Primer probe composition, kit and detection method for detecting human AR-V7 and AR gene expression

An AR-V7 and gene expression technology, applied in the field of primer-probe compositions for detecting human AR-V7 and AR gene expression, can solve the problems of high technical requirements, cumbersome operations, and high sample requirements, so as to overcome incompatibility and The effect of technical difficulty, convenient transportation and storage, and long storage time

Inactive Publication Date: 2020-08-07
WUXI SHENRUI BIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are still many problems in the use of CTC samples for AR-V7 detection: (1) The sample requirements are high, and the samples must be separated from CTC within a few hours of collection; (2) The operation is cumbersome and the technical requirements are high, and the laboratory needs to be equipped with a CTC separation platform. CTC isolation must be completed before RNA extraction, which is difficult to carry out in clinical laboratories; (3) The detection sensitivity is poor. Due to the heterogeneity of tumors, CTC isolation can only obtain part of the tumor cells, so that it cannot fully respond to patients disease state
The exosome method also requires very professional centrifugation equipment and very professional centrifugation procedures, which are difficult to carry out in clinical laboratories
At the same time, the representativeness of exosome samples and the quality control of exosome isolation are far from uniform internationally.

Method used

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  • Primer probe composition, kit and detection method for detecting human AR-V7 and AR gene expression
  • Primer probe composition, kit and detection method for detecting human AR-V7 and AR gene expression
  • Primer probe composition, kit and detection method for detecting human AR-V7 and AR gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] First of all, the application discloses a primer probe composition for detecting human AR-V7 and AR gene expression, including:

[0041] AR-V7 forward primer has a nucleotide sequence as shown in SEQ ID NO.1;

[0042] AR-V7 reverse primer has a nucleotide sequence as shown in SEQ ID NO.2;

[0043] The AR-V7 probe has a nucleotide sequence as shown in SEQ ID NO.3;

[0044] The AR forward primer has a nucleotide sequence as shown in SEQ ID NO.4;

[0045] AR reverse primer, having the nucleotide sequence shown in SEQ ID NO.5;

[0046] The AR probe has a nucleotide sequence as shown in SEQ ID NO.6;

[0047] GAPDH forward primer has a nucleotide sequence as shown in SEQ ID NO.7;

[0048] The GAPDH reverse primer has a nucleotide sequence as shown in SEQ ID NO.8;

[0049] The GAPDH probe has the nucleotide sequence shown in SEQ ID NO.9.

[0050] Wherein, the AR-V7 probe, the AR probe, and the 3' end of the GAPDH probe are marked with a fluorescent quenching group, and t...

Embodiment 2

[0053] Based on a primer probe composition for detecting human AR-V7 and AR gene expression disclosed in Example 1, this example also discloses a kit for detecting human AR-V7 and AR gene expression, which includes : the primer probe composition disclosed in Example 1, DNA polymerase, dNTPs, reaction buffer, a positive control group containing a positive quality control product, and a negative control group containing a negative quality control product.

[0054] The kit uses qRT-PCR or dPCR to detect human AR-V7 and AR on the test sample.

[0055] The detection sample is human peripheral whole blood or blood cells obtained by centrifugation of human peripheral whole blood.

[0056] The positive quality control is the pseudoviral RNA of the AR-V7 target amplification region or the RNA extracted from a known AR-V7 positive human cell line; the negative quality control is the pseudovirus RNA that does not contain the AR-V7 target amplification region. Viral RNA, RNA from human c...

Embodiment 3

[0059] Based on the technical solutions disclosed in Example 1 and Example 2, this example also discloses a detection method for human AR-V7 and AR gene expression, using the method for detecting human AR-V7 and AR as described in Example 2 The kit for gene expression uses qRT-PCR or dPCR to detect the test sample; and the qRT-PCR or dPCR adds a pre-reaction step of UNG enzyme during the execution of the amplification program. In this example, the role of the pre-reaction step of adding UNG enzyme is to prevent PCR products from contaminating the reaction system. At the same time, in this embodiment, the detection method also includes a sample pre-amplification step, the sample pre-amplification step and the pre-reaction step of adding UNG enzyme (that is, in the same reaction tube) or separately (that is, in two Different reaction tubes), and the effect is similar to each other.

[0060] The detection target regions of the primers and probes in the kits used in the detection...

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Abstract

The invention provides a primer probe composition, a kit and a detection method for detecting human AR-V7 and AR gene expression. The primer probe composition comprises an AR-V7 forward primer with anucleotide sequence as shown in SEQ ID NO. 1, an AR-V7 reverse primer with a nucleotide sequence as shown in SEQ ID NO. 2, an AR-V7 probe with a nucleotide sequence as shown in SEQ ID NO.3, an AR forward primer with a nucleotide sequence as shown in SEQ ID NO. 4, an AR reverse primer with a nucleotide sequence as shown in SEQ ID NO. 5, an AR probe with a nucleotide sequence as shown in SEQ ID NO.6 and an internal reference primer probe. According to the invention, the specific primers and probes are used for respectively amplifying AR full length and AR-V7 spliceosome molecules in blood cellRNA, and has the advantages of rapidness, high sensitivity, simplicity and convenience in operation, reduction of sample pollution in the intermediate process, low misoperation risk and the like.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a primer probe composition, a kit and a detection method for detecting human AR-V7 and AR gene expression. Background technique [0002] Prostate cancer is one of the most common malignant tumors in European and American men. The incidence of prostate cancer in the United States ranks first, and the mortality rate is second only to lung cancer. Although the incidence of prostate cancer in my country is lower than that in Europe and the United States, it has an increasing trend in recent years. According to statistics, the incidence of prostate cancer in my country in 2013 was 8.58 / 100,000, which is the sixth highest incidence of cancer in men. In 2018, the incidence of prostate cancer was 13.6 / 100,000, significantly higher than in 2013. Prostate cancer is an androgen-dependent tumor, so the main treatment for prostate cancer is Androgen Deprivation Thetapy (ADT). However,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/106C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/101C12Q2545/113C12Q2563/107C12Q2521/531
Inventor 朱桂春于垚垚姚鲁帅李涛盛青松
Owner WUXI SHENRUI BIO PHARMA
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