Multiplex RT-PCR detection of four aphid-borne strawberry viruses in Fragaria spp. in combination with a plant mRNA specific internal control.
The principal aphid-borne viruses infecting Strawberry (Fragaria spp.) Strawberry crinkle virus (SCV), Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV) can cause serious crop losses. In this paper, a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) method is described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which can be used as an indicator of the effectiveness of the extraction and RT-PCR. In total, 18 strawberry isolates infected naturally were analysed by this method. Every combination of RNA virus was able to be detected and a full complement of all four viruses were found together in three isolates, all taken from wild strawberry (Fragaria chiloensis (L.) Duch.) in Chile. The upper detection limit for the four viruses was at an extract dilution of 1/200. The broad applicability of the RNA specific internal control primers-which produced a PCR fragment of the expected size in 25 of 27 plant species tested-combined with improvements, made in extraction methods described provides potentially a standard method for comparable RT-PCR analyses in a wide variety of plant species.
Thompson
J R
JR
BBA, Institut für Pflanzenschutz im Obstbau, Schwabenheimer Strasse 101, D-69221, Dossenheim, Germany. jeremy.thompson@urz.uni-heidelberg.de
Wetzel
S
S
Klerks
M M
MM
Vasková
D
D
Schoen
C D
CD
Spak
J
J
Jelkmann
W
W
D000818:Animals; D001042:Aphids / Q000821:virology; D001532:Atropa belladonna / Q000201:enzymology / Q000235:genetics; D031985:Fragaria / Q000821:virology*; D009245:NADH Dehydrogenase / Q000235:genetics; D010935:Plant Diseases / Q000821:virology; D010942:Plant Viruses / Q000235:genetics / Q000302:isolation & purification*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012822:Silicon Dioxide
D012333:RNA, Messenger; D018749:RNA, Plant; D012367:RNA, Viral; D012822:Silicon Dioxide; D009245:NADH Dehydrogenase