| BACKGROUND:Chronic kidney disease(CKD)is one of the chronic diseases affecting human health in the world,and there is still a lack of accurate early diagnosis methods.mRNA detection could be an effective method for noninvasive diagnosis of early chronic kidney disease(CKD),Previously,through bioinformatics and gene expression screening,we found a series CKD related urinary mRNA,however,SYBR two step method of PCR detection of urine mRNA is time-consuming which limit the clinical validation of candidate biomarkers of large sample.The aim of this study was to develop one step fluorescence quantitative PCR Taqman kit,to improve the detection sensitivity,specificity and sensitivity.Besides,clinical sample validation study was perfrormed using the kit to find new diagnostic markers for CKD.METHODS:Based on the screening the specific expression of mRNA for patients with chronic kidney disease(CKD)in a previous study,we selected three mRNA gene,PODXL IL1RL1 and CCL2.The gene sequence was downloaded from NCBI for the 3 target genes and B2M(internal control)and Taqman probe for fluorescence quantitative PCR kit was established.The sensitivity,specificity and reproducibility of kits were evaluated.Urine samples were collected,and the urine levels of PODXL IL1RL1 and CCL2 mRNA were measured using the kit,and the diagnostic efficacy of the markers was evaluated.RESULTS:Based on big data analysis,we constructed the characteristic transcriptome expression profile of common CKD(DKD,HN,FSGS,MN and Michigan).In addition,we identified 176 differential expressed genes related with glomeruli and 50 differential expressed genes related with the tubules for CKD compared to controls.By optimizing the components and reaction conditions of the PCR system,the TaqMan probe one-step fluorescence quantitative PCR kit developed by us can realize effective amplification and quantitative detection of four kinds of mRNA in urine,including PODXL,IL1RL1,CCL2 and B2M(internal reference gene).The variation coefficient of this kit was less than 0.5%,with good detection stability,and the corresponding mRNA expression level could be detected from the urine sediment mRNA of the system as low as 0.5ng/10 ul.The whole detection process can be completed within 1 hour,which greatly reduces the detection time compared with the traditional two step PCR method.The constructed one-step kit was used for clinical verification of the previously screened differentially expressed genes,and it was found that the levels of PODXL mRNA,IL1RL1 mRNA and CCL2 mRNA in the urine of DKD patients were higher than those of the healthy control(HC)group.There was no difference in the expression level of the three genes in GN patients compared with the normal group.There were significant differences in PODXL mRNA,IL1RL1 mRNA between HC group and DKD stage III and DKD stage IV group.However,there was no difference in the expression level of CCL2 mRNA between HC group and DKD group.The levels of PODXL mRNA,IL1RL1 mRNA and CCL2 mRNA in the urine of DKD patients were not correlated with eGFR and serum creatinine,but were significantly positively correlated with 24-hour urine protein and ACR.The area under ROC curve(AUC)of PODXL to distinguish DKD from control group was 0.7879(p<0.0001)(specificity 87.5%,sensitivity 61.22%),and the AUC of IL1RL1 was 0.8249(p<0.0001)(specificity 93.75%,sensitivity 59.18%).The AUC of CCL2 was 0.7181(p=0.001)(specificity 81.25%,sensitivity 57.14%).CONCLUSIONS:The one-step real-time fluorescence quantitative detection method based on TaqMan probe constructed in this study has good sensitivity,specificity and reproducibility for urine gene expression detection by RT-PCR.This kit can be used for validation of urinary mRNA biomarker with large samples of CKD,and it was found that PODXL,IL1RL1 and CCL2 mRNA in urine may become new diagnostic markers for DKD. |
