CN106435032A - Double RT-PCR (reverse transcription-polymerase chain reaction) primer, kit and method for amplifying North America and European porcine blue ear disease viruses - Google Patents
Double RT-PCR (reverse transcription-polymerase chain reaction) primer, kit and method for amplifying North America and European porcine blue ear disease viruses Download PDFInfo
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Abstract
The invention discloses double RT-PCR (reverse transcription-polymerase chain reaction) primer, kit and method for amplifying North America and European porcine blue ear disease viruses; the method has the advantages of high specificity, high sensitivity, low time and labor consumption and the like, and is of important guidance significance to molecular epidemiology, genetic evolution, vaccine research and development and the like for studying porcine blue ear disease. In addition, denaturing and annealing temperature in the method only takes 20 s, amplification can be performed as well just with one PCR apparatus, and the method may also act as a method for identifying and detecting North America and European porcine blue ear disease viruses and helps quickly classify porcine blue ear viruses.
Description
Technical field
The invention belongs to detection field, and in particular to a kind of for while expanding North America type and Europe class PRRS virus
Duplex RT-PCR primer, test kit and method.
Background technology
Pig blue-ear disease is also called Porcine reproductive and respiratory syndrome（Porcine Reproductive and Respiratory
Syndrome, PRRS), its cause of disease is porcine reproductive and respiratory syndrome virus（Porcine Reproductive and
Respiratory Syndrome Virus, PRRSV).From 1987 since the U.S. finds the disease, the disease just spreads rapidly
To global pig industry, huge economic loss is brought to pig industry.Up to the present, pig blue-ear disease not only gives China
Live pig industry brings catastrophic impact, and breaks out the such as state such as Vietnam, Korea of country in China's periphery, it is seen that pig indigo plant ear
The Epidemic Scope of disease is increasing.The pig of the various age levels of various kinds can all infect reproductive and respiratory syndrome, and main clinic symptoms are showed
Breeding difficulty for sow and within a monthly age piglet respiratory symptom.The breeding difficulty of sow is mainly in-pig morning
Produce, miscarry, producing weak son, stillborn fetuses, mummy tire etc., and the respiratory symptom of other pigs especially piglet is mainly cough, plays spray
Sneeze, dyspnea etc..OIE is classified as B class infectious disease, and the high-pathogenicity blue ear disease for having broken out since 2006 is classified as by China
" a class infectious disease ".PRRSV is according to its antigenicity and genomic constitution feature, the Europe class being classified as with LV as representative in the world
（I type）With the North America type with ATCC-2332 as representative（II type）.
PRRSV belongs to the single strand plus RNA virus for having cyst membrane of Tao Shi viraleses Arteriviridae Arteriviruses,
Full-length genome about 15kb, containing including 10 open reading frame including ORF5a, the envelope protein of wherein ORF5 coding virus
GP5（E protein）Even if GP5 albumen is the maximum albumen of variation, but it is than more conservative, is that virus replication is requisite main
Structural protein, immunogenicity is preferable, can induce generation neutralizing antibody earlier than other albumen, and neutralizing antibody occur with
Afterwards, antiserum mainly recognizes GP5 albumen, and GP5 albumen also has the activity of inducing cell apoptosis.The ORF5 full length gene of North America type
For 603bp, the ORF5 full length gene of Europe class is 606bp, by amplification and analysis to ORF5 gene, can be to a certain degree
The feature of upper reflection PRRSV.In addition, the outburst of China's Europe class reproductive and respiratory syndrome presents more and more serious trend, to reproductive and respiratory syndrome
Prevention and control bring further difficult, while also reminding our the prevention and control to reproductive and respiratory syndrome need more new knowledge.Currently, same foster
Grow the same head pig in field and already there is the situation for not only having infected Europe class reproductive and respiratory syndrome but also having infected North America type reproductive and respiratory syndrome, clinically substantially can not
Distinguishing, huge challenge is brought to prevention with treatment.Therefore, develop while expanding North America type and Europe class PRRS virus
The method of ORF5 full-length gene order and test kit, the molecular epidemiology to grasp pig blue-ear disease, genetic evolution, vaccine research and development
Etc. aspect have important directive significance.
