CN108070641B - Primers and probes for detecting AR-V7 and AR in vesicles based on qPCR or digital PCR technology - Google Patents

Abstract

The invention provides a set of primers and probes for detecting AR-V7 and AR in vesicles, which are developed based on qPCR or digital PCR technology, and the sequences are respectively shown in SEQ ID NO. 1-6. The primer and the probe are utilized to establish a vesicle-based non-invasive, rapid and high-sensitivity qPCR or digital PCR detection method for AR-V7 and full-length AR. The detection method aims at body fluid samples (including blood, urine, prostatic fluid and the like), clinical samples are easy to obtain, medication guidance suggestions can be provided, and the occurrence of drug resistance can be repeatedly monitored.

Description

Primers and probes for detecting AR-V7 and AR in vesicles based on qPCR or digital PCR technology

Technical Field

The invention relates to a molecular biology detection technology, in particular to a primer and a probe for detecting AR-V7 and AR in vesicles based on a qPCR or digital PCR technology.

Background

The Extracellular Vesicles (EVs; hereinafter Vesicles are referred to as Extracellular Vesicles) refer to vesicular bodies with a double-layer membrane structure, which are shed from cell membranes or secreted by cells, and have diameters of 30-1000nm, and mainly comprise MicroVesicles (MVs) and exosomes (exosomes), and the MicroVesicles are Vesicles shed from cell membranes after cells are activated or damaged. Extracellular vesicles are of great interest in disease diagnosis, particularly exosomes, due to their unique biological characteristics.

The exosome is a membrane vesicle with the particle size of 30-150 nm secreted into the extracellular environment after an intracellular multivesicular body and a cell membrane are fused, is an important medium for intercellular information transfer, and plays an important role in antigen presentation, apoptosis, inflammatory reaction, tumorigenesis development and metastasis processes. It is widely distributed in body fluid, including blood, saliva, urine, milk, hydrothorax and ascites, etc.; contains various inclusion substances such as DNA, RNA, protein and the like, and can be used as noninvasive diagnosis markers of various diseases such as tumors and the like.

The use of enzalutamide (an inhibitor of AR translocation and signal transduction) and abiraterone (an inhibitor of androgen biosynthesis) in anti-androgen therapy for prostate cancer can lead to the development of resistance. Studies have shown that Androgen Receptor (AR) splice variant 7 messenger RNA (AR-V7) has a correlation with resistance to anti-androgen therapy.

The results of examining AR-V7 in circulating tumor cells of 31 patients receiving enzalutamide treatment and 31 prostate cancer patients receiving abiraterone treatment by Antonarakis ES (N Engl J med.2014.) showed that among the patients receiving enzalutamide, the response rate of PSA (prostate specific antigen) was lower in AR-V7 positive patients than in negative patients (0% vs 53%, P ═ 0.004), the progression-free survival of PSA was shorter (median time 1.4 months vs.6.0 months; P <0.001), the progression-free survival of clinical or imaging was shorter (median time 2.1 months vs.6.1 months; P <0.001), and the overall survival was also shorter (median time 5.5 months vs. not observed; P ═ 0.002). Similarly, PSA response rates were lower in patients receiving abiraterone treatment for AR-V7 positive patients than in negative patients (0% vs. 68%; P ═ 0.004), and PSA progression-free survival was shorter (median time 1.3 months vs. not observed; P <0.001), clinical or imaging progression-free survival was shorter (median time 2.3 months vs. not observed; P <0.001), and overall survival was also shorter (median time 10.6 months vs. not observed; P ═ 0.006). The results indicate that AR-V7 detected in circulating tumor cells of castration-resistant prostate cancer patients is likely to be associated with resistance to enzalutamide and abiraterone.

At present, a mature AR-V7 detection method does not exist, the conventional immunohistochemistry and fish aiming at a tissue sample cannot solve the problem well because only the variable shearing of a transcript is carried out, and the collection of the prostate cancer tissue depends on puncture, so that the method is an invasive method. The use of Circulating Tumor Cells (CTCs) for RNA detection in the above-mentioned literature is limited by the number of CTCs themselves. CTCs are not collected in all prostate cancer patients, even though they can be detected, with a low number of CTCs in most samples, low sensitivity and high cost for detection of AR-V7 itself.

