CN109722490A - A kind of primer combination, kit and its application for pig blue-ear disease poison detection of nucleic acids - Google Patents
A kind of primer combination, kit and its application for pig blue-ear disease poison detection of nucleic acids Download PDFInfo
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- CN109722490A CN109722490A CN201811620347.0A CN201811620347A CN109722490A CN 109722490 A CN109722490 A CN 109722490A CN 201811620347 A CN201811620347 A CN 201811620347A CN 109722490 A CN109722490 A CN 109722490A
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Abstract
The present invention provides a kind of primer combinations for pig blue-ear disease poison detection of nucleic acids, belong to technical field of biological.In the present invention, the primer combination includes upstream internal primers F IP, downstream inner primer BIP, upstream outer primers F 3 and downstream outer primer B3；Each primer sequence is successively as shown in No:1~4 SEQ ID.Primer combination provided by the invention designs to obtain using 6 specific positions in target gene pig blue-ear disease poison NSP2 genetic fragment, in 55~65 DEG C of isothermal duplications, can pass through strand replacement reaction massive amplification target sequence under the action of Bst archaeal dna polymerase.The present invention by being added colored indicator in the reaction system, the difference of positive and negative result is obvious, can be by color change interpretation result, and this method high sensitivity, the minimum PRRS virus that 10 copies can be detected, 10 times of the RT-PCR detection reagent high sensitivity of remolding sensitivity routine.
Description
Technical field
The invention belongs to technical field of biological, and in particular to a kind of primer sets for pig blue-ear disease poison detection of nucleic acids
Conjunction, kit and its application.
Background technique
Pig blue-ear disease poison (Porcinere productive and respiratory syndrome, PRRS) is artery
Scorching Viraceae Arterivirus member is found in 1987 earliest, has spread North America and Europe, passed in the world
It broadcasts.Guo Baoqing etc. (1996) isolates PRRSV from domestic doubtful PRRS infection swinery for the first time, to confirm the virus at me
State exists.The viral transmission speed is fast, especially in technological progress and developed country, since swinery is intensive, flowing is frequent, more
Easily cause prevalence.The infection of pig blue-ear disease poison will cause sick pig fever, anorexia, and pregnant sow late abortion, produces stillborn foetus, is weak premature labor
Tire and the mummification of fetus, the diseases such as various age pig (especially piglet) respiratory disorders, cause huge economic loss to pig breeding industry.
The detection method of existing PRRS virus nucleic acid has antibody test and antigen detection.Antibody test often lags behind
Antigen detection, thus cannot quick discovery epidemic disease promptly and accurately, lead to the eruption and prevalence of epidemic disease, cause huge economic damage
It loses.And virus purification time length, environmental facility require high, technical level requirement height, sensitivity in existing antigen detection method
Also it is difficult to meet actual demand.
Summary of the invention
In view of technical problem present in background technique, the purpose of the present invention is to provide one kind for pig blue-ear disease poison
Primer combination, kit and its application of detection of nucleic acids.
The present invention provides a kind of primer combinations for pig blue-ear disease poison detection of nucleic acids, and the primer combination includes upstream
Internal primer FIP, downstream inner primer BIP, upstream outer primers F 3 and downstream outer primer B3；
The nucleotide sequence of the upstream internal primers F IP is as shown in SEQ ID No:1；
The nucleotide sequence of the downstream inner primer BIP is as shown in SEQ ID No:2；
The nucleotide sequence of the upstream outer primers F 3 is as shown in SEQ ID No:3；
The nucleotide sequence of the downstream outer primer B3 is as shown in SEQ ID No:4.
It include Primer mix in the kit the present invention provides a kind of pig blue-ear disease poison kit for detecting nucleic acid,
Buffer, reverse transcriptase, Bst archaeal dna polymerase, stabilizing solution, MnCl2Solution, colored indicator and DEPC water；The Primer
Mix includes above-mentioned primer combination.
Preferably, in the Primer mix, the concentration of FIP and BIP are respectively 7~9 μm of ol/L；F3's and B3 is dense
Degree is respectively 0.8~1.2 μm of ol/L.
