Rapid diagnosis of Mycoplasma pneumonia infection by denaturation bubble-mediated strand exchange amplification: comparison with LAMP and real-time PCR

Abstract

M. pneumoniae infection is often ignored due to its similar clinical symptom with respiratory tract infections caused by bacteria or viruses, and thus leading to misdiagnosis and delayed treatment. It is critical to develop a rapid, sensitive and specific diagnosis method. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was established, which is an isothermal method with only a primer pair and one Bst DNA polymerase. Notably, colorimetric SEA assay was developed with simple visual readout, making instrument-independent in detection step. The method could detect as low as 1.0 × 10⁴ copies/mL genomic DNA within 60 min. Considering that more than 80% infected patients have 1.0 × 10⁵-1.0 × 10⁷ copies/mL M. pneumonia DNA, SEA is available for the practical diagnosis of M. pneumoniae in clinical specimens. Through comparing 224 sputum specimens, excellent performance of SEA assay with 90.48% sensitivity and 100% specificity relative to real-time PCR was observed. Compared with LAMP, a comparable sensitivity and low false positive rate was observed for SEA method. Therefore, SEA is a promising method for detecting M. pneumoniae directly from clinical specimens, which is especially suitable for point-of-care testing in primary care facilities and resource-limited settings with minimal equipment and technological expertises.

Figure 1

Schematic illustration of SEA assay for M. pneumoniae infection in clinical specimens.

Figure 2

The feasibility of SEA to detect M. pneumoniae. (A) The real-time fluorescence curves. (B) Native PAGE of the corresponding amplification products (Supplementary Fig. S3. Lane 1, lane 2 and lane 6) (C) Colorimetric SEA assay using a pH sensitive dye as the indicator. 1 represented the vector plasmid DNA containing 16S rDNA sequence of M. pneumoniae M129, 2 represented genomic DNA extracted from sputum specimen infected by M. pneumoniae, 3 represented no template control, and M represented the 20-bp ladder marker (TaKaRa).

Figure 3

The sensitivity of SEA to detect M. pneumoniae. (A) The real time fluorescence curves with target sequence concentration of 1.0 × 10⁸ (1), 1.0 × 10⁷ (2), 1.0 × 10⁶ (3), 1.0 × 10⁵ (4), 1.0 × 10⁴ (5) and 0 (6) copies/mL. (B) The linear relationship between the threshold time (T_t) value and the logarithm of target sequence concentration (copies/mL).

Figure 4

Distribution of threshold time (T_t) values of M. pneumoniae detection in clinical samples by SEA, LAMP and real-time PCR assay. For real-time PCR, T_t value was the corresponding time of cycle threshold (C_t). The Box-plot contained the mean, inter quartile range, non-outlier range, and outlier.