CN108070641A - It is used to detect the primer and probe of AR-V7 and AR in vesica based on qPCR or digital pcr technology - Google Patents
It is used to detect the primer and probe of AR-V7 and AR in vesica based on qPCR or digital pcr technology Download PDFInfo
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Abstract
The present invention provides a set of primer and probe that are used to detect AR V7 and AR in vesica based on qPCR or digital pcr technological development, and sequence is respectively such as SEQ ID NO:Shown in 16.A kind of noninvasive, quick, highly sensitive AR V7 based on the vesica and qPCR of overall length AR or digital pcr detection method are established using the primer and probe.This detection method is directed to humoral specimen (including blood, urine and prostatic fluid etc.), and clinical sample easily obtains, and can not only provide medication guide opinion, additionally it is possible to which the appearance of drug resistance situation is monitored repeatedly.
Description
Technical field
The present invention relates to molecular Biological Detection technologies, specifically, are related to a kind of based on qPCR or digital pcr technology
For detecting the primer and probe of AR-V7 and AR in vesica.
Background technology
Extracellular vesica (Extracellular Vesicles, EVs；Following vesica represents extracellular vesica) refer to from
The vesica shape corpusculum with double membrane structure for coming off on cell membrane or being secreted by cell, diameter are extracellular from 30-1000nm etc.
Vesica is mainly made of microcapsule bubble (MicroVesicles, MVs) and excretion body (exosomes), microcapsule bubble be cell-stimulating or
The vesicles to come off after damage from cell membrane.Due to the biological property of extracellular vesica uniqueness, have in medical diagnosis on disease
Important meaning, particularly excretion body therein.
Excretion body is a kind of by being secreted into extracellular environment grain size after extracellular compartment and cell membrane fusion between 30
The film vesicles of~150nm, be cell-tocell transfer important medium, antigen presentation, Apoptosis, inflammatory reaction,
Important function has been played in tumor development and transfer process.It is widely distributed in body fluid, including blood, saliva, urine,
Milk and Pleural effusions etc.；It, can be as noninvasive the examining of a variety of diseases such as tumour comprising a variety of inclusions such as DNA, RNA and protein
Disconnected marker.
The miscellaneous Shandong amine of grace (inhibitor of AR transpositions and signal transduction) and Ah's bit are used in prostate cancer anti androgenic therapy
Imperial (inhibitor of androgen biosynthesis) has the appearance of drug resistance phenomenon.Research shows androgen receptor (AR) splice variant 7
MRNA (AR-V7) has correlation with anti androgenic therapy drug resistance.
Antonarakis ES (N Engl J Med.2014.) etc. enter to have organized 31 patients for receiving the miscellaneous Shandong amine treatment of grace
The patients with prostate cancer for receiving abiraterone treatment with 31, is detected the AR-V7 in its circulating tumor cell, as a result shows
Show, in the patient for receiving the miscellaneous Shandong amine of grace, AR-V7 positive patients compared with negative patient PSA (prostate specific antigen) response rate more
Shorter (median time vs.6.0 months 1.4 months of low (0%vs53%, P=0.004), PSA progression free survival phases；P<
0.001), shorter (median time vs.6.1 months 2.1 months of clinical or iconography progression free survival phase；P<0.001), simultaneously
Overall survival is also shorter, and (5.5 months vs. of median time are not observed；P=0.002).It is similar therewith, receive abiraterone treatment
Patient in AR-V7 positive patient PSA response rates be less than negative patient (0%vs.68%；P=0.004), and PSA gets nowhere
Life cycle is shorter, and (1.3 months vs. of median time are not observed；P<0.001), clinical or iconography progression free survival phase is shorter
(2.3 months vs. of median time are not observed； P<, and also shorter (the 10.6 months vs. of median time of Overall survival 0.001)
It does not observe；P=0.006).The result shows that detected in the circulating tumor cell of castration-resistant prostate cancer patient
AR-V7 is likely to Shandong amine miscellaneous with grace and the drug resistance of abiraterone is related.
