kinds of virus were detected,they are NLSYV,NLV,NYSV,NDV,NMV.The highest detection rate for Zhangzhou narcissus sample was NDV,followed by NMV;To Pingtan samples,the two highest detection rate virus were NYSV and NMV,respectively.Among them,the highest detection rate is NDV,reached 77.14%;The detection rate of NMV and NYSV were 65.71%and 22.86%,respectively.The total prevalence of NLSYV was 45.70%.The lowest detection rate was NLV,only 2.86%.The infection rate reached as high as 77.14%,we can see that multiple virus mixed infection was a common phenomenon.From the "Zhangzhou Narcissus" and "Pingtan Narcissus" samples,we cloned the CP gene of NYSV 6;Nia-Pro gene of NDV Y9 and CP gene of NMV X1.The fragments of cloned NYSV,NDV and NMV were detected and analyzed,the results were as follows:? The size of NYSV6 CP gene fragments was 75 1bp.Compared with the reported sequence of GenBank of Zhangzhou NYSV-zhangzhou(EU430294.1)CP gene,there were 4 different bases,the similarity was 99%.? The size of NDV Y9 Nia-Pro gene fragments was 623bp.Compared with the reported sequence of GenBank of Marijiniup 2(JQ395041.1)complete genome,there were 8 different bases,the similarity was 99%.? The size of NMV X1 gene fragments was 483bp.Compared with the reported sequence of GenBank of New Zealand NMV strain(AY225449.1)complete genome,there were 10 different bases,the similarity was 98%.The system can realize the synchronous detection of field samples of NMV,NDV and NYSV,saved the required reagent and detection time greatly,reduced the test cost,improved the detection efficiency.