CN109852728A - A kind of RCA method detecting herpes simplex virus-2 (HSV-2) - Google Patents
A kind of RCA method detecting herpes simplex virus-2 (HSV-2) Download PDFInfo
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Abstract
The invention discloses a kind of RCA methods for detecting herpes simplex virus-2 (HSV-2), complete in accordance with the following steps: (1) extraction of specimen dna；(2) hybridization of capture probe, the cyclisation connection and digestion of (3) padlock probe, (4) RCA amplification, then with agarose gel electrophoresis, electrophoresis result is observed by gel imager.Padlock probe combination RCA technology of the present invention is a kind of detection herpes simplex virus-2 (HSV-2), new method, pass through padlock probe specific recognition target-gene sequence, RCA expand amplification detection signal.This method is not required to special installation, easy to operate, the good, high sensitivity of specificity, has unique advantage in terms of viral diagnosis.
Description
Technical field
The present invention relates to field of biotechnology, specifically, being related to a kind of detection herpes simplex virus-2 (HSV-2)
RCA method.
Background technique
Herpe simplex is a kind of virus dermatopathy caused by herpes simplex virus, and Chinese medicine is known as herpes simplex.It can cause people
The a variety of diseases of class, such as gingivostomatitis, keratoconjunctivitis, encephalitis and genital system infection and neonatal infection.In infection host
Afterwards, latent infection is often established in nerve cell, will appear asymptomatic toxin expelling again after activation, propagation chain is maintained in crowd,
Circulation in cycles.Herpe simplex belongs to herpetoviridae a Chordopoxvirinae, and about 180 nanometers of virus particle size.According to antigen
The virus is divided into 1 type and 2 types at present by the difference of property.Amphitypy viral nucleotide sequences have 50% homology, have between type common anti-
Original also has specific antigen.The primary infection of HSV-2 mainly causes genital herpes, and male shows as the vesiculovirus of penis and bursts
Ulcer damage, women are the vesiculovirus ulcerative lesions of uterine neck, vulva, vagina, and complication includes damaging and aseptic brain outside genitals
Film is scorching.HSV-2 virus is the main pathogen of genital herpes, once infection, patient will carry this viral and periodical all the life
There is genital herpes damage in ground, and HSV-2 infection also will increase the risk of HIV-1 propagation, and currently without for HSV-2's
Effective vaccine comes out.High positive rate and the route of transmission common with HIV-1 due to HSV-2, the correlative study for HSV-2 are got over
To be more taken seriously.
Have much for the inspection method of herpes simplex virus-2 (HSV-2), including virus purification culture, complement combine examination
It tests, neutralization test, immunofluorescence and enzyme-linked immunosorbent assay and DNA detection etc., but is directed to herpes simplex virus-2 at present
(HSV-2) the main still Serologic detection of detection method and PCR detection, respectively for there are antibody and etiology nucleic acid in serum
It is detected.The specificity and sensitivity of Serologic detection be not high, often results in false positive results；PCR method high sensitivity, spy
It is anisotropic strong, but reaction condition requires height, and there is also certain false positive reactions.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of side RCA for detecting herpes simplex virus-2 (HSV-2)
Method, the detection method is easy to operate, the detection time period is short, high specific and sensitivity are able to detect by rolling-circle replication method
Herpes simplex virus-2 (HSV-2) out.
