CN107619884A - A kind of RCA methods for detecting Epstein-Barr virus - Google Patents
A kind of RCA methods for detecting Epstein-Barr virus Download PDFInfo
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- CN107619884A CN107619884A CN201711002584.6A CN201711002584A CN107619884A CN 107619884 A CN107619884 A CN 107619884A CN 201711002584 A CN201711002584 A CN 201711002584A CN 107619884 A CN107619884 A CN 107619884A
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Abstract
The invention discloses a kind of RCA methods for detecting Epstein-Barr virus, complete in accordance with the following steps：（1）The extraction of specimen dna；（2）RCA, then with 1.0% agarose gel electrophoresis, electrophoresis result is observed by gel imager.Padlock probe combination RCA technologies of the present invention are a kind of new methods for detecting Epstein-Barr virus, RCA amplification amplification detection signals, are a kind of new methods for detecting Epstein-Barr virus by padlock probe specific recognition target-gene sequence.This method is not required to special installation, simple to operate, the good, high sensitivity of specificity, has unique advantage in terms of Viral diagnosis.
Description
Technical field
The present invention relates to biological technical field, specifically, is related to a kind of RCA methods for detecting Epstein-Barr virus.
Background technology
Epstein-Barr virus (Epstein-Barr virus, EBV) is found from African Burkitt ' s lymthomas culture cell
A kind of virion, it is the DNA virus of the thermophilic lymphocyte virus category of herpesviral γ subfamilies, can be established in bone-marrow-derived lymphocyte
Subclinical infection is played, stimulates hyperplasia and the conversion of cell, it includes Burkitt ' s with specific lymph source property and epithelial origin disease
Lymthoma, lymphogranulomatosis (HD), non Hodgkin lymphom (NHL), nasopharyngeal carcinoma (NPC) have substantial connection.EBV feels
Contaminate the epithelial cell and bone-marrow-derived lymphocyte in human oropharyngeal portion.EB has all been detected in southern china nasopharyngeal carcinoma patient groups mostly
Viral genome is present.EBV is widely distributed, more in sporadic, can also cause prevalence.Virus carrier and patient are the infections sources.
Oral close contact is major transmission path, and it is more to fall ill using the age group of 15~30 years old, once can obtain after being ill it is more lasting
Immunity.
Detection EBV method has Epstein-Barr virus partition method, ELISA method and PCR methods.Though Epstein-Barr virus partition method comparison is accurate,
Virus purification culture is difficult, time-consuming, needs special condition of culture；It is ELISA method high sensitivity, simple and quick, but non-spy
The opposite sex is higher, often results in false positive results；PCR method high sensitivity, high specificity, but reaction condition requires high, and
In the presence of certain false positive reaction.Rolling circle amplification (rolling circle amplification, RCA) is that a kind of constant temperature is linear
Amplification technique, received significant attention with the advantages that its fast simple, high sensitivity and high specificity.Padlock probe is by 5 ' Side Templates
Land, middle catenation sequence area and 3 ' Side Template lands composition.When 5 ' ends and 3 ' ends are with target sequence complete complementary, probe
Two sections form phosphodiester bond in the presence of DNA ligase and are connected cyclization, annular template are provided for RCA amplifications, after cyclisation
Probe can pass through primer and carry out rolling-circle replication and ramification amplification.If conversely, existing on target sequence, change XOR is no to be examined
The target gene of survey, probe can not be connected to ring-type, and duplication also can not just be carried out.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of RCA methods for detecting Epstein-Barr virus, detection method behaviour
Make that simple, the detection time cycle is short, high specific and sensitivity, by rolling-circle replication method, be capable of detecting when Epstein-Barr virus.
