CN109295255A - A kind of nucleic acid rapid detection method for African swine fever virus - Google Patents
A kind of nucleic acid rapid detection method for African swine fever virus Download PDFInfo
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- CN109295255A CN109295255A CN201811085469.4A CN201811085469A CN109295255A CN 109295255 A CN109295255 A CN 109295255A CN 201811085469 A CN201811085469 A CN 201811085469A CN 109295255 A CN109295255 A CN 109295255A
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a kind of nucleic acid rapid detection methods for African swine fever virus, including following detection method: material prepares, primer dilution, amplified reaction, and amplification curve and solubility curve obtain, determine；The beneficial effects of the present invention are: high specific: the high degree of specificity that 6 primer pairs answer the identification of 8 specific regions of target sequence to ensure that detection is reacted；It is highly sensitive: to detect the high sensitivity of reaction, 10 copies can be detected；Easy to operate: as long as sample to be tested, the special primer of design and reaction solution are mixed, upper machine, which expands 30 minutes, can be obtained testing result；It can be detected out whether sample has infected African swine fever virus using a reaction tube, filled up the vacancy of African swine fever nucleic acid rapid detection method；Kit can be used in multiple detection platforms, be not only used in Gene Detecting instrument and quantitative PCR apparatus, can also use such as metal bath in the equipment that other can provide steady temperature.
Description
Technical field
The invention belongs to biology techniques fields, and in particular to a kind of quick side of detection of the nucleic acid for African swine fever virus
Method.
Background technique
African swine fever (African swine fever, ASF) is by African swine fever virus (African swine fever
Virus, ASFV) caused by pig a kind of strong, highly contagious disease.ASF has the spies such as high incidence, high mortality
Point, once occurring, economic loss is huge；Lack effective vaccine and specific treatment means at present, so that ASF is become harm at present and support
One of most serious epidemic disease of pig industry.
ASFV is African swine fever virus section (Asfarviridae) African swine fever virus category (Asfi virus) unique member,
Some viral characteristics are similar to Iridoviridae and Poxviridae；The diameter of virion is 175-215 nanometers, is in 20 face bodies pair
Claim, there is cyst membrane.Genome is bifilar linear DNA, size 170-190kb.In pig body, African swine fever virus can be in several types
It is proliferated in the cytoplasm of type, is especially replicated in reticuloendothelial cell and mononuclear macrophage.The virus can increase in turicata
It grows, and becomes main communication media.The quick diagnosis in laboratory is relied only on, to morbidity to the control of ASF at present
Animal is catched and killed, and takes effective quarantine measures and stringent health measure.
Currently, ASFV is in China's the first explosion, domestic multiple areas are popular, epidemic situation continuous enlargement.ASFV lacks in China
Effective vaccine and quick Pathogen test technology are epidemic situation accurate measurements and the existing major issue of control.Therefore, novel disease is utilized
Original learns detection technique, establishes technology of the rapid detection method for ASFV cause of disease Rapid identification to be effective, accurate prevention and control epidemic situation and closes
Key.
Summary of the invention
It is above-mentioned to solve the purpose of the present invention is to provide a kind of nucleic acid rapid detection method for African swine fever virus
The ASFV proposed in background technique lacks effective vaccine and quick Pathogen test technology in China, is epidemic situation accurate measurements and control
Existing major issue overcomes the deficiencies of the prior art and provide a kind of quick, accurate, sensitive method detection based on nucleic acid
Whether swinery infects the primer sets of African swine fever virus, and the nucleic acid rapid detection method of the above-mentioned primer sets of application.
To achieve the above object, the invention provides the following technical scheme: a kind of nucleic acid for African swine fever virus is quick
Detection method, including following detection method:
Step 1: material prepares: whether detection pig infects the rapid amplifying primer sets of African swine fever, and primer sets include: to draw
The dry powder of object 1 and 2, the dry powder of primer 3 and 4, the dry powder of primer 5 and 6, each 1OD；Reaction solution composition: reaction concentrate (archaeal dna polymerase,
Buffer, dNTP, MgSO4, Betain, fluorescent dye)；Construct the African swine fever virus pUC-ASF plasmid sample completed；It is negative
Control group: target gene reaction solution is free of；
Step 2: primer dilution: primer 1 and 2 in kit is diluted to 40uM, primer 3 and 4 is diluted to 5uM, primer 5
20uM is diluted to 6；
Step 3: amplified reaction: reaction concentrate 15ul, primer sets 6ul, calligraphy or painting model, building standard plasmid DNA1ul, nothing
Bacterium nuclease-free water 3ul；
Step 4: amplification curve and solubility curve obtain: the reaction solution in step 3 mixed, quantitative PCR apparatus is put into, if
60 circulations are set, obtain amplification curve and solubility curve after the completion of circulation；Or in Gene Detecting system, 65 DEG C, 30 points
Clock obtains amplification curve and solubility curve；
Step 5: determine: being determined according to the obtained amplification curve of step 4 and solubility curve.