Preferably, the buffer includes the dNTPs of 13~15mmol/L, the MgSO of 75~85mmol/L4, 180~
(the NH of the Tris of 220mmol/L, 90~110mmol/L4)2SO4, the KCl of 450~550mmol/L and 0.8~1.2% volume
The Tween-20 of concentration.
Preferably, the reverse transcriptase is AMV reverse transcriptase, and the Rate activity of the AMV reverse transcriptase is 4~6U/ μ L；Institute
The Rate activity for stating Bst archaeal dna polymerase is 8~12U/ μ L.
Preferably, the stabilizing solution is the aqueous solutions of betaine of 4~6mol/L.
Preferably, the MnCl2The concentration of solution is 8~20mmol/L.
Preferably, the colored indicator is 4- (2- pyridylazo) resorcinol solution of 8~20mmol/L concentration.
It preferably, further include positive control and/or negative control in the kit；The positive control includes pig indigo plant ear
Virus N SP2 genetic fragment；The negative control does not include pig blue-ear disease poison NSP2 genetic fragment.
The present invention also provides the application of the combination of above-mentioned primer or kit in detection pig blue-ear disease poison, the applications
Not for the purpose of the diagnosis of disease.
Preferably, the application include in pannage, drinking-water pig blue-ear disease poison detection or to pig breeding environment (such as
Pig house) in pig blue-ear disease poison detection.
The utility model has the advantages that the present invention provides a kind of primer combination for pig blue-ear disease poison detection of nucleic acids, the primer sets
Close includes upstream internal primers F IP, downstream inner primer BIP, upstream outer primers F 3 and downstream outer primer B3；Each primer sequence
Leie time is as shown in No:1~4 SEQ ID.Primer combination provided by the invention utilizes target gene pig blue-ear disease poison NSP2 gene piece
6 specific positions in section design to obtain, and, in 55~65 DEG C of isothermal duplications, can lead under the action of Bst archaeal dna polymerase
Cross strand replacement reaction massive amplification target sequence.The present invention by being added colored indicator, positive and negative fruiting area in the reaction system
It is unobvious, can be by color change interpretation result, and this method high sensitivity, the minimum pig blue-ear disease that 10 copies can be detected are sick
Poison, 10 times of the RT-PCR detection reagent high sensitivity of remolding sensitivity routine.In addition, combined using primer provided by the invention or
Kit detects pig blue-ear disease poison, simple and convenient, after the reactive components such as enzyme, primer are mixed, keeps the temperature one hour
Identification can be completed；Colored indicator is added before reaction, reaction solution color variation interpretation result is observed by the naked eye after reaction, directly
It is convenient to see, and can effectively avoid uncap after reaction caused by Aerosol Pollution.The present invention utilizes multiple only on target-gene sequence
A plurality of primer is designed in vertical region, can guarantee the high degree of specificity of amplified reaction.
Detailed description of the invention
Fig. 1 is the structure corresponding relationship of primer of the present invention combination and target gene.
Specific embodiment
The present invention provides a kind of primer combinations for pig blue-ear disease poison detection of nucleic acids, and the primer combination includes upstream
Internal primer FIP, downstream inner primer BIP, upstream outer primers F 3 and downstream outer primer B3；
The nucleotide sequence of the upstream internal primers F IP is as shown in SEQ ID No:1；
The nucleotide sequence of the downstream inner primer BIP is as shown in SEQ ID No:2；
The nucleotide sequence of the upstream outer primers F 3 is as shown in SEQ ID No:3；
The nucleotide sequence of the downstream outer primer B3 is as shown in SEQ ID No:4.
