CN109504806A - A kind of African swine fever fluorescence PCR detection reagent and its detection method - Google Patents
A kind of African swine fever fluorescence PCR detection reagent and its detection method Download PDFInfo
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- CN109504806A CN109504806A CN201811639635.0A CN201811639635A CN109504806A CN 109504806 A CN109504806 A CN 109504806A CN 201811639635 A CN201811639635 A CN 201811639635A CN 109504806 A CN109504806 A CN 109504806A
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Abstract
The present invention relates to African swine fever detection technique fields, and disclose a kind of African swine fever fluorescence PCR detection reagent and its detection method, detection reagent includes a pair of PCR specific primer, a specific probe, an internal standard probe and PCR mixed liquor, and detection reagent is using highly conserved African swine fever virus VP72 gene as target gene.The African swine fever fluorescence PCR detection reagent and its detection method, have African swine fever fluorescence PCR detection reagent using it is simple, do not need a variety of special instruments, detection time is shorter, and detection efficiency is higher, and does not need multi agent cooperation, the advantages that saving labour.
Description
Technical field
The present invention relates to African swine fever detection technique field, specially a kind of African swine fever fluorescence PCR detection reagent and its
Detection method.
Background technique
African swine fever is a kind of strong, bleeding sexually transmitted disease of the pig as caused by African swine fever virus, and case fatality rate is close
100%, nineteen twenty-one Montgomery due to larger to pig breeding industry harm, and there is no effectively in African Kenya's first discovery
Vaccine control, therefore it is classified as the legal of pig by OIE and reports first of infectious disease, and the highest attention by countries in the world.By
There is complicated immune evasion mechanism in African swine fever virus, lack typical neutralizing antibody, and be used for without effective vaccine
Epidemic prevention, so establishing one kind, fast and accurately detection method is just particularly important, although China does not find this disease also at present,
But with international exchange increasingly frequently, incoming risk increasingly increases.
Existing African swine fever virus detection operation is excessively cumbersome and inconvenient, and detection is not accurate enough, and qualification rate is lower, and detects
Time-consuming more long, African swine fever virus detection efficiency is lower, and multi agent cooperation is needed to detect, labor intensive.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, the present invention provides a kind of African swine fever fluorescence PCR detection reagent and its detection sides
Method, have African swine fever fluorescence PCR detection reagent using it is simple, do not need a variety of special instruments, detection time is shorter, detection effect
The advantages that rate is higher, and does not need multi agent cooperation, saves labour solves African swine fever virus detection inconvenience, detection accurately
Property is lower, and detection efficiency is lower, the problem of lavishing labor on.
(2) technical solution
For realize above-mentioned African swine fever fluorescence PCR detection reagent using it is simple, do not need a variety of special instruments, detection time
Shorter, detection efficiency is higher, and does not need multi agent cooperation, saves the purpose of labour, the invention provides the following technical scheme: one
Kind African swine fever fluorescence PCR detection reagent, the detection reagent include a pair of PCR specific primer, specific probe, one
Internal standard probe and PCR mixed liquor, the detection reagent is using highly conserved African swine fever virus VP72 gene as target gene.
Preferably, the PCR specific primer is SFU and ASFL.
A kind of detection method of African swine fever fluorescence PCR detection reagent, comprising the following steps:
Step 1 makes sample sample；
Step 2, detection reagent prepare；
Step 3 mixes the sample and detection reagent of production, carries out PCR detection amplification
Step 4, recording detection data；
Step 5, Determination data, and judge testing result.
Preferably, sample makes in the step 1, first from pig withdraw blood, then carries out to cell in blood
Broken centrifugal treating is finally taken out serum as sample is detected, can be detected immediately after sample collection；It, can if cannot detect immediately
2~8 DEG C of refrigerated overnights are placed in save；If you need to long-term preservation, sample should be frozen in -80 DEG C hereinafter, avoid multigelation, then sharp
Reagent, which is extracted, with RNA extracts sample DNA.
Preferably, during detection reagent prepares in the step 2, kit is taken out from refrigerator first, is taken from kit
Reagent needed for testing out, sufficiently thawing mixes and brief centrifugation is to remove tube wall attaching liq, then calculates needed for secondary experiment
The stoichiometric number (n) wanted, and the various reagents amount required for time experiment, stoichiometric number are calculated according to reaction system as shown in the table
(n) calculation formula is；
The n=negative control number+pre- allowance+sample number of positive control number+error.
