Establishment and Clinical Application of a RPA-LFS Assay for Detection of Capsulated and Non-Capsulated Haemophilus influenzae

Abstract

A recombinase polymerase amplification-lateral flow strip assay was established for detection of the outer membrane protein P6 (omp6) and the capsule encoding gene bexA of Haemophilus influenzae and the detection limit, sensitivity, and specificity were determined. Specific primers and probes were designed based on the published nucleotide sequences of omp6 and bexA. The minimum detection limit was determined with standard strains and the practical applicability of the RPA-LFS assay was assessed by detection of 209 clinical samples. The results confirmed that the RPA-LFS assay was both specific and sensitive for the detection of capsulated and non-capsulated H. influenzae with a detection limit of 1 CFU/µL. The detection rate of the 209 clinical samples was 97.1%, while the detection rate of capsulated H. influenzae was 63.2%. The detection results were consistent with the traditional culture method and dual polymerase chain reaction (PCR), confirming the applicability of the RPA-LFS assay.

Real-time multiple cross displacement amplification assay for rapid and sensitive detection of Haemophilus influenzae

“…convenience, and low cost (Wang et al, 2015;Wang et al, 2016;Wang et al, 2019;Li et al, 2020). The results of the MCDA reaction can be visually judged by chromogenic agent preadded in the reaction vessels or further tested using lateral flow biosensors (Wang et al, 2018;Cao et al, 2021;Wang et al, 2022). The whole process is simple, rapid, efficient, and affordable.…”

“…In previous reports, the MCDA reaction is commonly recommended to be carried out in a thermostatic water bath or heater in consideration of its characteristics of simplicity, convenience, and low cost ( Wang et al., 2015 ; Wang et al., 2016 ; Wang et al., 2019 ; Li et al., 2019 ; Li et al., 2020 ). The results of the MCDA reaction can be visually judged by chromogenic agent preadded in the reaction vessels or further tested using lateral flow biosensors ( Wang et al., 2018 ; Li et al., 2019 ; Cao et al., 2021 ; Wang et al., 2022 ). The whole process is simple, rapid, efficient, and affordable.…”

“…The p6 sequence, found in all strains with or without capsule, was popularly used as the molecular identification marker for H. influenzae in previous studies ( Tian et al., 2012 ; Fan et al., 2018 ; Albuquerque et al., 2019 ). Additionally, the sequence has been considered as a very specific region for the diagnosis of H. influenzae ( Wang et al., 2022 ). Here, in the present study, we designed a set of 10 primers targeting the p6 sequence for the real-time MCDA assay.…”

Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications

“…This biosensor had the advantages of high sensitivity and specificity, short reaction time, and simple equipment, which was very suitable for detection in primary hospitals and on-site (Wang P et al, 2021). The RPA-lateral flow strips (RPA-LFS) method established by Wang et al could be used to distinguish capsulated and non-capsulated Haemophilus influenzae, with a detection limit of 1 cfu/ml (Wang et al, 2022). In addition, the RPA-LFS for the detection of Pseudomonas aeruginosa (Yang et al, 2021) and Vibrio parahaemolyticus (Jiang et al, 2020) had also been experimentally verified.…”

“…The RPA-lateral flow strips (RPA-LFS) method established by Wang et al. could be used to distinguish capsulated and non-capsulated Haemophilus influenzae, with a detection limit of 1 cfu/μl ( Wang et al., 2022 ). In addition, the RPA-LFS for the detection of Pseudomonas aeruginosa ( Yang et al., 2021 ) and Vibrio parahaemolyticus ( Jiang et al., 2020 ) had also been experimentally verified.…”