Nucleic acid hybridization techniques for the detection of bluetongue virus

Thesis/Dissertation · Sun Jan 01 04:00:00 EST 1989

OSTI ID:5475123

Schoepp, R J

Virus isolation, antigen detection, and in situ hybridization were compared in their abilities to detect in cell culture, the five serotypes of bluetongue virus (BTV) occurring in the United States, serotypes 2, 10, 11, 13, and 17. For isolation, virus was propagated in baby hamster kidney (BHK-21) cell culture. For antigen detection, two techniques, indirect fluorescent-antibody (IFA) and enzyme immunocytoassay (EICA) were used. For in situ hybridization, a complementary DNA (cDNA) of the L3 RNA genome segment of BTV, serotype 17 (BTV-17) labeled with {sup 35}S was used as a group-specific probe. Virus isolation was the most sensitive technique, often detecting input virus and then detecting virus throughout the course of the study. IFA and EICA were of similar sensitivity and detected BTV antigen shortly after detection of virus by isolation. A direct-blot hybridization technique using a {sup 32}P-labeled, strand-specific RNA transcript probe was developed, optimized, and used to detect BTV in pools of infected Culicoides variipennis midges. The technique was able to detect as few as one infected Culicoides midge in a pool of 100 and as little as 3.5 log{sub 10} TCID{sub 50} per ml of virus. A sandwich hybridization technique was developed and used to detect BTV in pools of infected Culicoides variipennis midges. The sandwich hybridization technique used a single-stranded DNA catcher sequence bound to a solid support and a {sup 32}P-labeled, single-stranded RNA detector sequence. Sandwich hybridization was compared to direct blot hybridization using a strand-specific RNA transcript probe or a cDNA probe. Sandwich hybridization was able to detect as few as one infected Culicoides midge in a pool of 50; however, the technique was approximately tenfold less sensitive than direct blot hybridization.

• Research Organization: Colorado State Univ., Fort Collins, CO (United States)
• OSTI ID: 5475123
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ANIMALS
ANTIGENS
ARTHROPODS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOASSAY
BODY
DAYS LIVING RADIOISOTOPES
DIAGNOSIS
DISEASES
DNA HYBRIDIZATION
ENZYME ACTIVITY
ENZYME IMMUNOASSAY
EVEN-ODD NUCLEI
FLUORESCENCE
HAMSTERS
HYBRIDIZATION
IMMUNOASSAY
INFECTIOUS DISEASES
INFECTIVITY
INSECTS
INVERTEBRATES
ISOTOPES
KIDNEYS
LIGHT NUCLEI
LUMINESCENCE
MAMMALS
NUCLEI
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANS
RADIOISOTOPES
RNA
RODENTS
SENSITIVITY ANALYSIS
SULFUR 35
SULFUR ISOTOPES
TRANSCRIPTION
VERTEBRATES
VIRAL DISEASES