Content of the invention
It is an object of the invention to carrying out design of primers, sequence alignment with methods such as molecular biology, bioinformatics
And reaction system optimization, build a kind of for while expanding North America type and Europe class PRRS virus ORF5 full-length gene order
Duplex RT-PCR method and test kit, realize the quick of North America type and Europe class PRRS virus ORF5 full-length gene order
Expanding effect.
The technical solution used in the present invention is：
A kind of duplex RT-PCR for expanding North America type and Europe class PRRS virus ORF5 full-length gene order simultaneously draws
Thing, its nucleotide sequence is as follows：
NA-ORF5-F: 5,-GTTTTAGCCTGTCTTTTTGC-3,（SEQ ID NO：1）；
NA-ORF5-R:5,- GCTGAGTACACHCCCCAAAG-3,（SEQ ID NO：2）；
EU-ORF5-F: 5,-ACCATYGCTTGTTTGTTCGCCAT-3,（SEQ ID NO：3）；
EU-ORF5-R: 5,-CTTTCTCACAGCGTATGCTCG-3,（SEQ ID NO：4）.
A kind of double RT- for expanding North America type and Europe class PRRS virus ORF5 full-length gene order simultaneously
PCR kit, the test kit includes above-mentioned primer.
A kind of double RT- for expanding North America type and Europe class PRRS virus ORF5 full-length gene order simultaneously
PCR method, comprises the following steps：
1) viral nucleic acid is extracted from sample；
2) with nucleic acid as template, RT-PCR amplified reaction is carried out with primer pair described above and obtains amplified production；
3) RT-PCR amplified production being entered row agarose gel electrophoresis analysis, result is observed under gel imaging system, according to bar
The ORF5 full-length gene order of North America type and Europe class PRRS virus is determined with size.
Preferably, step 2）Middle RT-PCR amplification reaction system is：PrimeScript 1 step Enzyme Mix 1 μ
25 μ L of L, 2 × 1 step Buffer, each 1 μ L of primer NA-ORF5-F, NA-ORF5-R, EU-ORF5-F and EU-ORF5-R,
6 μ L of viral nucleic acid template, excess water, cumulative volume is 50 L.
Preferably, step 2）Middle RT-PCR response procedures：50 DEG C of reverse transcription 30min；95 DEG C of denaturations 5min；95 DEG C of degeneration
20s, 58 DEG C of annealing 20s, 72 DEG C of extension 60s, 35 circulations altogether；72 DEG C extend 5min eventually.
Preferably, step 3）In under gel imaging system observe result, if the fragment only containing 900bp size,
Judge to amplify North America type PRRS virus ORF5 full-length gene order in detected sample；If only big containing 1036bp
Little fragment, then judge to amplify Europe class PRRS virus ORF5 full-length gene order in detected sample；If with
The fragment of Shi Hanyou above two size, then judge in detected sample while amplifying Europe class and North America type pig blue-ear disease
Virus O RF5 full-length gene order.
A kind of quick differentiation North America type and the duplex RT-PCR primer of Europe class PRRS virus, the nucleotides sequence of primer
Row are as follows：
NA-ORF5-F： 5,-GTTTTAGCCTGTCTTTTTGC-3,（SEQ ID NO：1）；
NA-ORF5-R： 5,-GCTGAGTACACHCCCCAAAG-3,（SEQ ID NO：2）；
EU-ORF5-F： 5,-ACCATYGCTTGTTTGTTCGCCAT-3,（SEQ ID NO：3）；
EU-ORF5-R： 5,-CTTTCTCACAGCGTATGCTCG-3,（SEQ ID NO：4）.
A kind of quick differentiation North America type and the duplex RT-PCR test kit of Europe class PRRS virus, the test kit contains
Above-mentioned primer.
