Reagent kit for detecting human prostatic cancer micro-metastasis by using real-time fluorescence quantitative RT-PCR technology

A real-time fluorescence quantitative and RT-PCR technology, which is applied in biological testing, microbial determination/inspection, measuring devices, etc., to achieve the effects of reducing false positive rate, improving detection sensitivity, and improving specificity

Inactive Publication Date: 2007-07-11
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These research results show that real-time quantitative RT-PCR technology can effectively detect micrometastasis of prostate cancer, but there is no relevant detection kit available, so the development of this kit is of great significance for the detection of micrometastasis of prostate cancer

Method used

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  • Reagent kit for detecting human prostatic cancer micro-metastasis by using real-time fluorescence quantitative RT-PCR technology
  • Reagent kit for detecting human prostatic cancer micro-metastasis by using real-time fluorescence quantitative RT-PCR technology
  • Reagent kit for detecting human prostatic cancer micro-metastasis by using real-time fluorescence quantitative RT-PCR technology

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Embodiment 1 Fluorescent real-time quantitative RT-PCR detects the application of PSA and PSMAmRNA in human peripheral blood mononuclear cells

[0063] 1. Materials

[0064] Trizol reagent, UNIQ-10 column, N 6 Random primers, dNTPs were purchased from Shanghai Bioengineering Co., Ltd., pMD 18-Tvector cloning system, Taq DNA polymerase was purchased from Dalian Bao Biotechnology Co., Ltd., M-MLV reverse transcriptase was purchased from Promega Company of the United States, and 2400 PCR instrument was purchased from From Perkin Elmer Company, MJ Opticon 2 fluorescent real-time quantitative PCR instrument was purchased from MJ Company of the United States.

[0065] 2. Design and synthesis of primers and probes

[0066] Using the full-length cDNA sequences of PSA, PSMA, and GAPDH (GeneBank accession numbers are X05332, M99487, and NM 002046) as templates, use Primer Express TM (V1.0, Perkin Elmer, USA) software analyzes primers and TaqMan probe sites, and simultaneously ...

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Abstract

The prostate cancerometastasis agent box uses a timely RT-PCR technique, starting from the erythrocytoschisis to separate the surrounding blood individual cell, extracting the overall RNA of each cell through Trizol, reversely transcribing the single cell RNA into CDNA No. 1 chain, achieving timely quantitative RT-PRC inspection through TaqMan detective needle. It improves the sensitivity and astopic feature of PSA and PSMAmRNA inspection, effectively getting over the defects of normal RT-PCR extension, with the precision could detecting one prostate within 107 single cells. This inspection method, design lead, detective needle and clinical sample inspection result provides reliable reference for the development of scheduled quantitative PCR inspection agent box.

Description

technical field [0001] The invention relates to a kit for detecting micrometastasis of human prostate cancer, which mainly involves isolating mononuclear cells from peripheral blood of patients, extracting total RNA, reverse transcribing the mRNA therein to obtain cDNA, and using real-time fluorescent quantitative RT-PCR technology to obtain cDNA through quantitative It is a kit for detecting the expression of prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) mRNA in prostate cells dissociated in peripheral blood to detect micrometastasis of prostate cancer, which belongs to biotechnology field. Background technique [0002] Metastasis of malignant tumors is a major problem in clinical treatment and an important cause of death from malignant tumors. Therefore, early diagnosis is the key to the correct choice of clinical treatment. Currently commonly used clinical imaging methods, such as transrectal ultrasound, CT, and MRI, can only detect nodul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/00G01N33/48C12Q1/68
Inventor 张琚石建党张立国杨睿符蓉杜小玲王克明徐勇强万明
Owner NANKAI UNIV
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