Evaluation of a novel micro/nanofluidic chip platform for the detection of influenza A and B virus in patients with influenza-like illness

Abstract

We introduced a novel micro/nanofluidic chip platform (MNCP), which is based on an isothermal nucleic acid amplification method. This study aimed to evaluate the MNCP method for influenza A and B viruses detecting and subtyping using throat swab samples from patients with influenza-like illness (ILI). A total of 266 throat swab samples from 266 non-repeated patients with ILI were tested for influenza A and B viruses using three methods, MNCP, a rapid influenza diagnostic test (RIDT), and real-time reverse transcription polymerase chain reaction (rRT-PCR). The results of MNCP were compared to those obtained by rRT-PCR and RIDT and the performance of MNCP was further evaluated. Compared with rRT-PCR results, the rates of sensitivity, specificity, overall concordance, and the kappa value of MNCP were 98.89%, 96.97%, 97.65%, and 0.95 for influenza A virus; 94.95%, 99.38%, 97.68%, and 0.95 for influenza B virus, respectively. Subtypes of influenza A viruses, e.g., A(H1N1)pdm09, A(H3N2), and A(not subtyped), and influenza B viruses could be distinguished in one MNCP assay within 1 h. Compared with rRT-PCR and MNCP, RIDT showed poor clinical sensitivity for influenza virus detection. This study showed MNCP is rapid, sensitive and versatile detecting system with potential for clinical application in pathogen diagnosis for patients with ILI.

Keywords: Influenza; Influenza-like illness; Nucleic acid sequence-based amplification; Rapid influenza diagnostic test.

Fig. 1

Flowchart of the study. MNCP: micro/nanofluidic chip platform; RIDT: rapid influenza diagnostic test; rRT-PCR: real-time reverse transcription PCR

Fig. 2

Principles of the MNCP based on NASBA amplification. a The isothermal cycle of the NASBA amplification. b Signal reporting of the amplification product. AMV-RT: avian myeloblastosis virus reverse transcriptase; MNCP: micro/nanofluidic chip platform; NASBA: nucleic acid sequence-based amplification

Fig. 3

The workflow of MNCP. The steps were as follows: (1) Nucleic acid was extracted from throat swab samples. (2) A mixture including nucleic acid, basic NASBA reaction reagents, primers, and molecular beacon probes was loaded into the micro/nanofluidic chip. (3) The loaded chip was placed into the RTisochip-A detector (CapitalBio, Beijing, China). (4) RNA was amplified based on the NASBA and the fluorescence intensity data were collected and analyzed using the software. (5) Results of detecting and subtyping of influenza A and B viruses were obtained. Fluorescent curves were plotted by detection time (X-axis) against fluorescent intensity (Y-axis) for influenza A(H1N1)pdm09, A(H3N2), and influenza B virus, respectively. A batch of the MNCP tests took about 1 h. MNCP: micro/nanofluidic chip platform; NASBA: nucleic acid sequence-based amplification

Fig. 4

Box and whisker plots of the viral concentration distribution of samples with RIDT/MNCP positive results and RIDT negative and MNCP/rRT-PCR positive results determined by rRT-PCR (log 10 copies/ml). a Influenza A virus. b Influenza B virus. The box showed the median (thick line and number) and interquartile range (box length). The whiskers represented 1.5 times interquartile ranges or the highest/lowest points). MNCP: micro/nanofluidic chip platform; RIDT: rapid influenza diagnostic test; rRT-PCR: real-time reverse transcription PCR