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Virol J. 2016 Jul 7;13:125. doi: 10.1186/s12985-016-0580-9.

Development and evaluation of a non-ribosomal random PCR and next-generation sequencing based assay for detection and sequencing of hand, foot and mouth disease pathogens.

Author information

1
Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam. anhnt@oucru.org.
2
Oxford University Clinical Research Unit, 764 Vo Van Kiet Street, Ward 1, District 5, Ho Chi Minh City, Vietnam.
3
Children's Hospital 2, Ho Chi Minh City, Vietnam.
4
Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
5
Children's Hospital 1, Ho Chi Minh City, Vietnam.
6
Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

Abstract

BACKGROUND:

Hand, foot and mouth disease (HFMD) has become a major public health problem across the Asia-Pacific region, and is commonly caused by enterovirus A71 (EV-A71) and coxsackievirus A6 (CV-A6), CV-A10 and CV-A16. Generating pathogen whole-genome sequences is essential for understanding their evolutionary biology. The frequent replacements among EV serotypes and a limited numbers of available whole-genome sequences hinder the development of overlapping PCRs for whole-genome sequencing. We developed and evaluated a non-ribosomal random PCR (rPCR) and next-generation sequencing based assay for sequence-independent whole-genome amplification and sequencing of HFMD pathogens. A total of 16 EV-A71/CV-A6/CV-A10/CV-A16 PCR positive rectal/throat swabs (Cp values: 20.9-33.3) were used for assay evaluation.

RESULTS:

Our assay evidently outperformed the conventional rPCR in terms of the total number of EV-A71 reads and the percentage of EV-A71 reads: 2.6 % (1275/50,000 reads) vs. 0.1 % (31/50,000) and 6 % (3008/50,000) vs. 0.9 % (433/50,000) for two samples with Cp values of 30 and 26, respectively. Additionally the assay could generate genome sequences with the percentages of coverage of 94-100 % of 4 different enterovirus serotypes in 73 % of the tested samples, representing the first whole-genome sequences of CV-A6/10/16 from Vietnam, and could assign correctly serotyping results in 100 % of 24 tested specimens. In all but three the obtained consensuses of two replicates from the same sample were 100 % identical, suggesting that our assay is highly reproducible.

CONCLUSIONS:

In conclusion, we have successfully developed a non-ribosomal rPCR and next-generation sequencing based assay for sensitive detection and direct whole-genome sequencing of HFMD pathogens from clinical samples.

KEYWORDS:

Enterovirus A; FR26RV-Endoh primer; Hand, foot and mouth disease; Next-generation sequencing; Random PCR

PMID:
27388326
PMCID:
PMC4937578
DOI:
10.1186/s12985-016-0580-9
[Indexed for MEDLINE]
Free PMC Article

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