Accurate, rapid and low-cost diagnosis of Mycoplasma pneumoniae via fast narrow-thermal-cycling denaturation bubble-mediated strand exchange amplification

Abstract

Mycoplasma pneumoniae is a strong infectious pathogen that may cause severe respiratory infections. Since this pathogen may possess a latent period after infection, which sometimes leads to misdiagnosis by traditional diagnosis methods, the establishment of a rapid and sensitive diagnostic method is crucial for transmission prevention and timely treatment. Herein, a novel detection method was established for M. pneumoniae detection. The method, which improves upon a denaturation bubble-mediated strand exchange amplification (SEA) that we developed in 2016, is called accelerated SEA (ASEA). The established ASEA achieved detection of 1% M. pneumoniae genomic DNA in a DNA mixture from multiple pathogens, and the limit of detection (LOD) of ASEA was as low as 1.0 × 10^-17 M (approximately 6.0 × 10³ copies/mL). Considering that the threshold of an asymptomatic carriage is normally recommended as 1.0 × 10⁴ copies/mL, this method was able to satisfy the requirement for practical diagnosis of M. pneumoniae. Moreover, the detection process was finished within 20.4 min, significantly shorter than real-time PCR and SEA. Furthermore, ASEA exhibited excellent performance in clinical specimen analysis, with sensitivity and specificity of 96.2% and 100%, respectively, compared with the "gold standard" real-time PCR. More importantly, similar to real-time PCR, ASEA requires only one pair of primers and ordinary commercial polymerase, and can be carried out using a conventional fluorescence real-time PCR instrument, which makes this method low-cost and easy to accomplish. Therefore, ASEA has the potential for wide use in the rapid detection of M. pneumoniae or other pathogens in large numbers of specimens. Graphical abstract.

Keywords: ASEA; Clinical diagnosis; Mycoplasma pneumoniae; Rapid pathogen detection; Sputum specimens.

Graphical abstract

Fig. 1

Schematic illustration of M. pneumoniae detection by ASEA. The initial cycling was mediated mainly by the invasion of primers into denaturation bubbles formed between the duplex of M. pneumoniae genomic DNA, whereas the later cycling was triggered by both melting of amplicons and invasion of primers into denaturation bubbles

Fig. 2

T₁ and T₂ optimization of the ASEA method for detecting M. pneumoniae. (a) T1 optimization at the T₂ value of 61 °C. ASEA reactions were carried out at T₁ values of (1) 76 °C, (2) 75 °C, (3) 74 °C, (4) 73 °C and (5) 72 °C. 1′–5′ represent the corresponding NTC. (b) T₂ optimization at the T₁ value of 74 °C. ASEA reactions were carried out at T₂ values of (1) 61 °C, (2) 60 °C, (3) 59 °C, (4) 58 °C and (5) 57 °C. 1′–5′ represent the corresponding NTC. (c) T₁ optimization at the T₂ value of 58 °C. ASEA reactions were carried out at T₁ values of (1) 76 °C, (2) 75 °C, (3) 74 °C, (4) 73 °C and (5) 72 °C. 1′–5′ represent the corresponding NTC

Fig.3

(a) Feasibility and specificity of ASEA for M. pneumoniae detection with genomic DNA of M. pneumoniae and nine other common pathogens as template. 1 represents genomic DNA of M. pneumoniae, and 2–10 represent genomic DNA of S. pneumoniae, MRSA, E. coli, P. aeruginosa, M. tuberculosis, L. pneumophila, S. maltophilia, H. influenzae and A. baumannii, respectively. (b) Anti-jamming capability of ASEA for M. pneumoniae detection and (c) agarose gel electrophoresis image of corresponding ASEA products. 1 represents genomic DNA extract of M. pneumoniae, and 2–6 represent a mixture of the genomic DNA of the above pathogens with 50%, 10%, 5%, 1% and 0% genomic DNA of M. pneumoniae, respectively. The white dashed line frames the bands of 36-bp amplicons. M and NTC represent a 20-bp DNA ladder and no target control, respectively

Fig. 4

Sensitivity of ASEA for M. pneumoniae detection. (a) The fluorescence curves of ASEA reactions with the template of M. pneumoniae genomic DNA at concentrations of (1) 1.0 × 10⁻¹¹ M, (2) 1.0 × 10⁻¹² M, (3) 1.0 × 10⁻¹³ M, (4) 1.0 × 10⁻¹⁴ M, (5) 1.0 × 10⁻¹⁵ M, (6) 1.0 × 10⁻¹⁶ M, (7) 1.0 × 10⁻¹⁷ M and (8) 1.0 × 10⁻¹⁸ M. (b) Linear relationship between the threshold time and the negative logarithmic values of the concentration of M. pneumoniae genomic DNA (−lgC_DNA). Error bars represent the standard deviations of three independent measurements