Therefore, the development of a low-cost, simple, rapid and sensitive method for detecting AR-V7 is urgently needed.

Disclosure of Invention

The invention aims to provide a primer, a probe and a detection kit for detecting AR-V7 and AR in vesicles based on qPCR or digital PCR technology.

It is another object of the invention to provide a vesicle-based method for detection of AR-V7.

In order to realize the purpose of the invention, the primers and the probes for detecting AR-V7 and AR in the vesicle, which are developed based on qPCR or digital PCR technology, are respectively (SEQ ID NO: 1-6):

AR-V7 forward primer: 5'-AATGTTATGAAGCAGGGATGACTCT-3'

AR-V7 reverse primer: 5'-CTTTCTTCAGGGTCTGGTCATTT-3'

AR-V7 probe: 5'-AAAATTCCGGGTTGGCA-3'

AR forward primer: 5'-GGAATTCCTGTGCATGAAAGC-3'

AR reverse primer: 5'-CGATCGAGTTCCTTGATGTAGTTC-3'

An AR probe: 5'-CTGGGTGTCACTATGGA-3'

The invention also provides a kit containing the primer and the probe and used for detecting AR-V7 and AR by qPCR or digital PCR.

The kit is used for detection of AR-V7 and AR in vesicles (including exosomes) extracted from bodily fluids, including blood, urine, prostatic fluid, and the like. The separation and extraction method of the vesicle comprises but is not limited to ultracentrifugation, gradient density centrifugation, membrane affinity method, polymer precipitation, chromatography and immunomagnetic bead capture method.

The qPCR reaction system and the reaction program matched with the kit are as follows:

qPCR reaction system:

PCR amplification procedure: 5min at 95 ℃; 5s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; 5min at 72 ℃.

In the invention, AR-V7 and AR are independent reaction systems; the cDNA template is obtained by reverse transcription of RNA extracted from vesicles.

The digital PCR reaction system and the reaction program matched with the kit are as follows:

digital PCR reaction System:

and (3) amplification procedure: 10min at 96 ℃; at 56 ℃ for 2min, at 98 ℃ for 30s, for 39 cycles; 60 ℃ for 2 min.

The invention provides a vesicle-based non-invasive, rapid and high-sensitivity AR-V7 and AR full-length detection method, a primer probe and a kit. The primer probe and the kit can be used for qPCR detection and ddPCR (digital PCR) detection. The detection method aims at body fluid samples (including blood, urine, prostatic fluid and the like), clinical samples are easy to obtain, medication guidance suggestions can be provided, and the occurrence of drug resistance can be repeatedly monitored.

The invention also provides a method for rapidly detecting the variable shearing of AR-V7, which comprises the following steps:

(1) sample sources vesicle (including exosome) RNA extracted from body fluids (blood, urine, prostatic fluid), including vesicles isolated by ultracentrifugation, Exoquick, ExoEasy and other different methods.

(2) Specific primers and probes for AR-V7 and the full-length AR are designed according to the sequence of the human genome.

(3) And carrying out reverse transcription on the vesicle RNA by using reverse transcriptase and a reagent to obtain a cDNA template.

(4) Preparing a fluorescent quantitative PCR amplification reaction system, and carrying out fluorescent quantitative PCR amplification; and judging the detection result according to the Ct value obtained by the fluorescent quantitative PCR instrument.

(5) In order to meet the requirement of higher detection sensitivity. The digital PCR amplification reaction system can be configured to carry out digital PCR amplification, and the detection result is judged according to the copy number obtained by the digital PCR instrument.

The invention provides a set of specific primer probes based on vesicle RNA for the first time, and utilizes qPCR or digital PCR to detect AR-V7. Compared with the prior art, the invention at least has the following advantages and beneficial effects:

establishing a non-invasive detection method based on a body fluid source sample without performing a puncture biopsy.

And (II) the system based on qPCR detection has low detection cost and high detection speed, and can be completed in only 90 minutes.

