CN109825649A - Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group and its diagnostic kit - Google Patents
Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group and its diagnostic kit Download PDFInfo
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Abstract
The invention discloses Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers groups, including two pairs of specific primers, it is primer PEDV-BF and PEDV-BR, primer PEDV-F and PEDV-R respectively, is respectively provided with the base sequence of sequence table SEQ .ID.NO.1 to SEQ.ID.NO.4.Accordingly, inventor still further developed corresponding diagnostic kit.The infection type of PEDV G1, G2 type can be identified in same reaction tube using the present invention, fast and convenient tool is provided for the accurate detection of swinery PEDV, solid foundation is established for clinical quickly identification detection PEDV G1, G2 type strain and laboratory epidemiological survey, facilitate the pig breeding industry accurate judgement cause of disease and formulate therapeutic scheme in time, reduces economic loss.Experimental study shows that specificity of the invention is good, and high sensitivity is reproducible, time-consuming short and at low cost.
Description
Technical field
The invention belongs to Porcine epidemic diarrhea virus detection technique field more particularly to a kind of Porcine epidemic diarrhea virus
G1/G2 type RT-PCR diagnostic primers group and its diagnostic kit.
Background technique
In recent years, develop with pig breeding industry to intensive, scale direction, that there is a situation where diarrhea is further serious for swinery.
Porcine epidemic diarrhea virus (PEDV) is the main pathogens of the viral intestines problem of suckling pig, has been divided by secular change
For G1 type strain and G2 type strain, G1 type strain vaccine is poor to G2 type strain effect, therefore the PEDV that makes a definite diagnosis promptly and accurately feels
Dye G1 type strain or G2 type strain are of great significance to pig breeding industry.
Pig epidemic diarrhea (porcine epidemic diarrhea, PED) is by Porcine epidemic diarrhea virus
The acute infectious intestinal disease of a boar caused by (porcine epidemic diarrhea virus, PEDV), clinical main table
It is now watery diarrhea and serious dehydration, 80~100% is up to the disease incidence of nursery-age pig, leads to piglet Deaths
One of main epidemic disease.The disease was reported in Britain in 1976 for the first time, had broken out epidemic disease in Asian countries such as China, Thailand within 2010
There is large-scale prevalence in the U.S. in 2013, cause huge economic loss to pig breeding industry in feelings.
Research shows that PEDV infection causes the diarrhea epidemic disease of pig, seriously endanger pig production, and according to clinical symptoms and
Epidemiology is difficult to distinguish PEDV G1, G2 type strain, it is necessary to carry out antidiastole by laboratory detection technology.Wherein, gene
It is longer that clone, sequence analysis etc. operate the more complicated and waiting time；ELISA cause of disease quick detection kit and conventional RT-PCR
The application of method is than wide, but serological method accuracy rate is lower, and time-consuming for common single PCR method.
It is examined therefore, it is necessary to establish and can quickly identify early stage of the RT-PCR of Porcine epidemic diarrhea virus G1, G2 type strain
Disconnected method reduces disease and endangers caused by pig breeding industry to take effective prevention and control measure as early as possible.
Summary of the invention
The technical problem to be solved in the present invention is to provide high specificity, susceptibility are high, reproducible, time-consuming short and at low cost
Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group and its diagnostic kit, convenient in clinical and scientific research to pig
The quick detection and epidemiological survey of epidemic diarrhea G1, G2 type strain.
In order to solve the above technical problems, the invention adopts the following technical scheme:
Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group, including two pairs of specific primers, are primer respectively
PEDV-BF and PEDV-BR, primer PEDV-F and PEDV-R, they are respectively provided with sequence table SEQ .ID.NO.1 extremely
The base sequence of SEQ.ID.NO.4.
Primer PEDV-BF, PEDV-BR, primer PEDV-F and PEDV-R molar ratio be 1:1:1:1.
Application of the above-mentioned Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group in terms of PCR amplification.
In PCR amplification, primer PEDV-F and PEDV-R are PED universal detector primer, for based on E gene order
It is designed at 1-24 and 208-231 of nucleotide；Primer PEDV-BF and PEDV-BR are G2 type specificity detection primer,
It is for G1 type and specific difference of the G2 type strain at S gene, at 164-184 and 982-1003, its nucleotide
Place's design.
The annealing temperature of PCR amplification is 55 DEG C.
Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic kit, contain following reagent: PCR standard items, the positive are right
According to product, negative controls, primer；Primer includes two pairs of specific primers, is primer PEDV-BF and PEDV-BR, primer respectively
PEDV-F and PEDV-R, they are respectively provided with the base sequence of sequence table SEQ .ID.NO.1 to SEQ.ID.NO.4.