Technical scheme is as follows: a kind of RCA method detecting herpes simplex virus-2 (HSV-2), according to as follows
Step is completed:
(1) the viral DNA extracts kit step process that sample to be measured is pressed to Qiagen company, obtains and contains herpesviral -2
(HSV-2) genome DNA sample；
(2) the genomic DNA sample containing herpesviral -2 (HSV-2) hybridization of capture probe: is sequentially added in reaction tube
Product, padlock probe, capture probe, mix well, and 55 ± 1 DEG C of water-baths hybridize 0.5-1.5h, are slowly cooled to room temperature, addition is dissolved in
In 2 × combination buffer the coated magnetic bead of Streptavidin (2 ×: the combination buffer of 2 times of working concentrations, Streptavidin
Coated magnetic bead is purchased from Shanghai bioengineering Co., Ltd, similarly hereinafter), it mixes, is placed at room temperature for 20-25min, Magnetic Isolation.With 1 ×
Combination buffer clean magnetic bead 2 times (1 ×: the combination buffer of 1 times of working concentration, similarly hereinafter), discard washing lotion.(hybridization meaning: will
Virus target gene to be detected is separated from viral genome, improves detection efficiency and accuracy)
(3) the cyclisation connection and digestion of padlock probe: hybrid product made from deionized water, step (2) and 10 ×
E.coli Buffer is mixed gently and is reacted 3-5min at 95 ± 1 DEG C, and after 45 ± 1 DEG C of incubation 35-40min, it is slow that 10 × BSA is added
Fliud flushing and E.coli DNA ligase mix, and 37 ± 1 DEG C carry out cyclisation connection reaction 35-40min；Add 10 ×
Exonuclease I exonuclease of I Buffer and Exonuclease mixes, and reacts 0.5-1.5h at 37 ± 1 DEG C, finally exists
I exonuclease 3-5min of Exonuclease (10 × E.coli used in reaction system is inactivated under the conditions of 95 ± 1 DEG C
Buffer, 10 × BSA buffer, E.coli DNA ligase, 10 × Exonuclease, I core of I Buffer and Exonuclease
Sour excision enzyme is purchased from Shanghai bioengineering Co., Ltd), circularizing probes connection product can be obtained；(cyclisation connection meaning:
Target sequence DNA is connected into the DNA of single stranded circle by E.coli DNA ligase, provides cricoid DNA mould for next step RCA amplification
Plate；Digestion meaning: I exonuclease of Exonuclease cuts the DNA of linear target DNA or connection error, prevents
Non-annularity DNA cloning expands RCA and generates interference)
(4) RCA is expanded: taking circularizing probes connection product, primer and 10 × phi29Buffer obtained in step (3), gently
It is light to mix, ice bath 1-2min is carried out after 3-5min is reacted at 95 ± 1 DEG C；Phi29DNA polymerase, dNTPs liquid is added, after mixing
37 ± 1 DEG C carry out RCA reaction 1.5-2.5h (used phi29DNA polymerase in reaction system, dNTPs and
Phi29buffer is purchased from Shanghai bioengineering Co., Ltd), obtain amplified production；
(5) agarose gel electrophoresis that amplified production made from step (4) is 1.0-2.0% with mass concentration is taken
Complete the detection to herpes simplex virus-2 (HSV-2).Electrophoresis result is observed by gel imager, as shown in Fig. 2, there are naked eyes
Bands visible, which amplifies, to be come and positive for herpes simplex virus-2 (HSV-2), otherwise is herpes simplex virus-2 (HSV-2) yin
Property.
Designed probe, primer for RCA reaction is in the step (2) (4):
Primer (specific binding target dna, enables the linear growth of amplified reaction product, improves detection sensitivity)
5'-ATTATTCACTTATTACATCGTATCCTGCTATTATCTACAG-3'SEQ NO1；
Probe is:
Padlock probe is (since the end of probe 5 ' and the connection at 3 ' ends need two sections of identification sequences of probe and target sequence completely mutual
It mends, by being locked on target dna for DNA probe specificity, therefore makes RCA reaction that there is high specific, while list can be distinguished
The mutation in one site)
5’-TCTAGAGGTAGTGCCTTTCAATGCAGATCAGAAGCCGCAATTACCACAATTCTC CTTAGGAACC
5 ' ends of CCTTAGATAGGACAACGAGGTAGACGAACAAACTTTCC-3 ' probe carry out phosphorylation modification, SEQ NO2；
Capture probe (probe of one section of special marking, can specificity combining target DNA on target sequence, then by
The absorption of affine magnetic bead specificity is conducive to enhance following amplification specificity and sensitivity)
Biotin-ATTATTCACTTATTACATCCATAAGAATCCACTAAACTTAGCCAACATGTTCTTATSEQ
NO3。
By adopting the above technical scheme, the present invention expands amplification detection by padlock probe specific recognition target-gene sequence, RCA
Signal is a kind of new method for detecting herpes simplex virus-2 (HSV-2).This method is not required to special installation, easy to operate, special
Property good, high sensitivity, in terms of viral diagnosis have unique advantage.
