Establishment And Application Of Method For Identification And Rapid Differentiation Of Non-tuberculosis Mycobacteria Based On Single-cell Raman Spectroscopy

Non-tuberculosis mycobacteria（NTM）are the collection of mycobacteria other than Mycobacterium tuberculosis complex（MTC）and Mycobacterium leprosy.In recent years,the incidence of human infection caused by NTM has been increasing.The clinical symptoms of NTM are very similar to MTC infection,yet their treatments are different.Due to the long culture cycle of NTM（more than one week）and difficult to identification,thus rapid and accurate detection methods of NTM are urgently needed.Single-cell Raman Spectroscopy（SCRS）is a spectroscopic detection technology based on the principle of light scattering to detect the composition characteristics of molecular chemical bonds in substances.SCRS has been applied to analyzing biological samples in recent years due to its advantages of label-free,culture-free,rapid,accurate and low cost.Heavy water（D₂O）labeling technology is the use of heavy water in the metabolic process of cells,causing the hydrogen（¹H）in the cell to be replaced by the isotope deuterium（²H）.SCRS is used to detect the ratio of deuterium atoms to hydrogen atoms（C-D ratio）in the cell during this process,which can quantify the metabolism of the cell.This labeling technology does not change the chemical composition of the cell and does not affect the cell.Metabolic ability,process reversibility and other advantages.But so far,there is no research on the application of SCRS to the rapid detection of NTM in China,and there is no report on the combination of SCRS and heavy water（D₂O）labeling technology to quickly identify NTM through the difference in metabolic rate.In this study,we proposed a method for the identification of NTM based on confocal single-cell Raman spectroscopy and Support Vector Machine.The results showed that the identification model using SVM as the NTM classifier had a higher accuracy（98.8%）for the identification of NTM and was more suitable for the identification of Mycobacterium species.In the process of SCRS detection after NTM culture with heavy water（D₂O）short time,we found that rapid-growing mycobacteria（RGM）and slow-growing mycobacteria（SGM）have difference in metabolic rate in a short time（8h）,the heavy water content can be detected by SCRS to distinguish the living cells of RGM and SGM in the sample,helping the clinic to quickly select drugs.This study we establishment a method for the identification of NTM based on SCRS,and a method for using heavy water labeling combined with SCRS to quickly detect samples and differentiate NTM living cells.Both methods can be used in rapid detection of NTM at different levels,and provide a new tool for assisting clinical diagnosis and treatment of NTM quickly and accurately.