Dataset Information

Microdroplet sandwich real-time rt-PCR for detection of pandemic and seasonal influenza subtypes.

ABSTRACT: As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR). Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic), H1N1s (seasonal), and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 10(4) copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies.

SUBMITTER: Angione SL

PROVIDER: S-EPMC3774678 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Microdroplet sandwich real-time rt-PCR for detection of pandemic and seasonal influenza subtypes.

Angione Stephanie L SL Inde Zintis Z Beck Christina M CM Artenstein Andrew W AW Opal Steven M SM Tripathi Anubhav A

PloS one 20130916 9

As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich pl ...[more]

PMID: 24066051

Similar Datasets

Project description:BACKGROUND:Influenza A virus is a leading causative pathogen of human acute respiratory infection. Recently, the co-circulation of pandemic (H1N1) 2009 and seasonal H3N2 viruses was reported, and sporadic cases with reassortant avian H7N9 virus are continually reported in China. We aimed to establish a multiplex one-step real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay to simultaneously detect and discriminate FluA subtypes, including human seasonal H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus, in one reaction tube. METHODS:Clinical samples, including throat swabs and sputum, were collected from the patients with influenza-like illness (ILIs). Total viral RNA from each sample or viral culture was extracted, and the specific detection of FluA virus and its subtypes was performed using a multiplex rRT-PCR assay. RESULTS:The limitation of detection (LOD) of the multiplex assay was 5.4 × 10-2 50% tissue culture infective dose (TCID50) per reaction or 4.8 × 101 copies per reaction for each virus of the three viruses. For simultaneously detecting the three viruses, the LOD was 1.8 × 10-2 TCID50 per reaction or 1.6 × 10 copies per reaction for testing the total FluA virus RNA and 5.6 × 10-2 TCID50 per reaction or 5.1 × 10 copies per reaction for the H3, H1, and H7 genes in one reaction tube. The multiplex assay specifically detected these viruses, and no cross-reaction with other pathogens was found. Moreover, the assay had reliable clinical sensitivity (100%) and valuable clinical specificity (>95%). The detection of FluA with the matrix (M) gene contributed to the further determination of these subtypes, and the Rnase P gene (RP) was considered an internal control to favourably evaluate the quality of the clinical samples. CONCLUSIONS:These findings indicate that the multiplex assay can simultaneously detect and discriminate FluA subtypes with reliable sensitivity and specificity, which is required for the early clinical diagnosis and viral surveillance of patients with FluA infection.

Dataset Information

Microdroplet sandwich real-time rt-PCR for detection of pandemic and seasonal influenza subtypes.

Publications

Microdroplet sandwich real-time rt-PCR for detection of pandemic and seasonal influenza subtypes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets