| Background:From later period of1980s, along with the epidemic of human immunodeficiency virus (HIV) infection, the drug resistance of mycobacterium tuberculosis (MTB), the increase of multi-drug-resistant strains and the increase of floating population, the global TB incidence rate showed a rising trend again, especially TB drug resistance making treatment go from bad to worse. The main reasons for the heavy burden of TB were inappropriate detection and low rate of cure. Sputum smear as an active TB detection method was simple and fast, but it only applied to sputum samples of whose bacteria number was greater than104. Therefore, most of patients were SNP-TB patients. Although infectious strength of SNP-TB was inferior to smear-positive TB, it couldn’t be neglected in the spread and pathogenic aspects of tuberculosis. At present SNP-TB and latent infection of MTB had become a major problem in TB prevention, control and treatment. If SNP-TB didn’t get timely diagnosis, detection and treatment, it would lead to the further spread of tuberculosis, making prevention and control work face more challenges. Therefore, there was an urgent need for new methods of rapid, sensitive, efficient diagnosis and differential diagnosis of SNP-TB patients.Reverse dot blot hybridization (RDBH) technology including DNA probes, nucleic acid hybridization and enzyme-linked color, could be used for the detection of MTB and its drug-resistance genes from sputum specimen of SNP-TB patients. This technology mainly researched TB from genomics. The Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS) as an core technology of proteomics research, could screen out the differential proteins of serum samples from tuberculosis, pneumonia and healthy controls, which provides a theoretical basis for looking for serum potential markers revelant to SNP-TB in clinical. This study would use PCR-RDBH technology to test MTB and its drug-resistance genes and simultaneously utilize mass spectrometry to screen serum potential markers of SNP-TB patients.Objective:To study the feasibility of PCR-RDBH technology testing MTB and drug-resistance genes from sputum specimen of SNP-TB patients and use quantitative proteomics technology to screen serum potential markers of SNP-TB patients.Methods:(1) The IS6110gene and drug-resistant genes of sputum specimens including50cases with SNP-TB patients and18non-TB patients were tested by PCR-RDBH technology. The results were analyzed and compared with that of acid fast stain smear and drug sensitive test respectively.(2) Multiple Affinity Removal System (MARS)-Human14was used to deplete14species of high-abundant proteins in serum. After concentrating low abundance proteins, stable isotope113,119,121iTRAQ labeling, strong cation exchange column (SCX) separation, reversed-phase chromatograph separation combined with MALDI-TOF/MS were to detect70serum samples, which included30SNP-TB patients,10pneumonia patients and30normal controls. Serum protein peaks were analyzed. Potential biological markers associated with diseases were screened and analyzed by bioinformatics analysis software.Results:(1) The IS6110gene of sputum specimens including50cases with SNP-TB patients and18non-TB patients were tested by PCR-RDBH technology. The positive rate reached up to76.0%(38/50) in detecting MTB of sputum specimens from SNP-TB patients, which was significantly higher than sputum smear test. Both of the specificity of18non-pulmonary tuberculosis samples between sputum smear test and PCR-RDBH detection were100％(18/18).(2) The drug-resistant genes from38sputum samples with positive IS6110gene of MTB were detected using PCR-RDBH. The result showed that5cases had drug-resistant gene mutations which might imply drug-resistance to INH, RFP, SM. The resistant gene mutations of2sample suggested the presence of INH, RFP, SM resistance, which were in accordance with the result of sputum cultures+drug sensitive tests.3cases were unable to provide the results of drug sensitive tests because of negative-cultrues. The detection result of1case was different from that of drug sensitive tests.(3) By comparing the results of three independent and repetitive experiments, serum12species of high-abundant proteins were deplet effectively by MARS column. iTRAQ labeling combined with MALDI-TOF-MS quantitative proteomics technology were employed to compare the differential protein expressions in the serum of SNP-TB patients, pneumonia patients and normal controls. The confidential levels of271proteins were above95%among a total of344identified proteins. Compared with the serum of normal controls, expression levels of13proteins were significantly up-regulated in the serum of SNP-TB patients (P＜0.05), protein expression levels of34proteins and2proteins were significantly up-regulated and down-regulated in the serum of pneumonia respectively (P<0.05).(4)13common proteins showing significantly difference in the serum of SNP-TB and pneumonia patients were found, among which, the ratio of SNP-TB/pneumonia were more than2in expression levels of4proteins who were respectively Fibronectin, Antithrombin-Ⅲ, histidine-rich glycoprotein and Alpha-2-antiplasmin.(5) GO classification analysis of13differential proteins found that most of them mainly involved in response to stress, biological regulation and immune system process; The protein-protein interaction network analysis by STRING found that Fibronectin, Antithrombin-Ⅲ, histidine-rich glycoprotein and Alpha-2-antiplasmin were key nodes of the whole network which had taken place interaction with more than four differential proteins in the corresponding network.Conclusion:(1) PCR-RDBH technology is sensitive and differential, which can detect MTB and drug-resistant genes of sputum samples directly.(2) MARS column can deplete most of high-abundant proteins and keep more small molecular weight proteins which is suitable to serum treatment before mass spectrometry analysis.(3) iTRAQ labeling combined with MALDI-TOF-MS have a higher sensitive, higher specificity and good repeatability, which can detect directly and rapidly a small quantity of serum samples. It is an ideal platform for screening and studying serum differential proteins of diseases.(4) Fibronectin, Antithrombin-III, histidine-rich glycoprotein and Alpha-2-antiplasmin which basically participate in the lipid and vascular metabolic pathways may become serum potential biomarkers of SNP-TB. They may provide the basis and lay the foundation for later diagnosis and differential diagnosis of SNP-TB. |
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