CN110358867A - African hog cholera virus fluorescent type RAA detection kit - Google Patents
African hog cholera virus fluorescent type RAA detection kit Download PDFInfo
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Abstract
The invention discloses a kind of African hog cholera virus fluorescent type RAA detection kits, including specific primer ASFV-F and ASFV-R, and RAA-exo probe ASFV-P, with tetrahydrofuran (THF residue), the base in a substitution target amplification subsequence is quenched in dT- fluorogen and dT-, and the reporter fluorescence dyestuff of label is FAM, fluorescent quenching group BHQ-1, exonuclease in reagent can crack probe in the site THF, to separate fluorophor and quenching group and generate fluorescence signal.Kit provided by the invention can realize the detection of African swine fever virus under 37~42 DEG C of constant temperatures in 20min, specificity is 100%, and detection sensitivity is 10copies/ μ l.Therefore, kit of the invention has the characteristics that easy to operate, quick, sensitive, provides effective technological means for the field quick detection screening of African swine fever virus.
Description
Technical field
The present invention relates to a kind of African hog cholera virus fluorescent type RAA detection kits, belong to technical field of bioengineering.
Background technique
African swine fever (African swine fever, ASF) is domestic pig and the open country as caused by African swine fever virus (ASFV)
A kind of strong, bleeding sexually transmitted disease of pig.Acute is died of illness characterized by high fever, reticuloendothelial system bleeding and high lethality rate
Rate is close to 100%.World Organization for Animal Health (OIE) be classified as must notifiable a kind of epidemic disease, pig breeding industry is endangered larger.
2018 Liaoning Province, the China Nian8Yue report African swine fever epidemic situation for the first time, indicate that African swine fever is passed to China, cause to my pig breeding industry
Massive losses, due to there is no effective vaccine control, so establishing one kind, fast and accurately detection method just seems especially heavy
It wants.
Recombinase-mediated chain replaces nucleic acid amplification technologies (Recombinase-aid Amplification, RAA), is one
Kind of constant temperature nucleic acid rapid amplifying technology, using the recombinase obtained from bacterium or fungi, at normal temperature, the recombinase can with draw
Object DNA combines closely, and the condensate of enzyme and primer is formed, when primer searches the complementation exactly matched therewith on template DNA
When sequence, with the help of single-stranded DNA binding protein, the duplex structure of template DNA is opened, and under the action of archaeal dna polymerase,
New DNA complementary strand is formed, amplified production is increased with exponential.Using the label of fluorescence probe, biotin labeling primer and
Exonuclease digestion, may be implemented interpretation of result at regular time and quantity, and usual 20min can be obtained by fluorescence detection result.
Currently, ASF there is no effective vaccine prevention and treatment method, in a short time, by early diagnosis and the anti-keyholed back plate of compartmentalization
Reason is to carry out anti-system, and using stringent monitoring plan, one it is found that take a wide range of mode of slaughtering to cut off the infection sources.OIE recommends
The Serology tests such as HAD, ELISA, IFA, time-consuming, complicated for operation, is not suitable for the quick inspection with African swine fever virus
It surveys.RAA technology has the advantages that easy to operate, quick and convenient, high sensitivity, reproducible, pollution rate is low, leads in genetic test
Domain has broad application prospects.
Based on this, the highly conserved p72 gene of African swine fever virus (coding B646L structural proteins) conduct is chosen in this research
Target gene, designs specific primer and probe establishes the special fluorescent type RAA detection kit and detection method of ASFV.
Summary of the invention
The purpose of the invention is to provide a kind of African swine fever fluorescent type RAA detection kit, African swine fever disease is realized
The quick detection of poison.The present invention extracts from DNA during the entire process of detecting ASFV and testing result occurs, only need 25min,
Detection time is greatly shortened, detection efficiency is improved.
According to the first aspect of the invention, a kind of African hog cholera virus fluorescent type RAA detection kit is provided, including special
Property primer ASFV-F and ASFV-R, and be marked with the RAA-exo probe ASFV-P of THF residue, the reporter fluorescence of probe label
Dyestuff is FAM, and the fluorescent quenching group of label is BHQ-1；Wherein specific primer and RAA-exo probe sequence are as follows:
ASFV-F:5′-TCGCCGAAGGGAATGGATACTGAGGGAATA-3′；
ASFV-R:5′-AGGGGATAAAATGACTGGATATAAGCACTTGGT-3′；
ASFV-P:5′-TGCGTATCATTTTCATCGGTAAGAATAGGTT[FAM-dT][THF]C[BHQ1-dT]
TTGGTGCGGCTTGTGC-3′。
Under concrete condition, kit of the invention further includes fluorescence RAA reaction member, Buffer A, Buffer B, the positive
Control and negative control.The fluorescence RAA reaction member contains recombinase, single strand binding protein, strand displacement DNA polymerization
Enzyme powder is lyophilized in enzyme, exonuclease；Buffer A is lysis buffer；Buffer B is magnesium acetate solution；Positive control be containing
There is the pUC57-p72 plasmid of African swine fever virus p72 gene, negative control is empty carrier pUC57 plasmid.
According to the second aspect of the invention, a kind of preparation side of African hog cholera virus fluorescent type RAA detection kit is provided
Method comprising the steps of:
(1) artificial synthesized African swine fever virus p72 full length gene sequence (accession number: MH713612.1) is cloned into pUC-57
On carrier, plasmid is extracted after colibacillus engineering pure culture as positive control；
(2) primer sets and probe preparation:
According to the conserved region of p72 gene, specific primer ASFV-F and ASFV-R and RAA-exo probe ASFV- is designed
P, with tetrahydrofuran (THF residue), the base in a substitution target amplification subsequence, THF residue is quenched in dT- fluorogen and dT-
The reporter fluorescence dyestuff of 5 ' extreme directions label be FAM, 3 ' extreme direction mark fluorescent quenching groups of THF residue are BHQ-1, examination
Exonuclease in agent can crack probe in the site THF, to separate fluorophor and quenching group and generate fluorescence signal；
(3) recombinase, single strand binding protein, strand displacement archaeal dna polymerase, exonuclease freeze-drying enzyme powder, lysis buffer,
Magnesium acetate solution and deionized water, for commercialization preparation.
According to the third aspect of the invention we, it provides and quickly detects African swine fever disease using above-mentioned African swine fever virus kit
The method of poison comprising the steps of:
1) 45.5 μ L lysis buffers, specific primer ASFV-F and ASFV-R, mark are added in fluorescence RAA reaction member
Note has the RAA-exo probe ASFV-P of THF residue, and 2 μ L positive controls/negative control/nucleic acid to be checked add 2.5 μ in reaction tube
L magnesium acetate solution, close the lid centrifugation, mixing of turning upside down, and is centrifuged 10 seconds, detects immediately；Response procedures are as follows: 39 DEG C 40 seconds；
39 DEG C of 30 seconds 40 circulations, acquire fluorescence signal, the achievable detection of 20min herein；
2) result judgement
1. positive control: thering is typical amplification curve to occur, appearance time is less than 15min (value≤30 Ct), for effectively knot
Fruit；
2. negative control: no amplification curve occurs or appearance time >=20min (value >=40 Ct), is effective result；
3. tested sample:
If a. (value < 35 Ct) appearance time≤17.5min, it is judged as positive；
If b. (value >=40 Ct) appearance time >=20min, it is judged as negative；
If c. (value < 40 35 < Ct) 17.5min < appearance time < 20min, be judged to it is suspicious, need to repeat detection be confirmed；Again
Secondary testing result is still (value < 40 35 < Ct) 17.5min < appearance time < 20min, should refer to negative control appearance time (Ct
Value), if negative control appearance time >=20min (value >=40 Ct), it is judged as positive.
By many experiments, optimize reaction condition, the best primed probe concentration of reagent is 20 μm of ol/L ASFV-F/ASFV-
R each 1 μ l, 20 μm of 0.5 μ l of ol/L ASFV-P.
Kit provided by the invention can be used in detecting African swine fever virus, can be used for the Nasal swabs of pig, blood,
Serum, blood plasma, the African swine fever virus detection in tissue.
Target gene can be achieved in isothermal amplification when kit provided by the invention detects under the conditions of 37~42 DEG C
Effectively amplification can realize the detection of African swine fever virus in 20min, and specificity is 100%, and detection sensitivity is
10copies/μl。
Therefore, kit of the invention has the characteristics that easy to operate, quick, sensitive, is the scene of African swine fever virus
Quickly detection screening provides effective technological means, has simultaneously for control African swine fever virus diffusion in China extremely important
Meaning.
Specific embodiment
The present invention is described in further detail below, the given examples are served only to explain the present invention, is not intended to limit
The scope of the present invention.