A kind of quick differentiation North America type and the duplex RT-PCR detection method of Europe class PRRS virus, including following step
Suddenly：
1) viral nucleic acid is extracted from testing sample；
2) with nucleic acid as template, RT-PCR amplified reaction is carried out with above-mentioned primer pair and obtains amplified production；
3) RT-PCR amplified production is entered row agarose gel electrophoresis analysis, under gel imaging system, result is observed, determine disease
Malicious type.
Preferably, step 2）Middle RT-PCR amplification reaction system is：PrimeScript 1 step Enzyme Mix 1μ
25 μ L of L, 2 × 1 step Buffer, each 1 μ L of primer NA-ORF5-F, NA-ORF5-R, EU-ORF5-F and EU-ORF5-R, disease
The nucleic acid-templated 6 μ L of poison, excess water, cumulative volume is 50 L.
Preferably, step 2）Middle RT-PCR response procedures：50 DEG C of reverse transcription 30min；95 DEG C of denaturations 5min；95 DEG C of degeneration
20s, 58 DEG C of annealing 20s, 72 DEG C of extension 60s, 35 circulations altogether；72 DEG C extend 5min eventually.
Preferably, step 3）In under gel imaging system observe result, if the only fragment containing 900bp, judges
Contain North America type PRRS virus in detected sample；If only the fragment containing 1036bp, judges in detected sample
In contain Europe class PRRS virus；If while the fragment containing above two size, judges in detected sample
While containing Europe class and North America type PRRS virus.
The invention has the beneficial effects as follows：
The invention discloses one kind can be used for while expanding North America type and Europe class PRRS virus ORF5 full-length gene sequence
The duplex RT-PCR method of row and test kit, high, time saving and energy saving have the advantages that high specificity, sensitivity, to studying pig indigo plant ear
The aspects such as the molecular epidemiology of disease, genetic evolution, vaccine research and development have important directive significance.Additionally, the change in the method
Property and annealing temperature only need 20s, and while expanded by only needing to a PCR instrument device, also can be used as North America type and Europe
The differential diagnostic method of continent type PRRS virus, contributes to the fast typing of PRRS virus.
Duplex RT-PCR primer specificity of the present invention is strong, sensitivity height, and broad covered area.Wherein NA-ORF5-F and NA-
ORF5-R primer pair can cover more North America type PRRS virus to greatest extent（Including North America type pig blue-ear disease classics
Strain, variant and vaccine strain）；EU-ORF5-F and EU-ORF5-R primer pair can cover more Europe class pigs to greatest extent
Reproductive and respiratory syndrome virus（Including vaccine strain and street strain）.
The primer of the present invention can be also used for differentiating North America type and Europe class PRRS virus, be examined using duplex RT-PCR
Survey method, it is only necessary to which a PCR instrument device can be expanded simultaneously, it is adaptable to aquaculture quick discriminating North America type and Europe class pig
Reproductive and respiratory syndrome virus, are simultaneously applicable to PRRS virus and the research and development of the vaccine in later stage of scientific research difference typing etc. and grind
Study carefully.
Description of the drawings
Fig. 1 specific test result；Swimming lane M: DNA Marker 2000；Swimming lane 1：North America type+Europe class PRRSV；Swimming
Road 2：Europe class PRRSV；Swimming lane 3：North America type PRRSV；Swimming lane 4：Porcine circovirus 2 type；Swimming lane 5：Swine fever virus；Swimming lane 6：Pig
Foot and mouth disease viruses；Swimming lane 7：PRV (Pseudorabies virus)；Swimming lane 8：Pig parvoviral；Swimming lane 9：Pig storehouse cloth virus；Swimming lane 10：Pig fourth type
Coronavirus；Swimming lane 11：Negative control；
Fig. 2 duplex RT-PCR susceptibility results；
Fig. 3 clinical sample testing result.
Specific embodiment
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
Material and method
North America type PRRS virus live vaccine（TJM-F92 strain）Purchased from Xinjiang Tian Kang herding Bioisystech Co., Ltd, Europe
Type PRRS virus are by Institute of Animal Health,Guangdong Academy Of Agricultural Sciences's isolation identification.Porcine circovirus 2 type virus stain,
Swine fever virus strain, swine foot-and-mouth disease virus strain, porcine pseudorabies poison strain, pig parvoviral strain, pig storehouse cloth virus stain,
Pig fourth type Coronavirus Strain, Europe class and North America type PRRS virus positive recombinant plasmid are moved by Guangdong Academy of Agricultural Sciences
Thing health research is prepared to be preserved.