Primer combination provided by the invention is to utilize 6 specificity in target gene pig blue-ear disease poison NSP2 genetic fragment
Site is designed to obtain, and the primer combination is as shown in Figure 1 with the structure corresponding relationship of target gene.Wherein, upstream internal primers F IP
The area Shi You F2 (Forward Inner Primer) and the region F1C composition, the region F2c at the end F2 Qu Nengyu target gene 3' is complementary,
The area F1C is identical as the Flc regional sequence at the end target gene 5'.Downstream inner primer BIP (Backward Inner Primer) be by
The region B1c and B2 composition, the area B2 is complementary with the region B2c at the end target gene 3', the Blc regional sequence phase at the end B1C and target gene 5'
Together.Upstream outer primers F 3 (Forward Outer Primer) is by F3 district's groups at complementary with the region F3c of target gene.Under
Trip external primers B3 (Backward Outer Primer) is made of the region B3, can be complementary with the region B3c of target gene.This
The preparation method that above-mentioned primer is combined in invention is not particularly limited, and synthesizes to obtain using the conventional method of this field.This
The primer combination that invention provides can be under the action of Bst archaeal dna polymerase, and 55~65 DEG C of isothermal duplications are big by strand replacement reaction
Amount amplification target sequence, high sensitivity.
It include Primer mix in the kit the present invention provides a kind of pig blue-ear disease poison kit for detecting nucleic acid,
Buffer, reverse transcriptase, Bst archaeal dna polymerase, stabilizing solution, MnCl2Solution, colored indicator and DEPC water.In the present invention,
The Primer mix is for identifying pig blue-ear disease poison nucleic acid specificity site；The buffer be used for as reverse transcription and
Strand replacement reaction provides suitable reaction condition；Reverse transcription of the reverse transcriptase for viral nucleic acid obtains cDNA sequence, is
Strand replacement reaction provides template；The Bst archaeal dna polymerase mediated isothermality strand replacement reaction；The stabilizing solution is used for intensified response
Stability and specificity；The MnCl2Solution combines colored indicator before the reaction, makes indicator in orange red；Colour developing instruction
Agent provides visualization instruction for reaction, and indicator becomes yellow from orange red after positive reaction.DEPC water is for supplying reactant
Volume in system.
Kit of the present invention includes Primermix.The Primer mix includes above-mentioned primer combination, described
In Primer mix, the concentration of FIP and BIP are respectively preferably 7~9 μm of ol/L, more preferably 8 μm of ol/L；The concentration of F3 and B3
It is respectively preferably 0.8~1.2 μm of ol/L, more preferably 1 μm of ol/L.Specification of the Primer mix in the kit is excellent
It is selected as 90~110 μ L × 1 pipe, more preferably 100 μ L × 1 pipe.
Kit of the present invention includes buffer.In the present invention, the buffer preferably includes dNTPs, MgSO4,
Tris, (NH4)2SO4, KCl and Tween-20.The concentration of the dNTPs is preferably 13~15mmol/L, more preferably
14mmol/L.The MgSO4Concentration be preferably 75~85mmol/L, more preferably 80mmol/L.The concentration of the Tris is excellent
It is selected as 180~220mmol/L, more preferably 200mmol/L.(the NH4)2SO4Concentration be preferably 90~110mmol/L,
More preferably 100mmol/L.The concentration of the KCl is preferably 450~550mmol/L, more preferably 500mmol/L.It is described
The volumetric concentration of Tween-20 is preferably 0.8~1.2%, and more preferably 1%.Specification of the buffer in the kit
Preferably 90~110 μ L × 1 pipe, more preferably 100 μ L × 1 pipe.
