Improvement of a real-time RT-PCR assay for the detection of enterovirus RNA

Department of Microbiology, ZNA Hospitals, site Middelheim, Lindendreef 1, 2020 Antwerp, Belgium.
Virology Journal (Impact Factor: 2.18). 02/2009; 6(1):95. DOI: 10.1186/1743-422X-6-95
Source: PubMed


We describe an improvement of an earlier reported real-time RT-PCR assay for the detection of enterovirus RNA, based on the 5' exonuclease digestion of a dual-labeled fluorogenic probe by Taq DNA polymerase. A different extraction method, real-time RT-PCR instrument and primer set were evaluated. Our data show that the optimized assay yields a higher sensitivity and reproducibility and resulted in a significant reduced hands-on time per sample.

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Available from: Peggy Bruynseels, Apr 24, 2014
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    • "The molecular methods that are based on PCR and sequencing are faster and more accurate [8,9]. Recently, reverse transcription-PCR (RT-PCR) and real-time PCR assays have been used for enterovirus detection [10,11]. But there is still no advanced molecular detection and identification method for the CVA6 infection. "
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    ABSTRACT: Hand, foot and mouth disease (HFMD) is caused by members of the family Picornaviridae in the genus Enterovirus. It has been reported that coxsackievirus A6 (CVA6) infections are emerging as a new and major cause of epidemic HFMD. Sporadic HFMD cases positive for CVA6 were detected in the mainland of China in recent years. To strengthen the surveillance of CVA6 infections and outbreak control, the clinical diagnosis is urgently needed to distinguish the CVA6 infection disease from other infections. In order to develop a sensitive quantitative real-time RT-PCR assay for rapid detection of CVA6 RNA, primers and probe were designed to target the VP1 gene segment of CVA6. The conservation of the target segment was firstly analyzed by bioinformatic technology. The specificity of the real-time RT-PCR was further confirmed by detecting other related viruses and standard curves were established for the sensitivity evaluation. The pharyngeal swab samples from the EV71 and CVA16 unrelated HFMD patients were applied for CVA6 detection through the established method. Based on the primer–probe set to detect the target VP1 gene segment of CVA6, the quantitative real-time RT-PCR assay could discriminate CVA6 infection from other resemble viral diseases with a potential detection limit of 10 viral copies/ml. The specificity of the assay was determined by sequence alignment and experimentally tested on various related viruses. The standard curve showed that the amplification efficiency of templates with different concentrations of templates was almost the same (R2 >0.99). Evaluation of the established method with pharyngeal swabs samples showed good accordance with the results from serology diagnosis. This study is the first report developing a VP1 gene-based quantitative real-time RT-PCR for rapid, stable and specific detection of CVA6 virus. The real-time RT-PCR established in this study can be used as a reliable method for early diagnosis of CVA6 infection.
    Virology Journal 11/2012; 9(1):298. DOI:10.1186/1743-422X-9-298 · 2.18 Impact Factor
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    ABSTRACT: Human enterovirus 71 (EV 71) has caused large-scale outbreaks of hand-foot-and-mouth disease (HFMD), particularly in the Asian-Pacific region. In this study, we report a major outbreak of EV 71 infection in Korea and describe the clinical differences between EV 71 and non-EV 71 enterovirus infections. We prospectively enrolled patients with suspected viral infections during a recent 2-year period through a nationwide surveillance system. We identified 719 patients with suspected HFMD or herpangina using real-time PCR and genotyping based on VP1 sequence analysis. The major pathogen causing HFMD changed substantially from 2008 to 2009, with EV 71 becoming the most common cause of HFMD in Korea in 2009. We successfully identified the enteroviral genotypes for 218 of the 719 patients. Patients with EV 71 infections tended to be younger than those with non-EV 71 enteroviral infections and presented with HFMD and meningoencephalitis. In addition, the occurrence of fever, headache, and neck stiffness was significantly higher in patients with EV 71 infections. Multivariable analysis showed that for patients presenting with HFMD, fever, or a sore throat, each covariate was independently associated with EV 71 infection; the adjusted odds ratios (with 95% confidence intervals in parentheses) for these variables were 31.86 (10.04 to 101.09), 4.76 (1.71 to 13.25), and 0.18 (0.04 to 0.77), respectively. Our results indicate that EV 71 was a major cause of HFMD in Korea during the study period. In addition, we found that clinical symptoms may be helpful in the early identification of patients with EV 71 infections.
    Journal of clinical microbiology 05/2010; 48(7):2490-4. DOI:10.1128/JCM.02369-09 · 3.99 Impact Factor
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    ABSTRACT: OTHER THEMES PUBLISHED IN THIS IMMUNOLOGY IN THE CLINIC REVIEW SERIES Metabolic diseases, host responses, cancer, autoinflammatory diseases, allergy. The hypothesis that under some circumstances enteroviral infections can lead to type 1 diabetes (T1D) was proposed several decades ago, based initially on evidence from animal studies and sero-epidemiology. Subsequently, enterovirus RNA has been detected more frequently in serum of patients than in control subjects, but such studies are susceptible to selection bias and reverse causality. Here, we review critically recent evidence from human studies, focusing on longitudinal studies with potential to demonstrate temporal association. Among seven longitudinal birth cohort studies, the evidence that enterovirus infections predict islet autoimmunity is quite inconsistent in our interpretation, due partially, perhaps, to heterogeneity in study design and a limited number of subjects studied. An association between enterovirus and rapid progression from autoimmunity to T1D was reported by one longitudinal study, but although consistent with evidence from animal models, this novel observation awaits replication. It is possible that a potential association with initiation and/or progression of islet autoimmunity can be ascribed to a subgroup of the many enterovirus serotypes, but this has still not been investigated properly. There is a need for larger studies with frequent sample intervals and collection of specimens of sufficient quality and quantity for detailed characterization of enterovirus. More research into the molecular epidemiology of enteroviruses and enterovirus immunity in human populations is also warranted. Ultimately, this knowledge may be used to devise strategies to reduce the risk of T1D in humans.
    Clinical & Experimental Immunology 04/2012; 168(1):12-23. DOI:10.1111/j.1365-2249.2011.04555.x · 3.04 Impact Factor
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