Main agents
DNA/RNA extracts kit is purchased from Axygen, and PCR reagent is purchased from Beijing Tiangeng company and TaKaRa company, DNA
Marker 2000 is purchased from TaKaRa company.
1 design of primers of embodiment
According to GenBank Middle and North America type and the genome sequence of Europe class PRRS virus, 2 pairs of primers are separately designed, wherein
NA-ORF5-F and NA-ORF5-R primer pair can cover more North America type PRRS virus to greatest extent（Including North America
The classics strain of type pig blue-ear disease, variant and vaccine strain）；EU-ORF5-F and EU-ORF5-R primer pair can be covered to greatest extent
More Europe class PRRS virus（Including vaccine strain and street strain）.Its base sequence is as follows.
NA-ORF5-F: 5,-GTTTTAGCCTGTCTTTTTGC-3,（SEQ ID NO：1）；
NA-ORF5-R:5,- GCTGAGTACACHCCCCAAAG-3,（SEQ ID NO：2）；
EU-ORF5-F: 5,-ACCATYGCTTGTTTGTTCGCCAT-3,（SEQ ID NO：3）；
EU-ORF5-R: 5,-CTTTCTCACAGCGTATGCTCG-3,（SEQ ID NO：4）.
2 duplex RT-PCR detection method of embodiment
1）The extraction of RNA
DNA/RNA extracts kit description according to Axygen company carries out the extraction of viral nucleic acid.
2）Duplex RT-PCR amplified reaction
Be clinically separated and be accredited as North America type and Europe class mixed infection positive PRRS virus nucleic acid as double RT-
The template of PCR, sets up duplex RT-PCR method.Using 50 μ L RT-PCR reaction systems：PrimeScript 1 step
25 μ L of Enzyme Mix 1 μ L, 2 × 1 step Buffer, primer NA-ORF5-F, NA-ORF5-R, EU-ORF5-F and EU-
The each 1 μ L of ORF5-R, 6 μ L, RNase Free ddH of viral nucleic acid template214 μ L of O, cumulative volume is 50 L.
RT-PCR response procedures：50 DEG C of reverse transcription 30min；95 DEG C of denaturations 5min；95 DEG C of degeneration 20s, 58 DEG C of annealing
20s, 72 DEG C of extension 60s, 35 circulations altogether；72 DEG C extend 5min eventually.
As a result observe：Taking 50 μ L RT- PCR primer carries out 1.2% agarose gel electrophoresiies analysis, in gel imaging system
Lower observation result.
3 specific test of embodiment
According to the method for embodiment 2, using duplex RT-PCR method, the PRRSV and pig of North America type and Europe class mixed infection are justified
2 type virus of circovirus virus, swine fever virus, swine foot-and-mouth disease virus, PRV (Pseudorabies virus), pig parvoviral, pig storehouse cloth virus, pig fourth type
Coronavirus are detected.
As a result see Fig. 1.Swimming lane M: DNA Marker 2000；Swimming lane 1：North America type+Europe class PRRSV；Swimming lane 2：Europe
Type PRRSV；Swimming lane 3：North America type PRRSV；Swimming lane 4：Porcine circovirus 2 type；Swimming lane 5：Swine fever virus；Swimming lane 6：Schweineseuche disease
Poison；Swimming lane 7：PRV (Pseudorabies virus)；Swimming lane 8：Pig parvoviral；Swimming lane 9：Pig storehouse cloth virus；Swimming lane 10：The coronal disease of pig fourth type
Poison；Swimming lane 11：Negative control.