Kit of the present invention includes reverse transcriptase and Bst archaeal dna polymerase.In the present invention, the reverse transcriptase is excellent
It is selected as AMV reverse transcriptase, the Rate activity of the AMV reverse transcriptase is preferably 4~6U/ μ L, more preferably 5U/ μ L.The Bst
The Rate activity of archaeal dna polymerase is preferably 8~12U/ μ L, more preferably 10U/ μ L.The present invention is to the reverse transcriptase and Bst DNA
The source of polymerase is not particularly limited, this field conventional commercial product.In an embodiment of the present invention, the reverse transcription
Enzyme is AMV reverse transcriptase (Reverse Transcriptase) of the purchase from TAKARA company, and specification is 200U (5U/ μ L).Institute
Stating Bst archaeal dna polymerase is purchase from one hundred Ke Biotechnology Co., Ltd of Henan, and specification is 10000U (10U/ μ L).In the present invention
In, specification of the reverse transcriptase in the kit is preferably 15~25 μ L × 1 pipe, more preferably 20 μ L × 1 pipe；It is described
Specification of the Bst archaeal dna polymerase in the kit is preferably 15~25 μ L × 1 pipe, more preferably 20 μ L × 1 pipe
Kit of the present invention includes stabilizing solution, MnCl2Solution and colour developing indicator solution.In the present invention, the stabilization
Liquid is preferably alkali solution of beet.The concentration of the alkali solution of beet is preferably 4~6mol/L, more preferably 5mol/L.It is described
MnCl2The concentration of solution is preferably 8~20mmol/L, more preferably 10mmol/L.The colour developing indicator solution is preferably 4- (2-
Pyridylazo) resorcinol solution.The concentration of 4- (2- pyridylazo) resorcinol solution is preferably 8~20mmol/L,
More preferably 10mmol/L.The present invention is to the stabilizing solution, MnCl2The source of solution and colour developing indicator solution is not particularly limited,
This field conventional commercial product.In the present invention, specification of the stabilizing solution in the kit be preferably 400~
600 μ L × 1 pipe, more preferably 500 μ L × 1 pipe；The MnCl2Specification of the solution in the kit is preferably 400~600
μ L × 1 is managed, more preferably 500 μ L × 1 pipe；The specification of the colour developing indicator solution in the kit be preferably 70~90 μ L ×
1 pipe, more preferably 80 μ L × 1 pipe.
Kit of the present invention further includes DEPC water.The present invention is not particularly limited the source of the DEPC water, this
Field conventional commercial product.
It is also preferable to include positive control and/or negative controls for kit of the present invention.In the present invention, the positive is right
According to including pig blue-ear disease poison NSP2 genetic fragment, preferably synthetic pig blue-ear disease poison NSP2 genetic fragment；The negative control
It does not include pig blue-ear disease poison NSP2 genetic fragment, preferably free of contamination sterile water.The positive control and/or negative control exist
The accuracy of testing result can be used to prove in the present invention.
The present invention also provides the application of the combination of above-mentioned primer or kit in detection pig blue-ear disease poison, the applications
Not for the purpose of the diagnosis of disease.Preferably, the application includes detecting to the pig blue-ear disease poison in livestock feed, drinking-water, right
Pig blue-ear disease poison detection in livestock-raising site environment (such as pig house) or the health detection to Pork For Export food.
In the present invention, the system of the detection is preferably 25 μ L systems, and the 25 μ L system preferably includes 2 μ L and waits for test sample
Product, 5 μ L Primer, 2.5 μ L buffer (10 ×), 1.5 μ L stabilizers, 1 μ L reverse transcriptase, 1 μ L Bst archaeal dna polymerase,
1.25μL MnCl2Solution, 1.25 μ L colored indicators and 9.5 μ LDEPC water.
After the present invention prepares detection architecture, reacted.The temperature of the reaction is preferably 55~65 DEG C, more preferably 60
℃.The time of the reaction is preferably 45~60min, more preferably 50min.After reaction, the present invention takes out reaction tube, cold
But to room temperature, 2000r/min is centrifuged 10s, observes the color of centrifugate: if centrifugate color be it is orange red or orange, say
Without containing pig blue-ear disease poison in bright sample to be tested；If centrifugate color is yellow or light yellow, illustrate to contain in sample to be tested
There is pig blue-ear disease malicious.If positive and negative control reaction result is not inconsistent with above-mentioned judgment criteria, illustrate that testing result is invalid, it should
Again it detects.
Below with reference to embodiment to provided by the invention a kind of for the primer combination of pig blue-ear disease poison detection of nucleic acids, reagent
Box and its application are described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
A kind of pig blue-ear disease poison kit for detecting nucleic acid, including composition as shown in Table 1.