It is described as a preferred technical solution of the invention
Primer 1:AACGTTGCCAACGTAGTGGATCTGCTCACGATGCCAATCTCATC；
Primer 2: CTTGGAGCGATGATGATTATGCCTATGACCGAAGGATTG；
Primer 3:ATGGCATACTCATCCATCGATG；
Primer 4:AAGGCTCTTGCTGTTGATACC；
Primer 5:AAGCTGTAAGCTCTGCAGGACT；
Primer 6:CTGGGTAGCCACGGAAGGA.
As a preferred technical solution of the invention, the design of primers uses Primer according to P72 segment
Designer software Design primers group.
As a preferred technical solution of the invention, in the step 4, reaction solution is mixed, and is put into quantitative PCR apparatus,
65 DEG C of setting, 30s/ recycle 60 circulations.
As a preferred technical solution of the invention, in the step 5, according to obtained amplification curve and dissolution
Curve is determined that amplification curve has apparent " S " type curve and solubility curve peak type single and more sharp, then is determined
For positive findings；There is not obvious " S " type curve in amplification curve, then is determined as negative findings；Amplification curve has obvious " S " type
Curve, but solubility curve peak type is not single is determined as non-specific amplification；The testing result of negative control group is negative findings, then
It is that this experiment is effective, otherwise this experiment is invalid.
Compared with prior art, the beneficial effects of the present invention are:
(1) high specific: the height that 6 primer pairs answer the identification of 8 specific regions of target sequence to ensure that detection is reacted
Specificity；
(2) highly sensitive: to detect the high sensitivity of reaction, 10 copies can be detected；
(3) easy to operate: as long as sample to be tested, the special primer of design and reaction solution are mixed, upper machine is expanded 30 minutes
Testing result can be obtained；
(4) it can be detected out whether sample has infected African swine fever virus using a reaction tube, filled up African swine fever
The vacancy of nucleic acid rapid detection method；
(2) African swine fever virus nucleic acid rapid amplifying detection kit has used Embedded fluorescent dye, in quantitative PCR
It is reacted on instrument or special Gene Detecting instrument, realizes real time monitoring process, and can do and dissolve for amplified production
Tracing analysis carries out the analysis of specificity for product；
(6) kit can be used in multiple detection platforms, be not only used in Gene Detecting instrument and quantitative PCR apparatus,
Such as metal bath in the equipment that other can provide steady temperature can also be used.
Detailed description of the invention
Fig. 1 is the structural diagram of the present invention；
Fig. 2 is that the African swine fever virus plasmid sample that uses of the invention is template, is obtained respectively using primer sets augmentation detection
Obtain ASFV amplification curve diagram；
Fig. 3 is that the African swine fever virus plasmid sample that uses of the invention is template, is obtained respectively using primer sets augmentation detection
Obtain ASFV solubility curve figure；
Specific embodiment
A kind of nucleic acid rapid detection method for African swine fever virus, including following detection method:
Step 1: material prepares: the rapid amplifying primer sets whether detection pig infects African swine fever make according to P72 segment
With Primer designer software Design primers group, primer sets include: the dry powder of primer 1 and 2, the dry powder of primer 3 and 4, primer 5 and 6
Dry powder, each 1OD,
Primer 1:AACGTTGCCAACGTAGTGGATCTGCTCACGATGCCAATCTCATC；
Primer 2: CTTGGAGCGATGATGATTATGCCTATGACCGAAGGATTG；
Primer 3:ATGGCATACTCATCCATCGATG；
Primer 4:AAGGCTCTTGCTGTTGATACC；
Primer 5:AAGCTGTAAGCTCTGCAGGACT；
Primer 6:CTGGGTAGCCACGGAAGGA；6 primer pairs answer the identification of 8 specific regions of target sequence to guarantee inspection
Survey the high degree of specificity of reaction；Reaction solution composition: reaction concentrate (archaeal dna polymerase, Buffer, dNTP, MgSO4, Betain,
Embedded fluorescent dye)；Construct the African swine fever virus pUC-ASF plasmid sample completed；Negative control group: target gene is free of
Reaction solution；
Step 2: primer dilution: primer 1 and 2 in kit is diluted to 40uM, primer 3 and 4 is diluted to 5nM, primer 5
20uM is diluted to 6；
Step 3: amplified reaction: amplification total system 25ul, including reaction concentrate 15ul, primer sets 6ul react sample
1ul, sterile nuclease-free water 3ul；
Step 4: amplification curve and solubility curve obtain: the reaction solution in step 3 mixed, quantitative PCR apparatus is put into, if
60 65 DEG C, 30s/ circulation circulations are set, obtain amplification curve and solubility curve after the completion of circulation；Or Gene Detecting system
In, 65 DEG C, 30 minutes obtain amplification curve and solubility curve；
Step 5: determine: being determined that amplification curve has obviously according to the obtained amplification curve of step 4 and solubility curve
" S " type curve and solubility curve peak type it is single and more sharp, then be determined as positive findings；Amplification curve does not occur
Obviously " S " type curve, then be determined as negative findings；Amplification curve has obvious " S " type curve, but solubility curve peak type is not single sentences
It is set to non-specific amplification；The testing result of negative control group is negative findings, then is that this experiment is effective, otherwise this is tested
In vain；
Primer sets and reaction system sensitivity experiment:
The African swine fever virus plasmid 10 of building is diluted to 10 again0、101、102、103、104、105A every microlitre of copy,
Using Step 3: the system referred in step 4 and step 5 is detected respectively.