PCR standard items include PEDV G1 type strain standard items and PEDV G2 type strain standard items；Positive reference substance contains
The positive plasmid mixture of PEDV G1 type strain, PEDV G2 type strain；Negative controls are deionized water after sterilizing.
PEDV G1 type strain standard items and PEDV G2 type strain standard items are respectively provided with sequence table SEQ .ID.NO.5 extremely
The base sequence of SEQ.ID.NO.6.
In primer, the upstream primer PEDV-BF and primer PEDV-F of two pairs of specific primers are packaged together, two species specificity
The downstream primer PEDV-BR and PEDV-R of primer are packaged together.
Porcine epidemic diarrhea virus G1, G2 type strain identify detection there are aiming at the problem that, inventor be directed to PEDV E base
Cause, S gene order devise Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group, including two pairs of specific primers,
It is primer PEDV-BF and PEDV-BR, primer PEDV-F and PEDV-R respectively, they are respectively provided with sequence table SEQ .ID.NO.1 extremely
The base sequence of SEQ.ID.NO.4.Wherein, based on E gene order at 1-24 of nucleotide and the position 208-231
The primer PED-F/R of design is PED universal detector primer, for G1 type and specific difference of the G2 type strain at S gene,
The primer PED-BF/BR designed at 164-184 and 982-1003, its nucleotide is G2 type specificity detection primer.According to
This, inventor still further developed corresponding diagnostic kit, contain following reagent: PCR standard items, positive reference substance, negative controls,
Primer.The infection type of PEDV G1, G2 type can be identified in same reaction tube using the present invention, be the accurate inspection of swinery PEDV
Survey provides fast and convenient tool, quickly identifies detection PEDV G1, G2 type strain and laboratory epidemiological survey to be clinical
Solid foundation is established, facilitates the pig breeding industry accurate judgement cause of disease and formulates therapeutic scheme in time, reduce economic loss.Experimental study
Show that sensibility of the present invention is 1.937 × 101Copies/ μ l, specificity is good, and high sensitivity is reproducible, it is time-consuming short and at
This is low.
Detailed description of the invention
Fig. 1 is the result figure of the specific detection of 2 kinds of corresponding primers of the invention, in figure: swimming lane is from left to right successively are as follows: M:
DNA molecular quality standard；The mixed poison of 1:PEDV G1, G2 type strain；2:PEDV G2 type strain；3:PEDV G1 type strain；4: pig
Infectious gastroenteritis virus；5:A group of pigs rotavirus；6；Porcine circovirus 2 type；7: swine fever virus；8: swine influenza virus；9: pig
Reproductive and respiratory syndrome virus；10: porcine pseudorabies virus；11: pig astrovirus；12: negative control.
Fig. 2 is the result figure of sensitivity Detection of the present invention, in figure: swimming lane is from left to right successively are as follows: M:DNA molecular mass mark
It is quasi-；1-10:1.937 × 108L~1.937 × 10 copies/ μ-1copies/μl；11: negative control.
Fig. 3 is the result figure of repeatability detection of the invention, and in figure: swimming lane is followed successively by M:DNA molecular mass mark from left to right
It is quasi-；1-6:PEDV G2 type strain；7: negative control；8-13:PEDV G1 type strain.
Fig. 4 is to apply clinical diarrhea sample RT-PCR testing result figure of the invention, and in figure: swimming lane is followed successively by from left to right
M:DNA molecular mass standard；1:PEDV G1 type strain；2:PEDV G2 type strain；3-12: clinical sample；13: negative control.
Specific embodiment
Embodiment 1, the assembling of Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic kit
Kit contains following reagent: PCR standard items, positive reference substance, negative controls, primer；Primer includes two pairs
Specific primer is primer PEDV-BF and PEDV-BR, primer PEDV-F and PEDV-R respectively, they are respectively provided with sequence table
The base sequence of SEQ.ID.NO.1 to SEQ.ID.NO.4.
1 double PCR primer of table
PCR standard items include PEDV G1 type strain standard items and PEDV G2 type strain standard items；Positive reference substance contains
PEDV G1 type strain, PEDV G2 type strain positive plasmid mixture (concentration of plasmid mixture is about 1.937 ×
109copies/μL)；Negative controls are deionized water after sterilizing.
PEDV G1 type strain standard items and PEDV G2 type strain standard items are respectively provided with sequence table SEQ .ID.NO.5 extremely
The base sequence of SEQ.ID.NO.6.
