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A kind of primer probe composition and application thereof

A technology of primer probe and composition, which is applied in the biological field to achieve the effect of high sensitivity

Active Publication Date: 2022-04-12
QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no mature digital PCR kit capable of quantitatively detecting the expression of AR-V7 on the market. Therefore, the development of a primer-probe composition and related detection kits that can accurately quantify AR-V7 based on digital PCR technology has broad potential. Application prospects and huge market value

Method used

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  • A kind of primer probe composition and application thereof
  • A kind of primer probe composition and application thereof
  • A kind of primer probe composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] In this embodiment, the plasmid detection system of the present invention is used to detect the system of the present invention. The AR-V7 plasmid template and the AR-FL plasmid template constructed by genetic engineering are each 1 strain, and the digital PCR detection of the present invention is carried out as follows:

[0072] (1) Plasmid construction and processing:

[0073]According to the human AR-V7 and AR-FL gene sequences (NM_001348061.1; NM_000044.4) published in the NCBI database, the AR-V7 plasmid and the AR-FL plasmid were constructed respectively; Photometer NanoDrop One was used for quality control and its concentration was determined. The two plasmids were mixed according to the ratio of AR-V7 / AR-FL at 1%, 0.6% and 0.1%, and then the plasmid concentration was adjusted to 5000 copies / uL. Take 2uL as a digital PCR template for PCR amplification, and do 2 replicates for each ratio of template.

[0074] (2) Establish a digital PCR amplification reaction sys...

Embodiment 2

[0086] In this embodiment, pseudoviruses are used to detect the system of the present invention. One strain of AR-V7 pseudovirus and one AR-FL pseudovirus constructed by bioengineering (commissioned to Xiamen Zhishan Biotechnology Co., Ltd. to prepare, with SEQ ID NO.17 and SEQ ID NO.17) The plasmid sequence of ID NO.18 synthesizes RNA and then includes the phage protein shell to form armored RNA and simulate the virus structure);

[0087] Utilize the present invention to implement digital PCR to detect as follows:

[0088] (1) Pseudovirus construction and processing:

[0089] According to the human AR-V7 and AR-FL gene sequences (NM_001348061.1; NM_000044.4) published in the NCBI database, the AR-V7 pseudovirus and the AR-FL pseudovirus were constructed respectively; Viral RNA was quality controlled with a micro-volume ultraviolet spectrophotometer, and its concentration was measured. The two pseudoviral RNAs were mixed according to the ratio of AR-V7 / AR-FL at 1%, 0.6% and 0...

Embodiment 3

[0103] In this embodiment, fresh tissue sample RNA is used to detect the system of the present invention, and 4 cases of clinical prostate cancer fresh tissue samples to be tested by our company are taken; the digital PCR detection is implemented by the present invention as follows:

[0104] (1) Sample processing and extraction:

[0105] No less than 30 mg of sample was taken, and RNA was extracted with the QIAGEN kit RNeasy mini kit, and the extraction steps were operated according to the kit instructions; the concentration of the extracted RNA was detected with a micro-volume ultraviolet spectrophotometer.

[0106] (2) cDNA synthesis:

[0107] Using Takara Kit PrimeScript TM II 1st Strand cDNA Synthesis Kit, using the extracted RNA as a template to synthesize cDNA; the specific operation steps are operated according to the kit instructions. The concentration of the obtained cDNA was detected by a micro-ultraviolet spectrophotometer.

[0108] (3) Establish a digital PCR a...

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Abstract

The present invention provides a primer-probe composition and its application. The composition includes four probes and twelve primers, wherein the nucleotide sequence of the primers is shown in SEQ ID NO.1-12, The nucleotide sequence of the probe is shown in SEQ ID NO.13‑16; the present invention designs specific primers and probes for the region of interest of androgen receptor V7, based on the primer probe composition, to explore Experimental conditions, optimized detection system, the two complement each other to achieve high sensitivity and strong specificity detection, specific quantitative detection of human AR‑V7 expression, wide range of detection samples, high detection sensitivity, convenient operation, simple and efficient, and broad application prospects and huge market value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer probe composition and its application. Background technique [0002] Prostate cancer is one of the most common malignant tumors of the urinary system. Androgen receptor (AR) plays an important role in the occurrence and development of prostate cancer. It promotes the progression and metastasis of prostate cancer by regulating the expression of downstream genes. Surgical treatment is still the first-line treatment for early prostate cancer, and the main treatment for advanced prostate cancer is endocrine therapy, namely androgen deprivation therapy (ADT). The U.S. Food and Drug Administration has approved a combination of the CYP17A1 inhibitor abiraterone and the potent antiandrogen enzalutamide as second-line treatments for metastatic prostate cancer. However, during the treatment of prostate cancer, it often develops drug resistance and develops into castration-resistant p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6851
CPCC12Q1/6851C12Q2563/107C12Q2531/113
Inventor 程雪涛吴子侠金丽琴张亚飞
Owner QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD