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

Abstract

Haemophilus influenzae is an opportunistic pathogen usually causing bacteremia, meningitis, and pneumonia in children. Here, we developed a method based on multiple cross displacement amplification (MCDA) method and real-tme fluorescence technique for rapid detection of H. influenzae. A set of 10 primers was designed for the H. influenzae real-time MCDA reaction, and a core primer was modified with a restriction endonuclease recognition sequence, a fluorescent, and a quencher according to the principle of the real-time MCDA assay. The H. influenzae real-time MCDA reactions were performed using a fluorescence instrument at 63°C for 40 min. The H. influenzae real-time MCDA assay can specifically detect H. influenzae without any cross-reaction with other bacteria as our results confirmed. The sensitivity of our assay is as low as 10 CFU per reaction. To validate its feasibility, our assay was applied to 40 DNA extracted from sputum samples. The results obtained from H. influenzae real-time MCDA were compared with that of H. influenzae-loop-mediated isothermal amplification (H. influenzae-LAMP) and MCDA-based lateral flow biosensor (MCDA-LFB). The positive rate of the real-time MCDA assay was 62.5%, which was consistent with the H. influenzae-MCDA-LFB assay, but was more sensitive than H. influenzae-LAMP (57.5%). Furthermore, the H. influenzae real-time MCDA assay takes only 40 min, which was less than that of a traditional PCR test. Taken together, the H. influenzae real-time MCDA assay reported here offers a new and valuable diagnostic tool for the reliable and rapid detection of H. influenzae.

Keywords: Haemophilus influenzae; PCR; loop mediated isothermal amplification; multiple cross displacement amplification; rapid diagnosis; real-time.

Figure 1

The location of the primer sequences used in this study on the targeting outer membrane protein (OMP) P6 gene of H. influenzae. Right and left arrows show sense and complementary sequences, respectively. The colored text indicates the position of primers, including two displacement primers (F1 and F2), two cross primers (CP1 and CP2), and six amplification primers (C1, C2, D1, D2, R1, and R2).

Figure 2

Effectiveness of the primer set for the H. influenzae-MCDA reaction. The DNA templates extracted from H. influenzae strains were effectively amplified with MCDA reaction at 65°C, and there is no reaction for the blank controls (DW).

Figure 3

Temperature optimization for H. influenzae-real-time MCDA assay. MCDA reactions detecting H. influenzae were conducted using real-time turbidimeter, and the kinetic curves at different temperatures ranging from 60 to 67°C were acquired, showing that 63°C was the optimal temperature.

Figure 4

Sensitivity confirmation of H. influenzae-real-time MCDA assay. Sensitivity of the H. influenzae-real-time MCDA assay was analyzed by using continuous dilutions from 2×10⁸ to 2×10¹ CFU/ml. The real-time MCDA reactions with various levels of DNA templates were repeatedly tested twice. Signals (A) representing DNA levels showed that the LoD of the H. influenzae-real-time MCDA was 10 CFU per tube. The sensitivity was also confirmed with indication of visual detection reagent (B) and LFB test (C) for the production of MCDA reactions. (C) TL, test line; CL, control line.

Figure 5

Specificity confirmation for H. influenzae-real-time MCDA assay. DNA templates from 2 H. influenzae strains and 28 non-H. influenzae strains were tested by the real-time MCDA assay. No reactions were recorded within the non-H. influenzae strains (A). The results were also confirmed by MCDA reactions, indicated by visual detection reagent (B) and LFB test (C). (B) Tube/Strips 1 and 2 represented two H. influenzae strains, and the other ones represented non-H. influenzae strains. (C) TL, test line; CL, control line.

Figure 6

Application of H. influenzae-real-time MCDA assay in clinical specimens. Forty NPS samples were detected by the H. influenzae-real-time MCDA assay and the results showed that 25 samples tested positive. The results were confirmed by the MCDA-LFB test simultaneously.