And (III) the sensitivity of the detection based on the digital PCR is high and can reach more than 98 percent (the sensitivity of the conventional immunohistochemical detection is only 60 to 70 percent), and the variable shearing of AR-V7 can be detected as low as a single copy.

Drawings

FIG. 1 shows the results of screening experiments for specific primers in example 1 of the present invention.

FIG. 2 shows the results of the specificity verification experiment of the primers in example 2 of the present invention.

FIG. 3 shows the results of qPCR amplification experiments for vesicles of cell line in example 3 of the present invention.

FIG. 4 shows the result of digital PCR detection of vesicles of the cell line in example 4 of the present invention.

FIG. 5 shows the results of digital PCR assay of clinical patients in example 5 of the present invention; wherein, A is a drug resistant patient, and B is a beneficiary patient.

FIG. 6 shows the qPCR test results of clinical patients in example 5 of the present invention; wherein 3 curves with a lower number of cycles correspond to the drug resistant patients in fig. 5 and 3 curves with a higher number of cycles correspond to the benefit patients in fig. 5.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 establishment of vesicle-based AR-V7 and full-length AR detection method

The invention takes an 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 detect AR-V7 and full-length AR simultaneously. The method for detecting the full length of the AR-V7 and the AR comprises the following steps:

1. specific primer probe design was performed based on the published human AR-V7(NCBI sequence No. NM — 001348061.1) and AR full-length (NCBI sequence No. NM — 000044.4) gene sequences from the NCBI database.

The primers and probes were designed as follows (SEQ ID NOS: 1-6):

AR-V7 forward primer: 5'-AATGTTATGAAGCAGGGATGACTCT-3'

AR-V7 reverse primer: 5'-CTTTCTTCAGGGTCTGGTCATTT-3'

AR-V7 probe: 5'-AAAATTCCGGGTTGGCA-3'

AR forward primer: 5'-GGAATTCCTGTGCATGAAAGC-3'

AR reverse primer: 5'-CGATCGAGTTCCTTGATGTAGTTC-3'

An AR probe: 5'-CTGGGTGTCACTATGGA-3'

In order to search for a proper primer pair aiming at AR-V7 and AR full-length detection, three pairs of AR-V7 primers and two pairs of AR full-length primers are respectively designed according to the gene sequences of AR-V7 and AR full-length based on the principle that double-ended primer annealing temperature is close and the GC content of the sequence cannot be too high or too low, and the specific steps are as follows:

AR-V7-1 forward primer: 5'-AATGTTATGAAGCAGGGATGACTCT-3'

AR-V7-1 reverse primer: 5'-CTTTCTTCAGGGTCTGGTCATTT-3'

AR-V7-2 forward primer: 5'-CCATCTTGTCGTCTTCGGAAATGTTATGAAGC-3'

AR-V7-2 reverse primer: 5'-TTTGAATGAGGCAAGTCAGCCTTTCT-3'

AR-V7-3 forward primer: 5'-AAGAGAAGTACCTGTGCGCC-3'

AR-V7-3 reverse primer: 5'-TCAGGGTCTGGTCATTTTGA-3'

AR-F-1 forward primer: 5'-GGAATTCCTGTGCATGAAAGC-3'

AR-F-1 reverse primer: 5'-CGATCGAGTTCCTTGATGTAGTTC-3'

AR-F-2 forward primer: 5'-AAGAGAAGTACCTGTGCGCC-3'

AR-F-2 reverse primer: 5'-TTCAGATTACCAAGTTTCTTCAG-3'

The results of PCR amplification using lysates of 4 cell lines such as AR-V7 prostate cancer cell line 22RV1 (known to express both AR-V7 and AR full length), lung cancer cell line H3122 (express AR full length, do not express AR-V7), esophageal cancer cell line YES2, KYSE30 (do not express both AR full length and AR-V7) using Ultrapure RNA Kit, reverse transcription using Promega Kit, 1ul of 2ng/ul cDNA template of general PCR system, 1ul of forward and reverse primers at a concentration of 0.2uM, 2 XTAQASM MasterMix 10ul, DEPC water to 25ul), and agarose gel electrophoresis using the amplified products are shown in FIG. 1. The results show that the primer pairs AR-V7-1 and AR-F-1 have good sensitivity and specificity respectively for AR-V7 and AR full length, while the primer pairs AR-V7-2 and AR-V7-3 have insufficient specificity of a plurality of amplified bands, and the brightness of the bands under the same conditions of AR-F-2 is insufficient, which shows that the sensitivity is not as good as that of AR-F-1. Therefore, the sequences of the primer pair AR-V7-1(SEQ ID NO:1-2) and AR-F-1(SEQ ID NO:4-5) were selected for subsequent experiments.

