RT-PCR Detection of Five Quarantine Plant RNA Viruses Belonging to Potyand Tospoviruses

Abstract

In order to detect quarantine plant viruses, we developed reverse transcription-polymerase chain reaction (RT-PCR) primer pairs for five single-stranded (ss) plant RNA viruses that are not currently reported in Korea but could be potential harmful plant viral pathogens. Three viruses such as Chilli veinal mottle virus (ChiVMV), Colombian datura virus (CDV), and Tobacco etch virus (TEV) belong to the genus Potyvirus while Chrysanthemum stem necrosis virus (CSNV) and Iris yellow spot virus (IYSV) are members of the genus Tospovirus. To design RT-PCR primers, we used reported gene sequences corresponding to the capsid protein and polyprotein for ChiVMV, CDV, and TEV while using nucleocapsid protein regions for CSNV and IYSV. At least two different primer pairs were designed for each virus. Fifteen out of 16 primer pairs were successfully applied in detection of individual quarantine virus with high specificity and efficiency. Taken together, this study provides a rapid and useful protocol for detection of five quarantine viruses.

Characterization of three full Iris yellow spot virus genes of a garlic-infecting isolate from Zimbabwe using next-generation sequencing technology

“…RT-PCR was performed using the RevertAid RT Reverse Transcription Kit (Thermo Fischer Scientific, USA) targeting the N gene. The resultant cDNA was used for PCR amplification using the KAPA2G Fast Hot Start ReadyMix Kit (KAPA Biosystems, USA) and N gene-specific primers (IYSV_2F: 5'-GGCGGTCCTCTCATCTTACTG-3' and IYSV-NCP2_R: 5'-GAAGTTCCAGGAGTGCATTTAGTC-3') (Lee et al, 2011) under the following cycling conditions: initial denaturation at 95°C for 2 min; 35 cycles of 94°C for 15 s (denaturation step), 57°C for 15 s (annealing step), and 72°C for 15 s (extension step), followed by a 5-min incubation period at 72°C. PCR products were analyzed by *Corresponding author.…”

Development and Optimization of a Reverse Transcription Hemi-Nested PCR Primer for the Detection of Potato Mop-Top Virus at Quarantine Inspection Sites in Korea

“…However, the disadvantages of ELISA [9,10] in quarantine viruses make it unreliable for the detection and accurate diagnosis of quarantine viruses [7,11]. To speed the diagnosis and shorten the inspection period, polymerase chain reaction (PCR) method has been widely used [12][13][14][15][16]. The use of PCR to diagnose PMTV has been reported [17][18][19].…”

Development of PCR Diagnostic System for Detection of the Seed-Transmitted Tobacco Ringspot Virus in Quarantine

“…However, its false positive reaction makes it difficult to conduct a precise diagnosis and quarantine examination, so methods with a higher detection capability are needed to detect a very tiny amount of viruses in a few infected seeds among a huge number of seeds [6,7]. In this study, the polymerase chain reaction (PCR) method, which had been found in a study to have a high degree of safety and was thoroughly studied recently as a quarantine examination method, was reviewed [8][9][10][11][12]. However, the following important problems were found.…”