The design of a kind of African swine fever fluorescent type RAA detection kit primer of embodiment 1 and probe
RAA nucleic acid amplification technologies have certain difference for design of primers and Standard PCR design of primers, there is two few nucleosides
Acid partners primer, respectively the upstream and downstream nucleotide sequence of one target set nucleic acid object of specific recognition；Length is in 30-35 nucleosides
Between sour (nt), without palindromic sequence, continuous single base repetitive sequence and internal secondary structure area in sequence；Primer Tm not as
Major consideration when design；Best primer pair need to be filtered out by assay optimization.Probe implementation sequence not with specific primer
Recognition site overlapping, length 46-52nt, sequence avoid palindromic sequence, internal secondary structure and continuously repeat base；It is shared
Four decorating sites, distance 5 ' hold >=one tetrahydrofuran (THF) of medium position label of 35nt, as exonuclease
Recognition site；One fluorophor of marker upstream in the site THF, marker downstream one is quenched base and closes, and the spacing of two groups is
2-4nt；3 ' ends of THF distance >=l5nt, and 3 ' end marks, one modification group.
According to the African swine fever of 24 genotype Reference strains of the African swine fever virus included on GenBank and China's separation
Strain p72 gene order (being shown in Table 1), screens conservative region, design primer and probe, primer ASFV-F ASFV-R and probe
ASFV-P is as follows:
ASFV-F:5′-TCGCCGAAGGGAATGGATACTGAGGGAATA-3′；
ASFV-R:5′-AGGGGATAAAATGACTGGATATAAGCACTTGGT-3′；
ASFV-P:5′-TGCGTATCATTTTCATCGGTAAGAATAGGTT[FAM–Dt][THF]C[BHQ1-Dt]
TTGGTGCGGCTTGTGC-3′。
1 African swine fever Reference strains of table
A kind of building of the African swine fever fluorescent type RAA detection kit recombinant vector of embodiment 2
The ASFV logged on artificial synthesized GenBank with reference to gene II type China isolated strain (accession number:
MH713612.1) p72 full length gene is cloned on pUC57 carrier as positive control.
A kind of foundation of the African swine fever fluorescent type RAA detection reagent reaction system of embodiment 3
Isothermal amplification reactions are carried out using the pUC57-p72 plasmid constructed in embodiment 2 as template.Multiple groups primer is screened to visit
Needle combination, finally using primer combination of probe described in embodiment 1 as optimal combination.Experimental stage reaction system are as follows: in RAA
42.5 μ l lysis buffers are added in reaction member, each 1 μ l of 20 μm of ol/L ASFV-F/ASFV-R, 20 μm of ol/L ASFV- is added
Then 0.5 μ l of P is added 2 μ l pUC57-p72 Plasmid DNA as template, 2.5 μ L magnesium acetate solutions is added in reaction tube
(280mM) starting reaction, close the lid centrifugation, mixing of turning upside down, and is centrifuged 10 seconds, detects immediately.Response procedures are as follows: 39 DEG C 40
Second；39 DEG C of 30 seconds 40 circulations, acquire fluorescence signal herein.
A kind of foundation of the African swine fever fluorescent type RAA detection kit detection method of embodiment 4
(1) on the basis of embodiment 2, the detection method of kit is that 45.5 μ l are added in fluorescence RAA reaction member
Lysis buffer, 2 μ L positive controls/negative control/nucleic acid to be checked add 2.5 μ L magnesium acetate solutions (280mM) in reaction tube,
Close the lid centrifugation, mixing of turning upside down, and is centrifuged 10 seconds, detects immediately.Response procedures are as follows: 39 DEG C 40 seconds；39 DEG C 30 seconds 40
Circulation, acquires fluorescence signal herein.
(2) result judgement
1. positive control: thering is typical amplification curve to occur, appearance time is less than 15min (value≤30 Ct), for effectively knot
Fruit
2. negative control: no amplification curve occurs or appearance time >=20min (value >=40 Ct), is effective result
3. tested sample:
If a. (value < 35 Ct) appearance time≤17.5min, it is judged as positive；
If b. (value >=40 Ct) appearance time >=20min, it is judged as negative；
If c. (value < 40 35 < Ct) 17.5min < appearance time < 20min, be judged to it is suspicious, need to repeat detection be confirmed.Again
Secondary testing result is still (value < 40 35 < Ct) 17.5min < appearance time < 20min, should refer to negative control appearance time (Ct
Value), if negative control appearance time >=20min (value >=40 Ct), it is judged as positive.
The verifying of 5 kit sensitivity of embodiment
The concentration of recombinant plasmid pUC57-p72, adjustment concentration to 10 are surveyed with ultraviolet absorption method6Copies/ μ l, uses ddH2O pairs
Positive plasmid carries out 10 times of doubling dilutions, dilution range 10-1~10-6, detected respectively as template.By kit
Reaction system reagent preparation and setting response procedures.The results show that the minimum detectability of this kit is 10copies/ μ l.
The verifying of 6 kit specificity of embodiment
Extract swine fever virus nucleic acid, foot and mouth disease virus nucleic acid, pig breeding and respiratory disorder syndrome virus nucleic acid, pseudo- mad dog
Viral nucleic acid, respectively template carry out constant-temperature amplification detection, by kit system reagent preparation and setting response procedures.As a result it shows
Show, testing result is feminine gender, this kit specificity is good.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (4)
1. a kind of African hog cholera virus fluorescent type RAA detection kit, including specific primer ASFV-F and ASFV-R, Yi Jibiao
Note has the RAA-exo probe ASFV-P of THF residue, and the reporter fluorescence dyestuff of probe label is FAM, the fluorescent quenching group of label
For BHQ-1；Wherein specific primer and RAA-exo probe sequence are as follows:
ASFV-F:5′-TCGCCGAAGGGAATGGATACTGAGGGAATA-3′；
ASFV-R:5′-AGGGGATAAAATGACTGGATATAAGCACTTGGT-3′；
ASFV-P:5′-TGCGTATCATTTTCATCGGTAAGAATAGGTT[FAM-dT][THF]C[BHQ1-dT]
TTGGTGCGGCTTGTGC-3′。
2. kit according to claim 1 further includes fluorescence RAA reaction member, Buffer A, Buffer B, the positive
Control and negative control.The fluorescence RAA reaction member contains recombinase, single strand binding protein, strand displacement DNA polymerization
Enzyme powder is lyophilized in enzyme, exonuclease；Buffer A is lysis buffer；Buffer B is magnesium acetate solution；Positive control be containing
There is the pUC57-p72 plasmid of African swine fever virus p72 gene, negative control is empty carrier pUC57 plasmid.
3. a kind of preparation method of African hog cholera virus fluorescent type RAA detection kit according to claim 2, feature
It is comprising the steps of:
(1) artificial synthesized African swine fever virus p72 full length gene sequence (accession number: MH713612.1) is cloned into pUC-57 carrier
On, plasmid is extracted after colibacillus engineering pure culture as positive control；
(2) primer sets in claim 1 and probe preparation:
According to the conserved region of p72 gene, specific primer ASFV-F and ASFV-R and RAA-exo probe ASFV-P are designed, is used
Tetrahydrofuran (THF residue), dT- fluorogen and dT- are quenched the base replaced in target amplification subsequence, and the 5 ' of THF residue
The reporter fluorescence dyestuff of extreme direction label is FAM, and 3 ' extreme direction mark fluorescent quenching groups of THF residue are BHQ-1, in reagent
Exonuclease can the site THF crack probe, to separate fluorophor and quenching group and generate fluorescence signal；
(3) enzyme powder, lysis buffer, acetic acid is lyophilized in recombinase, single strand binding protein, strand displacement archaeal dna polymerase, exonuclease
Magnesium solution and deionized water, for commercialization preparation.
4. the method for quickly detecting African swine fever virus using African swine fever virus kit as claimed in claim 2, feature
It is comprising the steps of:
1) it is added 45.5 μ L lysis buffers in fluorescence RAA reaction member, 2 μ L positive controls/negative control/nucleic acid to be checked,
Add 2.5 μ L magnesium acetate solutions in reaction tube, close the lid centrifugation, mixing of turning upside down, and is centrifuged 10 seconds, detects immediately；Reaction
Program are as follows: 39 DEG C 40 seconds；39 DEG C of 30 seconds 40 circulations, acquire fluorescence signal, the achievable detection of 20min herein；
2) result judgement
1. positive control: thering is typical amplification curve to occur, appearance time is less than 15min (value≤30 Ct), is effective result；
2. negative control: no amplification curve occurs or appearance time >=20min (value >=40 Ct), is effective result；
3. tested sample:
If a. (value < 35 Ct) appearance time≤17.5min, it is judged as positive；
If b. (value >=40 Ct) appearance time >=20min, it is judged as negative；
If c. (value < 40 35 < Ct) 17.5min < appearance time < 20min, be judged to it is suspicious, need to repeat detection be confirmed；It examines again
Surveying result is still (value < 40 35 < Ct) 17.5min < appearance time < 20min, should refer to negative control appearance time (Ct value),
If negative control appearance time >=20min (value >=40 Ct), it is judged as positive.
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| CN112760416A (en) * | 2020-12-29 | 2021-05-07 | 肇庆大华农生物药品有限公司 | African swine fever virus fluorescent PCR detection reagent, kit and detection method |
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