The utility model has the advantages that padlock probe combination RCA technology of the present invention be it is a kind of detection herpes simplex virus-2 (HSV-2) it is new
Method, by padlock probe specific recognition target-gene sequence, RCA expands amplification detection signal.Padlock probe is by 5 ' Side Template knots
Close area, intermediate catenation sequence area and 3 ' Side Template combined areas composition.When 5 ' ends and 3 ' ends are with target sequence complete complementary, probe two
Section forms phosphodiester bond under the action of DNA ligase and is connected cyclization, annular template is provided for RCA amplification, after cyclisation
Probe can carry out rolling-circle replication and ramification amplification by primer.Conversely, if there is variation on target sequence or without to be detected
Target gene, probe can not be connected to ring-type, and duplication also can not just carry out.
The present invention has prominent meaning: (1) diversity relative to existing detection technique simultaneously.Universal primer in RCA
The a plurality of different padlock probe of amplification that efficiency can be waited, overcomes many problems in the amplification methods such as PCR, such as reactant and
The phenomenon that amplified production reacts to each other and interferes；(2) high specific.When detecting target sequence using padlock probe, due to probe
The connection at 5 ' ends and 3 ' ends needs the two sections of identification sequences and target sequence complete complementary of probe, therefore the reaction of RCA has Gao Te
The opposite sex can distinguish the mutation of single site；(3) highly sensitive.The amplification efficiency of linear RCA can achieve 105Times, and index
The amplification efficiency of RCA can reach 109Times；(4) high-throughput.PCR etc. traditional nucleic acid amplification technologies are because its amplified production is spread
Feed liquor phase and the amplified signal that persistent accumulation cannot be generated, therefore be not suitable for carrying out nucleic acid amplification on chip, but RCA then may be used
To solve this problem, because of its ring-shaped sequence that will form closure on target, the signal energy that RCA is generated is ensured that
It enough focuses on a bit, amplification in situ and slide glass amplification is realized with this.
This method is not required to special installation, easy to operate, the good, high sensitivity of specificity, has in low virus load context of detection
There is unique advantage.
Detailed description of the invention
Fig. 1 be embodiment 1RCA detect various concentration template electrophoresis result (swimming lane 1,2,3,4 respectively represents final concentration of
5nM, 500pM, 50pM, 5pM template group RCA product electrophoresis result).
Fig. 2 be 2 different specimens of embodiment carry out RCA detection electrophoresis result (swimming lane 1,2,3,4,5,6,7,8,9,10,
11,12 be the 1st to No. 12 sample RCA product group electrophoresis result).
Specific embodiment
The present invention is further illustrated With reference to embodiment:
Embodiment 1
The detection of the minimal detectable concentration of RCA amplified reaction is completed in accordance with the following steps:
(1) known herpes simplex virus-2 (HSV-2) positive sample is pressed to the viral DNA extracts kit of Qiagen company
Step processing on operational manual, obtains the DNA (virus genom DNA) of sample, and it is dense to survey DNA with ultramicrospectrophotometer
It spends (NanoDrop 2000C), and is diluted to the template that detection final concentration is respectively 5pM, 50pM, 500pM and 5nM；
(2) 6 μ of dilution various concentration in step (1) hybridization of capture probe: are sequentially added into hybridization reaction pipe
L DNA sample, 1 μm of 1 μ l of ol/L padlock probe, 1 μm of 1 μ l of ol/L capture probe, mix well, and 55 DEG C of water-baths hybridize 1h, slowly
Be cooled to room temperature, be added 40 μ l be dissolved in the coated magnetic bead of Streptavidin in 2 × combination buffer (2 ×: 2 times of working concentrations, magnetic
Pearl is purchased from Shanghai bioengineering Co., Ltd), it mixes, is placed at room temperature for 20min, Magnetic Isolation.Magnetic is cleaned with 1 × combination buffer
Pearl 2 times (1 ×: 1 times of working concentration), discard washing lotion.