1 pig blue-ear disease poison kit for detecting nucleic acid of table composition
| Ingredient names (20test/Kit) | Specification × quantity |
| PCR8 connecting leg with cover | The row of 0.2ml × 3 |
| Primer mix | 100 μ L × 1 pipe |
| 10×buffer | 100 μ L × 1 pipe |
| Positive control | 1 pipe |
| Reverse transcriptase | 20ul × 1 is managed |
| Bst archaeal dna polymerase | 20ul × 1 is managed |
| Stabilizing solution | 500ul × 1 is managed |
| MnCl2 | 500ul × 1 is managed |
| Colored indicator | 80 μ L × 1 pipe |
| DEPC water | 1mL × 1 is managed |
| Specification | 1 part |
In table 1, Primer mix is the mixture of primers F IP, BIP, F3, B3, and wherein FIP/BIP concentration is 8 μ
Mol/L, F3/B3 concentration are 1 μm of ol/L.
In table 1, positive control is the pig blue-ear disease poison NSP2 genetic fragment of synthesis；Reverse transcriptase is the AMV of TAKARA
Reverse transcriptase (Reverse Transcriptase), specification 200U (5U/ul)；Bst archaeal dna polymerase is one hundred Ke's biology section of Henan
The Bst archaeal dna polymerase of skill Co., Ltd, specification are 10000U (10U/ul)；Stabilizing solution is the glycine betaine of 5mol/L；MnCl2
Concentration be 10mmol/L；Show that indicator is 4- (2- pyridylazo) resorcinol (PAR) of 10mmol/L concentration；DEPC
Water is autoclaved packing water after DEPC processing.
In table 1, the composition of 10 × buffer is as shown in table 2:
2 10 × buffer of table composition
Embodiment 2
Pig blue-ear disease poison detection of nucleic acids is carried out using kit described in embodiment 1
(1) 0.1g pork is taken, Trizol reagent is added and is ground, centrifugation obtains the lapping liquid supernatant of 200ul, extracts disease
Malicious RNA, and the viral RNA of extraction is dissolved in 20ul DEPC water, directly detected or stored -20 DEG C or less it is spare.
(2) nucleic acid amplification
1. reaction solution, primer, color developing agent etc. are melted using preceding room temperature and are mixed in kit.
2. setting required RT-LAMP reaction tube number as n (+1 negative control of+1 positive control of n=sample number), every tube reaction body
System is as shown in table 3.
Table 3: amplification system
| Reagent | Dosage |
| Primer mix | 5ul |
| 10x buffer | 2.5ul |
| Stabilizer | 1.5ul |
| Reverse transcriptase | 1ul |
| Bst enzyme | 1ul |
| MnCl2 | 1.25ul |
| Indicator | 1.25ul |
| DEPC water | 9.5ul |
| Total volume | 23ul |
Each reagent dosage is calculated by every pipe aequum × n, is added in a cleaning 1.5ml EP pipe, is uniformly mixed,
2000r/min is centrifuged 10s, and the above-mentioned mixed liquor of 23ul is separately added into 8 connecting leg of PCR.Move to sample treatment area sample-adding.
3. being sequentially added into negative control, template to be detected, each 2ul of positive control into above-mentioned each pipe, covers tightly pipe lid and do
Good label, moves to reaction zone.
4. 60 DEG C of isothermal reaction 50min.
5. result judges: taking out reaction tube, be cooled to room temperature, 2000r/min is centrifuged 10s, observes color change.In feminine gender
Compare reaction tube liquid be it is orange red, positive control reaction tube is under the conditions of light yellow: sample to be tested is in yellow, the sample knot
Fruit is that PRRS virus is positive；For sample to be tested in orange, which is that PRRS virus is negative；If yin and yang attribute pair
It is not inconsistent according to reaction result and above situation, then this testing result is invalid, should detect again.
(3) points for attention
1, this kit is only used for vitro detection, uses preceding PLEASE READ CAREFULLY kit specification full text.
2, experiment asks strict partition to operate:
One area: reagent area in preparation ----prepare required reagent；
2nd area: sample preparation area ----sample to be tested and control sample processing；
3rd area: reaction zone --- -- amplification and interpretation of result.
3, reaction temperature is at 60-65 DEG C, it is ensured that instrument true temperature.
4, each area's article prefecture is dedicated, avoids cross contamination.
5, in kit each reagent using it is preceding melt it is uniform.