As shown in Figure 1：Using the duplex RT-PCR detection method for having optimized to North America type and Europe class mixed infection
PRRSV the positive nucleic acid expanded, as a result obtain size be about 900bp and 1036bp fragment, with sequencing result 900bp and
The fragment of 1036bp matches.And porcine circovirus 2 type is viral, swine fever virus, swine foot-and-mouth disease virus, PRV (Pseudorabies virus), pig
Do not assume band after the nucleic acid amplifications such as parvovirus, pig storehouse cloth virus, pig fourth type coronavirus, show that the method has preferably
Specificity.
4 sensitivity testss of embodiment
Determine the template concentrations of North America type and Europe class PRRSV positive recombinant plasmid respectively, then doubling dilution is carried out, determine
The sensitivity of the method.As a result Fig. 2, wherein swimming lane M are seen：DNA Marker 2000；Swimming lane 1-9：Positive plasmid is according to 100Times,
101Again, 102Again, 103Again, 104Again, 105Again, 106Again, 107Again, 108Times doubling dilution result；Swimming lane 10：Negative control.
After testing, the duplex RT-PCR after optimization is respectively 23.71 pg/ to the sensitivity of North America type and Europe class PRRSV
Ml and 9.17pg/ml.
5 repeatability amplification test of embodiment
The PRRSV positive sample of nucleic acid that 10 parts are accredited as with North America type and the single or mixed infection of Europe class is expanded, as a result
Show and all amplification can accurately be drawn, further through nucleic acid sequencing, determine that the above results are completely correct, accuracy rate is
100%.
The amplification test of 6 clinical sample of embodiment
ORF5 full-length gene amplification is carried out to 11 parts therein with 21 parts of clinical samples for having determined PRRS virus type,
As a result as Fig. 3, wherein swimming lane M:DNA Marker 2000, swimming lane 1,2,3,4 is for confirming only to infect the sample of North America type PRRSV
Product, only amplify the about fragment of 900bp；Swimming lane 5,6,7,8 is only amplified for confirming only to infect the sample of Europe class PRRSV
The about fragment of 1036bp；Swimming lane 9,10,11 is for confirming the sample for not only having infected North America type but also having infected Europe class PRRSV, while expanding
Increase the fragment for about 900bp and 1036bp；Swimming lane 12 is negative control.It is consistent with expected resultss completely, therefore, the present invention sets
Primer NA-ORF5-F, NA-ORF5-R, EU-ORF5-F, EU-ORF5-R that meter is obtained can be very good to expand in clinical sample and feel
The ORF5 full-length gene order of the PRRS virus of dye, successfully construct can rapid amplifying PRRS virus North America type and
The dual RT-PCR method of Europe class ORF5 full-length gene order.Additionally, by amplification, it is also possible to distinguish North America type and Europe class
PRRS virus, for clinically viral identification.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, other any spirit without departing from the present invention and the change that is made under principle, modification, replacement, combine, simplify,
Equivalent substitute mode is all should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of South China, Institute of Animal Health,Guangdong Academy Of Agricultural Sciences
<120>A kind of duplex RT-PCR primer, test kit for expanding North America type and Europe class PRRS virus simultaneously
And method
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
gttttagcct gtctttttgc 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
gctgagtaca chccccaaag 20
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
accatygctt gtttgttcgc cat 23
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
ctttctcaca gcgtatgctc g 21
Claims (9)
1. a kind of for while expanding the duplex RT-PCR of North America type and Europe class PRRS virus ORF5 full-length gene order
Primer, its nucleotide sequence is as follows：
NA-ORF5-F: 5,-GTTTTAGCCTGTCTTTTTGC-3,；
NA-ORF5-R:5,- GCTGAGTACACHCCCCAAAG-3,；
EU-ORF5-F: 5,-ACCATYGCTTGTTTGTTCGCCAT-3,；
EU-ORF5-R: 5,-CTTTCTCACAGCGTATGCTCG-3,.
2. a kind of for while expanding the duplex RT-PCR of North America type and Europe class PRRS virus ORF5 full-length gene order
Test kit, it is characterised in that：The test kit is comprising the primer described in claim 1.