Technical scheme is as follows：A kind of RCA methods for detecting Epstein-Barr virus, are completed in accordance with the following steps：
(1) sample to be measured is pressed to the viral DNA extracts kit step process of Qiagen companies, obtains the DNA of sample；
(2) hybridization of capture probe：6 μ lDNA samples, 1 μm of ol/L locking-type are sequentially added in 32 μ l hybridization buffers
μ l of probe 1, the μ l of capture probe 1, fully mix, 55 DEG C of water-baths hybridize 1h, are slowly cooled to room temperature, and add 40 μ Streptavidins
Coated magnetic bead (being dissolved in 2 × combination buffer), mix, room temperature places 20min, Magnetic Isolation.It is clear with 1 × combination buffer
Wash magnetic bead 2 times, discard washing lotion；
(3) the cyclisation connection and digestion of padlock probe：Hybrid product made from μ l of deionized water 16, step (2) and 10 ×
The μ l of E.coli Buffer 2, mix, 95 DEG C of 5min, after 45 DEG C are incubated 40min, add the μ l and 0.2 μ l of 10 × BSA buffer solutions 2
E.coli DNA ligases, mix, 37 DEG C carry out being cyclized coupled reaction 40min；Add the Buffer of 10 × Exonuclease I
The μ l of 2 I exonuclease of μ l and Exonuclease 2, mix, 37 DEG C of reaction 1h, finally inactivated under the conditions of 95 DEG C
The exonucleases of Exonuclease I；
(4) RCA is expanded：Take μ l of circularizing probes connection product 42 being worth in step (3), add 100 μm of μ l of ol/L primers 1
With 10 × phi29Buffer 5 μ l, 95 DEG C of 5min, ice bath 1min；μ l of phi29DNA polymerases 1, the μ l of dNTP liquid 1 are added, is mixed
37 DEG C carry out RCA reactions 2h afterwards；
(5) 1.0% agarose gel electrophoresis of amplified production made from step (4) is taken, is observed by gel imager
Electrophoresis result；
The designed primer for RCA reactions is in the step (2) (4)：
- CTTGTGCTAATCGCAGTAACCTAAT-3 ' the SEQ of primer 5 ' NO1；
Probe is：
Padlock probe
5’-TCACCACCCGGGACTTGTACCCGGACTGTCTGTATCTGCTAACCA AGAGCAACTACACGAATTCT
CGATTAGGTTAGCACAAGCGGGAAGACAACCACAGACACCGTCC-3 ', 5 ' ends of probe carry out phosphorylation modification SEQ
NO2；
Capture probe
Biotin-AAGAGGATCAAAACATGCGGACCACCAGCTGGTACTTGACCGAAGSEQ NO3。
Using above-mentioned technical proposal, the present invention passes through padlock probe specific recognition target-gene sequence, RCA amplification amplification detections
Signal, it is a kind of new method for detecting Epstein-Barr virus.This method is not required to special installation, simple to operate, the good, high sensitivity of specificity,
There is unique advantage in terms of Viral diagnosis.
The minimal detectable concentration of RCA amplified reactions：
It is respectively 5pM, 50pM, 500pM and 5nM template with the method detection final concentration of the present invention, RCA amplified productions are electric
Result of swimming is as shown in Figure 1.As a result show, RCA visible bright bands more than template final concentration 5pmol/L, show RCA most
Low detectable concentration is 5pmol/L.As a result prompt, circulation RCA can preferably meet the detection of low virus load sample.
Beneficial effect：Padlock probe combination RCA technologies of the present invention are a kind of new methods for detecting Epstein-Barr virus, are visited by locking-type
Pin specific recognition target-gene sequence, RCA amplification amplification detection signals.This method is not required to special installation, simple to operate, specific
Good, high sensitivity, has unique advantage in low virus load context of detection.
Brief description of the drawings
(the 1st, 2,3,4,5 swimming lane represents Marker, final concentration to the template electrophoresis result of Fig. 1 RCA detection various concentrations respectively
For 5nM, 500pM, 50pM, 5pM template group RCA products electrophoresis result).
(1 swimming lane is Marker to the electrophoresis result of Fig. 2 samples progress RCA detections, and 2,3,4,5,6,7,8,9,10,11,12 are
1st to No. 11 sample RCA product groups electrophoresis result).