Test result:
Primer sets and reaction system detection:
The use of African swine fever virus plasmid sample is template, uses the real-time amplification curve graph of primer sets augmentation detection respectively
With solubility curve figure, primer sets amplification curve can be clearly observed from figure in " S " type, solubility curve peak type is single, and right
According to group without amplification；
Primer sets and reaction system sensitivity experiment:
By 10 times of gradient dilutions of African swine fever virus plasmid of building, respectively 101、102、103、104、105、106、107
A every microlitre of copy is dense in sample using Step 3: the system referred in step 4 and step 5 detects each concentration
Degree is 103When copy, amplification curve can be still generated, and solubility curve peak type is single and sharp, illustrate African pig of the invention
Pest detection kit sensitivity can reach 103Copy.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (5)
1. a kind of nucleic acid rapid detection method for African swine fever virus, which is characterized in that including following detection method:
Step 1: material prepares: whether detection pig infects the rapid amplifying primer sets of African swine fever, and primer sets include: 1 He of primer
2 dry powder, the dry powder of primer 3 and 4, the dry powder of primer 5 and 6, each 1OD；Reaction solution composition: reaction concentrate (archaeal dna polymerase, Buffer,
DNTP, MgSO4, Betain, fluorescent dye)；Construct the African swine fever virus pUC-ASF plasmid sample completed；Negative control group:
Without target gene reaction solution；
Step 2: primer dilution: primer 1 and 2 in kit is diluted to 40uM, primer 3 and 4 is diluted to 5uM, and primer 5 and 6 is dilute
It is interpreted into 20uM；
Step 3: amplified reaction: reaction concentrate 15ul, primer sets 6ul, calligraphy or painting model, building standard plasmid DNA1ul, sterile nothing
Nuclease water 3ul；
Step 4: amplification curve and solubility curve obtain: the reaction solution in step 3 being mixed, quantitative PCR apparatus, setting 60 are put into
A circulation, circulation obtain amplification curve and solubility curve after the completion；Or in Gene Detecting system, 65 DEG C, 30 minutes are obtained
Obtain amplification curve and solubility curve；
Step 5: determine: being determined according to the obtained amplification curve of step 4 and solubility curve.
2. a kind of nucleic acid rapid detection method for African swine fever virus according to claim 1, it is characterised in that: institute
It states
Primer 1:AACGTTGCCAACGTAGTGGATCTGCTCACGATGCCAATCTCATC；
Primer 2: CTTGGAGCGATGATGATTATGCCTATGACCGAAGGATTG；
Primer 3:ATGGCATACTCATCCATCGATG；
Primer 4:AAGGCTCTTGCTGTTGATACC；
Primer 5:AAGCTGTAAGCTCTGCAGGACT；
Primer 6:CTGGGTAGCCACGGAAGGA.
3. a kind of nucleic acid rapid detection method for African swine fever virus according to claim 1, it is characterised in that: institute
Design of primers is stated according to P72 segment, uses Primer Designer software Design primers group.
4. a kind of nucleic acid rapid detection method for African swine fever virus according to claim 1, it is characterised in that: institute
It states in step 4, reaction solution mixes, and is put into quantitative PCR apparatus, and 65 DEG C of setting, 30s/ recycle 60 circulations.
5. a kind of nucleic acid rapid detection method for African swine fever virus according to claim 1, it is characterised in that: institute
It states in step 5, is determined according to obtained amplification curve and solubility curve, amplification curve has apparent " S " type curve, with
And solubility curve peak type is single and more sharp, then is determined as positive findings；There is not obvious " S " type curve in amplification curve,
Then it is determined as negative findings；Amplification curve has obvious " S " type curve, but solubility curve peak type is not single is determined as non-specific expansion
Increase；The testing result of negative control group is negative findings, then is that this experiment is effective, otherwise this experiment is invalid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110241258A (en) * | 2019-06-19 | 2019-09-17 | 湖南新南方养殖服务有限公司 | The method whether detection pig farm birds or its living environment carry African swine fever virus |
| CN110364225A (en) * | 2019-08-19 | 2019-10-22 | 中国科学院武汉病毒研究所 | A method of utilizing raw letter technology mining ASFV detection of nucleic acids sequence |
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Application publication date: 20190201 |