ABSTRACT
In 2013, Tomato chlorosis virus (ToCV) was identified in symptomatic tomato plants in Korea. In the present study, a loop-mediated isothermal amplification (LAMP) method was developed using four specific primers designed against ORF6 in ToCV RNA2 to detect ToCV rapidly and with high sensitivity. The optimized reaction involved incubation of a reaction mixture containing 2U Bst DNA polymerase and 4mM MgSO4 for 1h at 60-62 °C. Although specific and rapid detection of ToCV by LAMP was confirmed, false-positive reactions caused by carry-over contamination sometimes occurred because of the high sensitivity of LAMP compared with other detection methods. To prevent false-positive reactions, dUTP was substituted for dTTP and uracil-DNA glycosylase (UDG) was added to the LAMP reaction. First, the LAMP reaction was conducted successfully with substitution of dUTP for dTTP. Before the next reaction, LAMP products with incorporated dUTP were cleaved selectively by UDG without any effect on thymine-containing DNA (template DNA). This modified LAMP method complemented with UDG treatment to prevent carry-over contamination offers a potentially powerful method for detecting plant viruses.
Subject(s)
Crinivirus/isolation & purification , Nucleic Acid Amplification Techniques/methods , Plant Diseases/virology , Solanum lycopersicum/virology , Uracil-DNA Glycosidase/metabolism , Virology/methods , DNA Primers/genetics , False Positive Reactions , Korea , Sensitivity and Specificity , TemperatureABSTRACT
Basal stem rot symptoms were found on blueberry seedlings imported from the United States of America in 2008. The fungus obtained from the diseased seedlings was identified as Calonectria colhounii based on morphological and molecular characteristics. The consignments of the blueberry seedlings infected with C. colhounii were destroyed to prevent introduction of the fungus to Korea.
