| BackgroundTuberculosis is a still deadly infectous illness, the pathogen of which has latently infected in one-third of the global population. Even one-third of patients with tuberculosis have not been diagnosed and gotten any treatment. Nowadays detection of mycobacterium tuberculosis still relys on bacteriological tools, such as culture and sputum smear, but these methods can not meet the need of clinical patients. A major focus of the WHO’s global plan to stop tuberculosis is the development of a simple and cost-effective diagnositic method to improve case detection. So there is an urgent need to diagnose, treat tuberculosis early and control the epidemic by Point-of-Care Test with high sensitivity and low cost, based on rapid and specific molecular target. The objective of this study is to identificate and evaluate novel molecular targets for TB diagnosis through combining bioinformatics with the experimental method, which are high sensitive and specific, independent on instruments.Methods1. This study detected SNPs (single nucleotide polymorphism) and Indels (insertion/ deletion) of the essential genes in Mycobacterium tuberculosis through the analysis of the complete genome sequence of Mycobacterium tuberculosis strains, then screened essential genes with the incidence of SNPs and indels as low as possible, as potential molecular diagnostic targets.2. The diagnostic potentiality of clpP2 gene in Mycobacterium tuberculosis was evaluated in detailed by bioinformatics methods. Nucleotide composition, codon bias, variability, DNA polymorphism of clpP2 gene and possibility that HGT event of clpP2 gene occurs were analyzed. The gene evolution tree based on the clpP2 gene, was constructed and evaluated about its distinguish ability between Mycobacterium tuberculosis and non-tuberculosis mycobacteria. By homology comparison analysis, one of bioinformatics methods, species-specificity of clpP2 gene target was evaluated.3. Isothermal amplification method, an alternative method of polymerase chain reaction, can be used for Point-of-Care test. In this study, based on mycobacterium tuberculosis clpP2 gene target, visual loop mediated isothermal amplification inspection (also as clpP2-LAMP) was established. Optimization of clpP2-LAMP reaction conditions, evaluation of the sensitivity and specificity of the method, and the application in clinical specimens have been perfomed.4. Bioinformation speculated that ClpP2 protein of Mycobacterium tuberculosis for tuberculosis diagnosis should have some specificity. Mycobacterium tuberculosis ClpP2 protein was successfully expressed in prokaryotic expression system, and polyclonal antibody prepared. Subcellular localization of ClpP2 in M. bovis BCG strain Pasteur was investigated by SDS-PAGE and Western blot analysis. To investigate the influence of stress conditions on ClpP2 expression, the gene mRNA expression in M. bovis BCG was assessed with quantitative real-time PCR under limiting growth conditions. The immune characteristics of ClpP2 protein were analysed. Also, ClpP2 protein and antibody level in sera of patients with tuberculosis were determinated to application value was evaluated in clinical practice.Results1.615 essential genes in Mycobacterium tuberculosis H37Rv genomes were downloaded from the Database of essential genes. GMTV (Genome-wide Mycobacterium tuberculosis variation) database analysed these essential gene sequence for SNPs and indels. Essential genes with minimal levels of polymorphisms(the incidence of SNPs<1% and indels<1%), were regarded as diagnostic potential targets. At last 45 essential genes were screened.2. Comparised with the Mtb clpP2 gene sequence, the genome of non-Mtb pathogenic microorganism in the respiratory tract has not homologous sequence, then level of the amino acid sequence homology between them was 50% or so. The results indicated that clpP2 gene as a molecular diagnostic target has good specificity. We constructed clpP2 gene phylogenetic tree, the topology structure of which is similar to that of 16S rRNA, but its evolutionary distance is greated than that of 16S rRNA. So the phylogenetic tree based on clpP2 gene indicates that NAA targeting clpP2 gene can be used to distinguish Mycobacterium tuberculosis from non tuberculosis mycobacterium.3. Based on Mtb clpP2 gene, visual and convenient LAMP method for detecting tuberculosis was established successfully. The optimal reaction conditions of LAMP was found through some series tests. The sensitivity of clpP2-LAMP is higher than PCR by 100 times, and the assay was testified in 105 strains with a specifity 100%. The clpP2-LAMP method was used for detection of 152 clinical samples directly, whose results are 98% identity with that of "gold standard" culture method.4. The recombinant Mtb ClpP2 protease has successfully been cloned and expressed in Escherichia coli. Mtb ClpP2 protease has strong immunogenicity, and can stimulate the generation of polyclonal antibodies, and can be used for the detection and diagnostic test. Indirect ELISA showed that ClpP2 antigen and antibody levels were increased in patients with lung tuberculosis. The area under the curve of ROC for diagnosis tuberculosis on ClpP2 antigen is 0.911 with a sensitivity 72.2% and a specificity 91.3%.ConclusionMtb clpP2 gene is highly conservative, species specific, a new potential target for tuberculosis diagnosis by bioinformatics analysis, which is a complement to the known molecular markers. LAMP method for TB detection based on Mtb clpP2 gene, and serology diagnostic method based Mtb ClpP2 protease show high application value in clinical specimens. |
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