Urine nucleic acid maker and detection reagent kit for assisting in early screening of prostatic cancer

A technology for prostate cancer and detection reagents, applied in the fields of molecular biology and medical diagnostics, can solve the problems of interfering with the relative expression level of nucleic acid markers, subtle deviations in the working efficiency of detection systems, and inability to eliminate deviations in detection equipment, and achieve good clinical applications. Prospect, ideal working performance, effect of guaranteed reliability

Active Publication Date: 2020-10-16
诺迦(杭州)生物工程有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, internal reference genes cannot eliminate the bias introduced by detection equipment
For example, the quantitative systems of nucleic acid markers in the above-mentioned patents work in different detection tubes, and the slight deviation in the matching degree between the detection tube and the detection equipment directly leads to slight deviations in the working efficiency of each detection system. After exponential amplification by polymerase chain reaction, it will have a non-negligible impact on the detection results
[0006] In addition, the above-mentioned patents all use a single internal reference gene for the calibration of ge

Method used

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  • Urine nucleic acid maker and detection reagent kit for assisting in early screening of prostatic cancer
  • Urine nucleic acid maker and detection reagent kit for assisting in early screening of prostatic cancer
  • Urine nucleic acid maker and detection reagent kit for assisting in early screening of prostatic cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 The detection limit of multiple fluorescent RT-PCR reagents, the operation steps are as follows:

[0048] Sequence synthesis: For transmembrane serine protease 2-ETS transcription factor related genes fusion gene (T2E fusion gene), prostate cancer antigen 3 gene (PCA3 gene) 1, matrix metalloproteinase 2 gene (MMP-2 gene), prostate specific antigen The five genes (PSA gene) and the ETS transcription factor (SPDEF gene) containing the SAM pointing domain are added with T7 promoter sequence and T7 terminator sequence respectively upstream and downstream of the five gene segments. The sequence information is delivered to Shenggong Bioengineering (Shanghai) Co., Ltd. (hereinafter referred to as "Shanghai Shenggong") to synthesize gene fragments de novo and insert the pcDNA3.1(-) plasmid.

[0049] 2. Linear plasmid preparation: According to the restriction enzyme cutting site downstream of the insertion position, the circular pcDNA3.1(-) plasmid is cut into linear plasmi...

Embodiment 2

[0059] Example 2 Multi-gene detection of urine samples assists in the identification of benign and malignant prostate diseases, the operation steps are as follows:

[0060] 1. Sample collection: 30 mL of initial urine of subjects undergoing prostate massage was collected from the clinical laboratory. Sixty urine samples were diagnosed with cancer by fine needle aspiration of the prostate, and 60 urine samples were cancer-excluded.

[0061] 2. Exosomes enrichment: Ultracentrifugation is used to enrich the exosomes in urine samples. The steps are as follows: centrifugation at 300g for 10 minutes to retain the supernatant to remove cell pellets; centrifugation at 2000g for 10 minutes to retain the supernatant to remove dead cell pellets; centrifugation at 10,000g for 30 minutes to retain the supernatant to remove cell debris pellets; centrifuge at 100,000 g for 90 Minutes, remove the supernatant and retain the exosomal pellet; add 15mL PBS buffer, wash and resuspend the exosomal pell...

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Abstract

The invention discloses a urine nucleic acid maker and detection reagent kit for assisting in early screening of prostatic cancer. The nucleic acid marker comprises three prostatic cancer related genes of a T2E fusion gene, a PCA3 gene and an MMP-2 gene, and two internal reference genes of a PSA gene and an SPDEF gene. A multiple fluorescence reverse transcription polymerase chain reaction (RT-PCR) detection reagent comprising the five nucleic acid markers can be used for assisting in screening of prostatic cancer, an area under curve (AUC) of a testee operating characteristic (ROC) curve canachieve 0.81, and sensitivity predicted value and negative predicted value are respectively as high as 91.6% and 93.3%. The detection reagent kit is simple to operate, and quantitative detection of the nucleic acid markers and screening of the prostatic cancer can be completed in one pipe of the reagent. Unnecessary puncturing inspection of prostate can be notably reduced, and the reagent kit hasfavorable clinical application prospects.

Description

Technical field [0001] The invention relates to the fields of molecular biology and medical diagnostics, in particular to a urine nucleic acid marker and a detection kit for assisting in the early screening of prostate cancer. Background technique [0002] With the aging of our country's population structure and the changes in people's living habits, prostate cancer has quietly become one of the common tumors that seriously threaten the health of men in our country. The survey results in 2018 showed that the total number of prostate cancer patients in my country was nearly 145,000, and the compound growth rate in the last five years was nearly 10% higher. It is the tumor type with the fastest growing incidence. If prostate cancer is detected early and treated in time, the five-year survival rate can reach more than 95%, while the five-year survival rate of advanced prostate cancer that has metastasized is less than 30%. Only one-quarter to one-third of newly diagnosed patients i...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/166C12Q2600/16C12Q2537/143C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 孙刚朱晓进范春雷李铭夫
Owner 诺迦(杭州)生物工程有限公司
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