At present there has been no ripe AR-V7 detection methods, due to being only the variable sheer of transcript, conventional is directed to group
Problem can not be well solved by knitting sample immunohistochemistry, fish, while the acquisition of prostate cancer cancerous tissue is relied on and punctured, and is one
The invasive method of kind.RNA detections are carried out in above-mentioned document using circulating tumor cell (CTC) to be limited be subject to CTC numbers itself.
CTC can be collected in not all patients with prostate cancer, even if being able to detect that CTC, CTC in most of sample
Number it is less, it is relatively low and expensive to the detection sensitivities of AR-V7 in itself.
It would therefore be highly desirable to develop a kind of low cost, easy quick, sensitive AR-V7 detection methods.
The content of the invention
It is used to detect AR-V7 and AR in vesica based on qPCR or digital pcr technology the object of the present invention is to provide a kind of
Primer and probe, detection kit.
It is a further object of the present invention to provide a kind of AR-V7 detection methods based on vesica.
In order to realize the object of the invention, it is provided by the invention based on qPCR or digital pcr technological development for detecting capsule
The primer and probe of AR-V7 and AR in bubble, the primer and probe are respectively (SEQ ID NO:1-6)：
AR-V7 forward primers：5′-AATGTTATGAAGCAGGGATGACTCT-3′
AR-V7 reverse primers：5′-CTTTCTTCAGGGTCTGGTCATTT-3′
AR-V7 probes：5′-AAAATTCCGGGTTGGCA-3′
AR forward primers：5′-GGAATTCCTGTGCATGAAAGC-3′
AR reverse primers：5′-CGATCGAGTTCCTTGATGTAGTTC-3′
AR probes：5′-CTGGGTGTCACTATGGA-3′
The present invention also provides the reagents that AR-V7 and AR is detected for qPCR or digital pcr containing the primer and probe
Box.
The kit is used for the detection of AR-V7 and AR in vesica (including excretion body), and the vesica extracts from body fluid, wraps
Include blood, urine, prostatic fluid etc..The separating and extracting process of vesica includes but not limited to ultracentrifugation, gradient density centrifugal, film parent
With method, polymer precipitation, chromatography, immunomagnetic ca pture method.
The qPCR reaction system and response procedures mating with the kit be：
QPCR reaction systems：
PCR amplification program：95℃5min；95 DEG C of 5s, 60 DEG C of 30s, 72 DEG C of 60s, 35 Xun Huans；72℃5min.
In the present invention, AR-V7 and AR are each independent reaction system；The cDNA templates are extracted from vesica
RNA is obtained through reverse transcription.
The digital pcr reaction system and response procedures mating with the kit be：
Digital pcr reaction system：
Amplification program：96℃10min；56 DEG C of 2min, 98 DEG C of 30s, 39 Xun Huans；60℃ 2min.
A kind of method of noninvasive, quick, the highly sensitive AR-V7 and AR overall lengths detection based on vesica of present invention offer,
Primed probe and kit.Primed probe and kit in the present invention can both carry out qPCR detections or can carry out ddPCR (numbers
Word PCR) detection.This detection method is directed to humoral specimen (including blood, urine and prostatic fluid etc.), and clinical sample easily obtains,
Medication guide opinion can not only be provided, additionally it is possible to which the appearance of drug resistance situation is monitored repeatedly.
The present invention also provides a kind of methods of quick detection AR-V7 variable sheers, comprise the following steps：
(1) vesica (comprising excretion body) RNA that samples sources are obtained for body fluid (blood, urine, prostatic fluid) extraction,
Including ultracentrifugation, Exoquick, ExoEasy and the isolated vesica of other distinct methods.
(2) special primer and probe of AR-V7 and AR overall lengths are designed according to human genomic sequence.