Embodiment
With reference to embodiment, the present invention is further illustrated：
Embodiment 1
A kind of RCA methods for detecting Epstein-Barr virus, are completed in accordance with the following steps：
(1) sample to be measured is pressed to the viral DNA extracts kit step process of Qiagen companies, obtains the DNA of sample；
(2) hybridization of capture probe：6 μ lDNA samples, 1 μm of ol/L locking-type are sequentially added in 32 μ l hybridization buffers
μ l of probe 1, the μ l of capture probe 1, fully mix, 55 DEG C of water-baths hybridize 1h, are slowly cooled to room temperature, and add 40 μ Streptavidins
Coated magnetic bead (being dissolved in 2 × combination buffer), mix, room temperature places 20min, Magnetic Isolation.It is clear with 1 × combination buffer
Wash magnetic bead 2 times, discard washing lotion；
(3) the cyclisation connection and digestion of padlock probe：Hybrid product made from μ l of deionized water 16, step (2) and 10 ×
The μ l of E.coli Buffer 2, mix, 95 DEG C of 5min, after 45 DEG C are incubated 40min, add the μ l and 0.2 μ l of 10 × BSA buffer solutions 2
E.coli DNA ligases, mix, 37 DEG C carry out being cyclized coupled reaction 40min；Add the Buffer of 10 × Exonuclease I
The μ l of 2 I exonuclease of μ l and Exonuclease 2, mix, 37 DEG C of reaction 1h, finally inactivated under the conditions of 95 DEG C
The exonucleases of Exonuclease I；
(4) RCA is expanded：Take μ l of circularizing probes connection product 42 being worth in step (3), add 100 μm of μ l of ol/L primers 1
With 10 × phi29Buffer 5 μ l, 95 DEG C of 5min, ice bath 1min；μ l of phi29DNA polymerases 1, the μ l of dNTP liquid 1 are added, is mixed
37 DEG C carry out RCA reactions 2h afterwards；
(5) 1.0% agarose gel electrophoresis of amplified production made from step (4) is taken, is observed by gel imager
Electrophoresis result；
The designed primer for RCA reactions is in the step (2) (4)：
- CTTGTGCTAATCGCAGTAACCTAAT-3 ' the SEQ of primer 5 ' NO1；
Probe is：
Padlock probe
5’-TCACCACCCGGGACTTGTACCCGGACTGTCTGTATCTGCTAACCA AGAGCAACTACACGAATTCT
CGATTAGGTTAGCACAAGCGGGAAGACAACCACAGACACCGTCC-3 ', 5 ' ends of probe carry out phosphorylation modification SEQ
NO2；
Capture probe
Biotin-AAGAGGATCAAAACATGCGGACCACCAGCTGGTACTTGACCGAAG SEQ NO3。
11 clinical samples are detected by the inspection method of embodiment 1：
RCA product detections：
DNA is extracted to 11 samples by the inspection method of embodiment 1, DNA is then subjected to RCA detections, as a result shown
10 are the positive, and 1 is feminine gender.
The positive test symbol of Epstein-Barr virus is completely the same with conventional PCR method, and the result for illustrating detection is satisfied.
Claims (1)
- A kind of 1. RCA methods for detecting Epstein-Barr virus, it is characterised in that complete in accordance with the following steps：(1) sample to be measured is pressed to the viral DNA extracts kit step process of Qiagen companies, obtains the DNA of sample；(2) hybridization of capture probe：6 μ lDNA samples, 1 μm of ol/L padlock probe 1 are sequentially added in 32 μ l hybridization buffers μ l, the μ l of capture probe 1, fully mix, 55 DEG C of water-baths hybridize 1h, are slowly cooled to room temperature, and add 40 μ l Streptavidins coating Magnetic bead (being dissolved in 2 × combination buffer), mix, room temperature place 20min, Magnetic Isolation.Magnetic is cleaned with 1 × combination buffer Pearl 2 times, discards washing lotion；(3) the cyclisation connection and digestion of padlock probe：Hybrid product made from μ l of deionized water 