Preferably, the step 3 sequentially adds sample DNA and detection reagent in PCR reaction tube, reacts later in PCR
It is separately added into that the specimen dna to be measured handled well is 5 microlitres each, 25 microlitres of final volume/pipe in sample tube, covers PCR reaction lid and mix
Instantaneous low-speed centrifugal afterwards, is transferred in PCR instrument, the yin that negative control and positive control pipe then respectively add 5 microlitres of kits included
Property control or positive reference substance, final volume be 25 microlitres/pipe, cover PCR reaction lid mix after instantaneous low-speed centrifugal, be transferred to
In PCR instrument, the PCR reaction tube containing sample is taken later, is placed on real-time fluorescence quantitative PCR instrument sample cell corresponding position, and
Placement order is recorded, PCR amplification is then carried out.
Preferably, recording detection data in the step 4, then carries out data statistics, records the detection knot of detection reagent
Fruit.
Preferably, in the step 5, testing result is obtained according to detection data, if testing result is positive, sample
Middle detection African swine fever virus DNA, if testing result is negative, without detection African swine fever virus DNA in sample.
(3) beneficial effect
Compared with prior art, the present invention provides a kind of African swine fever fluorescence PCR detection reagent and its detection method, tools
It is standby following the utility model has the advantages that
The African swine fever fluorescence PCR detection reagent and its detection method, fluorescence PCR detection reagent are special by a pair of PCR
Property primer, a specific probe, an internal standard probe and PCR mixed liquor composition, PCR mixed liquor separately packs, and avoids its length
Time mixing failure, is improved the holding time of detection reagent, and special by the design of the highly conserved region of African swine fever virus
Property primer and probe, in the reaction system in the case where genomic templates containing African swine fever virus, PCR reaction is able to carry out and release
Fluorescence signal is put, real-time monitoring and output are carried out to respective channel signal during PCR using fluorescence quantitative PCR instrument, realize inspection
The qualitative analysis of result is surveyed, while detection reagent carries out record and the comparison of detection data by one man operation, uses profession
Detecting instrument is less, and during the entire process of detection African swine fever, extracts from DNA and testing result occur, then only need 2-2.5h,
Greatly reduce manual operations and shorten the time, improves African swine fever virus detection efficiency.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
A kind of African swine fever fluorescence PCR detection reagent, detection reagent include a pair of PCR specific primer, a specificity
Probe, an internal standard probe and PCR mixed liquor, detection reagent are using highly conserved African swine fever virus VP72 gene as target base
Cause.
PCR specific primer is SFU and ASFL.
A kind of detection method of African swine fever fluorescence PCR detection reagent, comprising the following steps:
Step 1 makes sample sample；
Step 2, detection reagent prepare；
Step 3 mixes the sample and detection reagent of production, carries out PCR detection amplification
Step 4, recording detection data；
Step 5, Determination data, and judge testing result.
Sample makes in step 1, first from pig withdraw blood, then carries out at broken centrifugation to cell in blood
Reason is finally taken out serum as sample is detected, can be detected immediately after sample collection；If cannot detect immediately, 2~8 DEG C can be placed in
Refrigerated overnight saves；If you need to long-term preservation, sample should be frozen in -80 DEG C hereinafter, avoid multigelation, then be extracted using RNA
Reagent extracts sample DNA.
During detection reagent prepares in step 2, kit is taken out from refrigerator first, is taken out needed for experiment from kit
Reagent melts mixing and brief centrifugation sufficiently to remove tube wall attaching liq, then calculates the stoichiometric number required for time experiment
(n), the various reagents amount required for time experiment, stoichiometric number (n) calculation formula and according to reaction system as shown in the table are calculated
For；
The n=negative control number+pre- allowance+sample number of positive control number+error.
Step 3 sequentially adds sample DNA and detection reagent in PCR reaction tube, divides in PCR reaction sample tube later
It is not added that the specimen dna to be measured handled well is 5 microlitres each, 25 microlitres of final volume/pipe, covers instantaneous low speed after PCR reaction lid mixes
Centrifugation, is transferred in PCR instrument, the negative control or sun that negative control and positive control pipe then respectively add 5 microlitres of kits included
Property reference substance, final volume be 25 microlitres/pipe, cover PCR reaction lid mix after instantaneous low-speed centrifugal, be transferred in PCR instrument, it
The PCR reaction tube containing sample is taken afterwards, is placed on real-time fluorescence quantitative PCR instrument sample cell corresponding position, and record placement order,
Then PCR amplification is carried out.
Then recording detection data in step 4 carries out data statistics, record the testing result of detection reagent.
In step 5, testing result is obtained according to detection data, African pig is detected if testing result is positive, in sample
Pestivirus DNA, if testing result is negative, without detection African swine fever virus DNA in sample.
