PCA3 mRNA/ACPP mRNA RT-PCR detection primer and detection kit thereof

A detection kit, -CTCCTCAACATGAGAGCTGC-3 technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of poor diagnostic sensitivity and specificity of prostate cancer, so as to improve sensitivity and specificity, improve detection sensitivity, and diagnose The effect of high accuracy

Inactive Publication Date: 2015-09-02
谭巍
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The present invention proposes a PCA3mRNA / ACPP mRNA RT-PCR detection primer and detection kit, which solves the problem of poor diagnostic sensitivity and specificity for prostate cancer in the prior art

Method used

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  • PCA3 mRNA/ACPP mRNA RT-PCR detection primer and detection kit thereof
  • PCA3 mRNA/ACPP mRNA RT-PCR detection primer and detection kit thereof
  • PCA3 mRNA/ACPP mRNA RT-PCR detection primer and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] ACPPmRNA RT-PCR detection

[0046] 1. Design and synthesis of ACPPmRNA gene primers: Refer to the ACPPmRNA gene sequence provided by Genebank, and use the Genamic expression software to design 5' and 3' primers:

[0047] Upstream primer: 5'-CTCCTCAACATGAGAGCTGC-3'

[0048] Downstream primer: 5'-TATATACTCTCCAAGTTCATA-3'

[0049] After bioinformatics analysis and BLAST search, the above primers are highly specific and 100% specific, no homologous sequences were found, and they are very good primers.

[0050] 2. LNCaP cell culture and application: LNCaP cells are human prostate cancer cells, which can highly express ACPPmRNA, and can be used to study RT-PCR reaction conditions and conduct related experiments.

[0051] Recovery of LNCaP cells: first heat the water bath to 37°C, take out the cryopreservation tube and place it in a water bath (37°C) and shake until it melts and becomes a suspension. 75% alcohol sterilized the cryopreservation tube mouth, sucked out the cel...

Embodiment 2

[0087] Preparation of PCA3mRNA / ACPP mRNA RT-PCR Detection Kit

[0088] Reagent composition:

[0089]

[0090] The production steps are as follows:

[0091] 1) Prepare positive control:

[0092] LNCaP cell culture and application: LNCaP cells are human prostate cancer cells, which can highly express ACPPmRNA and PCA3mRNA, and can be used to study RT-PCR reaction conditions and conduct related experiments.

[0093] Recovery of LNCaP cells: first heat the water bath to 37°C, take out the cryopreservation tube and place it in a water bath (37°C) and shake until it melts and becomes a suspension. 75% alcohol sterilized the cryopreservation tube mouth, sucked out the cell suspension with a straw, poured it into a centrifuge tube and added 10 times more culture medium dropwise, mixed and centrifuged at 1000gX5min, and removed the supernatant. After appropriate dilution with the culture medium, the culture bottle was inoculated and placed in a 5% CO2 incubator at 37°C for static...

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Abstract

The invention provides a PCA3 mRNA/ACPP mRNA RT-PCR detection primer and a detection kit thereof. The detection primer comprises an upstream primer represented by 5'-CTCCTCAACATGAGAGCTGC-3' and a downstream primer represented 5'-TATATACTCTCCAAGTTCATA-3'. The detection kit comprises an RT-PCR reaction solution, MMLV RT/Hot start Taq Mix, a positive reference substance and a negative reference substance. The PCA3 mRNA/ACPP mRNA RT-PCR detection kit prepared in the invention is better than PCA3 mRNA/PSA mRNA RT-PCR combination detection kits, and the detection sensitivity of the detection kit prepared in the invention increases to above 90%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a PCA3mRNA / ACPP mRNA RT-PCR detection primer and a detection kit. Background technique [0002] ACPP (acid prostatic phosphatase,) encodes a non-specific tyrosine phosphatase that dephosphorylates a variety of substrates under acidic (PH4-6) conditions. ACPP substrates include alkyl, aryl, and acyl orthophosphate monoesters and phosphorylated proteins. Under the regulation of androgen, prostate epithelial cells synthesize ACPP and secrete it into semen. The expression of ACPP mRNA in the prostate is more than 100 times that of any other tissue. IHC staining with ACPP antibody showed obvious immunohistochemical staining reactions in both intracellular and intercellular in prostate tissue, proving that ACPP is a prostate-specific gene. ACPP protein can be detected in serum. In a pathological state, prostate epithelial cells will be shed and enter the blood and urine. Damaged prost...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 谭巍史镜宇王广银黄云坚马小伟
Owner 谭巍
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