Development of a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for clinical Zika diagnosis.
Int J Infect Dis
; 85: 167-174, 2019 Aug.
Article
em En
| MEDLINE
| ID: mdl-31202908
ABSTRACT
OBJECTIVE:
The nucleic acid-based polymerase chain reaction (PCR) assay is commonly applied to detect infection with Zika virus (ZIKV). However, the time- and labor-intensive sample pretreatment required to remove inhibitors that cause false-negative results in clinical samples is impractical for use in resource-limited areas. The aim was to develop a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for ZIKV diagnosis directly from clinical samples.METHODS:
The combination of inhibitor-tolerant polymerases, polymerase enhancers, and dirRT-qPCR conditions was optimized for various clinical samples including blood and serum. Sensitivity was evaluated with standard DNA spiked in simulated samples. Specificity was evaluated using clinical specimens of other infections such as dengue virus and chikungunya virus.RESULTS:
High specificity and sensitivity were achieved, and the limit of detection (LOD) of the assay was 9.5×101 ZIKV RNA copies/reaction. The on-site clinical diagnosis of ZIKV required a 5µl sample and the diagnosis could be completed within 2h.CONCLUSIONS:
This robust dirRT-qPCR assay shows a high potential for point-of-care diagnosis, and the primer-probe combinations can also be extended for other viral detection. It realizes the goal of large-scale on-site screening for viral infections and could be used for early diagnosis and the prevention and control of viral outbreaks.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Reação em Cadeia da Polimerase Via Transcriptase Reversa
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Zika virus
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Infecção por Zika virus
Tipo de estudo:
Diagnostic_studies
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Screening_studies
Limite:
Adult
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Child
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Female
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Humans
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Male
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article