(3) the cyclisation connection and digestion of padlock probe: every pipe is separately added into miscellaneous made from deionized water 16ul, step (2)
Product and 10 × E.coli Buffer2 μ l are handed over, is mixed gently, reacts 5min at 95 DEG C, after 45 DEG C of incubations 40min, addition 10 ×
2 μ l of BSA buffer and 0.4 μ l E.coli DNA ligase mix, and 37 DEG C carry out cyclisation connection reaction 40min；Add 10
I I exonuclease of Buffer2 μ l and Exonuclease of × Exonuclease, 1.8 μ l is mixed, 37 DEG C of reaction 1h, finally
Inactivated under the conditions of 95 DEG C I exonuclease 5min of Exonuclease (used enzyme in reaction system, BSA buffer and
Buffer is purchased from Shanghai bioengineering Co., Ltd), obtain circularizing probes connection product；
(4) RCA is expanded: being taken 42 μ l of circularizing probes connection product obtained in step (3), is separately added into 100 μm of ol/L and draws
1 μ l and 10 × phi29Buffer1 μ l of object, mixes gently, 5min is reacted at 95 DEG C, then carry out ice bath 1-2min；It is added
1 μ l of phi29DNA polymerase, 1 μ l of dNTPs liquid, after mixing 37 DEG C carry out RCA reaction 2h (used enzyme in reaction system,
DNTPs and buffer is purchased from Shanghai bioengineering Co., Ltd)；
(5) taking amplified production mass concentration made from step (4) is 1.0% agarose gel electrophoresis, be can be completed
Detection to the minimal detectable concentration of herpes simplex virus-2 (HSV-2).Electrophoresis result, such as Fig. 1 are observed by gel imager
It is shown.
Designed primer, probe for RCA reaction is in the step (2) (4):
Primer 5 '-ATTATTCACTTATTACATCGTATCCTGCTATTATCTACAG-3 ' SEQ NO1；
Probe is:
Padlock probe
5’-TCTAGAGGTAGTGCCTTTCAATGCAGATCAGAAGCCGCAATTACCACAATTCTC CTTAGGAAC
5 ' ends of CCCTTAGATAGGACAACGAGGTAGACGAACAAACTTTCC-3 ' probe carry out phosphorylation and repair
Decorations, SEQ NO2.
Capture probe
Biotin-ATTATTCACTTATTACATCCATAAGAATCCACTAAACTTAGCCAACATGTTCT
TAT SEQ NO3。
The minimal detectable concentration sample of herpes simplex virus-2 (HSV-2) is examined by the inspection method of embodiment 1
It surveys, obtains:
It is respectively the template of 5pM, 50pM, 500pM and 5nM, RCA amplified production electricity with method detection final concentration of the invention
Result of swimming is as shown in Figure 1.The results show that RCA is showed no amplified production, template final concentration below template final concentration 5pmol/L
The visible bright band of 50pmol/L or more shows that the minimal detectable concentration of RCA is 50pmol/L.As a result it prompts, rolling circle amplification
RCA method can preferably meet the detection of low virus load sample.
Embodiment 2
A kind of specific detection for the RCA method detecting herpes simplex virus-2 (HSV-2), is completed in accordance with the following steps:
(1) sample to be measured is handled by the step on the viral DNA extracts kit operational manual of Qiagen company, is obtained
Obtain the DNA (virus genom DNA) of sample；
(2) 6 μ l DNA samples, 1 μm of 1 μ of ol/L padlock probe the hybridization of capture probe: are sequentially added in hybridization reaction pipe
L, 1 μm of 1 μ l of ol/L capture probe, mixes well, and 55 DEG C of water-baths hybridize 1h, are slowly cooled to room temperature, and 40 μ l are added and are dissolved in 2 × knot
Close buffer in the coated magnetic bead of Streptavidin (2 ×: 2 times of working concentrations, magnetic bead be purchased from Shanghai bioengineering Co., Ltd),
It mixes, is placed at room temperature for 20min, Magnetic Isolation.Clean magnetic bead 2 times with 1 × combination buffer (1 ×: 1 times of working concentration), it discards
Washing lotion.
