Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/8/2006
Publication Date: 1/1/2007
Citation: Hashimoto, Y., Valles, S.M., Strong, C.A. 2007. DETECTION AND QUANTITATION OF SOLENOPSIS INVICTA VIRUS IN FIRE ANTS BY REAL-TIME PCR. Journal of Virological Methods. 140(1-2):132-139. Interpretive Summary: The red imported fire ant was introduced into the United States in the 1930s and currently infests about 300 million acres. It causes approximately $6 billion in damage annually in livestock and agricultural production and poses a serious threat to human health. USDA-ARS scientists at the Center for Medical, Agricultural and Veterinary Entomology (Gainesville, FL) have recently discovered a new RNA virus in the fire ant. Studies suggest that the virus (SINV) may be an excellent biological control agent for fire ants. In order to develop the virus as a control agent for fire ants, a more thorough understanding of the virus is necessary. To accomplish this goal, scientists must be able to quantify the level of infection and track its progression. USDA-ARS scientists at the Center for Medical, Agricultural and Veterinary Entomology (Gainesville, FL) have developed a real-time PCR method capable of quantifying the number of genome copies of the virus in individual fire ants. This method will assist in epidemiological studies and aid in the development of the virus as a fire ant control agent.
Technical Abstract: A quantitative real-time PCR (QPCR) method was developed to detect and quantify the amount of Solenopsis invicta virus (SINV) infecting individual ants of Solenopsis invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting the SINV RNA-dependent RNA polymerase (RdRp) for cDNA synthesis. SINV RdRp cDNA was then quantified by QPCR using SYBR Green dye and a standard curve generated from SINV RdRp plasmid clones. The optimized reaction was comprised of 12.5 'l of SYBR Green SuperMix, oligonucleotide primers, p517 and p519 (160 nM), MgCl2 (3 mM), 1 'l of the reverse transcription reaction (2 mM Tris-HCl, pH 8.3, 3 mM KCl, 0.75 mM MgCl2, 0.04 'M p523, 0.04 mM dNTP mix, 1 U SsRT), and 10.7 'l of H2O. 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 CT and starting SINV RdRp copy number was observed within a dynamic range of 5 to 5x106 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 109 copies/worker ant) and pupae exhibited the lowest (4.2 x 102 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.