Biomarker for diagnosing bladder cancer and monitoring recurrence and detection kit

A biomarker and detection reagent technology, applied in the fields of biotechnology and medicine, can solve the problems of unsatisfactory sensitivity and specificity, and achieve the effect of avoiding pain and high sensitivity

Active Publication Date: 2019-04-05
北京恩泽康泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to urine cytology, the current diagnosis and detection of bladder cancer recurrence through urine examination mainly include urinary

Method used

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  • Biomarker for diagnosing bladder cancer and monitoring recurrence and detection kit
  • Biomarker for diagnosing bladder cancer and monitoring recurrence and detection kit
  • Biomarker for diagnosing bladder cancer and monitoring recurrence and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] The separation method of embodiment 1 urine vesicle

[0088] 1. UC method: 40ml human morning urine sample, centrifuged at low speed to remove cells, centrifuged at 17000g for 30min to remove cell debris and apoptotic bodies, etc., ultracentrifuged at 100000g for 2h, removed supernatant, resuspended in PBS and centrifuged again at 100000g for 2h, resuspended in 100ul PBS Vesicles were obtained from the suspended pellet.

[0089] 2. Ultrafiltration method: After the sample is thawed, centrifuge at 17000×g for 30min; after centrifugation, the sample passes through 0.45um0.22um in turn; prepare two ultrafiltration centrifuge tubes, and take 15ml of the sample filtered by the filter membrane in each ultracentrifuge tube, Centrifuge at 4500×g for 15 minutes at room temperature; carefully remove the centrifuge tube, discard the liquid at the bottom of the ultrafiltration tube, then resuspend the retentate with 5ml of the remaining filtered urine and 10ml of 1XPBS, invert 3-4 ...

Embodiment 2

[0094] Example 2 Screening of differentially expressed genes

[0095] In order to screen urinary vesicle markers related to bladder cancer, 15 cases of bladder cancer patients (including newly diagnosed bladder cancer patients and patients with postoperative recurrence) and controls (normal subjects and patients without postoperative recurrence) were selected. The morning urine should not be less than 50ml, and the vesicles in the urine were separated by classical ultracentrifugation method and RNA was extracted, and the obtained RNA was subjected to miRNA and long RNA library sequencing (including mRNA, LncRNA and CircRNA). The obtained data were analyzed by bioinformatics, and the differentially expressed RNAs in bladder cancer patients and controls were compared, and correlation analysis was performed with the miRNA sequencing and transcriptome sequencing data published in TCGA (https: / / cancergenome.nih.gov / ), The screened differential RNAs from urine vesicles derived from ...

Embodiment 3

[0098] Example 3 Verify the primers of selected gene markers by real-time fluorescent PCR

[0099] 1. Design and synthesis of primers and probes

[0100] Primers and probes for 11 bladder cancer markers and 3 internal reference genes listed in Table 1 were designed in combination with Primer Premier 5 software and Primer-BLAST (NCBI). The design principles are as follows: 1) The amplified fragment is less than 150bp; 2) At least one primer spans the exon-exon boundary; 3) The Tm value of the probe is at least 5°C higher than that of the primer. The designed primers and probe sequences were synthesized by the company, in which the 5' end of the probe was labeled with FAM group, and the 3' end was labeled with BHQ1.

[0101] Table 2 Bladder cancer markers and primer probe sequences (SEQ ID NO: 1-42) of internal reference genes

[0102]

[0103]

[0104] *:Reference gene.

[0105] 2. Reverse transcription and qPCR detection

[0106] Using Takara's PrimeScript TM RT rea...

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Abstract

The invention provides a biomarker for diagnosing bladder cancer and monitoring recurrence and a detection kit. The biomarker is a gene UBE2C and at least one of genes BIRC5, UBE2C, CDK1, MMP11, TPX2,CDC20, MYBL2, TK1, FOXM1, CCNB1 and UCA1. The biomarker and a combination thereof, provided by the invention, can be used for accurately confirming whether an individual has the bladder cancer or notand has the possibility of recurrence or not, and has relatively high sensitivity; when the ACU of the performance of a recurrence monitoring model reaches 0.955 and the sensitivity and the NPV are 100 percent, the specificity can reach 80 percent or more; when the AUC of the performance of a diagnosis model reaches 0.99 and the sensitivity and the NPV are 98 percent or more, the specificity reaches 92 percent. The invention provides a noninvasive detection method based on urine detection and the pain of a patient frequently utilizing a cystoscope can be effectively avoided.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and in particular relates to a biomarker and a detection kit for bladder cancer diagnosis and recurrence monitoring. Background technique [0002] Bladder cancer is the most common tumor in the genitourinary system in my country. The peak incidence is between 60 and 69 years old. In recent years, with the acceleration of industrialization and changes in personal diet, smoking and other living habits, the incidence of bladder cancer has been increasing year by year and becoming younger. trend. Cystoscopy and cytology are still the main techniques for the diagnosis of bladder cancer, but the former is invasive and expensive, while the latter has low sensitivity. Transurethral resection of bladder tumor (TUR-BT) can simultaneously diagnose and treat bladder cancer, and clarify the pathological stage. It is the first choice for the treatment of bladder cancer. -70% of patients will relapse...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/158
Inventor 孔关义孙圣坤陈苏红秦宏亮刘翔陈亚庆李鸿艳
Owner 北京恩泽康泰生物科技有限公司
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