A One-Step Reverse Transcription-Polymerase Chain Reaction System for the Simultaneous Detection and Identification of Multiple Tospovirus Infections

Abstract

A one-step reverse transcription-polymerase chain reaction (RT-PCR) method has been developed for the simultaneous detection and identification of multiple tospoviruses that infect plants. The RT-PCR system is composed of six primers in a single tube: a universal degenerate primer and five virus species-specific primers. Amplifications resulted in an 848-bp PCR product for Watermelon silver mottle virus, 709-bp for Tomato spotted wilt virus, 589-bp for Impatiens necrotic spot virus, 511-bp for Melon yellow spot virus, and a 459-bp amplicon for Iris yellow spot virus. This system enables the simultaneous detection of at least three types of tospovirus infections, in addition to their species identities, from five possible tospoviruses studied, on the basis of their S RNA combinations. This multiplex RT-PCR system was applied to the detection of tospovirus in ornamental crops cultivated in fields and shows potential for epidemiological studies.

Immunocapture and simple-direct-tube-reverse transcriptase polymerase chain reaction (RT-PCR) for detection of Rice yellow mottle virus

“…Several variations of RT-PCR have been developed, including nested- (Kiliç and Yardimci, 2012), one step- (Uga and Tsuda, 2005), multiplex- (Grieco and Gallitelli, 1999), real-time (Osman et al, 2007), immunocupture- (Wetzel et al, 1992) and simple-direct-tube RT-PCR (Suehiro et al, 2005). Immuno-capture-RT PCR is designed for highly specific detection of templates present in very low amounts (Wetzel et al, 1992 (Wetzel et al, 1992;Harper et al, 1999;Hema et al, 2003;Mansilla et al, 2003;Ling et al, 2007;Zein et al, 2008;Silva et al, 2011;Ha et al, 2012;Kiliç, and Yardimci;.…”

Detection of Plantago asiatica mosaic virus in lily hybrid plants (Lilium spp.) in Costa Rica grown from imported bulbs

“…RNA was extracted (RNeasy Plant Mini Kit, Q i a g e n , G e r m a n y ) a n d c D N A w a s o b t a i n e d b y retrotranscription (Maxima H Minus First Strand cDNA Synthesis Kit, Thermo Scientific, Lithuania) primed with random hexamers. cDNAs were tested by PCR (DreamTaq PCR Master Mix, Thermo Scientific, Lithuania) with five different primer pairs for orthotospovirus detection (Eiras et al 2001;Uga and Tsuda 2005). An amplicon under 500 bp was obtained for both plants, T188 and T189, using primer pair IYSV-837 / Tos-R15 (Uga and Tsuda 2005) and sequenced (Macrogen, Korea) in both directions.…”

“…cDNAs were tested by PCR (DreamTaq PCR Master Mix, Thermo Scientific, Lithuania) with five different primer pairs for orthotospovirus detection (Eiras et al 2001;Uga and Tsuda 2005). An amplicon under 500 bp was obtained for both plants, T188 and T189, using primer pair IYSV-837 / Tos-R15 (Uga and Tsuda 2005) and sequenced (Macrogen, Korea) in both directions. The resulting sequences (T188, KY807780; T189, KY807781) had top BLAST hits in GenBank (National Center for Biotechnology Information, USA) to isolates of PlAMV.…”

Improved multiplex reverse transcription-polymerase chain reaction to detect and identify five tospovirus species simultaneously

“…To simultaneously diagnose these causal five tospovirus diseases, Uga and Tsuda (2005) developed a one-step multiplex reverse transcription-polymerase chain reaction (multi-PCR) method using a degenerate primer and species-specific primers.…”