16, step (2) and 10 × The μ l of E.coli Buffer 2, mix, 95 DEG C of 5min, after 45 DEG C are incubated 40min, add the μ l and 0.2 μ l of 10 × BSA buffer solutions 2 E.coli DNA ligases, mix, 37 DEG C carry out being cyclized coupled reaction 40min；Add 10 × Exonuclease I The μ l of 2 I exonucleases of μ l and Exonuclease of Buffer 2, mix, 37 DEG C of reaction 1h, finally inactivated under the conditions of 95 DEG C The exonucleases of Exonuclease I；(4) RCA is expanded：Take μ l of circularizing probes connection product 42 being worth in step (2), add 100 μm of μ l of ol/L primers 1 and 10 × phi29 Buffer 5 μ l, 95 DEG C of 5min, ice bath 1min；μ l of phi29 archaeal dna polymerases 1, the μ l of dNTP liquid 1 are added, after mixing 37 DEG C carry out RCA reactions 2h；(5) 1.0% agarose gel electrophoresis of amplified production made from step (4) is taken, electrophoresis is observed by gel imager As a result；The designed primer for RCA reactions is in the step (2) (4)：- CTTGTGCTAATCGCAGTAACCTAAT-3 ' the SEQ of primer 5 ' NO1；Probe is：Padlock probe5’-TCACCACCCGGGACTTGTACCCGGACTGTCTGTATCTGCTAACCAAGAGCAACTACACGAATTCTCGATT AGGTTAGCACAAGCGGGAAGACAACCACAGACACCGTCC-3 ', 5 ' ends of probe carry out phosphorylation modification SEQ NO2；Capture probeBiotin-AAGAGGATCAAAACATGCGGACCACCAGCTGGTACTTGACCGAAGSEQ NO3。
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Cited By (7)
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| CN109136333A (en) * | 2018-09-20 | 2019-01-04 | 北京倍科为生物技术有限公司 | Identify ebv infection lymphocyte subgroup and infected cell accounts for the method and its application of the cell subset ratio |
| CN109825645A (en) * | 2019-03-13 | 2019-05-31 | 湖北朗德医疗科技有限公司 | A kind of detection nerpes vinrus hominis -6(HHV-6) RCA method |
| CN109825646A (en) * | 2019-03-13 | 2019-05-31 | 湖北朗德医疗科技有限公司 | A kind of RCA method detecting herpes simplex virus -1 (HSV-1) |
| CN109825644A (en) * | 2019-03-13 | 2019-05-31 | 湖北朗德医疗科技有限公司 | A kind of RCA method detecting human herpes virus 7 (HHV-7) |
| CN109852728A (en) * | 2019-03-13 | 2019-06-07 | 湖北朗德医疗科技有限公司 | A kind of RCA method detecting herpes simplex virus-2 (HSV-2) |
| CN109897915A (en) * | 2019-03-13 | 2019-06-18 | 湖北朗德医疗科技有限公司 | A kind of RCA method detecting cytomegalovirus (HCMV) |
| WO2023021518A1 (en) * | 2021-08-18 | 2023-02-23 | Yeda Research And Development Co. Ltd. | Ultrafast molecular inversion probe-based targeted sequencing assay for low variant allele frequency |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136333A (en) * | 2018-09-20 | 2019-01-04 | 北京倍科为生物技术有限公司 | Identify ebv infection lymphocyte subgroup and infected cell accounts for the method and its application of the cell subset ratio |
| CN109825645A (en) * | 2019-03-13 | 2019-05-31 | 湖北朗德医疗科技有限公司 | A kind of detection nerpes vinrus hominis -6(HHV-6) RCA method |
| CN109825646A (en) * | 2019-03-13 | 2019-05-31 | 湖北朗德医疗科技有限公司 | A kind of RCA method detecting herpes simplex virus -1 (HSV-1) |
| CN109825644A (en) * | 2019-03-13 | 2019-05-31 | 湖北朗德医疗科技有限公司 | A kind of RCA method detecting human herpes virus 7 (HHV-7) |
| CN109852728A (en) * | 2019-03-13 | 2019-06-07 | 湖北朗德医疗科技有限公司 | A kind of RCA method detecting herpes simplex virus-2 (HSV-2) |
| CN109897915A (en) * | 2019-03-13 | 2019-06-18 | 湖北朗德医疗科技有限公司 | A kind of RCA method detecting cytomegalovirus (HCMV) |
| WO2023021518A1 (en) * | 2021-08-18 | 2023-02-23 | Yeda Research And Development Co. Ltd. | Ultrafast molecular inversion probe-based targeted sequencing assay for low variant allele frequency |
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