Establishment of cross-priming amplification for point-of-care detection of Mycobacterium tuberculosis and non-tuberculosis mycobacteria

Abstract

ObjectiveTo simplify sputum sample preparation steps and achieve point-of-care testing (POCT) of Mycobacterium tuberculosis (MTB) and non-tuberculosis mycobacteria (NTM) using Cross-Priming Amplification (CPA) technology on portable devices, overcoming the challenges of existing nucleic acid detection technologies that cannot be widely promoted in grassroots settings in China. MethodsEvaluate the liquefying ability of high-concentration guanidine thiocyanate (GTC) for sputum and the effectiveness of MTB inactivation; establish a rapid detection system for MTB and NTM based on CPA technology using EasyNAT integrated detection tubes, with the left amplification zone specific to MTB CPA amplification and the right amplification zone specific to both MTB and NTM CPA amplification. Suspected pulmonary tuberculosis (PTB) patients or patients diagnosed as suspected NTM pulmonary infections specimens collected from the Second Hospital of Longyan, Fujian Province, from September 2022 to September 2023, acid-fast bacilli (AFB) smear, quantitative real-time PCR (RT-PCR) and CPA-POCT were performed. The kappa coefficients was used to evaluate the consistency between the RT-PCR and CPA-POCT. ResultsThe liquefaction effect of 6M GTC on sputum was equivalent to 4 % NaOH, and no MTB growth was observed in the Lowenstein-Jensen medium of sputum samples treated with 6M GTC incorporating the H37Rv strain. The newly established CPA-POCT method showed good agreement with RT-PCR with a positive compliance rate of 86.27 %, a negative compliance rate of 89.36 %, an overall compliance rate of 87.75 %, and a Kappa coefficients of 0.786 (P < 0.05). Conclusion6M GTC can liquefy sputum and render MTB non-viable, eliminating the need for Mycobacterium nucleic acid testing in BSL-2 laboratories; the newly established CPA method can rapidly and accurately distinguish MTB and NTM in the form of POCT, with simple and fast operation, suitable for promotion and application in grassroots medical institutions and remote rural areas.