RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens.
Biomed Environ Sci
; 33(11): 829-838, 2020 Nov 20.
Article
em En
| MEDLINE
| ID: mdl-33771236
ABSTRACT
OBJECTIVE:
To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases.METHODS:
A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID 50 per µL and copies per µL, and the newly established methods were tested in clinical specimens collected in recent years.RESULTS:
The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID 50 per µL and 10 virus copies per µL, and for the complete VP1 gene was 1 CCID 50 per µL and 100 virus copies per µL, using serially-diluted virus stocks of five serotypes. As a proof-of-concept, 25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons.CONCLUSION:
This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Epidemiologia Molecular
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Enterovirus Humano B
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Reação em Cadeia da Polimerase Via Transcriptase Reversa
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Enterovirus Humano A
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Enterovirus Humano C
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Enterovirus Humano D
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Proteínas do Capsídeo
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Tipagem Molecular
Limite:
Humans
Idioma:
En
Revista:
Biomed Environ Sci
Assunto da revista:
SAUDE AMBIENTAL
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
China