(3) vesica RNA is subjected to reverse transcription using reverse transcriptase and reagent and obtains cDNA templates.
(4) fluorescent quantitative PCR reaction system is configured, carries out fluorescent quantitative PCR；According to fluorescence quantitative PCR instrument
Obtained Ct values judge testing result.
(5) it is the requirement of satisfaction more high detection sensitivity.Configurable digital pcr amplification reaction system, carries out digital pcr expansion
Increase, testing result is judged according to the copy number that digital pcr instrument obtains.
The present invention provides a set of specific primer probe based on vesica RNA for the first time, is carried out using qPCR or digital pcr
The detection of AR-V7.Compared with prior art, the present invention at least has following advantages and advantageous effect：
(1) a kind of non-invasive detection methods based on body fluid source sample are established, without carrying out aspiration biopsy.
(2) the system testing cost based on qPCR detections is low, and detection speed is fast, it is only necessary to can be completed within 90 minutes.
(3) high sensitivity based on digital pcr detection, up to more than 98%, (conventional immunohistochemistry detection sensitivity is only
For 60%~70%), it can detect down to the AR-V7 variable sheers singly copied.
Description of the drawings
Fig. 1 is the screening experiment result of specific primer in the embodiment of the present invention 1.
Fig. 2 is the specificity verification experimental result of primer in the embodiment of the present invention 2.
Fig. 3 is that the qPCR of cell line vesica in the embodiment of the present invention 3 expands experimental result.
Fig. 4 is the digital pcr testing result of cell line vesica in the embodiment of the present invention 4.
Fig. 5 is clinical patients digital pcr testing result in the embodiment of the present invention 5；Wherein, A is drug resistance patient, and B is benefit
Patient.
Fig. 6 is clinical patients qPCR testing results in the embodiment of the present invention 5；Wherein, 3 few curves of period correspond to
Drug resistance patient in Fig. 5,3 curves more than period, which correspond in Fig. 5, benefits patient.
Specific embodiment
Following embodiment is not limited to the scope of the present invention for illustrating the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular Cloning:A Laboratory Manual, 2001) or the condition according to manufacturer's specification suggestion.
The foundation of AR-V7 and overall length AR detection method of the embodiment 1 based on vesica
The present invention builds AR-V7 fluorescence quantitative PCR detections system and number using AR-V7 positive cell lines 22RV1 as template
Word PCR detection architectures, while detect AR-V7 and overall length AR.The method of detection AR-V7 and AR overall lengths comprises the following steps：
1st, according to the mankind AR-V7 (NCBI sequence number NM_001348061.1) and AR overall lengths of ncbi database announcement
(NCBI sequence number NM_000044.4) gene order carries out specific primer probe design.
Following (the SEQ ID NO of the primer and probe of design:1-6)：
AR-V7 forward primers：5′-AATGTTATGAAGCAGGGATGACTCT-3′
AR-V7 reverse primers：5′-CTTTCTTCAGGGTCTGGTCATTT-3′
AR-V7 probes：5′-AAAATTCCGGGTTGGCA-3′
AR forward primers：5′-GGAATTCCTGTGCATGAAAGC-3′
AR reverse primers：5′-CGATCGAGTTCCTTGATGTAGTTC-3′
AR probes：5′-CTGGGTGTCACTATGGA-3′
In order to explore the suitable primer pair for the detection of AR-V7 and AR overall lengths, approached based on both-end primer annealing temperature,