In primer, the upstream primer PEDV-BF and primer PEDV-F of two pairs of specific primers are packaged together, two species specificity
The downstream primer PEDV-BR and PEDV-R of primer are packaged together.
The reagent of kit is stored in -20 DEG C, reduces multigelation to the greatest extent.
The application method of kit specifically can be as follows:
Negative control and positive control are set up in detection as the case may be every time；
Standard items are diluted to 10 with sterile ultrapure water1~108copies/μL。
RNA virus sample extraction: according to products instruction, with viral RNA extraction agent box AxyPrep Body
Fluid Viral DNA/RNA Miniprep Kit (Axygen) extracts viral nucleic acid from clinical diarrhea sample.
Reverse transcription reaction: 8 μ of total serum IgE template of previous step preparation is sequentially added in autoclaved 0.2mL PCR pipe
L, 5 × Reversa Transcriptase M-MLV Buffer (TaKaRa) 2.5 μ L, dNTP Mixture (TaKaRa) 1 μ L,
0.5 μ L, Reversa Transcriptase M-MLV (TaKaRa) of Oligo dT, 0.25 μ L, Ribolock RNase
0.25 μ L of Inhibitor (Vazyme), total volume are 12.5 μ L, and reaction condition is 42 DEG C of reaction 1h, and record reaction to be reversed terminates
Afterwards, gained reaction solution is cDNA template.
The detection of nucleic acid: the cDNA template for taking 3 μ L to prepare carries out RT-PCR reaction, wherein Green Taq Mix
(Vazyme) 12.5 μ L, 0.5 μ L of upstream primer mixture, downstream primer mixture 0.5 μ L, ddH28.5 μ L of O, total volume 25
μ L, response procedures are first 94 DEG C of initial denaturation 5min, and the reaction conditions of 34 circulations are 94 DEG C of denaturation 40s, 55 DEG C of annealing 40s, 72
DEG C extend 50s, last 72 DEG C of extensions 10min；PCR product is detected with 1% agargel electrophoresis, through gel imager at
As post analysis.
As a result report: under conditions of negative control and positive control are set up, according to the quantity of target fragment and size come
Judge the type of PEDV strain.
Embodiment 2, present invention detection dual RT-PCR specificity experiments
Step 1: primer synthesizes
Two pairs of specific primer sequences (being shown in Table 1) of virus designed by the present invention are limited by Shanghai outstanding person Lee's biotechnology
Company's synthetic primer, synthetic quantity are every pipe primer 1OD.
Step 2: dual RT-PCR quick detection kit specific detection
According to double PCR reaction system, Green Taq Mix (Vazyme) 12.5 μ L, 0.5 μ L of upstream primer mixture,
Downstream primer mixture 0.5 μ L, ddH28.5 μ L of O is separately added into 3 μ L positive sample cDNA template 1:PEDV G1, G2 type strain
Mixed poison；2:PEDV G2 type strain；3:PEDV G1 type strain；4: transmissible gastro-enteritis virus；5:A group of pigs rotavirus；
6；Porcine circovirus 2 type；7: swine fever virus；8: swine influenza virus；9: porcine reproductive and respiratory syndrome virus；10: pseudorabies
Virus；11: pig astrovirus；12: deionized water negative control after sterilizing.Reaction life ProFlex PCR amplification instrument into
Row, response procedures are as follows: first 94 DEG C of initial denaturation 5min, the reaction conditions of 34 circulations are 94 DEG C of denaturation 40s, 55 DEG C of annealing 40s, 72
DEG C extend 50s, last 72 DEG C of extensions 10min.10 μ L PCR products are taken, point sample is in 1% agargel electrophoresis plate hole, 150V
Voltage, electrophoresis about 20min, judgement of taking pictures after gel imager ultraviolet imagery.
The result is shown in Figure 1, PEDV G1, G2 type strain it is mixed poison obtain 2 purpose bands, size be respectively 840bp and
246bp；PEDV G2 type strain obtains 2 purpose bands, and size is respectively 840bp and 246bp；PEDV G1 type strain obtains 1
Purpose band, size 246bp.And the testing result of TGEV, PRoVA, CSFV, PRRSV, PRV, PCV2, SI, PAstV are equal
Without band, without cross reaction, show that structure of the present invention identifies Porcine epidemic diarrhea virus G1, G2 type RT-PCR built and quickly detects
Kit specificity is preferably
Embodiment 3, present invention detection dual RT-PCR sensitivity experiments
Step 1: primer synthesizes
With the first step in embodiment 2.
