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PCA3 gene detection method and primer in human urine

A urine, amplification primer technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc. High repetition stability and damage reduction effect

Inactive Publication Date: 2015-04-22
上海润达榕嘉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, 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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  • PCA3 gene detection method and primer in human urine
  • PCA3 gene detection method and primer in human urine
  • PCA3 gene detection method and primer in human urine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] A method for detecting PCA3 gene in human urine:

[0032] 1. Collection and processing of urine samples

[0033] 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.

[0034] 2. Total RNA Extraction

[0035] Thaw the above urine sediment sample added with Trizonl at 4°C.

[00...

Embodiment 2

[0067] A kit for the diagnosis of prostate cancer, comprising:

[0068] (1) Collect urine sediment reagents and utensils containing prostate cells, including: digital rectal examination urine collection tube, PBS washing solution, 1.5ml centrifuge tube, Trizol reagent;

[0069] (2) Reagents for extracting total RNA in urine, including: chloroform, isopropanol, 75% ethanol, absolute ethanol, DEPC water;

[0070] (3) Reagents for detecting the expression of PCA3 and PSA genes, including reverse transcription reaction solution and fluorescent PCR reaction solution. Wherein the reverse transcription reaction solution includes 5X Buffer, reverse transcriptase, dNTP and DEPC water; the fluorescent PCR reaction solution includes 5X Buffer, dNTP, TaqMen enzyme, primers and DEPC water as shown in SEQ ID NO:3-8;

[0071] (4) Calculation formula of urine PCA3 score in the sample: PCA3 score=(PCA3 mRNA / PSA mRNA)×1000.

Embodiment 3

[0073] Utilize the test kit of embodiment 2 to detect clinical sample:

[0074] The initial urine after prostate massage was collected from male patients sent to urology department. Among them, 37 cases of prostate cancer, aged 39-78 years, with an average age of 70.6 years; 68 cases of benign prostatic hyperplasia, aged 59-87 years, with an average age of 68.2 years; Patients with urinary calculus of prostate disease were used as normal controls, aged 30-43 years old, with an average of 36.3 years old.

[0075] Urine total RNA was extracted according to the method described in Example 1, and the urine PCA3 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. Judgment based on scoring results.

[0076] The PCA3 score of the control group was 13.5﹢8.93, that of the benign prostatic hyperplasia group was 30.56﹢38.91, and that of the prostate cancer group was 158.89﹢118.25. There ...

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Abstract

The invention relates to the technical field of gene detection and provides a method for extracting total RNA from urine of a subject and detecting PCA3 mRNA and PSA mRNA and a primer for performing specific amplification on PCA3 mRNA and PSA mRNA. The primer comprises two groups of PCR amplified nucleic acid primers for detecting the PCA3 mRNA and PSA mRNA, the length of each primer is 18-26bp, and the PCA3 mRNA fragment and PSA mRNA fragment are respectively and specifically amplified. According to the amplification result, the ratio of PCA3 mRNA to PSA mRNA is measured, and the PCA3 grade is calculated, so that the aims of realizing early diagnosis of prostatic cancer and reducing prostate puncture as much as possible are achieved.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and primers for RNA extraction and PCA3 gene detection in human urine. Background technique [0002] Prostate cancer is one of the common malignant tumors in the male genitourinary system. In 2012, the incidence rate of prostate cancer in my country's tumor registration areas was 9.92 / 100,000, ranking sixth in the incidence rate of male malignant tumors. The age of onset is at a low level before the age of 55, and gradually increases after the age of 55. The incidence rate increases with age, and the peak age is 70-80 years old [1] . [0003] At present, serum prostate cancer specific antigen (PSA) level is mainly used for early detection of prostate cancer clinically. [2] . PSA is a serine protease secreted by prostate epithelial cells, and its half-life is about 3.15 days. The normal range of PSA of 0-4ng / ml commonly used in clinical practice is the standard...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/158
Inventor 钱学庆
Owner 上海润达榕嘉生物科技有限公司
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