Establishment and application of the Telosma mosaic virus and Cucumber mosaic virus Duplex RT-PCR System in passionfruit

Abstract: 【Objective】Passion fruit is rich in nutrition with unique flavor and more than 130 kinds of aromatic substances, covering most of the aroma in fruits. Passionfruit is an important raw material for juice making. At present, there are a large number of cultivated purple (P. edulis) , yellow (P. edulis var.flavicarpa) and hybrid passionfruits in China. Passionfruit has been planted as a new fruit species in Fujian Province in recent years. However, severe passionfruit virus disease leads to tree decline as well as yield and quality decline, which seriously hinders the development of passionfruit industry. Cucumber mosaic virus, East Asian Passiflora virus, Passionfruit mottle virus, Papaya leaf curl Guangdong virus, Euphorbia leaf curl virus and Telosma mosaic virus have been found in China. Among them, Cucumber mosaic virus is the main infection virus. Therefore, it is of great significance to establish a rapid and efficient virus detection system for passionfruit.【Methods】From June to September 2018, 34 samples of passionfruit leaf were collected from different areas of Fujian Province. The samples were frozen in liquid nitrogen and stored in-80 ℃ refrigerator. According to the conserved region of TeMV, CMV coat protein gene in NCBI database, a pair of specific primers for the two viruses were designed using Primer5.0 software. Extraction of total RNA from passionfruit leaves was carried out using Trizol Kit (TRANS) according to the instruction manual. Synthesis of cDNA was done using reverse transcription Kit (TaKaRa) . With the cDNA from the reverse transcription as a template, TeMV and CMV specific primers were used for RT-PCR amplification. The amplified product was recovered and ligated to the pMD18-T vector, then transformed into Escherichia coli DH5α. The positive clones were collected and sequenced after PCR detection. Based on single RT-PCR technology, the concentration of primer combination, annealing temperature and cycle times of Duplex RT-PCR system were optimized.【Results】The results of single RT-PCR amplification showed that when annealing temperature was 57 ℃, the band specificity was strongest, and the specific band was brighter and clearer than other bands. Therefore, 57 ℃ was the best annealing temperature for TeMV and CMV. The nucleotide sequences of TeMV and CMV subjected to Blast alignment using NCBI database. The results showed that the amplified products were the partial sequences of TeMV and CMV of passionfruit. The optimization results of Duplex RT-PCR showed that the best primer combinations of TeMV and CMV were 2.0 mmol·L-1 and 8.0 mmol·L-1, respectively with the optimum annealing temperature of 57 ℃ and the optimum cycle number of 35. The sensitivity analysis results showed that when the total RNA solution was diluted to 10-5 the target bands amplified by the two viruses, 323 bp and 227 bp, could still be detected. Using healthy tissue culture seedlings as control group, 34 samples of passionfruit collected from different areas of Fujian Province were detected by using the established dual RT-PCR detection technique. Both TeMV and CMV could be detected at the same time.【Conclusion】In this study, a virus detection system for simultaneous detection of TeMV and CMV in passionfruit was established. When the optimized detection system is used to detect passionflower samples collected in the field, the detection effect is good. It provides technical support for rapid detection of passionflower virus diseases.