6, it avoids generating bubble when being loaded, dispensing, whether each reaction tube of inspection covers tightly pipe lid before reacting, and avoids leaking dirty
Dye.
7, reaction tube is cooled to room temperature observation after completing reaction, avoids opening pipe lid, amplified production is effectively prevent to escape dirt
Dye.
8, a reaction tube is sealed in special container after analysis, and fixed point abandons.Waste and primary in experimentation
Property consumptive material sterilizing after abandon.
9, workbench and each article periodically sterilize.
10, work clothes, band disposable glove, it is proposed that use the disposable pipette tips with filter core please be dress in experimentation.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>one hundred Ke's albumen of Xuchang and Co., Ltd, genetic engineering research institute
<120>a kind of primer combination, kit and its application for pig blue-ear disease poison detection of nucleic acids
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gtgaatgagc cgacaccacc tgggagtctg acgagagca 39
<210> 2
<211> 42
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttgccgtctt cagatggtgt ggggtctaag agccttcctg ct 42
<210> 3
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ccaaaggtcc ccggatgt 18
<210> 4
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
acgaggctaa aaacctggc 19
Claims (10)
1. a kind of primer for pig blue-ear disease poison detection of nucleic acids combines, which is characterized in that the primer combination includes in upstream
Portion primers F IP, downstream inner primer BIP, upstream outer primers F 3 and downstream outer primer B3；
The nucleotide sequence of the upstream internal primers F IP is as shown in SEQ ID No:1；
The nucleotide sequence of the downstream inner primer BIP is as shown in SEQ ID No:2；
The nucleotide sequence of the upstream outer primers F 3 is as shown in SEQ ID No:3；
The nucleotide sequence of the downstream outer primer B3 is as shown in SEQ ID No:4.
2. a kind of pig blue-ear disease poison kit for detecting nucleic acid, which is characterized in that it include Primer mix in the kit,
Buffer, reverse transcriptase, Bst archaeal dna polymerase, stabilizing solution, MnCl2Solution, colored indicator and DEPC water；The Primer
Mix includes primer combination described in claim 1.
3. kit according to claim 2, which is characterized in that in the Primer mix, the concentration of FIP and BIP
Respectively 7~9 μm of ol/L；The concentration of F3 and B3 is respectively 0.8~1.2 μm of ol/L.
4. kit according to claim 2, which is characterized in that the buffer includes the dNTPs of 13~15mmol/L,
The MgSO of 75~85mmol/L4, (the NH of the Tris of 180~220mmol/L, 90~110mmol/L4)2SO4, 450~
The Tween-20 of the KCl of 550mmol/L and 0.8~1.2% volumetric concentration.
5. kit according to claim 2, which is characterized in that the reverse transcriptase is AMV reverse transcriptase, the AMV
The Rate activity of reverse transcriptase is 4~6U/ μ L；The Rate activity of the Bst archaeal dna polymerase is 8~12U/ μ L.
6. kit according to claim 2, which is characterized in that the stabilizing solution is that the glycine betaine of 4~6mol/L is water-soluble
Liquid.
7. kit according to claim 2, which is characterized in that the MnCl2The concentration of solution is 8~20mmol/L.
8. kit according to claim 2, which is characterized in that the colored indicator is 8~20mmol/L concentration
4- (2- pyridylazo) resorcinol solution.
9. kit according to claim 2, which is characterized in that further include positive control and/or yin in the kit
Property control；The positive control includes pig blue-ear disease poison NSP2 genetic fragment；The negative control does not include pig blue-ear disease poison
NSP2 genetic fragment.
10. kit described in the combination of primer described in claim 1 or claim 2~9 any one is in detection pig indigo plant ear
Application in virus, the application is not for the purpose of the diagnosis of disease.
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| CN201811620347.0A CN109722490A (en) | 2018-12-28 | 2018-12-28 | A kind of primer combination, kit and its application for pig blue-ear disease poison detection of nucleic acids |
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Cited By (1)
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| CN113293236A (en) * | 2021-06-29 | 2021-08-24 | 龙岩学院 | Porcine reproductive and respiratory syndrome virus RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection primer group and kit |
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Application publication date: 20190507 |