2. Sample processing and RNA extraction: the applicable range of the sample comprises body fluids such as blood, urine (more than 20 ml) and prostatic fluid (5 ml); blood was taken at a concentration of 10 ml/urine of 20ml or more/prostatic fluid of 5ml, and vesicles were isolated by ultracentrifugation, ExoQuck, or ExoEasy. After obtaining vesicles, total RNA was extracted using an RNA extraction Kit such as TRIzol Reagent, Ultrapure RNA Kit, and mirneasyMini Kit, and the extracted RNA was dissolved in DEPC-treated ddH₂O, used for carrying out reverse transcription operation.

3. Reverse transcription of RNA: and carrying out reverse transcription on the obtained RNA by adopting a reverse transcription kit such as Promega or SuperScript IV and the like according to the designated operation of the kit to obtain a cDNA template.

4. Fluorescent quantitative pcr (qpcr) detection: taking 1ul of the 2ng/ul cDNA template, respectively adding 1ul of forward primer, reverse primer and 2ul of probe (the concentration is 0.2uM), adding 10ul of 2 xTaq MasterMix, and supplementing DEPC water to 25 ul; the PCR amplification conditions were: pre-denaturation at 95 ℃ for 5min for 1 cycle; 5s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; 5min 1 cycles at 72 ℃.

5. Digital PCR detection: taking 3ul of 2ng/ul cDNA template, respectively adding 1ul forward primer, 1ul reverse primer and 2ul probe (the concentration is 0.2uM), adding 7.5ul Quantstudio 3D Master Mix, and supplementing DEPC water to 15 ul; the amplification conditions were: pre-denaturation at 96 ℃ for 10min for 1 cycle; at 56 ℃ for 2min, at 98 ℃ for 30s, for 39 cycles; 2min at 60 ℃ for 1 cycle.

Example 2 specificity verification of primers

Lysates of 4 cell lines, such as prostate cancer cell line 22RV1 (known to express both AR-V7 and AR full length), lung cancer cell line H3122 (express AR full length, do not express AR-V7) and esophageal cancer cell line YES2, KYSE30 (do not express both AR full length and AR-V7), were each subjected to RNA extraction using Ultrapure RNA Kit, reverse transcription using Promega Kit, PCR amplification using a general PCR system (2ng/ul cDNA template 1ul, 1ul forward and reverse primers at a concentration of 0.2uM, 2 × Taq MasterMix 10ul, and DEPC water to 25ul), and gel electrophoresis using the amplified products, the results are shown in fig. 2. The results show that the specific primers designed in the invention can only amplify corresponding bands from an AR-V7 positive cell line, and the AR-V7 primers and the AR full-length primers are consistent with the expected results, which indicates that the primer sequences have good specificity in the whole genome range.

Example 3 qPCR amplification experiments of cell line vesicles

Cell supernatants of the prostate cancer cell line 22RV1 and the esophageal cancer cell line KYSE30 were respectively taken for vesicle extraction, RNA extraction was performed on the obtained vesicles using an Ultrapure RNA Kit, reverse transcription was performed using a Promega Kit, qPCR amplification was performed using a qPCR system (1 ul of cDNA template, 1ul of forward and reverse primers at a concentration of 0.2uM and 2ul of probe at a concentration of 0.2uM, respectively, 2 XTaq MasterMix 10ul was added, DEPC water was supplemented to 25ul) (the reaction conditions were the same as in example 1), and agarose gel electrophoresis was performed using the amplified product, the results of which are shown in FIG. 3. The results show that corresponding products can be obtained by amplifying in the vesicle RNA of the positive cell line 22RV1, and the corresponding vesicle RNA of the negative cell line KYSE30 is negative, which indicates that the primer sequence has good specificity in the vesicle RNA.

