A Rapid Detection of Haemophilus influenzae Using Multiple Cross Displacement Amplification Linked With Nanoparticle-Based Lateral Flow Biosensor

Abstract

Haemophilus influenzae is a major human pathogenic bacterium, resulting in a series of diseases, such as pneumonia, bacteremia, meningitis. However, it is hard to diagnose H. influenzae quickly. In this study, the multiple cross displacement amplification (MCDA) and nanoparticle-based lateral flow biosensor (LFB) (MCDA-LFB) were combined to detect H. influenzae, which has been proven to be reliable, rapid, and not complicated. On the basis of H. influenzae outer membrane protein P6 gene, 10 specific primers were designed. The best MCDA condition was 61°C for 1 h. The sensitivity of H. influenzae-MCD-LFB assay showed, in the pure cultures, the minimum concentration of genomic DNA templates was 100 fg. The specificity of H. influenzae-MCD-LFB assay showed only H. influenzae templates were detected, and no cross-reactivity was found in non-H. influenzae isolates and other Haemophilus species. In 56 sputum samples, with MCDA-LFB method and PCR detection, 21 samples were positive, which was in consistent with the traditional culture method. The accuracy of diagnosis of MCDA-LFB, in comparison with the traditional culture method and PCR detection, can reach 100%, indicating that the MCDA-LFB assay gains an advantage over the cultured-based method for target pathogen detection. In conclusion, the MCDA-LFB assay is suitable for the sensitive, rapid, and specific detection of H. influenzae, which might be used as a potential diagnostic tool for H. influenzae in basic and clinical laboratories.

Keywords: Haemophilus influenzae; MCDA-LFB; nanoparticle-based biosensor; potential diagnostic tool; rapid detection.

Figure 1

The MCDA primers designed based on the OMP P6 gene. The location of the 10 primers were marked with different colors. Right arrows and left arrows indicated sense and complementary sequences.

Figure 2

Identification and confirmation of H. influenzae-MCDA products. (A) The visible color changes of amplification products of H. influenzae-MCDA assay were analyzed by VDR. (B) The products of H. influenzae-MCDA were visually detected with Lateral flow biosensor. Tube 1/biosensor 1, positive amplification of H. influenzae strain; tube 2/biosensor 2, negative control of Staphylococcus aureus strain; tube 3/biosensor 3, negative control of Escherichia coli strain; tube 4/biosensor 4, blank control (double-distilled water, DW).

Figure 3

Amplification temperature optimization for H. influenzae-MCDA assay. The MCDA reaction for detection of H. influenzae was monitored by real-time turbidimeter, and the corresponding curves of concentrations of DNA were showed in the pictures. The threshold value was 0.1, and the turbidity of > 0.1 was considered positive. Eight kinetic graphs (A–H) were acquired at a series of temperatures from 5 to 65°C (with 1°C intervals) with DNA templates of H. influenzae at the level of 1 pg per reaction. Signal 1, positive amplification of H. influenzae strain; signal 2, negative control of Staphylococcus aureus strain.

Figure 4

Sensitivity of H. influenzae-MCDA-LFB assay with serially diluted genomic DNA templates. Two measurement assays, namely, real-time turbidity (A) and later flow biosensor (B), were applied for detecting MCDA products. The serial dilutions of target DNA templates (10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg) were used for sensitivity analysis. Turbidity signals (A) / Biosensors (B) 1–8, respectively, represent the DNA levels of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg per reaction. The DNA levels of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg per reaction generated the positive reactions.

Figure 5

Specificity of H. influenzae-MCDA-LFB assay with DNA templates from distinct strains. The MCDA reactions were conducted with different genomic DNA templates and were analyzed by means of visual mode including VDR (A) and later flow biosensor (B). 1–7, H. influenzae strains; 8–9, H. parainfluenzae; 10, H. haemolyticus; 11, H. parahaemolyticus; 12, Enteroadhesive E. coli;13, Enterohemorrhagic E. coli; 14, Enteropathogenic E. coli; 15, Enteroinvasive E. coli; 16, Enterotoxigenic E. coli; 17–18, Listeria monocytogenes; 19–20, Pseudomonas aeruginosa; 21, Streptococcus suis; 22–23, Klebsiella pneumoniae; 24, Staphylococcus aureus; 25, Staphylococcus epidermidis; 26, Staphylococcus haemolyticus; 27, Acinetobacter baumannii; 28, Enterococcus faecalis.

Figure 6

Sensitivity of MCDA-LFB assay for detecting H. influenzae in clinical samples. Later flow biosensor was applied for detecting MCDA amplicons. The numbers 2, 5, 8, 11, 18, 22–25, 27, 28, 32, 33, 37–39, 41, 47, 50, 54, 55 represented the positive results. Other numbers represented the negative results.

Figure 7

PCR assay for detecting H. influenzae in clinical samples. The numbers 2, 5, 8, 11, 18, 22–25, 27, 28, 32, 33, 37–39, 41, 47, 50, 54, 55 represented the positive results. Other numbers represented the negative results.