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Liquid-phase chip method for detecting PCA3 gene and PSA gene and diagnostic reagent kit thereof

A diagnostic kit and liquid-phase chip technology, which is applied in the determination/examination of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of insufficient sensitivity, cumbersome operation, and poor repeatability.

Active Publication Date: 2011-01-12
邵棠
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Among the molecular biology methods widely used at home and abroad to detect specific genes related to prostate diseases, fluorescent quantitative PCR has the limitation of detection throughput, so it cannot really meet the needs of clinical diagnosis and detection.
The traditional solid-phase biochip (Biochip) technology has outstanding weaknesses such as poor repeatability, insufficient sensitivity, and cumbersome operations.

Method used

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  • Liquid-phase chip method for detecting PCA3 gene and PSA gene and diagnostic reagent kit thereof
  • Liquid-phase chip method for detecting PCA3 gene and PSA gene and diagnostic reagent kit thereof
  • Liquid-phase chip method for detecting PCA3 gene and PSA gene and diagnostic reagent kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1: Liquid-phase chip method for detecting PCA3 gene in urine with different primer and probe combinations

[0121] The specific detection method includes the following steps:

[0122] 1. Preparation of microsphere mixture for detecting PCA3 gene

[0123] 1. Synthesize oligonucleotide probes according to the following sequence:

[0124] For the mRNA of PCA3 gene:

[0125] Probe (1) 5'-AminolinkerC12 ATTTCTCACCTCTGTATCATC-3', as shown in SEQ ID NO.1;

[0126] Probes (2), (3), (5), (6) are the same as probe (1), as shown in SEQ ID NO.1;

[0127] Probe (4) 5'-AminolinkerC12 CTCACCTCTGTATCATCAG-3', as shown in SEQ ID NO.2; probe (7) 5'-AminolinkerC12 ATCTCTGTGCTTCCTTTTGT-3', as shown in SEQ ID NO.3;

[0128] Probe (8) is the same as probe (7), as shown in SEQ ID NO.3

[0129] Probe (9) 5'-AminolinkerC12 CAAATCTGTAATCCCGTTCA-3', as shown in SEQ ID NO. 4; Probe (10) 5'-AminolinkerC12 TATGTGTCAAGAGGAGAGCC-3', as shown in SEQ ID NO. 5;

[0130] β-actin gene: 5'-AminolinkerC12 TCATTGTAG...

Embodiment 2

[0250] Example 2: Liquid-phase chip method for detecting PSA gene in urine with different primer and probe combinations

[0251] The specific detection method includes the following steps:

[0252] 1. Preparation of microsphere mixture for detecting PSA gene

[0253] 1. Synthesize oligonucleotide probes according to the following sequence:

[0254] For the mRNA of PSA gene:

[0255] Probe A: 5'-AminolinkerC12 CAGAATCACCCGAGCAGGTG-3', as shown in SEQ ID NO.6;

[0256] Probe B: 5'-AminolinkerC12 GGGGTCAAGAACTCCTCTGG-3', as shown in SEQ ID NO.7;

[0257] Probe C: 5'-AminolinkerC12 CACGCTTTTGTTCCTGATGC-3', as shown in SEQ ID NO. 8;

[0258] Probe D: same as PSA probe A, as shown in SEQ ID NO.6;

[0259] Probe E: same as PSA probe B, as shown in SEQ ID NO.7;

[0260] β-actin gene: 5'-AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3', as shown in SEQ ID NO.41;

[0261] 2. Couple the oligonucleotide probes containing amino modification to 6 kinds of carboxyl microspheres numbered 11, 13, 15, 21, 25, 66

[0262]...

Embodiment 3

[0304] Example 3: Liquid-phase chip method for combined detection of PCA3, PSA and PCA3 / PSA ratio in urine

[0305] The specific detection method includes the following steps:

[0306] 1. Preparation of microsphere mixture for detecting PSA gene

[0307] 1. Synthesize oligonucleotide probes according to the following sequence:

[0308] PCA3: 5'-AminolinkerC12 ATTTCTCACCTCTGTATCATC-3', as shown in SEQ ID NO.1;

[0309] PSA: 5'-AminolinkerC12 CACGCTTTTGTTCCTGATGC-3', as shown in SEQ ID NO.8;

[0310] β-actin gene: 5'-AminolinkerC12 TCATTGTAGAAGGTGTGGTG-3', as shown in SEQ ID NO.41;

[0311] 2. Coupling the oligonucleotide probes containing amino modification to 3 kinds of carboxyl microspheres numbered 11, 13, and 66 respectively

[0312] 2.1 Take out a small portion of fresh dry powdered EDC stored at -20°C and equilibrate to room temperature;

[0313] 2.2 Use dH 2 O respectively dissolve the oligonucleotide probes of PCA3, PSA, β-actin, the concentration is 1mM (1nmol / μl);

[0314] 2.3 Vorte...

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Abstract

The invention discloses a liquid-phase chip method for detecting a PCA3 gene and a PSA gene and a diagnostic reagent kit thereof. A primer and a probe are designed for mRNA of the PCA3 gene and / or the PSA gene; a mixture, formed by the covalent bonding of the probe and microsphere, is hybridized with a reverse transcription P CR amplification product, after streptavidin phycoerythrin is added fluorescent signals of different microspheres can be detected to determine whether a sample to be detected contains the PCA3 and PSA genes and the expression condition thereof, and simultaneously, the PCA3 / PSA ratio can be obtained. The liquid-phase chip method and the reagent kit have the advantages of high sensitivity, high throughput, quick and accurate detection and the like, and can perform qualitative and quantitative detection on the PCA3 and PSA genes related to different degrees of prostate diseases including prostate cancer and benign prostatic hyperplasia and perform specific diagnosison the prostate cancer.

Description

Technical field [0001] The present invention relates to the technical field of in vitro diagnostic detection, in particular to a liquid-phase chip detection method for specific genes PCA3 and PSA related to prostate diseases and a diagnostic kit thereof. Background technique [0002] PCA3 (prostate cancer antigen 3) gene, also known as DD3 (differential display code 3) gene, is a gene that functions as a non-coding RNA discovered by Bussemakers in 1999 and consists of 4 exons and 3 introns. . The PCA3 gene is only expressed in prostate tissue, and its expression is extremely low or not expressed in normal prostate and benign prostatic hyperplasia (BPH) tissues, while it is highly expressed in prostate cancer (PCa) cells. Therefore, the PCA3 gene is currently considered to be the most specific gene for prostate cancer. [0003] Although prostate specific antigen (PSA) has been widely used in the screening, diagnosis, staging, efficacy observation and prognosis of prostate cancer, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
CPCC12Q1/6837C12Q1/6886C12Q2600/158
Inventor 邵棠
Owner 邵棠
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