Detection method and primers for TMPRSS2-ERG gene in human urine

A urine, amplification primer technology, applied in biochemical equipment and methods, microbial determination/examination, DNA/RNA fragments, etc., can solve the problems of uneven detection efficiency and reliability, and improve the diagnostic accuracy , good reliability, high repeatability effect

Inactive Publication Date: 2015-05-13
SHANGHAI RUNDARONGJIA BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the detection reagents and amplification primers used in the detection process of this method are very random, resulting in uneven detection efficiency and reliability.

Method used

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  • Detection method and primers for TMPRSS2-ERG gene in human urine
  • Detection method and primers for TMPRSS2-ERG gene in human urine
  • Detection method and primers for TMPRSS2-ERG gene in human urine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] A method for detecting TMPRSS2-ERG gene in human urine:

[0034] 1. Collection and processing of urine samples

[0035] The traditional prostate massage method is used, that is, the examiner makes a digital rectal examination, touches the prostate on the front wall of the rectum, massages symmetrically from the left and right sides to the central groove three times, and then massages the central groove from the bottom of the prostate to the tip three times, and then instructs the patient to urinate and collect Initial urine 50ml, immediately put into cold water to cool. Centrifuge at 2500r / min at 4°C for 5 minutes, collect the urine sediment, and add an appropriate amount of pre-cooled PBS washing solution twice. Collect the urine sediment in a 1.5ml centrifuge tube, add Trizol reagent and repeatedly blow and mix, and put it in a -80°C refrigerator for later use.

[0036] 2. Total RNA Extraction

[0037] Thaw the above urine sediment sample added with Trizonl at 4°C....

Embodiment 2

[0068] A kit for the diagnosis of prostate cancer, comprising:

[0069] (1) Kit for collecting urine sediment containing prostate cells, including: digital rectal examination urine collection tube, PBS washing solution, 1.5ml centrifuge tube, Trizol reagent;

[0070] (2) total RNA extraction kit in urine, including: chloroform, isopropanol, 75% ethanol, absolute ethanol, DEPC water;

[0071] (3) A kit for detecting the expression of TMPRSS2-ERG and PSA genes, including a reverse transcription reaction solution and a fluorescent PCR reaction solution. Wherein the reverse transcription reaction solution includes 5×Buffer, reverse transcriptase, dNTP and DEPC water; the fluorescent PCR reaction solution includes 5×Buffer, dNTP, TaqMen enzyme, primers and DEPC water as shown in SEQ ID NO:3-8;

[0072] (4) Calculation formula of urinary TMPRSS2-ERG score in the sample: TMPRSS2-ERG score=(TMPRSS2-ERG mRNA / PSA mRNA)×100,000.

Embodiment 3

[0074] Utilize the test kit of embodiment 2 to detect clinical samples:

[0075] The initial urine after prostate massage was taken from male patients sent to the Department of Urology, including 52 cases of prostate cancer, aged 49-78 years, with an average age of 65.6 years; 27 cases of urinary stones without prostate disease were used as normal controls, aged 37-46 years old, with an average of 41.3 years old.

[0076] Urine total RNA was extracted according to the method described in Example 1, and the urinary TMPRSS2-ERG score was detected by fluorescent PCR method. Each sample was repeated 3 times, and positive, negative, and blank controls were made at the same time. Make judgments based on the scoring results.

[0077] The TMPRSS2-ERG score of the control group was 4.0±1.9, and that of the prostate cancer group was 40.2±11.5. TMPRSS2-ERG scores were significantly different between the two groups, P<0.001.

[0078] The kit uses TMPRSS2-ERG score of 40.0 as the criti...

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Abstract

The invention relates to the technical field of gene detection and provided a method for extracting total RNA from urine of a subject and detecting TMPRSS2-ERG mRNA and PSA mRNA as well as primers which specifically amplify the TMPRSS2-ERG mRNA and PSA mRNA, wherein the primers comprise two groups of PCR amplification nucleic acid primers for detecting the TMPRSS2-ERG mRNA and PSA mRNA. The lengths of the primers are 17-24bp, and the primers specifically amplify a TMPRSS2-ERG mRNA fragment and a PAS mRNA fragment. The ratio of the TMPRSS2-ERG mRNA / PAS mRNA is measured and a TMPRSS2-ERG score is calculated by virtue of an amplification result, so that the purpose of early diagnosis of prostatic cancer and estimation of prognosis is realized.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and primers for extracting total RNA in human urine and detecting TMPRSS2-ERG gene. Background technique [0002] Prostate cancer is the most common malignant tumor of the male reproductive system, which seriously threatens men's health. With the advent of China's aging society, the incidence of prostate cancer will further increase. Therefore, research on the prevention, diagnosis and treatment of prostate cancer is very important. Significance. Tumors are often accompanied by chromosomal structural abnormalities, among which fusion genes caused by chromosomal translocations are the most common. TMPRSS2-EST type gene fusions are most common in prostate cancer and show diversity of fusion types, with TMPRSS2-ERG occurring most frequently [1] . [0003] Transmembrane protease serine 2 (Transmembrane protease serine 2, TMPRSS2) belongs to the type II transmembran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/158
Inventor 钱学庆
Owner SHANGHAI RUNDARONGJIA BIOLOGICAL TECH CO LTD
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