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Use of multiplex real‐time PCR (TaqMan) for the detection of potato viruses *
Author(s) -
Boonham N.,
Walsh K.,
Mumford R.A.,
Barker I.
Publication year - 2000
Publication title -
eppo bulletin
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.327
H-Index - 36
eISSN - 1365-2338
pISSN - 0250-8052
DOI - 10.1111/j.1365-2338.2000.tb00923.x
Subject(s) - taqman , multiplex , virology , biology , real time polymerase chain reaction , gene , genetics
Certain viruses affect the quality of potato tubers for either table use or processing. Visual discrimination of these viruses is problematic because of variable symptoms, but is important if proper controls are to be implemented. Work at the Central Science Laboratory has concentrated on the detection of Potato mop‐top pomovirus (PMTV), Tobacco rattle tobravirus (TRV) (both associated with the disease spraing) and the tuber necrotic strain of Potato Y potyvirus (PVY NTN ), the symptoms of which can often be confused with spraing. A nucleic acid‐based approach has been adopted as TRV is often found as naked RNA with no associated coat protein, and accurate discrimination of PVY strains is impossible by serology. The multiplex TaqMan assay developed in this work streamlines the testing, replacing two separate tests currently used (a TRV RT‐PCR and a PMTV enzyme‐linked immunosorbent assay) with a single‐tube assay, which has no post‐PCR manipulations. The assay has been shown to be more sensitive than either of the tests which it replaces, allowing 100‐ and 10000‐fold increases in sensitivity for TRV and PMTV detection respectively. The test reliably detected over 40 different isolates of TRV and PMTV obtained from a wide range of cultivars and locations, including samples where existing tests failed. A PCR‐based method capable of discriminating strains of PVY was also developed.