In conclusion the African swine fever fluorescence PCR detection reagent and its detection method, pass through the height of African swine fever virus
Conservative region designs specific primer and probe, in the reaction system in the case where genomic templates containing African swine fever virus, PCR
Reaction is able to carry out and discharge fluorescence signal, is supervised in real time using fluorescence quantitative PCR instrument to respective channel signal during PCR
Survey and output, realize the qualitative analysis of testing result, while detection reagent is by one man operation, carry out detection data record and
It compares, the detecting instrument using profession is less, and during the entire process of detection African swine fever, extracts appearance detection knot from DNA
Fruit then only needs 2-2.5h, greatly reduces manual operations and shortens the time, improves African swine fever virus detection efficiency.
It should be noted that the terms "include", "comprise" or its any other variant are intended to the packet of nonexcludability
Contain, so that the process, method, article or equipment for including a series of elements not only includes those elements, but also including
Other elements that are not explicitly listed, or further include for elements inherent to such a process, method, article, or device.
In the absence of more restrictions, the element limited by sentence "including a ...", it is not excluded that including the element
Process, method, article or equipment in there is also other identical elements.
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 (8)
1. a kind of African swine fever fluorescence PCR detection reagent, it is characterised in that: the detection reagent includes that a pair of PCR specificity is drawn
Object, a specific probe, an internal standard probe and PCR mixed liquor, the detection reagent are with highly conserved African swine fever
Viral VP72 gene is target gene.
2. a kind of African swine fever fluorescence PCR detection reagent according to claim 1, it is characterised in that: the PCR specificity
Primer is SFU and ASFL.
3. a kind of detection method of African swine fever fluorescence PCR detection reagent, it is characterised in that: the following steps are included:
Step 1 makes sample sample；
Step 2, detection reagent prepare；
Step 3 mixes the sample and detection reagent of production, carries out PCR detection amplification
Step 4, recording detection data；
Step 5, Determination data, and judge testing result.
4. a kind of detection method of African swine fever fluorescence PCR detection reagent according to claim 3, it is characterised in that: institute
It states sample in step 1 to make, first from pig withdraw blood, broken centrifugal treating then is carried out to cell in blood, finally
Serum is taken out as sample is detected, can be detected immediately after sample collection；If cannot detect immediately, 2~8 DEG C of refrigerated overnights can be placed in
It saves；If you need to long-term preservation, sample should be frozen in -80 DEG C hereinafter, avoid multigelation, then extracted reagent using RNA and extracted
Sample DNA.
5. a kind of detection method of African swine fever fluorescence PCR detection reagent according to claim 3, it is characterised in that: institute
It states in step 2 during detection reagent prepares, takes out kit from refrigerator first, reagent needed for testing is taken out from kit, is filled
Divide and melt mixing and brief centrifugation to remove tube wall attaching liq, then calculates stoichiometric number (n) required for working as time experiment, and root
The various reagents amount required for time experiment is calculated according to reaction system as shown in the table, stoichiometric number (n) calculation formula is；
The n=negative control number+pre- allowance+sample number of positive control number+error.
6. a kind of detection method of African swine fever fluorescence PCR detection reagent according to claim 3, it is characterised in that: institute
It states step 3 to sequentially add in PCR reaction tube sample DNA and detection reagent, be separately added into PCR reaction sample tube later
The specimen dna to be measured handled well is 5 microlitres each, 25 microlitres of final volume/pipe, covers instantaneous low-speed centrifugal after PCR reaction lid mixes, turns
It moves on in PCR instrument, the negative control or positive control that negative control and positive control pipe then respectively add 5 microlitres of kits included
Product, final volume are 25 microlitres/pipe, cover instantaneous low-speed centrifugal after PCR reaction lid mixes, are transferred in PCR instrument, take contain later
Have the PCR reaction tube of sample, be placed on real-time fluorescence quantitative PCR instrument sample cell corresponding position, and record placement order, then into
Row PCR amplification.
7. a kind of detection method of African swine fever fluorescence PCR detection reagent according to claim 3, it is characterised in that: institute
Recording detection data in step 4 is stated, data statistics is then carried out, records the testing result of detection reagent.
8. a kind of detection method of African swine fever fluorescence PCR detection reagent according to claim 3, in the step 5,
Testing result is obtained according to detection data, if testing result is positive, African swine fever virus DNA is detected in sample, if detection
As a result it is negative, then without detection African swine fever virus DNA in sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111254220A (en) * | 2020-02-06 | 2020-06-09 | 成都导胜生物技术有限公司 | Composition and kit for detecting African swine fever virus DNA |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111254220A (en) * | 2020-02-06 | 2020-06-09 | 成都导胜生物技术有限公司 | Composition and kit for detecting African swine fever virus DNA |
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Application publication date: 20190322 |