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Objective To screen the specific cytokines of tuberculous pleural effusion(plTB)by using liquid array technique to establish a diagnostic model and discuss its application value.Methods A total of 86 patients with plTB(plTB group)were included,including 41 patients in the confirmed plTB group and 45 patients in the clinically diagnosed plTB group.There were 42 other patients with pleural effusion in the control group.Seventeen cytokines in pleural effusion were analyzed by liquid array technology.Interleukin(IL)-1β,IL-2,IL-4,IL-5,IL-6,IL-8,IL-9,IL-10,gamma-interferon-induced protein 10(IP-10),IL-15,IL-17F,IL-27,tumor necrosis factor(TNF)-α,monocyte chemotactic protein-1(MCP-1),the expression levels of macrophage inflammatory protein-3a(MIP-3α),macrophage colony-stimulating factor(M-CSF)and β-interferon(IFN-β)were detected.Difference factors between the confirmed plTB group and the control group were screened,and the receiver operating characteristic(ROC)curve was drawn in the confirmed plTB patients.IP-10,IL-27 and MCP-1 with AUC>0.850 and specificity>80%were combined to diagnose plTB,and were compared with adenylate deaminase(ADA)and T-SPOT.TB in pleural effusion to evaluate the diagnostic efficacy.Results The levels of IL-2,IP-10,IL-27,TNF-α and MCP-1 were higher in the confirmed plTB group than those in the control group(P＜0.05).The sensitivity and specificity of IP-10,IL-27 and MCP-1 in the diagnosis of plTB were 87.8%and 81.0%.The sensitivity of three-factor combined diagnosis in 45 patients with plTB was still as high as 86.7%,and there was no significant difference in sensitivity compared with that in the diagnosed plTB group(P＞0.05).In the plTB group,the sensitivity of IP-10,IL-27 and MCP-1 combined detection was 87.2%,which was higher than that of T-SPOT.TB(81.4%)and ADA(54.7%).Conclusion The application of liquid array technology to the joint detection of pleural effusion IP-10,IL-27 and MCP-1 can provide help for the diagnosis of plTB.
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Objective:To explore the feasibility of U6 and Cel-miR-39 as reference genes for quantitative detection of microRNA (miRNA) in cerebrospinal fluid (CSF) of tuberculous meningitis (TBM), and validate the difference of miRNAs between tuberculous and viral meningitis (VM).Methods:The remaining CSF specimens after routine examination were collected in Beijing Chest Hospital of Capital Medical University. A total of 36 TBM and 34 VM patients were enrolled based on the information in the medical records. Total RNA were extracted from the CSF samples, and Taqman based real-time quantitative PCR (RT-CR) analysis were performed to determine the concentration of the miRNAs in CSF. GeNorm, NormFinder and Bestkeeper software were used for stability analysis of the two reference genes. 2 -ΔCt method was used to determine the relative gene expression. Accordance of repeated tests was analyzed by Pearson correlation test. Continuous variables were compared by the t-test. Results:Among the 70 samples, the average cycle threshold (Ct) value of U6 was 30.40±3.30, while the average Ct value of Cel-miR-39 was 21.49±0.70. The expression level of Cel-miR-39 was higher than that of U6. Correlation analysis showed good accordance of the repeated tests among the reference genes and target genes analysis in the randomly selected 10 samples ( r>0.931, P<0.001). Based on the analyses results of the three software, including GeNorm, NormFinder and Bestkeeper, Cel-miR-39 presented better stability in RT-PCR analysis and was more suitable as a reference gene for miRNA quantitative determination in CSF sample of TBM patients. The relative expression levels of the three target miRNAs were calculated using Cel-miR-39 as the reference gene, and miR-126-3p (1.13±0.41 vs 3.34±0.82, t=2.452, P=0.016), miR-130a-3p (0.56±0.10 vs 2.59±0.70, t=2.960, P=0.004) and miR-151a-3p (0.64±0.25 vs 2.11±0.33, t=3.536, P<0.001) were showed significant lower expression levels in CSF in TBM group than that in VM group. Conclusions:Cel-miR-39 can be used as a reference gene for quantitative detection of miRNAs in CSF of TBM patients. Significant differences were detected in expression level of miR-126-3p, miR-130a-3p and miR-151a-3p between TBM and VM group.
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Objective·To construct recombinant mycobacteriophage TM4-RpfE to lay a foundation for experimental research about how to eradicate Mycobacterium tuberculosis in combination with anti-tuberculosis drugs,and how to shorten treatment for tuberculosis ultimately.Methods·Electrotransformation was used to introduce pJV53 plasmid into Mycobacterium smegmatis to prepare recombinant engineering bacteria.After amplification of hsp60-RpfE fusion gene by overlap PCR,a long gene fragment (homologous +hsp60-RpfE+homologous,HHRH) was amplified by multi-step overlap PCR.The DNA of mycobaeteriophage TM4 and HHRH fragment were cotransfected into the recombinant engineering bacteria by electrotransformation,then the recombinant phage from the single primary plaques were confirmed by PCR and sequencing.SDS-PAGE was used to analyze the protein expression in recombinant phage.Results·The hsp60-RpfE fusion gene at the length of 901 bp and HHRH fragment at the length of 1 873 bp were identified by overlap PCR.The PCR product produced 955 bp and 301 bp DNA bands in the first generation plaques colony.SDS-PAGE analysis showed a specific protein band at 21 000 in the recombinant phages.Conclusion·The recombinant mycobacterium phage TM4-RpfE was successfully constructed and the expression of target gene RpfE was initially verified.
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Objecflve To evaluate the performance of two rapid and low-cost metheds(MTT test,and rosazurin mierotitre assay)for the detection of resistance to first-line drugs in Mycobacterium tuberculosis.Methods sixty-four Myeobaeterium tuberculosis clinical isohtes were tested by the MTT test and the rosazuxin microtitre assay(REMA)respectively,and the results were compared with those obtained with the absolute concentration method on L(o)wenstein Jensen medium.Results The MTT test and the resazurin microtitre assay showed a good agreement compared with the absolute concentration method for all first-line drugs tested.The sensitibity,specificity and accuracy of the MTT test were 94.8%,96.0%,95.3%,for RFP;93.8%,93.8%,93.8% for INH;92.9%,96.O%,95.3% for EMB,90.6%,87.5%,89.1% for SM,respectively.The sensitivity,specificity and accuracy of the resazurin microtitre assay were 92.3%,96.0%,93.8%,for RFP;90.6%,90.6%,90.6% for INH;92.9%,94.0%,93.8% for EMB,87.5%,87.5%,87.5% for SM,respectively.The Kappa value of the MTT test and the absolute concentration method for the detection of resistance to RFP,INH,EMB,SM were 0.857,0.831,0.714,0.792.respeedvely;The Kappa value of the regazurin mierotitre assay and the absolute concentration method for the detection of resistance to RFP,INH,EMB,SM were 0.871,0.826,0.826,0.750,respectively.The Kappa value of the MTT test and the resazurin microtitre assay for the detection of resistance to RFP,INH,EMB,SM wefe 0.889,0.875.0.787,0.844,respectively.Conclusions Both MTT test and the resazurin microtitre assays are simple,rapid,low-cost and sensitive for rapid detection of resistance to first-line drugs.They could be promising methods for susceptibility assay of the first-line antituberculosis drugs in low-resource countries.