Sequence G/C content cannot be too high or too low design of primers principle, separately designed according to the gene order of AR-V7 and AR overall lengths
Three pairs of AR-V7 primers and two pairs of AR overall length primers, it is specific as follows：
AR-V7-1 forward primers：5′-AATGTTATGAAGCAGGGATGACTCT-3′
AR-V7-1 reverse primers：5′-CTTTCTTCAGGGTCTGGTCATTT-3′
AR-V7-2 forward primers：5′- CCATCTTGTCGTCTTCGGAAATGTTATGAAGC-3′
AR-V7-2 reverse primers：5′- TTTGAATGAGGCAAGTCAGCCTTTCT-3′
AR-V7-3 forward primers：5′-AAGAGAAGTACCTGTGCGCC-3′
AR-V7-3 reverse primers：5′-TCAGGGTCTGGTCATTTTGA-3′
AR-F-1 forward primers：5′-GGAATTCCTGTGCATGAAAGC-3′
AR-F-1 reverse primers：5′-CGATCGAGTTCCTTGATGTAGTTC-3′
AR-F-2 forward primers：5′-AAGAGAAGTACCTGTGCGCC-3′
AR-F-2 reverse primers：5′-TTCAGATTACCAAGTTTCTTCAG-3′
Utilize AR-V7 prostate cancer cell lines 22RV1 (known to express AR-V7 and AR overall lengths simultaneously), lung cancer cell line
H3122 (expression AR overall lengths do not express AR-V7) and esophageal carcinoma cell line YES2, KYSE30 (not expressing AR overall lengths and AR-V7)
Deng the lysate of 4 cell line, RNA extractions are carried out using Ultrapure RNA Kit, and are carried out using Promega kits
Reverse transcription, using regular-PCR system 2ng/ul cDNA template 1ul, be separately added into forward primer that 1ul concentration is 0.2uM and
Reverse primer adds in 2 × Taq MasterMix 10ul, mends DEPC water to 25ul) PCR amplification is carried out, and obtained with amplification
Product is into row agarose gel electrophoresis, and the results are shown in Figure 1.The result shows that primer pair AR-V7-1 and AR-F-1 are directed to AR- respectively
V7 and AR overall lengths have good sensitivity and specificity, and there are multiple amplified bands are special for primer pair AR-V7-2, AR-V7-3
Property it is inadequate, the brightness of band is inadequate under AR-F-2 equal conditions, show sensitivity be not so good as AR-F-1.Therefore, in subsequent experimental
Choose primer pair AR-V7-1 (SEQ ID NO:1-2) and AR-F-1 (SEQ ID NO:Sequence 4-5) is tested.
2nd, the extraction of sample process and RNA：The sample scope of application includes blood, urine (more than 20ml) and prostatic fluid
Body fluid such as (5ml)；Take more than blood 10ml/ urines 20ml/prostatic fluid 5ml, using ultracentrifugation, Exoquick or
ExoEasy carries out the separation of vesica.TRIzol Reagent, Ultrapure RNA Kit, miRNeasy are used after obtaining vesica
The RNA such as Mini Kit extracts kits extract total serum IgE, and the RNA of extraction is dissolved in the ddH of DEPC processing2In O, for carrying out
Reverse transcription operates.
3rd, RNA reverse transcriptions：Using Reverse Transcriptase kits such as Promega or SuperScript IV to RNA obtained above
By kit operation is specified to carry out reverse transcription, obtain cDNA templates.
4th, quantitative fluorescent PCR (qPCR) detects：Above-mentioned 2ng/ul cDNA template 1ul are taken, the forward direction for being separately added into 1ul is drawn
Object and reverse primer and 2ul probes (concentration is 0.2uM) add in 2 × Taq MasterMix 10ul, mend DEPC water extremely
25ul；PCR amplification condition is：95 DEG C of pre-degeneration 5min, 1 Xun Huan；95 DEG C of 5s, 60 DEG C of 30s, 72 DEG C of 60s, 35 Xun Huans；72
DEG C 5min 1 is cycled.
5th, digital pcr detects：Take 2ng/ulcDNA template 3ul, be separately added into 1ul forward primers, 1ul reverse primers and
2ul probes (concentration is 0.2uM) add in Quantstudio 3D Master Mix 7.5ul, mend DEPC water to 15ul；Expand
Increasing condition is：96 DEG C of pre-degeneration 10min, 1 Xun Huan；56 DEG C of 2min, 98 DEG C of 30s, 39 Xun Huans；60 DEG C of 2min, 1 Xun Huan.
