Reverse transcription recombinase polymerase amplification with lateral flow dipsticks for detection of influenza A virus and subtyping of H1 and H3

Abstract

Three reverse transcription recombinase polymerase amplification assays with lateral flow dipsticks (RT-RPA-LFD) were developed for identification of the matrix and hemagglutinin (HA) genes to detect influenza A virus and distinguish subtypes H1 and H3. Assessment of the assays' specificity showed that there was no cross-reactivity with other targets. Their limits of detection were 123.6 copies per reaction for the matrix gene, 677.1 copies per reaction for the H1 HA gene, and 112.2 copies/reaction for the H3 HA gene. Of 111 samples tested by RT-RPA-LFD assays, 27 were positive for influenza A virus, 14 were positive for H1, and 10 were positive for H3. Compared to the results obtained from real-time RT-PCR assays, the sensitivity of RT-RPA-LFD assays was 75%, 93.33% and 71.43% for the matrix, H1, and H3, with 100% specificity. The sensitivity of RT-RPA-LFD assays is lower than that of real-time RT-PCR, comparable or better than that of conventional RT-PCR, and much better than that of RIDTs. In conclusion, these assays offer an efficient and reliable tool for identification and subtyping of influenza A virus (subtype H1 and H3) in the resource-limited setting.

Keywords: Detection; Influenza A virus; Lateral flow dipstick; Recombinase polymerase amplification; Subtyping.

Fig. 1

Specificity of RT-RPA-LFD assays. (A) Nucleic acid extracted from H1N1, H3N2, influenza B virus (Flu B), respiratory syncytial virus (RSV) subgroup A and B, human metapneumovirus (hMPV) and herpes simplex virus 1 (HSV-1) was detected by three RT-RPA-LFD assays corresponding to the matrix, H1, and H3. (B) Quantitatively measurement of results of RT-RPA-LFD assays.

Fig. 2

Probit regression of RT-RPA-LFD assays (matrix, H1, and H3) using the data of 8 independent assays.

Fig. S1

Fig. S2