Step 2: dual RT-PCR quick detection kit sensitivity Detection
Double PCR sensitivity Detection reaction system: Green Taq Mix (Vazyme) 12.5 μ L, upstream primer mixture
0.5 μ L, downstream primer mixture 0.5 μ L, ddH28.5 μ L of O is separately added into the PEDV G1 type strain of 3 μ L, 10 times of doubling dilutions
Strain, PEDV G2 type strain positive plasmid melange (1.937 × 108L~1.937 × 10 copies/ μ-1Copies/ μ l) mould
Plate.Reaction is carried out in life ProFlex PCR amplification instrument, response procedures are as follows: first 94 DEG C of initial denaturation 5min, 34 circulations it is anti-
Answering condition is 94 DEG C of denaturation 40s, 55 DEG C of annealing 40s, 72 DEG C of extensions 50s, last 72 DEG C of extensions 10min.10 μ L PCR products are taken,
The detection of 1% agargel electrophoresis.
As a result see Fig. 2, the experimental results showed that, dual RT-PCR reaction is 1.937copies/ μ l to PEDV detection lower limit.
As it can be seen that identify PEDV G1, the sensitivity of G2 type strain higher for dual RT-PCR of the present invention.
Embodiment 4, present invention detection dual RT-PCR repeated experiment
Step 1: primer synthesizes
With the first step in embodiment 2.
Step 2: dual RT-PCR quick detection kit repeatability detects
Double PCR repeatability detects reaction system: Green Taq Mix (Vazyme) 12.5 μ L, upstream primer mixture
0.5 μ L, downstream primer mixture 0.5 μ L, ddH28.5 μ L of O is separately added into 3 μ L positive sample cDNA templates: 1-6:G2 type poison
Strain；7: deionized water negative control after sterilizing；8-12:G1 type strain.Reaction is carried out in life ProFlex PCR amplification instrument, instead
Answer program are as follows: the reaction condition of first 94 DEG C of initial denaturation 5min, 34 circulations are 94 DEG C of denaturation 40s, and 55 DEG C of annealing 40s, 72 DEG C are prolonged
Stretch 50s, last 72 DEG C of extensions 10min.Take 10 μ L PCR products, the detection of 1% agargel electrophoresis.
As a result see Fig. 3, the experimental results showed that, RT-PCR reaction is uniform to the qualification result of PEDV G1, G2 type strain steady
It is fixed.As it can be seen that the repeatability of dual RT-PCR identification PEDV G1 of the present invention, G2 type strain is preferably.
Embodiment 5, clinical detection clinic diarrhea sample of the present invention.
Step 1: primer synthesizes
With the first step in embodiment 2
Step 2: sample collection
Clinical 92 parts of sample of the diarrhea in the area such as Nanning, Qiezhou, Guigang, Chongzuo, Yulin.
Step 3: total serum IgE extracts in a small amount
According to products instruction, with viral RNA extraction agent box AxyPrep Body Fluid Viral DNA/
RNA Miniprep Kit (Axygen) extracts viral nucleic acid from clinical diarrhea sample.
Step 4: reverse transcription synthesizes cDNA
The 8 μ L of total serum IgE template of previous step preparation is sequentially added in autoclaved 0.2mL PCR pipe.5×
Reversa Transcriptase M-MLV Buffer(TaKaRa)2.5μL。dNTP Mixture(TaKaRa)1μL。Oligo
DT Mix (Oligo dT and PRoV-R mixture) 0.5 μ L.Reversa Transcriptase M-MLV(TaKaRa)0.25μ
L.Ribolock RNase Inhibitor(Vazyme)0.25μL.Total volume is 12.5 μ L, and reaction condition is 42 DEG C of reaction 1h,
After reaction, gained reaction solution is cDNA template for record to be reversed.
Step 5: present invention clinic diarrhea sample dual RT-PCR detection.
1, dual RT-PCR detection architecture of the present invention is as follows: the cDNA template for taking 3 μ L to prepare carries out double PCR reaction,
Wherein 12.5 μ L of Green Taq Mix (Vazyme), 0.5 μ L of upstream primer mixture, downstream primer mixture 0.5 μ L, ddH2O
8.5 μ L, total volume are 25 μ L；Response procedures are that first 94 DEG C of initial denaturation 5min, the reaction condition of 34 circulations are 94 DEG C of denaturation
40s, 55 DEG C of annealing 40s, 72 DEG C of extensions 50s, last 72 DEG C of extensions 10min；PCR product is carried out with 1% agargel electrophoresis
Detection, is imaged post analysis through gel imager.
Step 6: testing result such as Fig. 4
Experimental result is shown, detects 55 parts of PEDV positive sample using the present invention, wherein 2 parts of PEDV G1 type strain, accounts for
3.6% (2/55) of PEDV strain, accounts for 96.36% (53/55) of PEDV strain by 53 parts of strain of PEDV G2 type.
