Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing

Abstract

Moraxella catarrhalis (M. catarrhalis) was an important pathogen closely associated with respiratory tract infections. We employed the loop-mediated isothermal amplification (LAMP) coupled with nanoparticle-based lateral flow biosensor (LFB) and fluorescence testing technique for formulating two diagnostic methods for M. catarrhalis detection, termed M. catarrhalis-LAMP-LFB assay and M. catarrhalis-LAMP-FRT, respectively. The M. catarrhalis-LAMP-LFB system incorporated the use of biotin-14-dCTP and a forward loop primer (LF) with a hapten at the 5' end. This design in LAMP reaction enabled the production of double-labeled products that could be effectively analyzed using the lateral flow biosensor (LFB). For the M. catarrhalis-LAMP-FRT assay, the LF was modified with a sequence at 5' end, and a fluorophore, as well as a black hole quencher, were strategically labeled at the 5' end and within the middle of the new LF. The restriction endonuclease Nb.BsrDI could accurately recognize and cleave the newly synthesized double-strand terminal sequences, resulting in the separation of the fluorophore from the black hole quencher and releasing fluorescence signals. Both assays have been proven to be highly sensitive and specific, capable of detecting genomic DNA of M. catarrhalis at concentrations as low as 70 fg, with no cross-reactivity observed with non-M. catarrhalis pathogens. Furthermore, both methods successfully identified M. catarrhalis in all clinical samples within 1 h that were confirmed positive by real-time PCR, exhibiting superior sensitivity than conventional culture methods. Herein, the newly developed two LAMP-based assays were rapid and reliable for M. catarrhalis detection and hold significant promise for deployment in point-of-care (POC) settings.

Keywords: Moraxella catarrhalis; loop-mediated isothermal amplification; nanoparticle-based lateral flow biosensor; restriction endonuclease; visual detection.

FIGURE 1

Schematic illustration of the M. catarrhalis-LAMP-LFB&FRT assays. (A) The detection principle of M. catarrhalis-LAMP-LFB assay. In the M. catarrhalis-LAMP-LFB system, a FAM-labeled loop primer (LF^#) and biotin-14-dCTP were utilized, leading to generation of double-labeled amplicons (FAM and biotin labeled), which were captured by the immobilized anti‐FAM of LFB and visualized via reaction between biotin and SA‐GNPs (streptavidin‐coated dyed (crimson red) polymer nanoparticles), resulting in a red color line occurred in the TL region of LFB. The remaining SA‐GNPs were captured by the immobilized biotin‐BSA (biotinylated bovine serum albumin) at the CL region, leading to a red color line occurred in the CL region that indicated the usefulness of the LFB. (B) The detection principle of M. catarrhalis-LAMP-FRT assay. In the M. catarrhalis-LAMP-FRT system, an additional restriction endonuclease Nb.BsrDI and a modified loop primer (LF*) were utilized. The primer LF* was modified by adding a short sequence (TGCAATG) at 5′ end which could be recognized by restriction endonuclease. Nb.BsrDI and a fluorophore and a black hole quencher 1 (BHQ1) at the 5′ end and the middle of new primer. When reacted with LF*, the M. catarrhalis-LAMP-FRT system generated plenty of Ss-containing target amplicons that were then cleaved by restriction endonuclease Nb.BsrDI, which could seperate the fluorophore FAM from BHQ1, resulting in emission of fluorescence signals that could be observed by unaided eyes under blue light. (C) Sequences and locations of primers used in this study. Right arrows and left arrows indicate sense and complementary sequences that were used.

FIGURE 2

Feasibility of the M. catarrhalis-LAMP-LFB assay and M. catarrhalis-LAMP-FRT assay for M. catarrhalis detection was confirmed by real-time turbidity (A), color change (B), and LFB (C) and visual inspection under blue light (D) with genomic DNA of M. catarrhalis as positive control, that of Neisseria meningitides, Klebsiella pneumoniae, and Staphylococcus aureus as negative control, and distilled water (DW) as blank control. 1–5 indicated reaction results with genomic DNA of M. catarrhalis, Neisseria meningitides, Klebsiella pneumoniae, Staphylococcus aureus and DW, respectively. TL, test line; CL, control line.

FIGURE 3

Analytical sensitivity evaluation of M. catarrhalis-LAMP-LFB&FRT assays Serial dilutions of genomic DNA of M. catarrhalis (from 70 pg to 7 fg per microliter) were employed to evaluation the analytical sensitivity of M. catarrhalis-LAMP-LFB assay (A) and M. catarrhalis-LAMP-FRT assay (B). TL, test line; CL, control line.

FIGURE 4

Analytical specificity of M. catarrhalis-LAMP-LFB&FRT assays. The specificity of M. catarrhalis-LAMP-LFB assay (A) and M. catarrhalis-LAMP-FRT assay (B) were analyzed using 28 non-M. catarrhalis pathogens and 4 M. catarrhalis strains. 1-4 represented the 4 M. catarrhalis strains; 5–32 represented the 28 non-M. catarrhalis pathogens (Supplementary Table S2). TL, test line; CL, control line.

FIGURE 5

Clinical validation of M. catarrhalis-LAMP-LFB&FRT assays with clinical specimens. A total of 48 clinical samples were tested by M. catarrhalis-LAMP-LFB assay (A) and M. catarrhalis-LAMP-FRT assay (B) to confirm the clinical feasibility. TL, test line; CL, control line.

FIGURE 6

Clinical performance comparison of the M. catarrhalis-LAMP-LFB&FRT assays, real-time PCR and culture methods. +, positive result; -, negative result.