Accurate, rapid and low-cost diagnosis of Mycoplasma pneumoniae via fast narrow-thermal-cycling denaturation bubble-medi
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RESEARCH PAPER
Accurate, rapid and low-cost diagnosis of Mycoplasma pneumoniae via fast narrow-thermal-cycling denaturation bubble-mediated strand exchange amplification Chen Yang 1 & Yang Li 1 & Jie Deng 1 & Mengzhe Li 1 & Cuiping Ma 2 & Chao Shi 1 Received: 17 July 2020 / Revised: 23 September 2020 / Accepted: 26 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
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.
Keywords ASEA . Mycoplasma pneumoniae . Rapid pathogen detection . Sputum specimens . Clinical diagnosis Abbreviations 2019-nCoV 2019 Novel coronavirus ASEA Accelerated strand exchange amplification CAP Community-acquired pneumonia LOD Limit of detection
NTC SEA T1 T2 Tm Tt
No template control Strand exchange amplification First temperature Second temperature Melting temperature Threshold time
Chen Yang and Yang Li contributed equally to this work. * Chao Shi [email protected] 1
Qingdao Nucleic Acid Rapid Testing International Science and Technology Cooperation Base, College of Life Sciences, Department of Pathogenic Biology, School of Basic Medicine, and Department of Clinical Laboratory, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao 266071, Shandong, China
2
Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Provincial Key Laboratory of Biochemical Engine
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