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United States Department of Agriculture

Agricultural Research Service

Research Project: VECTOR COMPETENCE AND PROTECTION OF U.S. LIVESTOCK AND WILDLIFE FROM ARTHROPOD-BORNE DISEASES Title: An Improved Method for Detection of Bluetongue Virus in Culicoides (Biting Midges)

Authors
item Kato, Cecilia
item Mayer, Richard

Submitted to: United States Animal Health Association Proceedings
Publication Type: Proceedings
Publication Acceptance Date: December 17, 2005
Publication Date: January 20, 2006
Citation: Kato, C.Y., Mayer, R.T. 2006. An improved method for detection of bluetongue virus in culicoides (biting midges). United States Animal Health Association Proceedings.

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 rapid (less than 6 h start-to-finish), and sensitive assay method that enables the extraction of nucleic acids from individual Culicoides specimens in a 96-well plate format by using specialized beads for mechanical disruption of insect tissues, and the novel use of infrared (IR) dye-labeled primers for virus detection of BTV in biting midges that is also adaptable to other arboviruses. This new method is especially useful for epidemiologic studies involving arboviruses in that it allows for sensitive and specific detection of virus in 96 individual insect specimens by RT-PCR in less than 6 h, as well as the ability to perform virus isolations on the original samples. A comparison of PCR methods for BTV detection is discussed.

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 rapid (less than 6 h start-to-finish), and sensitive assay method that enables the extraction of nucleic acids from individual Culicoides specimens in a 96-well plate format by using specialized beads for mechanical disruption of insect tissues, and the novel use of infrared (IR) dye-labeled primers for virus detection of BTV in biting midges that is also adaptable to other arboviruses. This new method is especially useful for epidemiologic studies involving arboviruses in that it allows for sensitive and specific detection of virus in 96 individual insect specimens by RT-PCR in less than 6 h, as well as the ability to perform virus isolations on the original samples. A comparison of PCR methods for BTV detection is discussed.

Last Modified: 9/23/2014
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