Matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS)

MALDI—TOF-MS mainly produces different track spectra under the action of magnetic field according to the bacterial protein, and compares them with the spectrum in the database to obtain the result of bacterial species identification. The combination of QPCR and MALDI-TOF remains a suitable method for timely diagnosis of mycobacterium species. This may ultimately help detect cases missed by phenotypic testing and enhance NTM diagnostic capabilities to effectively improve patient management [11]. The 16SrRNA gene fragment in molecular detection technology is sensitive, and its main defect lies in that some non-tuberculate mycobacterium species cannot be detected. However, as its relevant database is the most complete at present, it is recommended for routine use, and the data obtained by this method for NTM strain identification are of clinical value and epidemiological significance. Therefore, rapid identification of Mycobacterium tuberculosis complex and NTM is the key to appropriate treatment [12].

Gene chip technology

The common chip technologies are DNA microarray method and Liquid chip (Suspension array). The known sequence amplified by FLUORESCENCE labeling PCR is used for qualitative and quantitative analysis by hybridizing signal with oligonucleotide probe on the chip. The fluxes of the two microarray methods were higher in the detection of drug resistance genes and identification of bacterial strains. Although the identification ability was weaker than that of direct gene sequence analysis, the synthesis of nucleic acid probes was prone to errors or impurities, which reduced the specificity of hybridization. However, this technology is simple to operate, capable of identifying common clinical NTM, and can solve most clinical problems [13]. The whole process of the chip method used in this study only takes 4 hours [14]. After initial screening by chip hybridization, 16S rRNA gene sequencing was selected for confirmation. Although the selected 16S rRNA gene identification ability is relatively low, due to its most complete relevant database at present, it is recommended for routine use [15]. Currently, there are few detection items available for solid-phase chip, so it is difficult to be widely used.

Sanger Sequencing

The clinical radiological characteristics of this disease and tuberculosis (TB) were confirmed and identified by intra transcriptional spacer gene sequencing in 16S-23S rRNA. Patients with active disease were treated by NTM species and followed up to determine clinical, radiological, and microbiological outcomes [16]. With the development of molecular biology technology, DNA sequencing identification method has become the gold standard for bacterial species identification. DNA sequencing identification method for mycobacterium species identification is based on certain specificity of certain homologous DNA fragments of different mycobacteria. This method has high resolution and short detection time. DNA sequence number of clustering analysis is the first to use DNA sequence method for the identification of mycobacteria strains, the appearance of DNA sequence database makes DNA sequence contrast identification of mycobacterium species no longer need to professional and technical personnel can also be measured, some open public information database platform to enrich the database itself, such as America's national centre for biological information (NCBI), However, some scholars found that NCBI has some identification error sequences, incomplete sequences and unnamed sequences, which can affect the accuracy of identification results [17]. Due to the shortcomings of traditional detection methods such as low sensitivity and long time, misdiagnosis or missed diagnosis is easy to be caused to some patients. Therefore, whether to provide rapid, sensitive, specific, reliable, simple and suitable for clinical application of laboratory examination technology has become the focus of research at home and abroad. First-generation sequencing has the advantages of sensitivity, specificity and rapidity, which can improve the detection rate of TUBERCULOSIS bacilli and play an important role in promoting the prevention and treatment of tuberculosis in China and the world.

Metagenomics Next Generation Sequencing (NGS)

NGS can be analyzed by genomics method to extract DNA from the environment, which can cover the entire genome instantly without prior knowledge of specific pathogens. It is suitable for mycobacterium species that are not easy to find, but its accuracy depends on accurate extraction of high concentration OF DNA or RNA and efficient removal of host genes. Although second-generation sequencing can make up for the limitations of traditional identification technology, it is difficult to popularize widely due to its high price, high requirements for identification operation and limited research environment. A rapid, simple, specific and sensitive tuberculosis diagnostic test can accurately identify the existing technology can't detection of mycobacterium tuberculosis strains, the itself patients with lung basic diseases such as tuberculosis, the defects in advance by means of screening for genetic genes or gene screening to regular follow-up, prevent prompt action, it is of great help and significance to clinical diagnosis and treatment [18].

Prospect

China is a country with a high burden of TB, and the burden of non-tuberculous mycobacterium (NTM) disease is also increasing worldwide, but its diagnosis is still delayed and mistaken for multi-drug resistant tuberculosis (MDR-TB) [19] The proportion of diseases caused by non-tuberculous mycobacterium is increasing, long-term ineffective treatment and continuous TB transmission. It seriously affects the prevention, control and management of TUBERCULOSIS and poses a major threat to human health. However, the lack of an organized monitoring system for NTM, such as that for TUBERCULOSIS, still delays its diagnosis and is easily misdiagnosed as multidrug-resistant tuberculosis (MDR-TB) [20].

Existing application of species identification technology, the traditional test method of overall existence one-sided sex, although the sensitivity is high, the operation is simple and the price is low, but time-consuming, specificity and reliability is low, can't close relatives of accurate ruled out identification of strains of mycobacterium tuberculosis, however, because of its low cost, and still is the core of all diagnostic methods, it is widely used in clinical practice. With increasing attention paid to Non-Tuberculous Mycobacterium (NTM) disease, especially in health infection outbreaks, researchers are increasingly developing molecular biotechnology and gene bank testing systems to detect and control NTM at low cost. Maldi-tof-ms [21], PCR amplification and dot hybridization are relatively efficient and reliable identification methods with high resolution, good repeatability and short detection time. These new technologies greatly improve the accuracy of NTM detection. In addition, it has been applied in some large hospitals and laboratories [22]. However, comprehensive data on patient susceptibility, dominant species and drug resistance profile are also needed in order to improve treatment plan and management of NTM. Although only a limited number of antibiotic NTM show sensitivity, the resistance of isolates is highly variable, so more caution should be taken in the empirical treatment of NTM infection [23]. At present, there are still many non-tuberculous mycobacterium disease-causing strains that cannot be identified with model specificity. Therefore, rapid identification of NTM from MTBC and specific identification of NTM are essential for correct treatment and correct patient management.

Therefore, the Sanger sequencing mentioned in this paper can solve the problems of time-consuming, complex operation and low cost. In the future, a simple, rapid, convenient and reliable routine laboratory NTM species identification technology will be established. This method can be used to identify the most common NTM from lung samples in developing countries [24]. Due to the complex identification mechanism of non-tuberculous mycobacteria, which is not completely clear and difficult to determine, there is still a lack of very effective and accurate methods. The above methods have shown the advantages of detection in experimental studies, but more studies are needed to explore the clinical application and specific implementation methods. There is still a long way to go in the research and selection of methods to identify non-tuberculous mycobacteria. It is believed that in the near future, people can conquer the disease caused by non-tuberculous mycobacteria, which seriously affects human health.