Example 4 digital PCR detection of cell line vesicles

The supernatants of prostate cancer cell lines 22RV1, H2231 and NTC cell lines were taken for vesicle extraction, the vesicles obtained were RNA extracted using Ultrapure RNA Kit, and were reverse transcribed using Promega Kit, and AR-V7 and AR full-length copy number detection were performed using digital PCR system (taking 2ng/ul cDNA template 3ul, adding 1ul forward primer, 1ul reverse primer and 2ul probe at 0.2uM, adding Quantstudio 3D Master Mix 7.5ul, and adding DEPC water to 15ul) (reaction conditions were the same as in example 1), with the results shown in FIG. 4. The results show that: firstly, the primer probe has good specificity, and nonspecific amplification does not exist completely; secondly, the whole system of the invention can be effectively used for detecting AR-V7 and the full length of AR.

Example 5 clinical sample testing

1ml of each of the plasma samples of patients who had drug resistance and benefited from treatment with the prostatic cancer antiandrogen was taken for separation and extraction of vesicles (including exosomes), the obtained vesicles were subjected to RNA extraction using the Ulrapure RNA Kit, reverse transcription was performed using the Promega Kit, and AR-V7 detection was performed using the digital PCR reaction system (the reaction system and the reaction conditions were the same as in example 1). The FAM signal values showed that 49 copies of AR-V7 were detected in 1ml plasma vesicle RNA from drug resistant patients (FIG. 5-A), while expression of AR-V7 was not detected in benefit patients (FIG. 5-B). The digital PCR detection system in the invention can be used for detecting AR-V7 of clinical patients.

The cDNA templates of the two prostate cancer patients were taken, and AR-V7 detection was performed using a qPCR reaction system (the reaction system and reaction conditions were the same as in example 3), and 3 parallel controls were set for each sample, and the detection results are shown in fig. 6. The threshold criteria were positive ct values (cycle number) less than 30 and negative ct values (cycle number) greater than 30, consistent with the above digital PCR assay results.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Reference to the literature

Antonarakis E S,Lu C,Wang H,et al.AR-V7 and resistance toenzalutamide and abiraterone in prostate cancer[J].New England Journal ofMedicine,2014,371(11):1028-38。

Sequence listing

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Claims (1)

1. A kit for qPCR or digital PCR detection of AR-V7 and AR in vesicles extracted from blood, urine, prostatic fluid, for detection of AR-V7 and AR in vesicles;

the primers and probes used for detecting AR-V7 and AR in vesicles were:

AR-V7 forward primer: 5'-AATGTTATGAAGCAGGGATGACTCT-3'

AR-V7 reverse primer: 5'-CTTTCTTCAGGGTCTGGTCATTT-3'

AR-V7 probe: 5'-AAAATTCCGGGTTGGCA-3', respectively;

AR forward primer: 5'-GGAATTCCTGTGCATGAAAGC-3'

AR reverse primer: 5'-CGATCGAGTTCCTTGATGTAGTTC-3'

An AR probe: 5'-CTGGGTGTCACTATGGA-3', respectively;

the qPCR reaction system and the reaction program matched with the kit are as follows:

qPCR reaction system:

wherein AR-V7 and AR are independent reaction systems; the cDNA template is obtained by reverse transcription of RNA extracted from the vesicle;

PCR amplification procedure: 5min at 95 ℃; 5s at 95 ℃, 30s at 60 ℃, 60s at 72 ℃ and 35 cycles; 5min at 72 ℃;

the digital PCR reaction system and the reaction program matched with the kit are as follows:

digital PCR reaction System:

wherein AR-V7 and AR are independent reaction systems; the cDNA template is obtained by reverse transcription of RNA extracted from the vesicle;

and (3) amplification procedure: 10min at 96 ℃; at 56 ℃ for 2min, at 98 ℃ for 30s, for 39 cycles; 60 ℃ for 2 min.

Images