Establishment and Application of a Multiplex RT-PCR Detection Method for Virus in Passion Fruit
[Objective]Passion fruit is a tropical and subtropical fruit with significant eco-nomic value.However,the viral diseases due to its asexual reproduction pose a serious threat to the development of passion fruit industry.This study aimed to enhance field monitoring of passion fruit virus disease by establishing a multiplex RT-PCR assay capable of detecting telosma mosaic virus(TeMV),East Asian passiflora virus(EAPV),and cucumber mosaic virus(CMV).[Methods]Three pairs of primers were designed based on conserved motifs within the CP gene sequences of TeMV,EAPV,and CMV available on GenBank.The primers concentration,number of cycles and an-nealing temperature in the multiplex RT-PCR reaction system were optimized.this study further veri-fied the detection sensitivity of multiplex RT-PCR assay.This optimized RT-PCR system was ap-plied to detect viruses in 40 passionfruit leaf samples collected from fields in Guangxi.[Results]After optimization and screening experiments,a 20 µL PCR reaction system was established for suc-cessful detection of TeMV,EAPV,and CMV.The optimal reaction conditions included primer vol-umes of 2 µL TeMV,2 µL EAPV and 3 µL CMV;35 cycles with annealing temperature 54.9℃.Veri-fication showed the detection sensitivity could be up to 104 copies/µL.The amplified product sizes were 496 bp,279 bp,and 157 bp,respectively,for TeMV,EAPV,and CMV.The virus detection re-sults for the field samples of passionfruit leaves showed that TeMV,EAPV and CMV were 97.5%(39/40),95%(38/40)and 2.5%(1/40),respectively.The results detection with single RT-PCR were 100%(40/40),95%(38/40)and 2.5%(1/40),respectively.The coincidence between the results of multiple RT-PCR and single RT-PCR was 99.16%(119/120).The detection results showed that TeMV and EAPV tended to be combined in infection,and the combined infection rate was as high as 95%(38/40).[Conclusion]An optimized multiplex RT-PCR assay system was established in this study,and it can be used to detect TeMV,EAPV and CMV rapidly,sensitively and reliably at the same time,which provides the foundation for field control,quarantine planning and diagnosis of these three passion fruit viruses.
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