VereFlu™: an integrated multiplex RT-PCR and microarray assay for rapid detection and identification of human influenza A and B viruses using lab-on-chip technology

Abstract

Threatening sporadic outbreaks of avian influenza and the H1N1 pandemic of 2009 highlight the need for rapid and accurate detection and typing of influenza viruses. In this paper, we describe the validation of the VereFlu™ Lab-on-Chip Influenza Assay, which is based on the integration of two technologies: multiplex reverse transcription (RT)-PCR followed by microarray amplicon detection. This assay simultaneously detects five influenza virus subtypes, including the 2009 pandemic influenza A (H1N1), seasonal H1N1, H3N2, H5N1 and influenza B virus. The VereFlu™ assay was clinically validated in Singapore and compared against reference methods of real-time PCR, virus detection by immunofluorescence of cell cultures and sequencing. A sensitivity and specificity of 96.8% and 92.8%, respectively, was demonstrated for pandemic H1N1; 95.7% and 100%, respectively, for seasonal H1N1; 91.2% and 97.6%, respectively, for seasonal H3N2; 95.2% and 100%, respectively, for influenza B. Additional evaluations carried out at the World Health Organization (WHO) Collaborating Centre, Melbourne, Australia, confirmed that the test was able to reliably detect H5N1. This portable, fast time-to-answer (3 hours) device is particularly suited for diagnostic applications of detection, differentiation and identification of human influenza virus subtypes.

Evaluation of Alere i Influenza A&B for Rapid Detection of Influenza Viruses A and B

“…Similarly, culture has increased sensitivity over both RIDTs and DFAs but requires skilled technologists and specialized laboratory settings and has a long turnaround time (2 to 14 days) (17). NAAT are highly sensitive and are gradually replacing culture as the gold standard, but these tests are generally expensive; depending on the manufacturer, require highly skilled molecular technologists; and have turnaround times of up to 24 h from receipt to results (1,7,(18)(19)(20)(21)(22). As a result, specimens with negative RIDTs are usually tested subsequently by more sensitive culture or molecular assays.…”

“…As a result, specimens with negative RIDTs are usually tested subsequently by more sensitive culture or molecular assays. PCR-based molecular assays have shown excellent clinical utility for the detection and identification of influenza viruses; numerous FDA-cleared commercial devices are now available (18,23,24).…”

Lab-on-Chip-Based Platform for Fast Molecular Diagnosis of Multidrug-Resistant Tuberculosis

“…In addition, the LoC can be adapted for other diagnostic or research needs, thus providing a S531L S315T1 WT 1 D516V S315T1 T-8C D516V S315T1 T-8C 1 WT WT WT WT WT WT 15 WT S315T1 WT WT S315T1 WT 1 S S WT WT WT 9 pos WT WT WT WT 4 WT WT WT PCNV PCNV PCNV 1 S S ND S315T1 WT MTBND MTBND MTBND 1 S S ND WT WT ND ND ND 1 multipurpose platform suitable for other relevant diseases (e.g., influenza, malaria, tropical diseases) (25,26). We thank Tanja Ubben and Tanja Struwe Sonnenschein for excellent technical assistance and Enrico Tortoli for valuable support.…”

Profiling In Situ Microbial Community Structure with an Amplification Microarray

“…Furthermore, while traditional sample preparation, amplification, and microarray hybridization chemistries can be incorporated into a consumable via microfluidic transfer steps (9)(10)(11)(12)(13)(14)(15), the engineering, manufacturing, and quality control challenges of such consumables (and control instruments) scale with the complexity of the microfluidic architecture. An alternative approach for simplifying microarray technology for routine monitoring applications, then, is to minimize the biochemistry and processing steps that precede and interface with the microarray itself.…”