(3) the cyclisation connection and digestion of padlock probe: hybrid product made from deionized water 16ul, step (2) and 10 ×
E.coli Buffer2 μ l, mixes gently, and reacts 5min at 95 DEG C, and after 45 DEG C of incubation 40min, 10 × BSA buffer, 2 μ l is added
It with 0.2 μ l E.coli DNA ligase, mixes, 37 DEG C carry out cyclisation connection reaction 40min；Add 10 × Exonuclease
I I exonuclease of Buffer2 μ l and Exonuclease, 2 μ l, mixes, and 37 DEG C of reaction 1h are finally inactivated under the conditions of 95 DEG C
(used enzyme, BSA buffer and buffer are purchased from upper marine growth to I exonuclease 5min of Exonuclease in reaction system
Engineering Co., Ltd), obtain circularizing probes connection product；
(4) RCA is expanded: taking 42 μ l of circularizing probes connection product obtained in step (3), 100 μm of 1 μ l of ol/L primer are added
It with 10 × phi29Buffer1 μ l, mixes gently, 5min is reacted at 95 DEG C, then carry out ice bath 1-2min；It is poly- that phi29DNA is added
1 μ l of synthase, 1 μ l of dNTPs liquid carry out RCA reaction 2h (enzyme, dNTPs and buffer used in reaction system for 37 DEG C after mixing
Purchased from Shanghai bioengineering Co., Ltd)；
(5) taking amplified production mass concentration made from step (4) is 1.0% agarose gel electrophoresis, be can be completed
Detection to herpes simplex virus-2 (HSV-2).Electrophoresis result is observed by gel imager, as shown in Fig. 2, there are naked eyes visible
Band, which amplifies, to be come and positive for herpes simplex virus-2 (HSV-2), otherwise negative for herpes simplex virus-2 (HSV-2).
Designed primer, probe for RCA reaction is in the step (2) (4):
Primer 5 '-ATTATTCACTTATTACATCGTATCCTGCTATTATCTACAG-3 ' SEQ NO1；
Probe is:
Padlock probe
5’-TCTAGAGGTAGTGCCTTTCAATGCAGATCAGAAGCCGCAATTACCACAATTCTC CTTAGGAAC
5 ' ends of CCCTTAGATAGGACAACGAGGTAGACGAACAAACTTTCC-3 ' probe carry out phosphorylation and repair
Decorations, SEQ NO2.
Capture probe
Biotin-ATTATTCACTTATTACATCCATAAGAATCCACTAAACTTAGCCAACATGTTCT
TAT SEQ NO3。
Herpes simplex virus-2 (HSV-2) had clinically been detected no to 12 by the detection method of embodiment 2
RCA detection is carried out with patient specimen, is obtained:
DNA is extracted to 12 samples by the inspection method of embodiment 2, DNA is then subjected to RCA detection, as the result is shown
10 are the positive, and 2 are feminine gender.
The positive test symbol and clinical detection result of herpes simplex virus-2 (HSV-2) are completely the same, illustrate detection
The result is that satisfied.
