The Diagnostic Value Of KLF2,GBP5 And DUSP3 Gene In Active Tuberculosis And Latent Tuberculosis Infection

Objectives: To investigate the diagnostic value of Krüppel-like transcription factor 2(KLF2),guanylate-binding protein(GBP5)and dual specificity protein phosphatase 3(DUSP3)and tuberculosis score(GBP5+DUSP3 / 2-KLF2)in active tuberculosis(ATB)and latent tuberculosis infection(LTBI)through different blood samples,to provide a new idea for the diagnosis of ATB and LTBI.Methods: From May 1,2019 to December 31,2020,the hospitalized patients in the Department of respiratory and critical care medicine,Affiliated Hospital of Qingdao University,Qingdao Huangdao tuberculosis prevention and Control Institute,and the normal physical examinees in the physical examination center of Affiliated Hospital of Qingdao University were selected as the research objects.Peripheral blood mononuclear cells(PBMC): 110 cases in ATB group,114 cases in other pulmonary diseases group(OPD),52 cases in LTBI group and 63 cases in healthy control group(HC);peripheral whole blood(WB): 62 cases in ATB group,103 cases in OPD group,58 cases in LTBI group,117 cases in HC group and 102 cases in ATB group after treatment.The real-time PCR(RT-PCR)was used to detect the relative expression of GBP5,DUSP3 and KLF2 genes in each group.T-A cloning was used to construct standard plasmids containing the target genes of GBP5,DUSP3 and KLF2,and the RNA standards of GBP5,DUSP3 and KLF2 genes were synthesized by T7 transcription in vitro.The standard curves of g GBP5,DUSP3 and KLF2 genes were constructed by RT-PCR after reverse transcription,and the absolute expression levels of target genes in PBMC of whether groups were determined.Receiver operating characteristic curve(ROC)was used to analyze the diagnostic efficacy of GBP5,DUSP3 and KLF2 genes and TB score in ATB and LTBI.Results: 1.By comparing the constructed pMD18-T-KLF2,pMD18-T-GBP5,pMD18-T-DUSP3 plasmid sequences with target gene sequences,the results showed that KLF2 and DUSP3 were completely consistent with the sequencing results,and there was only one base mutation in GBP5 gene.2.The melting curves of GBP5,DUSP3 and KLF2 gene standards showed a single peak state.The Intra-assay and Inter-assay were less than3%.The correlation coefficient of the standard curve was close to 1.0,and the amplification efficiency was close to 100%.3.In PBMC,the expression of GBP5,DUSP3 and KLF2 genes in ATB group was significantly higher than that in HC group(P <0.05),and the expression of DUSP3 and GBP5 genes in OPD group was significantly higher than that in HC group(P < 0.05).The expression of DUSP3 gene in LTBI group was significantly higher than that in HC group(P < 0.05).4.The area under the curve(AUC)of DUSP3 gene,TB score and KLF2 gene detected by relative quantitative detection in PBMC were 0.962,0.929 and 0.724 respectively when ATB versus HC,and the AUC scores of DUSP3 gene and TB score in ATB versus LTBI were 0.805 and 0.771,respectively.And the AUC scores of absolute quantitative detection of DUSP3 gene and TB score in ATB versus HC groups were 0.910 and 0.749,respectively.5.In peripheral whole blood,the relative expression of KLF2 gene in ATB group,LTBI group,OPD group and pulmonary tuberculosis treatment group was significantly lower than that in HC group(P < 0.05),while the relative expression of DUSP3 and GBP5 gene in ATB group,LTBI group and OPD group was significantly higher than that in HC group(P <0.05),but there was no significant difference when pulmonary tuberculosis treatment group compare with HC group(P > 0.05).6.In the results of peripheral whole blood detection,the AUC for distinguishing ATB and HC,KLF2,GBP5,DUSP3 and TB score were 0.979,0.763,0.859 and 0.999 respectively,the AUC for LTBI versus HC,KLF2,DUSP3,and TB score were 0.970,0.722 and 0.970 respectively,the AUC for distinguishing ATB and OPD,KLF2,DUSP3,and TB score were 0.814,0.743 and 0.892,and the AUC for ATB versus LTBI,DUSP3,and TB score were 0.780 and 0.847,respectively.Conclusion: When genes of Peripheral whole blood were detected by relative quantification RT-PCR,KLF2 gene and TB score have high diagnostic efficiency in differentiating ATB from OPD,and can be used to differentiate LTBI from HC group.TB score can distinguish ATB from LTBI.KLF2,GBP5,DUSP3 and TB score can be used to distinguish ATB and HC group.