Objective To establish clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas12a technology combined with loop-mediated isothermal amplification (LAMP) single-tube one-step method for rapid detection of Mycoplasma pneumoniae (Mp), and to explore its application value in clinical diagnosis.
Methods Mp LAMP primers were designed and optimized to design Cas12a CrRNA according to LMAP product targets. The sensitivity of CRISPER-LAMP single-tube one-step method for Mp DNA detection was analyzed; the Mp, influenza virus A (FluA), influenza virus B (FluB), Klebsiella pneumoniae (Kp), Streptococcus pneumoniae (Sp) and 50 clinical samples were detected, and the specificity of CRISPER-LAMP single-tube one-step method was evaluated.
Results Mp DNA could be visualized within 1 hour by CRISPER-LAMP single-tube one-step method, with a detection limit of 10 fg/μL and a detection limit of 100 fg/μL by polymerase chain reaction (PCR); the FluA, FluB, Kp and Sp were all negative detected by CRISPER-LAMP single-tube one-step method; the detection results of 50 clinical samples were consistent with the clinical diagnosis results, and the consistency between CRISPER-LAMP and PCR method was good (Kappa=1).
Conclusion CRISPER-LAMP single-tube one-step method for Mp detection is simple, rapid, sensitive and specific. It does not need any special equipment, and there is no aerosol pollution in the detection process. It is expected to become a new method for rapid detection of Mp.
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