Development and Validation of a Colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Eastern Equine Encephalitis Virus in Mosquitoes.
J Am Mosq Control Assoc
; 38(1): 7-18, 2022 03 01.
Article
em En
| MEDLINE
| ID: mdl-35276729
ABSTRACT
Eastern equine encephalitis virus (EEEV) is a highly pathogenic alphavirus that causes periodic outbreaks in the eastern USA. Mosquito abatement programs are faced with various challenges with surveillance and control of EEEV and other mosquito-borne illnesses. Environmental sampling of mosquito populations can be technically complex. Here we report the identification of biomarkers, development and validation of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of EEEV. Positive samples are easily visualized by a color change from pink to yellow. The assay was validated using EEEV from viral culture, experimentally spiked mosquito pools, and previously tested mosquito pools. The RT-LAMP assay detected viral titers down to approximately 10% of what would be present in a single infectious mosquito, based upon EEEV viral titers determined by previous competency studies. The RT-LAMP assay efficiently detected EEEV in combined aliquots from previously homogenized pools of mosquitoes, allowing up to 250 individual mosquitoes to be tested in a single reaction. No false positive results were obtained from RNA prepared from negative mosquito pools acquired from known and potential EEEV vectors. The colorimetric RT-LAMP assay is highly accurate, technically simple, and does not require sophisticated equipment, making it a cost-effective alternative to real time reverse transcriptase-polymerase chain reaction (RT-PCR) for vector surveillance.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Vírus da Encefalite Equina do Leste
/
Culicidae
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
J Am Mosq Control Assoc
Ano de publicação:
2022
Tipo de documento:
Article