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Titolo:
SENSITIVE AND SPECIFIC COLORIMETRIC DOT ASSAY TO DETECT EASTERN EQUINE ENCEPHALOMYELITIS VIRAL-RNA IN MOSQUITOS (DIPTERA, CULICIDAE) AFTER POLYMERASE CHAIN-REACTION AMPLIFICATION
Autore:
ARMSTRONG P; BOROVSKY D; SHOPE RE; MORRIS CD; MITCHELL CJ; KARABATSOS N; KOMAR N; SPIELMAN A;
Indirizzi:
UNIV FLORIDA,INST FOOD & AGR SCI,FLORIDA MED ENTOMOL LAB,200 9TH ST SE VERO BEACH FL 32962 UNIV FLORIDA,INST FOOD & AGR SCI,FLORIDA MED ENTOMOL LAB VERO BEACH FL 32962 HARVARD UNIV,SCH PUBL HLTH,DEPT TROP PUBL HLTH BOSTON MA 00000 YALE UNIV,SCH MED,DEPT ENTOMOL & PUBL HLTH,YALE ARBOVIRUS RES UNIT NEW HAVEN CT 06510 CTR DIS CONTROL,NATL CTR INFECT DIS,DIV VECTOR BORNE INFECT DIS FT COLLINS CO 80522
Titolo Testata:
Journal of medical entomology
fascicolo: 1, volume: 32, anno: 1995,
pagine: 42 - 52
SICI:
0022-2585(1995)32:1<42:SASCDA>2.0.ZU;2-2
Fonte:
ISI
Lingua:
ENG
Soggetto:
DIGOXIGENIN-LABELED PROBES; NUCLEOTIDE-SEQUENCE; OLIGONUCLEOTIDE PROBES; STRUCTURAL PROTEINS; DNA AMPLIFICATION; VIRUS; ENCEPHALITIS; DIAGNOSIS;
Keywords:
EASTERN EQUINE ENCEPHALOMYELITIS VIRUS; POLYMERASE CHAIN REACTION; MOSQUITO;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
27
Recensione:
Indirizzi per estratti:
Citazione:
P. Armstrong et al., "SENSITIVE AND SPECIFIC COLORIMETRIC DOT ASSAY TO DETECT EASTERN EQUINE ENCEPHALOMYELITIS VIRAL-RNA IN MOSQUITOS (DIPTERA, CULICIDAE) AFTER POLYMERASE CHAIN-REACTION AMPLIFICATION", Journal of medical entomology, 32(1), 1995, pp. 42-52

Abstract

A sensitive and specific colorimetric dot assay following polymerase chain reaction (PCR) method has been developed to detect 0.1 pg of eastern equine encephalomyelitis viral (EEEV) RNA. The assay is 250-fold more sensitive than analysis by electrophoresis and is based on converting a 291-nucleotide sequence of the viral coat protein amino terminus into a double-stranded DNA (dsDNA) and amplifying die DNA using a specific primer pair and PCR. The amplified complementary DNA (cDNA) is denatured, adsorbed onto a nylon strip, baked, and detected with a digoxigenin-labeled probe. Dots with viral cDNA are stained dark red, whereas controls do not stain or stain lightly. The assay is very specific and sensitive and detects only EEEV. RNA of Venezuelan equine encephalitis, St. Louis encephalitis, Keystone, Flanders, Tensaw, and western equine encephalitis viruses were net detected. EEEV (Ten Broeck) RNAwas detected at the 10-ng level, indicating that die prototype we used may have different nucleotides in the region where the primer pair binds. The PCR amplified EEEV cDNA that was 92% homologous to the consensus sequence of EEEV. The detection of EEEV in the liver of an infected Emu bird and in field-collected mosquitoes from Florida and Masschusetts that were analyzed concurrently as blind samples by tissue culture plaque assay and by PCR clot analysis proved that the assay is sensitive and can be used to detect infected mosquitoes. The assay can detect at least 1 infected mosquito in a pool of 1,000 uninfected mosquitoes.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 23/09/20 alle ore 22:13:15