AN IMPROVED, HIGH-THROUGHPUT METHOD FOR DETECTION OF BLUETONGUE VIRUS IN CULICOIDES (BITING MIDGES)

Authors: Kato, Cecilia; Mayer, Richard

Submitted to: United States Animal Health Association Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 7/4/2005
Publication Date: 11/5/2005
Citation: Kato, C.Y., Mayer, R.T. 2005. An improved, high-throughput method for detection of bluetongue virus in Culicoides (biting midges). Proceedings of the One hundred and ninth annual meeting of the United States Animal Health Association.

Interpretive Summary: Current protocols for RT-PCR assay of bluetongue virus (BTV) RNA in biting midges (Culicoides sp.) involve time consuming steps for sample (single insect) homogenization, RNA extraction, and RT-PCR assay. Further, current RT-PCR methods for assay of BTV RNA are either not very sensitive, require multiple steps for chemical denaturation, reverse transcriptase reaction, 1° and 2° PCR amplification, and/or Real Time PCR instrumentation. We report a new, rapid (less than 5 h start-to-finish), and sensitive assay method for detecting BTV in biting midges. The method enables the extraction of nucleic acids from individual Culicoides specimens in a 96 - well plate format, followed by the novel use of infrared (IR) dye-labeled primers for virus detection. Specialized beads are used for mechanical disruption of insect tissues in a homogenization buffer that is compatible with cell culture and RNA extraction. The process begins with a 1 min homogenization of the 96 insects (individuals) in a 96 well plate. One tenth of the homogenate from each specimen is saved for cell culture virus isolation, while the remainder of each sample is used for rapid RNA extraction. An aliquot of the extracted RNA is heat denatured, then used in a single step reverse transcriptase PCR (RT-PCR) reaction with IRdye-labeled primers. The RT-PCR products are separated by agarose gel electrophoresis and visualized by an infrared scanner). The adaptation of IR-dye-labeled primers and a one step RT-PCR increases the sensitivity of a highly selective primer set for BTV serotypes 2, 4, 6, 10, 11, 13, 16, and 17 by 100-fold (i.e., from 100 fg, Akita et al., 1992) to a sensitivity of 1 fg of purified BTV RNA. This new method is especially useful for epidemiologic studies involving arboviruses in that it allows for sensitive and specific detection of virus in 96 insect individual specimens by RT-PCR in less than 5 h, and the ability to perform virus isolations on the original samples.

Technical Abstract: Current protocols for RT-PCR assay of bluetongue virus (BTV) RNA in biting midges (Culicoides sp.) involve time consuming steps for sample (single insect) homogenization, RNA extraction, and RT-PCR assay. Further, current RT-PCR methods for assay of BTV RNA are either not very sensitive, require multiple steps for chemical denaturation, reverse transcriptase reaction, 1° and 2° PCR amplification, and/or Real Time PCR instrumentation. We report a new, rapid (less than 5 h start-to-finish), and sensitive assay method for detecting BTV in biting midges. The method enables the extraction of nucleic acids from individual Culicoides specimens in a 96 - well plate format, followed by the novel use of infrared (IR) dye-labeled primers for virus detection. Specialized beads are used for mechanical disruption of insect tissues in a homogenization buffer that is compatible with cell culture and RNA extraction. The process begins with a 1 min homogenization of the 96 insects (individuals) in a 96 well plate. One tenth of the homogenate from each specimen is saved for cell culture virus isolation, while the remainder of each sample is used for rapid RNA extraction. An aliquot of the extracted RNA is heat denatured, then used in a single step reverse transcriptase PCR (RT-PCR) reaction with IRdye-labeled primers. The RT-PCR products are separated by agarose gel electrophoresis and visualized by an infrared scanner). The adaptation of IR-dye-labeled primers and a one step RT-PCR increases the sensitivity of a highly selective primer set for BTV serotypes 2, 4, 6, 10, 11, 13, 16, and 17 by 100-fold (i.e., from 100 fg, Akita et al., 1992) to a sensitivity of 1 fg of purified BTV RNA. This new method is especially useful for epidemiologic studies involving arboviruses in that it allows for sensitive and specific detection of virus in 96 insect individual specimens by RT-PCR in less than 5 h, and the ability to perform virus isolations on the original samples.