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Primer and probe for detecting AR-V7 and AR in vesicle based on qPCR or digital PCR technology

A technology of AR-V7 and probe, which is applied in the field of primers and probes for detecting AR-V7 and AR in vesicles, can solve the limitation of CTC number in circulating tumor cells, low detection sensitivity of AR-V7, and can not be solved well problems and other problems, to achieve the effect of fast detection speed, high sensitivity and low detection cost

Active Publication Date: 2018-05-25
北京恩泽康泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no mature AR-V7 detection method. Due to the variable splicing of transcripts, conventional immunohistochemistry and fish for tissue samples cannot solve the problem well. At the same time, the collection of prostate cancer tissues relies on puncture. is an invasive method
In the above literature, the use of circulating tumor cells (CTCs) for RNA detection is limited by the number of CTCs themselves
Not all prostate cancer patients can collect CTCs. Even if CTCs can be detected, the number of CTCs in most samples is small, and the detection sensitivity of AR-V7 itself is low and expensive.

Method used

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  • Primer and probe for detecting AR-V7 and AR in vesicle based on qPCR or digital PCR technology
  • Primer and probe for detecting AR-V7 and AR in vesicle based on qPCR or digital PCR technology
  • Primer and probe for detecting AR-V7 and AR in vesicle based on qPCR or digital PCR technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Establishment of vesicle-based detection methods for AR-V7 and full-length AR

[0048] The invention uses the AR-V7 positive cell line 22RV1 as a template to construct an AR-V7 fluorescent quantitative PCR detection system and a digital PCR detection system, and simultaneously detects AR-V7 and full-length AR. The method for detecting the full length of AR-V7 and AR comprises the following steps:

[0049] 1. According to the human AR-V7 (NCBI sequence number NM_001348061.1) and AR full-length (NCBI sequence number NM_000044.4) gene sequences published by the NCBI database, specific primers and probes were designed.

[0050] The designed primers and probes are as follows (SEQ ID NO:1-6):

[0051] AR-V7 forward primer: 5′-AATGTTATGAAGCAGGGATGACTCT-3′

[0052] AR-V7 reverse primer: 5′-CTTTCTTCAGGGTCTGGTCATTT-3′

[0053] AR-V7 probe: 5′-AAAATTCCGGGTTGGCA-3′

[0054] AR forward primer: 5′-GGAATTCCTGTGCATGAAAGC-3′

[0055] AR reverse primer: 5′-CGATCGAGTTCCTTGATG...

Embodiment 2

[0073] Example 2 Specific verification of primers

[0074] Prostate cancer cell line 22RV1 (known to express both AR-V7 and full-length AR), lung cancer cell line H3122 (expressing full-length AR but not AR-V7), and esophageal cancer cell lines YES2 and KYSE30 (neither expressing AR The lysates of 4 cell lines, including full-length and AR-V7), were extracted with Ultrapure RNA Kit, and reverse-transcribed with Promega kit, using common PCR system (2ng / ul cDNA template 1ul, adding 1ul concentration 0.2uM forward primer and reverse primer, add 2×Taq MasterMix 10ul, add DEPC water to 25ul) for PCR amplification, and carry out agarose gel electrophoresis with the amplified product, the result is as follows figure 2 shown. The results show that using the specific primers designed in the present invention can only amplify the corresponding bands from AR-V7 positive cell lines, no matter the AR-V7 primer or the AR full-length primer is consistent with the expected result, indicati...

Embodiment 3

[0075] Example 3 qPCR amplification experiment of cell line vesicles

[0076] The cell supernatants of the prostate cancer cell line 22RV1 and the esophageal cancer cell line KYSE30 were collected to extract vesicles, and the obtained vesicles were extracted with Ultrapure RNA Kit, reverse-transcribed with Promega kit, and qPCR system (cDNA Template 1ul, add 1ul forward primer and reverse primer with a concentration of 0.2uM and 2ul probe with a concentration of 0.2uM, add 2×Taq MasterMix 10ul, add DEPC water to 25ul) for qPCR amplification (reaction conditions are the same as the implementation Example 1), and carry out agarose gel electrophoresis with the amplified product, the result is as follows image 3 shown. The results showed that the corresponding products could be amplified in the vesicle RNA of the positive cell line 22RV1, while the vesicle RNA of the corresponding negative cell line KYSE30 were all negative, indicating that in the vesicle RNA, the primer sequenc...

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Abstract

The invention provides a primer and a probe for detecting AR-V7 and AR in a vesicle based on a qPCR or digital PCR technology. The sequence is as shown in SEQ ID NO: 1-6. The primer and the probe areused for building a qPCR or digital PCR detecting method based on the noninvasive, rapid and high-sensitive AR-V7 and total length AR of the vesicle. The detecting method is capable of, in allusion toa body liquid sample (comprising blood, urine and prostatic fluid and so on), easily getting the clinic sample, providing the administration guiding opinions, and repeatedly monitoring the drug resistance circumstance.

Description

technical field [0001] The invention relates to molecular biology detection technology, in particular to a primer and a probe for detecting AR-V7 and AR in vesicles based on qPCR or digital PCR technology. Background technique [0002] Extracellular vesicles (Extracellular Vesicles, EVs; the following vesicles represent extracellular vesicles) refer to vesicle-like bodies with a double-membrane structure that are shed from the cell membrane or secreted by cells, with diameters ranging from 30-1000nm , Extracellular vesicles are mainly composed of microvesicles (MicroVesicles, MVs) and exosomes (exosomes). Microvesicles are small vesicles that are shed from the cell membrane after cell activation or injury. Due to the unique biological characteristics of extracellular vesicles, they are of great significance in disease diagnosis, especially the exosomes. [0003] Exosomes are a kind of membranous vesicles with a particle size of 30-150 nm secreted into the extracellular envi...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q2563/107C12Q2545/114C12Q2563/159
Inventor 赵立波孔关义郭静雅李志
Owner 北京恩泽康泰生物科技有限公司
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