The specificity verification of 2 primer of embodiment
Prostate cancer cell line 22RV1 (known while express AR-V7 and AR overall lengths), lung cancer cell line H3122 are taken respectively
(expression AR overall lengths do not express AR-V7) and esophageal carcinoma cell line YES2, KYSE30 (not expressing AR overall lengths and AR-V7) etc. 4
The lysate of a cell line is carried out RNA extractions using Ultrapure RNA Kit, and is inverted using Promega kits
Record, using regular-PCR system (2ng/ul cDNA template 1ul, be separately added into 1ul concentration be 0.2uM forward primer and reversely
Primer adds in 2 × Taq MasterMix 10ul, mends DEPC water to 25ul) carry out PCR amplification, and the product obtained with amplification
Into row agarose gel electrophoresis, the results are shown in Figure 2.The results show that using the specific primer designed in the present invention, only can
Amplify respective strap from the cell line of the AR-V7 positives, no matter AR-V7 primers or AR overall lengths primer all with expected results
Unanimously, show that primer sequence has specificity well in the range of full-length genome.
The qPCR amplification experiments of 3 cell line vesica of embodiment
The cell conditioned medium of prostate cancer cell line 22RV1 and esophageal carcinoma cell line KYSE30 is taken to carry out carrying for vesica respectively
It taking, obtained vesica carries out RNA extractions using Ultrapure RNA Kit, and carries out reverse transcription using Promega kits,
Using qPCR systems, (cDNA template 1ul are separately added into forward primer and reverse primer and 2ul concentration that 1ul concentration is 0.2uM
For the probe of 0.2uM, 2 × Taq MasterMix 10ul are added in, mend DEPC water to 25ul) carry out qPCR amplifications (reaction item
Part is with embodiment 1), and the product obtained with amplification, into row agarose gel electrophoresis, the results are shown in Figure 3.The results show that in sun
It can expand to obtain corresponding product in the vesica RNA of property cell line 22RV1, and corresponding negative cells system KYSE30
Vesica RNA is rendered as feminine gender, shows in vesica RNA, and primer sequence has specificity well.
The digital pcr detection of 4 cell line vesica of embodiment
Prostate cancer cell line 22RV1, H2231 and NTC cell line supernatant is taken to carry out the extraction of vesica, obtained capsule respectively
Bubble carries out RNA extractions using Ultrapure RNA Kit, and carries out reverse transcription using Promega kits, uses digital pcr
Reaction system (take 2ng/ul cDNA template 3ul, be separately added into concentration be the 1ul forward primers of 0.2uM, 1ul reverse primers and
2ul probes add in Quantstudio 3D Master Mix 7.5ul, mend DEPC water to 15ul) carry out AR-V7 and AR overall lengths
Copy number detects (reaction condition is with embodiment 1), and the results are shown in Figure 4.The result shows that：Primed probe tool first in the present invention
There is good specificity, be completely absent non-specific amplification；Secondly, the whole system in the present invention can be effectively used for
The detection of AR-V7 and AR overall lengths.
5 clinical sample of embodiment detects
Prostate cancer anti-androgen therapy drug resistance and each 1ml of the patients blood plasma's sample benefited is taken to carry out vesica and (include respectively
Excretion body) separation and Extraction, obtained vesica using Ultrapure RNA Kit carry out RNA extractions, and using Promega try
Agent box carries out reverse transcription, and AR-V7 inspections are carried out using digital pcr reaction system (reaction system and reaction condition are with embodiment 1)
It surveys.According to FAM signal values the results show that detected in drug resistance patient's 1ml blood plasma vesicas RNA 49 copy AR-V7 (Fig. 5-
A), benefit not detect the expression (Fig. 5-B) of AR-V7 in patient.Show that the digital pcr detection architecture in the present invention can be used
It is detected in the AR-V7 of clinical patients.
