Ultra-sensitive detection of two garlic potyviruses using a real-time fluorescent (Taqman) RT-PCR assay.
A method for the detection of Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), the two most prevalent garlic potyviruses, has been developed that combines IC-RT-PCR/RT-PCR with the use of TaqMan probes. Comparisons with ELISA results obtained with identical OYDV and LYSV infected samples showed sensitivity in detecting these viruses increased up to 10(6)-fold. OYDV and LYSV were detected using different fluorochromes in the probe, thus allowing unequivocal diagnosis for each of them. The polyvalence of the designed virus-specific primers and probes was shown through their application to the detection of three isolates from very different geographical areas and from different hosts. A second version of the method avoids the need for an immunocapture step through the performance of a TaqMan RT-PCR assay directly on extracts of garlic cloves. This modification on the proposed basic method allows the analysis of bulb samples in 3-4h but did not give reproducible results with leaves. Both versions of the new diagnostic method bear great potential for their implementation in virus-free certification schemes in garlic, a vegetatively propagated crop for which such a certification is critical for a high-quality product.
Lunello
Pablo
P
INIA, Department of Biotechnology, Autopista A-6, km 7, 28040 Madrid, Spain.
Mansilla
Carmen
C
Conci
Vilma
V
Ponz
Fernando
F
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D005737:Garlic / Q000821:virology*; D017800:Potyvirus / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods / Q000706:statistics & numerical data
D017931:DNA Primers; D004279:DNA, Viral; D005456:Fluorescent Dyes