The use of the polymerase chain reaction (PCR) for the detection of bean yellow mosaic virus in gladiolus

Abstract

A method is described for the improved detection of bean yellow mosaic virus (BYMV) in gladioli leaves. Specific sequences of BYMV RNA, present in total RNA extracts of infected plants were detected following amplification by the polymerase chain reaction (PCR). The viral RNA was initially reverse-transcribed into cDNA, then specific sequences were amplified by PCR using specific oligonucleotides as primers. Detectable amounts of virus RNA in BYMV-infected plant tissue by PCR were approximately three to four orders of magnitude lower as compared with those detectable by ELISA and molecular hybridisation. Combining PCR with molecular hybridisation (using a 32P-labelled transcript of viral sequences as a probe), further increased the sensitivity of this method to a gain of four to five orders of magnitude as compared with direct molecular hybridisation, and enabled the detection of up to single picogram quantities of the virus.

Improved detection of prunus necrotic ringspot virus by the polymerase chain reaction

“…The authenticity of the amplified PCR fragment was confirmed by comparing the PCR products obtained from RNA isolated from infected tissue with RNA isolated from purified virus and the cloned PNRSV as well as by hybridisation with a 32p-labelled PNRSV riboprobe. Similar results were shown earlier for PCR detection of bean yellow mosaic virus in gladiolus (Vunsh et al, 1990) and for potato leafroll virus in potato tubers (Spiegel and Martin, 1993). Low amounts of products, yielded an undefined diffuse band or no visible stained product leading to inconclusive results.…”

“…Two oligonucleotide primers were used in this study: primer #1-5~-[TATI"FCT CTAGTA-ACATCCC]-3 I, primer #2-5 I-[ATACTCGGCCTACA CATCTG]-3C cDNA synthesis, PCR amplification and hybridisation The reverse transcriptase (RT) and PCR reactions were performed following a single non-interrupted thermal cycling program (Sellner et al, 1992), Each reaction contained the RNA template (about 1 #g), virus specific primers (0.4 #M each), 100/tM dNTPs, 3 mM MgC12, 2.5 #1 of 10x reaction buffer giving a final concentration of 67 mM Tris-HC1 pH 8.8, 17 mM (NH4)2SO4, 1 mM Mercaptoethanol, 6/tM EDTA and 0.002% Gelatin. Reaction conditions were as previously described by Vunsh et al (1990). The total volume of 25/_tl was overlayed with 50/_tl of mineral oil and subjected in a thermocycler (Hybaid, UK) to the following program: 30 min at 42 ~ 3 min at 95 ~ then 40 cycles of 92 ~ for 30 sec, 54 ~ for 30 sec and 72 ~ for 1 min, and finally 5 min at 72 ~ PCR products were electrophoresed in a 1.2% agarose gel and stained with ethidium bromide (EtBr).…”

An appraisal of different methods for the detection of the walnut strain of cherry leafroll virus

“…PCR has already been used to detect plant DNA viruses (Rybicki et al, 1990) and RT-PCR to detect plant RNA viruses (Vunsh et al, 1990, Korschineck et al, 1991. Our results are in support of the potential value of RT-PCR as a simple and specific method for the detection of plant RNA viruses in infected sap.…”

“…Our results are in support of the potential value of RT-PCR as a simple and specific method for the detection of plant RNA viruses in infected sap. Similar tests using a reverse transcription step prior to cDNA amplification have been developed for detection of viroids (Puchta et al, 1989;Hadidi et al, 1990), for several animal RNA viruses including rhinovirus (Gama et al, 1989), HIV (Byrne et al, 1988;Hart et al, 1988;Murakawa et al, 1988), human picornaviruses (Hyypia et al, 1989), and for plant RNA viruses (Vunsh et al, 1990;Korschineck et al, 1991). These reports describe methods that use a previous step for RNA purification.…”

Serological and molecular characterisations of the Egyptian isolate ofBean common mosaic virus

“…PCR has been successfully used in the detection and characterisation of many plant viruses and viroids (Vunsh et al 1990). PCR using specific primers has proved to be a rabid, accurate and efficient method for detecting and determining genetic diversity among Potyviruses.…”