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Quantitative detection method, internal standard and kit for nucleic acid of androgen receptor splicing variant-7

A quantitative detection and internal standard technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of cumbersome operation process, cumbersome operation, long time, etc., and achieve simplified operation process, repeated Good performance and low cost

Pending Publication Date: 2020-12-04
上海思路迪医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The obvious disadvantages of ddPCR detection are the cumbersome operation process, long time and high cost
qPCR standard curve quantification, each test needs to configure and detect the standard curve, the operation is cumbersome

Method used

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  • Quantitative detection method, internal standard and kit for nucleic acid of androgen receptor splicing variant-7
  • Quantitative detection method, internal standard and kit for nucleic acid of androgen receptor splicing variant-7
  • Quantitative detection method, internal standard and kit for nucleic acid of androgen receptor splicing variant-7

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 AR-V7 QS primer pair and probe screening

[0067] 1.1 AR-V7 sequence is:

[0068] TTGTCCATCTTGTCGTCTTCGGAAATGT TATGAAGCAGGGATGA CTCTGGGAGAAAAATTCCGGGTTGGCAATTG.

[0069] The AR-V7 QS sequence is:

[0070] TTGTCCATCTTGTCGTCTTCGGAAATGT TGATAGCAAGAGGGTA CTCTGGGAGAAAAATTCCGGGTTGGCAATTG.

[0071] The upstream and downstream primers used to detect AR-V7 QS are:

[0072] AR-V7 QS forward primer (ie upstream primer): TTGTCCATCTTGTCGTCTTCG

[0073] AR-V7 QS reverse primer (i.e. downstream primer): CAATTGCCAACCCGGAATTT

[0074] The upstream and downstream primers used to detect AR-V7 are:

[0075] AR-V7 forward primer (ie upstream primer): TTGTCCATCTTGTCGTCTTCG

[0076] AR-V7 reverse primer (i.e. downstream primer): CAATTGCCAACCCGGAATTT

[0077] 1.2 AR-V7 probe: 5'-TATGAAGCAGGGATGA-3', the 5' end is labeled with FAM, and the probe sequence of the internal standard AR-V7 QS is designed by adjusting the base sequence according to the probe sequence of AR-V7, AR ...

Embodiment 2

[0092] Example 2 AR-V7 QS primer pair concentration screening

[0093] 2.1 Since AR-V7 and AR-V7 QS share primers, there will be competitive inhibition between the two. Therefore, we need to optimize and adjust the primer concentration to minimize the effect of this competitive inhibition. In this test, 22RV1 cell line RNA was used as the template, and the final concentrations of AR-V7 primer pairs and probes were 200nM and 100nM respectively. The probe concentration was kept constant at 100nM, and the effect of the primer concentration on the detection results was investigated.

[0094] 2.2 The AR-V7 detection process is shown in Example 1;

[0095] 2.3 Analysis of test results

[0096]With the same amount of internal standard and 22RV1 RNA sample, the concentration of primers increased, the detection Ct value of AR-V7 and AR-V7 QS did not change significantly, and the signal value of AR-V7 and AR-V7 QS detection increased with the concentration of primers. increase, AR-V...

Embodiment 3

[0097] Example 3 internal standard AR-V7 QS dosage screening

[0098] 3.1 Due to the competitive inhibition between the detection of AR-V7 and the internal standard AR-V7 QS, it is necessary to optimize and adjust the dosage of the internal standard AR-V7 QS to meet the needs of AR-V7 detection. If the AR-V7 QS is too low, the detection of high concentration AR-V7 will significantly inhibit the detection of AR-V7QS; if the AR-V7 QS is too high, the detection of low concentration AR-V7 may be affected by the detection of AR-V7 QS Impact. Based on this, we set up four template quantity gradients for AR-V7 QS (here the template quantity is quantified by ddPCR), and T1 (5000copies), T2 (500copies), T3 (50copies) and T4 (5copies) were tested in turn. .

[0099] 3.2 The AR-V7 detection process is shown in Example 1;

[0100] 3.3 Analysis of test results

[0101] For AR-V7, three different AR-V7 template amounts were used for detection (each concentration was detected twice in pa...

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Abstract

The invention discloses a quantitative detection method, an internal standard and a kit for nucleic acid of an androgen receptor splicing variant-7. The kit comprises a primer probe combination related to AR-V7 quantitative detection, the internal standard and a reagent related to AR-V7 RT-PCR method detection. The kit can complete the quantitative detection of AR-V7 in a single tube, the quantitative result is accurate, the conventional quantitative operation process is greatly simplified, the efficiency is improved, and the risk of detection errors is also reduced.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a quantitative detection method for human androgen receptor splice variant-7 nucleic acid, an internal standard and a kit. Background technique [0002] Prostate cancer is a common malignancy and the second leading cause of cancer death in men. In recent years, the incidence of prostate cancer in my country has shown a rapid upward trend year by year, from 5.8 / 100,000 in 2004 to 8 / 100,000 in 2009. Due to the relatively backward screening of prostate cancer susceptible population in my country, advanced prostate cancer accounts for about 70% of the first diagnosed prostate cancer, and the proportion of newly diagnosed advanced prostate cancer in my country is much higher than that in European and American countries (20-30%). Androgen deprivation therapy (Androgen deprivation therapy, ADT) is the most important treatment for this kind of prostate cancer patient...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/166C12Q2531/113C12Q2537/165C12Q2545/101C12Q2563/107
Inventor 王晓楠乔春晓张亚楠
Owner 上海思路迪医学检验所有限公司