Detection and quantitation of Solenopsis invicta virus in fire ants by real-time PCR.
A quantitative real-time PCR (QPCR) method was developed to detect and quantify the amount of Solenopsis invicta virus (SINV) infecting individual ants of S. invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting the SINV RNA-dependent RNA polymerase (RdRp) for cDNA synthesis. Dithiothreitol used in the cDNA synthesis step was found to significantly decrease the detection sensitivity for SINV RdRp and was therefore omitted. SINV RdRp cDNA was then quantified by QPCR using SYBR Green dye and a standard curve generated from SINV RdRp plasmid clones. A standard curve was successfully constructed from clones of the SINV RdRp region. A strong linear relationship [r2=0.998; y=(-3.63+/-0.37)x+(39.19+/-1.33)] between C(T) and starting SINV RdRp copy number was observed within a dynamic range of 5-5 x 10(6) copies. SINV RdRp copy number was determined with the optimized QPCR method in individual S. invicta ants taken from an infected field colony. Worker ants exhibited the highest RdRp copy number (2.1 x 10(9) copies/worker ant) and pupae exhibited the lowest (4.2 x 10(2) copies/pupa). Mean RdRp copy number was lowest in early larvae and pupae. Overall, SINV RdRp copy number increased through larval development, sharply declined during pupation, then sharply increased in adults.
Hashimoto
Yoshifumi
Y
Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608, USA.
Valles
Steven M
SM
Strong
Charles A
CA
D000818:Animals; D001000:Ants / Q000821:virology*; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D005456:Fluorescent Dyes; D018628:Gene Dosage; D016679:Genome, Viral; D007304:Insect Viruses / Q000235:genetics*; D010849:Picornaviridae / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000302:isolation & purification
D017931:DNA Primers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012367:RNA, Viral