Sequence table
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cccaagcttt tatacgtcaa taacagtact 30
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ttggtgaaaa ccagggtgtc aattcaactt ggtactgtgc tggccaacat ccaactgcta 60
gtggcgttca tggtatcttt cttagccata ttagaggtgg tcatggcttt gagattggca 120
tttcgcaaga gccttttgac cctagtggtt accagcttta tttacataag gctactaacg 180
ataacactaa tgctactgcg cgactgcgca tttgccagtt tcccagcatt aaaacattgg 240
gccccactgc taataatgat gttacaacag gtcgtaactg cctatttaac aaagccatcc 300
cagctcatat gagtgaacat agtgttgtcg gcataacatg ggataatgat cgtgtcactg 360
tcttttctga caagatctat catttttatt ttaaaaatga ttggtcccgt gttgcgacaa 420
agtgttacaa cagtggaggt tatgctatgc aatatgttta cgaacccacc tattacacgc 480
ttaatgttac tagtgctggt gaggatggta tttcttatca accctgtaca gctaattgca 540
ttggttatgc tgccaatgta tttgctactg agcccaacgg ccacatacca gaaggtttta 600
gttttaataa ttggtttctt ttgtccaatg attccacttt ggtgcatggt aaggtggttt 660
ccaaccaacc tttgttggtt aattgtcttt tggccattcc taagatttat ggactaggcc 720
aatttttctc attcaatcaa acgatgaatg gcgtttgtaa tggagcttct gcgcagcgtg 780
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ttcacttgtc accggttgtg taatagcgca gtttacacac ctatagggcg tttgtataga 180
gtttataagt cctacatgca aatagacccc ctccccagta ctgttattga cgtataaaag 240
cttggg 246
Claims (9)
1. Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group, it is characterised in that including two pairs of specific primers, divide
It is not primer PEDV-BF and PEDV-BR, primer PEDV-F and PEDV-R, they are respectively provided with sequence table SEQ .ID.NO.1 extremely
The base sequence of SEQ.ID.NO.4.
2. Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group according to claim 1, it is characterised in that:
The primer PEDV-BF, PEDV-BR, primer PEDV-F and PEDV-R molar ratio be 1:1:1:1.
3. Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic primers group answering in terms of PCR amplification described in claim 1
With.
4. application according to claim 3, it is characterised in that: in the PCR amplification, primer PEDV-F and PEDV-R are
PED universal detector primer, for the design at 1-24 of nucleotide and the position 208-231 based on E gene order；Draw
Object PEDV-BF and PEDV-BR are G2 type specificity detection primer, for G1 type and specificity of the G2 type strain at S gene
Difference designs at 164-184, its nucleotide and the position 982-1003.
5. application according to claim 4, it is characterised in that: the annealing temperature of the PCR amplification is 55 DEG C.
6. a kind of Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic kit, it is characterised in that contain following reagent: PCR
Standard items, positive reference substance, negative controls, primer；The primer includes two pairs of specific primers, is primer PEDV- respectively
BF and PEDV-BR, primer PEDV-F and PEDV-R, they are respectively provided with the alkali of sequence table SEQ .ID.NO.1 to SEQ.ID.NO.4
Basic sequence.
7. Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic kit according to claim 6, it is characterised in that:
The PCR standard items include PEDV G1 type strain standard items and PEDV G2 type strain standard items；The positive reference substance contains
The positive plasmid mixture of PEDV G1 type strain, PEDV G2 type strain；The negative controls are deionized water after sterilizing.
8. Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic kit according to claim 7, it is characterised in that:
The PEDV G1 type strain standard items and PEDV G2 type strain standard items are respectively provided with sequence table SEQ .ID.NO.5 extremely
The base sequence of SEQ.ID.NO.6.
9. Porcine epidemic diarrhea virus G1/G2 type RT-PCR diagnostic kit according to claim 6, it is characterised in that:
In the primer, the upstream primer PEDV-BF and primer PEDV-F of two pairs of specific primers are packaged together, two species-specific primers
Downstream primer PEDV-BR and PEDV-R be packaged together.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112941241A (en) * | 2021-04-16 | 2021-06-11 | 武汉科前生物股份有限公司 | Method for identifying porcine epidemic diarrhea virus type 2a and type 2b |
| CN114807437A (en) * | 2022-04-06 | 2022-07-29 | 南京农业大学 | Quadruple fluorescent quantitative PCR detection kit for detecting porcine epidemic diarrhea virus and porcine rotavirus and application thereof |
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