Hepatitis A virus subgenotyping based on RT-qPCR assays

BMC Microbiology

Sylvie Perelle³  Anne-Marie Roque-Afonso²  Sandra Martin-Latil³  Camélia Mokhtari¹  Audrey Fraisse³  Coralie Coudray-Meunier³ 

[1] AP-HP, Hôpital Paul Brousse, Virologie, Villejuif 94804, France;INSERM U785, Villejuif 94804, France;Université Paris-Est, ANSES, Food Safety Laboratory, Enteric viruses unit, 23 Avenue du Général de Gaulle, Maisons-Alfort, 94706, France

关键词: Genotyping;    RT-qPCR assays;    Hepatitis A virus;   

Others  :  1137722
DOI  :  10.1186/s12866-014-0296-1

 received in 2014-04-07, accepted in 2014-11-13,  发布年份 2014

【 摘 要 】

Background

The hepatitis A virus (HAV) is the most frequent cause of viral hepatitis worldwide and is recognized as one of the most widespread foodborne pathogens. HAV genotypes and subtypes differ in their geographic distribution and the incidence of HAV infection varies considerably among countries, and is particularly high in areas with poor sanitation and hygiene. Phylogenetic analyses are traditionally used in clinical microbiology for tracing the geographic origin of HAV strains. In food microbiology, this approach is complicated by the low contamination levels of food samples. To date, real-time reverse-transcription PCR has been one of the most promising detection methods due to its sensitivity, specificity and ability to deliver quantitative data in food samples, but it does not provide HAV subtyping information.

Results

Six subtype-specific RT-qPCR assays were developed for human HAV. The limit of detection of HAV was 50 genome copies/assay for subtype IIB, 500 genome copies assay for IA, IB, IIA and IIIB and 5000 genome copies/assay for IIIA. The specificity of the assays was evaluated by testing reference isolates and in vitro HAV RNA transcripts. No significant cross reactivity was observed. Subtyping results concordant with sequencing analysis were obtained from 34/35 clinical samples. Co-infection with a minor strain of a different subtype was suggested in 5 cases and a recombinant event in one case.

Conclusions

These RT-qPCR assays may be particularly useful for accurately tracing HAV in low-level contaminated samples such as food matrices but also to allow co-infection identification in human samples.

【 授权许可】

   
2014 Coudray-Meunier et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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