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Pretreatment method and application of large-volume urine

A pretreatment and large-volume technology, applied in biochemical equipment and methods, microbial measurement/testing, DNA preparation, etc., can solve the problems of increasing complexity, time-consuming, and reducing the detection sensitivity of patients with urothelial carcinoma

Pending Publication Date: 2022-04-15
THE THIRD XIANGYA HOSPITAL OF CENT SOUTH UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extraction of urine increases the complexity of the experiment, is time-consuming, and most importantly, since only a small portion of urine is taken for testing, this greatly reduces the risk of early or ultra-early urinary tract disease containing trace amounts of cancer-specific DNA. Sensitivity of detection in skin cancer patients

Method used

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  • Pretreatment method and application of large-volume urine
  • Pretreatment method and application of large-volume urine
  • Pretreatment method and application of large-volume urine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Urine pretreatment, removal of inhibitors and enrichment of cell-free DNA

[0037] Take two samples A and B of blood and urine from patients with non-urothelial carcinoma, each 300 mL. Each was divided into three portions, each 100mL. Numbered A-S1, A-S2, A-S3, B-S1, B-S2, B-S3 respectively.

[0038] The artificially synthesized positive TERT sequence to be detected is as follows:

[0039] TERT(-124 / -146):

[0040] GGAGAGGGCGGGGCCGCGGAAAGGAAGGGGAGGGGCTGGGAGGGCCCGGAAGGGGCTGGGCCGGGGACCCGGAAGGGGTCGGGACGGGGCGGGGTCCGCGCGGAGGAGGCGGAGCTGGAAGGTGAAGGGGCAGGACGGGTGCCCGGGTCCCCA, as shown in SEQ ID NO.1;

[0041] The sequences of QPCR primers and probes are as follows:

[0042] TERT124-F: GGAGAGGGCGGGGCCGCGGA, as shown in SEQ ID NO.2;

[0043] TERT124-R: GGGTCCCCGGCCCAGCCCCT, as shown in SEQ ID NO.3;

[0044] TERT124-P: FAM-AAGGAAGGGGAGGGGCTGG-BHQ1, as shown in SEQ ID NO.4;

[0045] TERT146-F: GGCTGGGCCGGGGACCCGGA, as shown in SEQ ID NO.5;

[0046] TERT146-R: TGGGGACCCGGGCAC...

Embodiment 2

[0074] In order to achieve the purpose of reducing costs, instead of using detection probes, mutation detection can be performed by gel electrophoresis.

[0075] Three hematuria samples A, B and C were taken from patients with non-urothelial carcinoma, 100 mL each.

[0076] 人工合成单阳性TERT(-124)和双阳性TERT(-124 / -146)待检测序列如下:TERT(-124):GGAGAGGGCGGGGCCGCGGAAAGGAAGGGGAGGGGCTGGGAGGGCCCGGAAGGGGCTGGGCCGGGGACCCGGGAGGGGTCGGGACGGGGCGGGGTCCGCGCGGAGGAGGCGGAGCTGGAAGGTGAAGGGGCAGGACGGGTGCCCGGGTCCCCA,如SEQ ID NO.11所示;

[0077] TERT(-124 / -146):

[0078] GGAGAGGGCGGGGCCGCGGAAAGGAAGGGGAGGGGCTGGGAGGGCCCGGAAGGGGCTGGGCCGGGGACCCGGAAGGGGTCGGGACGGGGCGGGGTCCGCGCGGAGGAGGCGGAGCTGGAAGGTGAAGGGGCAGGACGGGTGCCCGGGTCCCCA, as shown in SEQ ID NO.1;

[0079] The sequences of QPCR primers and probes are as follows:

[0080] TERT124-F: GGAGAGGGCGGGGCCGCGGA, as shown in SEQ ID NO.2;

[0081] TERT124-R: GGGTCCCCGGCCCAGCCCCT, as shown in SEQ ID NO.3;

[0082] TERT146-F: GGCTGGGCCGGGGACCCGGA, as shown in SEQ ID NO.5;

[...

Embodiment 3

[0103] 84 samples of clinically confirmed urothelial carcinoma with hematuria were tested.

[0104] A. Urine pretreatment:

[0105] Mix each 100mL portion of hematuria evenly and divide it into two 50mL BD tubes. Centrifuge at 800g in a refrigerated centrifuge for 10 minutes, remove the sediment, and transfer the supernatant to a new 50mL BD tube. Add 100 μL (20mg / mL) proteinase K, and digest in a water bath at 55°C for 30 minutes. Mix by inversion every 10 minutes.

[0106] B. Removal of inhibitors:

[0107] The urine pretreated samples were brought to room temperature. Add 35mg of PVPP (Ashland, #Polyplasdone XL, particle size 110-130um), and mix upside down on a vertical mixer for 30 minutes. Centrifuge at 1600 g for 5 minutes in a refrigerated centrifuge. Transfer the supernatant to a new 50 mL BD tube.

[0108] C. Cell-free DNA enrichment:

[0109] ① Prepare silica gel particles:

[0110] Take 250mg silica gel 60N (Kanto, #37565-79), 100mg powdered poly-lysine (p...

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Abstract

The invention discloses a pretreatment method and application of large-volume urine. According to the method, efficient and low-cost DNA enrichment can be carried out from a large amount of urine, and inhibitors, such as heme and urea, influencing subsequent molecular biological reaction in the hematuria are effectively removed, so that the enriched product is directly detected, and the purpose of quicker detection is achieved. The detection method and the detection kit provided by the invention can be used for accurately detecting 5 copies of urothelial carcinoma related mutant DNA molecules from 100mL of urine.

Description

technical field [0001] The invention relates to a pretreatment method and application of large-volume urine. The invention belongs to the technical field of gene detection. Background technique [0002] Urothelial carcinoma is a multisource malignant tumor originating from the urothelium and is the most common urinary system tumor. Urothelial carcinoma can be divided into non-muscle-invasive urothelial carcinoma and muscle-invasive urothelial carcinoma. However, 10-15% of patients with muscle-invasive urothelial carcinoma have metastasized at the time of diagnosis. The incidence of urothelial cancer in Western countries ranks fourth, second only to prostate cancer, lung cancer, and colorectal cancer. From the perspective of the site of occurrence, it includes renal pelvis cancer and ureter cancer in the upper urinary tract, and bladder cancer and urethral cancer in the lower urinary tract. Among them, upper urothelial carcinoma is rare, accounting for only 5-10% of uroth...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6806C12N15/10
Inventor 王龙徐根明李超王永利姚鲲刘建业鞠巍汤维李仁君李永祥
Owner THE THIRD XIANGYA HOSPITAL OF CENT SOUTH UNIV
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