CN108611439A - A kind of porcine reproductive and respiratory syndrome virus differentiates detection kit and its application - Google Patents
A kind of porcine reproductive and respiratory syndrome virus differentiates detection kit and its application Download PDFInfo
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- CN108611439A CN108611439A CN201810352091.3A CN201810352091A CN108611439A CN 108611439 A CN108611439 A CN 108611439A CN 201810352091 A CN201810352091 A CN 201810352091A CN 108611439 A CN108611439 A CN 108611439A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention relates to a kind of porcine reproductive and respiratory syndrome virus to differentiate detection kit and its application.The porcine reproductive and respiratory syndrome virus be include classic and/or high pathological form and/or NADC30 like type strains；The kit includes sense primer and downstream primer.Kit provided by the invention is quick, economical, can distinguish classic, high pathological form and NADC30 like type strains by a PCR reaction, have advantage and practical value easy to operate, at low cost in terms of above-mentioned three kinds of strains differentiate detection use.
Description
Technical field
The present invention relates to technical field of biological more particularly to a kind of mirror for porcine reproductive and respiratory syndrome virus
Kit and its application Jian Ce not used.
Background technology
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome,
PRRS), also known as pig blue-ear disease, is by porcine reproductive and respiratory syndrome virus (Porcine reproductive and
Respiratory syndrome virus, PRRSV) caused by one kind with pig breeding dysfunction and respiratory disease for main disease
The viral infectious of shape, the infectious disease can result in the clinical symptoms such as Sow abortion, piglet respiratory disorder, have higher dead
Die rate.The virus is divided into two genotype, i.e., using LV strains as the Europe class (1 type of gene) that represents and using VR2332 strains as generation
The North America type (2 type of gene) of table.1996, first Chinese was separated to using CH-1a as the classic strains of the PRRSV of representative；2006
Year, the high pathological form PRRSV caused by high pathological form PRRSV strains is broken out in China, and the areas strain NSP2 are discontinuous there are 30
Amino acid deletions；2014, the areas NSP2 occurs in China, and there are the NADC 30-like poison of 131 discontinuous amino acid deletions
Strain, the strains of pathogenic is between classic strain and high pathological form strain.There are three kinds of diseases for China's large-scale pig farm at present
The generation of poison and prevalence, serious economic loss is caused to China's pig breeding industry.Therefore, three kinds of different strains are distinguished, for
The accurate prevention and control of PRRS are of great significance.But PRRS has the characteristics that a kind of virus that there are different strains, and differentiated using PCR
Different Kinds of Pathogens needs to use different system and program so that substance PCR method operating procedure is more and actual amount is big, and operation is numerous
Trivial and cost is higher.
Invention content
For a kind of cumbersome, the of high cost technical problems of substance PCR when detecting virus difference strain, the present invention provides
A kind of porcine reproductive and respiratory syndrome virus differentiates detection kit.
And the present invention also provides a kind of detection methods of non-diagnostic purpose porcine reproductive and respiratory syndrome virus.
And the present invention also provides a kind of mentioned reagent box detection contain classic, high pathological form and NADC30-
Application in the product of like type porcine reproductive and respiratory syndrome virus.
To achieve the above object of the invention, the embodiment of the present invention uses the following technical solution：
A kind of porcine reproductive and respiratory syndrome virus discriminating detection kit, which is characterized in that the pig breeds and exhales
It includes classic and/or high pathological form and/or NADC30-like type strains to inhale syndrome virus to be；The kit includes upper
Primer and downstream primer are swum, wherein
The sequence of the sense primer is 5 '-CTCCTTTGATTGGAATGTTGTG-3 '；
The sequence of the downstream primer is 5 '-GATGGCTTGAGCTGAGTATTTTG-3 '.
This research according to classic strain, high pathological form strain and NADC 30-like type strain NSP2 genome differences,
It devises and differentiates detection primer, establish the PCR discriminating detection methods for distinguishing tri- kinds of strains of PRRSV and assemble reagent
Box.The primer that the kit includes be announced according to GenBank with relevant classic, the high cause of porcine reproductive and respiratory syndrome
Obtained by sick type, NADC30-like type strains sequence design, it can amplify simultaneously and the relevant a plurality of target DNA of strain to be measured
Segment, the specificity and sensibility of existing substance PCR, and compared with quick, economical, easily operated, reacted by a PCR
Simultaneously differentiate tri- kinds of strains of PRRSV, above-mentioned three kinds of strains individually or mixed infection context of detection have easy to operate, cost
Low advantage and practical value.
Preferably, the classic strain amplified fragments size is 1072bp, and the high pathological form strain amplified fragments are big
Small is 985bp, and the NADC30-like types strain amplified fragments size is 682bp.
Preferably, the kit further includes 5 × reverse transcription buffer, dNTP Mixture, reverse transcriptase, RNA enzyme suppression
Preparation and 2 × PCR Mix.
Preferably, 2 × PCR Mix are made of archaeal dna polymerase, 2 × PCR buffer and dNTP Mixture.
The reverse transcriptase is M-MLV reverse transcriptase.
Preferably, the kit further includes RNA extracts reagents.
Preferably, the RNA extracts reagents include Carrier RNA, chloroform, isopropanol, 75% ethyl alcohol.
And the present invention also provides a kind of discriminating detection sides of non-diagnostic purpose porcine reproductive and respiratory syndrome virus
Method carries out classic, high pathological form and NADC30-like type porcine reproductive and respiratory syndromes with above-mentioned kit in vitro tissue
The detection of virus.
And the present invention also provides a kind of mentioned reagent box detection containing classic and/or high pathological form and/or
Application in the product of NADC30-like type porcine reproductive and respiratory syndrome virus.It, can by the detection to these three strains
The discriminating detection of the porcine reproductive and respiratory syndrome caused for these three strains provides technical support, to assist its prevention and
Epidemiological survey.
Description of the drawings
Fig. 1 is classic strain PCR specific detection results in the embodiment of the present invention；
Fig. 2 is high pathological form strain PCR specific detection results in the embodiment of the present invention；
Fig. 3 is NADC30-like type strain PCR specific detection results in the embodiment of the present invention.
Description of the drawings：
11 classic PRRSV of 10DL2000Marker, 12 negative controls
21 high 22 negative controls of pathological form PRRSV of 20DL2000Marker
32 negative controls of 30DL2000Marker 31NADC30-like types PRRSV
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with specific embodiment, to this
Invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.
Embodiment 1
An embodiment of the present invention provides one kind differentiating detection kit for porcine reproductive and respiratory syndrome virus, specifically
Including：
(1) RNA extracts reagents：Carrier RNA, chloroform, isopropanol, 75% ethyl alcohol；
(2) Reverse Transcription：5 × reverse transcription buffer, dNTP Mixture of a concentration of 2.5mM, a concentration of 2.5U/ μ L
M-MLV reverse transcriptase, a concentration of 30U/ μ L RNase inhibitor；
(3) PCR reaction reagents：2 × PCR Mix are (by archaeal dna polymerase, 2 × PCR buffer and dNTP Mixture groups
At), sense primer, downstream primer, wherein the sequence of sense primer be 5 '-CTCCTTTGATTGGAATGTTGTG-3 ', downstream is drawn
The sequence of object is 5 '-GATGGCTTGAGCTGAGTATTTTG-3 ', and the sense primer and downstream primer are the freeze-drying of chromatographically pure
Powder；
(4) nuclease-free water.
Embodiment 2
An embodiment of the present invention provides a kind of porcine reproductive and respiratory syndrome virus of non-diagnostic purpose to differentiate detection side
Method, it is test serum to take lungs, lymph node, spleen, the kidney of sick dead pig, and detecting step is specific as follows：
(1) RNA is extracted
Test serum pre-processes：After test serum is shredded, according to 1:5 mass volume ratio is added physiological saline and grinds
Uniformly, 3000~5000rpm centrifugation 5~take supernatant after ten minutes, obtain sample to be tested.
Sample to be tested is carried out to the extraction of RNA：250 μ L samples to be tested are added in 750 μ L TRIzol LS Reagent,
Acutely oscillation 2min, is placed at room temperature for 5min.250 μ L chloroforms are added, acutely vibrate 1min, after being placed at room temperature for 5min, 4 DEG C
12000rpm centrifuges 15min, and upper strata aqueous phase is transferred to new centrifuge tube.It is added and the isometric isopropanol of water phase, mixing, room temperature
15min is stood, 4 DEG C of 12000rpm centrifuge 15min, gently remove supernatant, 75% ethyl alcohol 700 for being then added that DEPC handles~
800 μ L, 4 DEG C of 12000rpm centrifugation 5min abandon supernatant.It is dried by precipitation natural air drying or in 50 DEG C of drying boxes, with appropriate nothing
Nuclease water carry out RT-PCR immediately after fully dissolving or be stored in -80 DEG C it is spare.
(2) reverse transcription reaction
A concentration of 10 μM of downstream primers of 2 μ L, 4 μ 5 × reverse transcriptions of L buffering is added into above-mentioned 11 μ L RNA templates respectively
Liquid, 0.5 μ L reverse transcriptase, 0.5 μ L RNase inhibitors, 2 μ L dNTP Mixture to 20 μ L, 42 DEG C of 1~2h of water-bath to get
To cDNA solution, -20 DEG C save backup.
(3) PCR reacts
Above-mentioned cDNA solution is taken, PCR identifications are carried out.It is added in reaction tube 10 × Easy Taq Buffer, 2 μ L respectively,
2 2.0 μ L, Easy Taq DNA polymerase (5U/ μ L) of μ L, dNTPs (2.5mM) of cDNA, 0.5 μ L, 0.5 μ L sense primers
(10 μM), 0.5 μ L downstream primers (10 μM), finally plus nuclease-free water to volume is 20 μ L, mixing.
Response procedures are as follows：94 DEG C of 3min, 1 cycle；94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 70s, totally 30 recycle；72℃
1 cycle of 10min, PCR product store for future use in 4 DEG C of refrigerators.Simultaneously negative control is set up with sterile ultra-pure water.
After reaction, pcr amplification product is checked through 1% agarose gel electrophoresis, if there is the band of 1072bp sizes
Then sample to be tested contains classic strain；Sample to be tested contains high pathological form strain if the band for 985bp sizes occur；If going out
Then sample to be tested contains NADC30-like type strains to the band of existing 682bp sizes.The result is shown in Figure 1,2,3.
By result as it can be seen that the kit that this research institute provides can differentiate tri- kinds of poison of PRRSV simultaneously by a PCR reaction
Strain, above-mentioned three kinds of strains individually or the context of detection of mixed infection has quick, economic, advantage easy to operate.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modification, equivalent replacement or improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.
SEQUENCE LISTING
<110>Agricultural University Of Hebei
<120>A kind of porcine reproductive and respiratory syndrome virus differentiates detection kit and its application
<130> 2018.4.16
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>Sense primer
<400> 1
ctcctttgat tggaatgttg tg 22
<210> 2
<211> 23
<212> DNA
<213>Downstream primer
<400> 2
gatggcttga gctgagtatt ttg 23
Claims (9)
1. a kind of porcine reproductive and respiratory syndrome virus differentiates detection kit, which is characterized in that the pig breeding and breathing
Syndrome virus be include classic and/or high pathological form and/or NADC30-like type strains；The kit includes upstream
Primer and downstream primer, wherein
The sequence of the sense primer is 5 '-CTCCTTTGATTGGAATGTTGTG-3 '；
The sequence of the downstream primer is 5 '-GATGGCTTGAGCTGAGTATTTTG-3 '.
2. porcine reproductive and respiratory syndrome virus according to claim 1 differentiates detection kit, which is characterized in that institute
It is 1072bp to state classic strain amplified fragments size, and the high pathological form strain amplified fragments size is 985bp, described
NADC30-like type strain amplified fragments sizes are 682bp.
3. porcine reproductive and respiratory syndrome virus according to claim 1 differentiates detection kit, which is characterized in that institute
It further includes 5 × reverse transcription buffer, dNTP Mixture, reverse transcriptase, RNase inhibitor and 2 × PCR Mix to state kit.
4. porcine reproductive and respiratory syndrome virus according to claim 2 differentiates detection kit, which is characterized in that institute
2 × PCR Mix are stated to be made of archaeal dna polymerase, 2 × PCR buffer and dNTP Mixture.
5. porcine reproductive and respiratory syndrome virus according to claim 2 differentiates detection kit, which is characterized in that institute
It is M-MLV reverse transcriptase to state reverse transcriptase.
6. porcine reproductive and respiratory syndrome virus according to claim 2 differentiates detection kit, which is characterized in that institute
It further includes RNA extracts reagents to state kit.
7. porcine reproductive and respiratory syndrome virus according to claim 5 differentiates detection kit, which is characterized in that institute
It includes Carrier RNA, chloroform, isopropanol, 75% ethyl alcohol to state RNA extracts reagents.
8. a kind of detection method of the porcine reproductive and respiratory syndrome virus of non-diagnostic purpose, it is characterised in that：With claim 1
Any one of~6 kits carry out classic, high pathological form and the breeding of NADC30-like type pigs and exhale in vitro test serum
Inhale the discriminating detection of syndrome virus.
9. a kind of any one of claim 1~6 kit detection containing classic and/or high pathological form and/or
Application in the product of NADC30-like type porcine reproductive and respiratory syndrome virus.
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Cited By (1)
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Application publication date: 20181002 |