The cDNA templates of above-mentioned two patients with prostate cancer are taken, use qPCR reaction systems (reaction system and reaction condition
With embodiment 3) AR-V7 detections are carried out, each sample sets 3 parallel controls, and testing result is as shown in Figure 6.With ct values (Xun Huan
Number) it is less than 30 for the positive, ct values (period) are that feminine gender is threshold value standard more than 30, with above-mentioned digital pcr testing result one
It causes.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Bibliography
Antonarakis E S,Lu C,Wang H,et al.AR-V7 and resistance to
enzalutamide and abiraterone in prostate cancer[J].New England Journal of
Medicine,2014,371(11):1028-38。
Sequence table
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Claims (5)
1. the primer and probe that are used to detect AR-V7 and AR in vesica based on qPCR or digital pcr technological development, feature exist
In the primer and probe is respectively：
AR-V7 forward primers：5′-AATGTTATGAAGCAGGGATGACTCT-3′
AR-V7 reverse primers：5′-CTTTCTTCAGGGTCTGGTCATTT-3′
AR-V7 probes：5′-AAAATTCCGGGTTGGCA-3′；
AR forward primers：5′-GGAATTCCTGTGCATGAAAGC-3′
AR reverse primers：5′-CGATCGAGTTCCTTGATGTAGTTC-3′
AR probes：5′-CTGGGTGTCACTATGGA-3′.
2. the kit for qPCR or digital pcr detection AR-V7 and AR containing primer described in claim 1 and probe.
3. kit according to claim 2, which is characterized in that the kit is used for the inspection of AR-V7 and AR in vesica
It surveys, the vesica extraction autoblood, urine, prostatic fluid.
4. kit according to claim 2, which is characterized in that the qPCR reaction system mating with the kit and anti-
The program is answered to be：
QPCR reaction systems：
Wherein, AR-V7 and AR is each independent reaction system；The cDNA templates are the RNA extracted from vesica through reversion
What record obtained；
PCR amplification program：95℃5min；95 DEG C of 5s, 60 DEG C of 30s, 72 DEG C of 60s, 35 Xun Huans；72℃5min.
5. kit according to claim 2, which is characterized in that the digital pcr reaction system mating with the kit
And response procedures are：
Digital pcr reaction system：
Wherein, AR-V7 and AR is each independent reaction system；The cDNA templates are the RNA extracted from vesica through reversion
What record obtained；
Amplification program：96℃10min；56 DEG C of 2min, 98 DEG C of 30s, 39 Xun Huans；60℃2min.
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| CN112517093A (en) * | 2020-11-17 | 2021-03-19 | 四川大学 | Fish saliva automatic sample separation detection disc and detection method thereof |
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| CN108929907A (en) * | 2018-07-17 | 2018-12-04 | 张丽英 | A kind of detection method based on digital pcr platform ARV7 gene mutation |
| CN109321569A (en) * | 2018-10-29 | 2019-02-12 | 凯杰（苏州）转化医学研究有限公司 | A kind of primer combination of probe object and its application |
| CN109321569B (en) * | 2018-10-29 | 2022-04-12 | 迈杰转化医学研究(苏州)有限公司 | Primer probe composition and application thereof |
| CN110923299A (en) * | 2020-02-11 | 2020-03-27 | 上海思路迪医学检验所有限公司 | Primer, probe and kit for rapid quantitative detection of AR-V7 fluorescent qRT-PCR method |
| CN111500728A (en) * | 2020-05-13 | 2020-08-07 | 无锡市申瑞生物制品有限公司 | Primer probe composition, kit and detection method for detecting human AR-V7 and AR gene expression |
| CN112517093A (en) * | 2020-11-17 | 2021-03-19 | 四川大学 | Fish saliva automatic sample separation detection disc and detection method thereof |
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