Sequence table
<110>Hubei Lang De medical science and technology Co., Ltd
<120>a kind of RCA method for detecting herpes simplex virus-2 (HSV-2)
<160> 3
<170> PatentIn version 3.4
<210> 1
<211> 40
<212> DNA
<213>artificial sequence
<400> 1
5’-ATTATTCACTTATTACATCGTATCCTGCTATTATCTACAG-3’
<210> 2
<211> 102
<212> DNA
<213>artificial sequence
<400> 2
5’-TCTAGAGGTAGTGCCTTTCAATGCAGATCAGAAGCCGCAATTACCACAATTCTCCTTAGGAACCCCTTA
GATAGGACAACGAGGTAGACGAACAAACTTTCC-3’
<210> 3
<211> 56
<212> DNA
<213>artificial sequence
<400> 3
Biotin-ATTATTCACTTATTACATCCATAAGAATCCACTAAACTTAGCCAACATGTTCTTAT
Claims (4)
1. a kind of RCA method for detecting herpes simplex virus-2 (HSV-2), which is characterized in that complete in accordance with the following steps:
(1) the viral DNA extracts kit step process that sample to be measured is pressed to Qiagen company, obtains and contains herpesviral -2
(HSV-2) genome DNA sample；
(2) genome DNA sample, lock containing herpesviral -2 (HSV-2) hybridization of capture probe: are sequentially added in reaction tube
Formula probe, capture probe, mix well, 55 ± 1 DEG C of water-baths hybridization, cooled to room temperature after the completion of hybridization, and addition is dissolved in 2 ×
The coated magnetic bead of the Streptavidin of combination buffer mixes, is placed at room temperature for, Magnetic Isolation, cleans magnetic with 1 × combination buffer
Pearl for several times, discards washing lotion, obtains hybrid product；
(3) the cyclisation connection and digestion of padlock probe: hybrid product and 10 × E.coli made from deionized water, step (2)
Buffer is mixed gently, and 3-5min is reacted at 95 ± 1 DEG C, and after being then incubated at 45 ± 1 DEG C again, 10 × BSA buffering is added
Liquid and E.coli DNA ligase mix, and cyclisation connection reaction is carried out at 37 ± 1 DEG C；After the reaction was completed be added 10 ×
Exonuclease I exonuclease of I Buffer and Exonuclease mixes, and reacts at 37 ± 1 DEG C, exists after the reaction was completed
I exonuclease of Exonuclease is inactivated under the conditions of at 95 ± 1 DEG C, obtains circularizing probes connection product；
(4) RCA is expanded: circularizing probes connection product, addition primer and 10 × phi29 Buffer obtained in step (3) are taken,
3-5min is reacted after mixing at 95 ± 1 DEG C, then carries out ice bath；Phi29 archaeal dna polymerase, dNTPs liquid are added, after mixing
In 37 ± 1 DEG C of progress RCA reactions, amplified production is obtained；
(5) agarose gel electrophoresis for taking amplified production made from step (4) has naked eyes bands visible to amplify and to be list
Pure herpesviral -2 (HSV-2) is positive, otherwise negative for herpes simplex virus-2 (HSV-2), can be completed to herpe simplex disease
The detection of -2 (HSV-2) of poison；
The designed primer for RCA reaction is in the step (4):
5'-ATTATTCACTTATTACATCGTATCCTGCTATTATCTACAG-3'SEQ NO1；
Padlock probe in the step (2) are as follows:
5’-TCTAGAGGTAGTGCCTTTCAATGCAGATCAGAAGCCGCAATTACCACAATTCTC CTTAGGAACCCCTT
5 ' ends of AGATAGGACAACGAGGTAGACGAACAAACTTTCC-3 ' probe carry out phosphorylation modification SEQ NO2；
Capture probe in the step (2) are as follows:
Biotin-ATTATTCACTTATTACATCCATAAGAATCCACTAAACTTAGCCAACATGTTCTTAT SEQ NO3。
2. the RCA method of detection herpes simplex virus-2 (HSV-2) according to claim 1, which is characterized in that step
(2) padlock probe in, capture probe concentration be 0.5-1.5 μm of ol/L, and padlock probe, capture probe are relative to DNA sample
The 10-20% of product volume.
3. the RCA method of detection herpes simplex virus-2 (HSV-2) according to claim 1, which is characterized in that step
(3) 18-25% of 10 × E.coli Buffer relative to hybrid product volume made from step (2) in；10 × BSA buffer
18-25% relative to hybrid product volume made from step (2)；E.coli DNA ligase is obtained relative to step (2)
The 1.5-2.5% of hybrid product volume；10 × Exonuclease I Buffer is relative to hybrid product body made from step (2)
Long-pending 18-25%；18-25% of the Exonuclease I relative to hybrid product volume made from step (2).
4. the RCA method of detection herpes simplex virus-2 (HSV-2) according to claim 1, which is characterized in that step
(4) additive amount of primer is the 1.8-3% of circularizing probes connection product in；The additive amount of 10 × phi29 Buffer is probe ring
Change the 9.5-15% of connection product；The additive amount of phi29 archaeal dna polymerase is the 1.8-3% of circularizing probes connection product.
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