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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 × 10 4  copies/mL genomic DNA within 60 min. Considering that more than 80% infected patients have 1.0 × 10 5 −1.0 × 10 7  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.

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