3. a kind of for while expanding the duplex RT-PCR of North America type and Europe class PRRS virus ORF5 full-length gene order
Method, it is characterised in that comprise the following steps：
1) viral nucleic acid is extracted from sample；
2) with nucleic acid as template, RT-PCR amplified reaction is carried out with the primer pair described in claim 1 and obtains amplified production；
3) RT-PCR amplified production being entered row agarose gel electrophoresis analysis, result is observed under gel imaging system, according to bar
The ORF5 full-length gene order of North America type and Europe class PRRS virus is determined with size.
4. method according to claim 3, it is characterised in that step 2）Middle RT-PCR amplification reaction system is：
1 step Enzyme Mix of PrimeScript, 1 25 μ L of μ L, 2 × 1 step Buffer, primer NA-ORF5-F, NA-
The each 1 μ L of ORF5-R, EU-ORF5-F and EU-ORF5-R, 6 μ L of viral nucleic acid template, excess water, cumulative volume is 50 L.
5. method according to claim 3, it is characterised in that step 2）Middle RT-PCR response procedures：50 DEG C of reverse transcriptions
30min；95 DEG C of denaturations 5min；95 DEG C of degeneration 20s, 58 DEG C of annealing 20s, 72 DEG C of extension 60s, 35 circulations altogether；72 DEG C of ends
Extend 5min.
6. method according to claim 3, it is characterised in that step 3）In under gel imaging system observe result, such as
Fragment of the fruit only containing 900bp size, then judge to amplify North America type PRRS virus ORF5 total length in detected sample
Gene order；If the fragment only containing 1036bp size, judge to amplify Europe class pig blue-ear disease in detected sample
Virus O RF5 full-length gene order；If while the fragment containing above two size, judge in detected sample while
Amplify Europe class and North America type PRRS virus ORF5 full-length gene order.
7. a kind of duplex RT-PCR primer of quick differentiation North America type and Europe class PRRS virus, it is characterised in that primer
Nucleotide sequence as follows：
NA-ORF5-F：5,-GTTTTAGCCTGTCTTTTTGC-3,；
NA-ORF5-R：5,-GCTGAGTACACHCCCCAAAG-3,；
EU-ORF5-F：5,-ACCATYGCTTGTTTGTTCGCCAT-3,；
EU-ORF5-R：5,-CTTTCTCACAGCGTATGCTCG-3,.
8. the duplex RT-PCR test kit of a kind of quick differentiation North America type and Europe class PRRS virus, it is characterised in that：Should
Test kit contains the primer described in claim 7.
9. the duplex RT-PCR detection method of a kind of quick differentiation North America type and Europe class PRRS virus, it is characterised in that
Comprise the following steps：
1）Viral nucleic acid is extracted from testing sample；
2）With nucleic acid as template, RT-PCR amplified reaction is carried out with the primer pair described in claim 7 and obtains amplified production；
3）RT-PCR amplified production is entered row agarose gel electrophoresis analysis, under gel imaging system, result is observed, determine disease
Malicious type.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110735005A (en) * | 2019-11-26 | 2020-01-31 | 广西大学 | SIV and PRRSV multiple RT-PCR rapid detection kit and primer |
| CN111500598A (en) * | 2020-05-08 | 2020-08-07 | 华南农业大学 | Kit and method for detecting European PRRSV antibody titer |
| CN112094952A (en) * | 2020-10-28 | 2020-12-18 | 云南农业大学 | Complete set of primer pair for porcine reproductive and respiratory syndrome virus whole genome determination and application thereof |
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| CN110735005A (en) * | 2019-11-26 | 2020-01-31 | 广西大学 | SIV and PRRSV multiple RT-PCR rapid detection kit and primer |
| CN110735005B (en) * | 2019-11-26 | 2023-01-10 | 广西大学 | SIV and PRRSV multiple RT-PCR rapid detection kit and primer |
| CN111500598A (en) * | 2020-05-08 | 2020-08-07 | 华南农业大学 | Kit and method for detecting European PRRSV antibody titer |
| CN112094952A (en) * | 2020-10-28 | 2020-12-18 | 云南农业大学 | Complete set of primer pair for porcine reproductive and respiratory syndrome virus whole genome determination and application thereof |
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| CN106435032B (en) | 2020-01-03 |
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