CN106435014A - Multiple RT-PCR detection kit and primer group of porcine epidemic diarrhea virus virulent strain and vaccine strain - Google Patents
Multiple RT-PCR detection kit and primer group of porcine epidemic diarrhea virus virulent strain and vaccine strain Download PDFInfo
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Abstract
The invention discloses a multiple RT-PCR detection primer group of porcine epidemic diarrhea virus virulent strain and vaccine strain. The primer group comprises the following three pairs of specific primers: primers 1 and 2, primers 3 and 4, and primers 5 and 6. All the primers respectively have base sequences respectively represented by SEQ. ID. No.1 to SEQ. ID. No.6 in a sequence table. The invention also discloses a detection kit, and a corresponding multiple RT-PCR detection method. The method can be used to specifically detect PEDV and has no cross reaction to other important pathogens of pigs; the lower detection limit of a recombinant plasmid substance product is 2.62*10<3> copies; and a uniform and consistent result is obtained after repeated test. A clinic detection result shows that the primer group, the kit and the detection method have the characteristics of high specificity, high sensitivity and good repeatability, can simultaneously detect and distinguish PEDV field virus classical strains, variant strains and vaccine strains, and provide an effective technical platform for clinic and rapid discrimination detection and epidemiological investigation.
Description
Technical field
The invention belongs to RT-PCR detection technique field, more particularly to a kind of Porcine epidemic diarrhea virus virulent strain and vaccine
Strain multiple RT-PCR detection kit and its primer sets.
Background technology
Porcine epizootic diarrhea (porcine epidemic diarrhea, PED) be by Porcine epidemic diarrhea virus (PEDV)
Pig is caused to be particularly a kind of acute, the highly propagated intestinal tract disease of piglet, with acute enteritises, watery diarrhea, vomiting and tight
Be dehydrated as principal character again, its M ＆ M is all very high, suckling pig sickness rate up to 100%, mortality rate up to
80%-100%, brings about great losses to pig industry.
Exist while due to clinically PEDV open country poisons allusion quotation strain, variant, a large amount of uses of attenuated live vaccines are added, is given
Clinical monitoring and diagnosis bring very big difficulty.Set up PEDV street strain, the discriminating detection method of vaccine strain is to detect and diagnose
The sick important prerequisite.
Content of the invention
The technical problem to be solved in the present invention is to provide a kind of high specificity, sensitivity high, reproducible pig popularity
Diarrhea viruses virulent strain and vaccine strain multiple RT-PCR detection kit and its primer sets, for PEDV open country poisons allusion quotation strain, variation
The quick discriminating of strain and vaccine strain is detected.
For solving above-mentioned technical problem, the present invention is employed the following technical solutions：Porcine epidemic diarrhea virus virulent strain and epidemic disease
Seedling strain multiple RT-PCR detection primer group, including three pairs of specific primers, is primer 1 and 2, primer 3 and 4, primer 5 and 6 respectively,
They are respectively provided with the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.6.
Primer 1, primer 2, primer 3, primer 4, primer 5, the mol ratio of primer 6 are 1: 1: 1: 1: 1: 1.
Porcine epidemic diarrhea virus virulent strain and vaccine strain multiple RT-PCR detection kit, the test kit contains following examination
Agent：Reverse transcription RT reactant liquor, PCR reactant liquor, sterilizing distilled water, PCR reactant liquor includes three pairs of specific primers, is primer respectively
1 and 2, primer 3 and 4, primer 5 and 6, they are respectively provided with the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.6.
The every 15 μ L of pipe of reverse transcription RT reactant liquor, including：5 × Prime Scrip II buffer, 4.0 1.0 μ L of μ L, dNTP
Containing each dNTP 10mM, 6 aggressiveness 50pmol/ μ L of random primer, 1.5 μ L, RNase inhibitor 40U/ μ L, 0.5 μ L, RNase H- type
Reverse transcription 200U/ μ L, 1.0 μ L, sterilize 7 μ L of distilled water；The every 45 μ L of pipe of PCR reactant liquor, including：2×Taq PCR
25 μ L of MasterMix, primer 1 and each 1 μ L of 2 25pmol/ μ L, primer 3 and each 0.75 μ L of 4 25pmol/ μ L, primer 5 and 6
The each 1.25 μ L of 25pmol/ μ L, sterilize 14 μ L of distilled water.
The problem for existing is detected for current Porcine epidemic diarrhea virus, according to the gene sequence of PEDV street strain and vaccine strain
Row feature, designs primer at ORF3 absent region two ends, and in order to distinguish street strain, (strain of wild poisons allusion quotation and variant exist with vaccine strain
This regional sequence is identical)；Primer is designed in S gene delection and insert region, in order to distinguish wild poison variant, classical strain and vaccine
Strain (classical strain is identical in this regional sequence with vaccine strain), thus, inventor devise Porcine epidemic diarrhea virus virulent strain and
Vaccine strain multiple RT-PCR detection primer group, including three pairs of specific primers, is primer 1 and 2, primer 3 and 4, primer, 5 and respectively
6, they are respectively provided with the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.6.Accordingly, inventor also prepares detection
Test kit, and establish corresponding multiple RT-PCR detection method, the method can be and important with other of pig with specific detection PEDV
Cause of disease swine foot-and-mouth disease virus (FMDV), swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pig transmissible
Marcy agent (TGEV), porcine rotaviruses (PRoV), porcine circovirus 2 type (PCV2), porcine pseudorabies virus (PRV) are no handed over
Fork reaction；Detection lower limit to plasmid standard of recombinating is 2.62 × 103Copy；Repeat to test the result for obtaining uniformity.
Clinical detection result shows, the present invention have high specificity, sensitivity high, reproducible the features such as, can detect simultaneously and distinguish
PEDV open country poisons allusion quotation strain, variant and vaccine strain, are that the detection of clinical quick discriminating and Epidemiological study are provided effectively
Technology platform.
Description of the drawings
Fig. 1 is multiplex PCR specific test result figure, in figure：M：DNA molecular quality standard, 1 four kinds of recombiant plasmid mixing
Thing, 2p-PEDV-SJ/Y, 3p-PEDV-SB, 4p-PEDV-ORF3J/B, 5p-PEDV-ORF3Y, 6FMDV, 7CSFV, 8PRRSV,
9TGEV, 10PRoV, 11PCV2,12PRV, 13 negative control.
Fig. 2 is multiplex PCR sensitivity testss result figure, in figure：M DNA molecular quality standard；1-9 plasmid concentration is respectively
2.62×109Copy/μ L-2.62 × 101Copy/μ L；10 negative controls.
Fig. 3 is multiplex PCR replica test result figure, in figure：M DNA molecular quality standard；Under 1-5 the same terms 5
Secondary repeat reaction；6 negative controls.
Specific embodiment
In following examples, PEDV vaccine CV777 strain, wild poisons allusion quotation strain, variant, the weak poison China strain of TGEV vaccine,
PRoV vaccine G5 type NX strain, PRRSV vaccine TJM-F92 strain, FMDV vaccine O/MYA/BY/2010 strain, CSFV vaccine C strain, PCV2
Vaccine virus LG strain, PRV vaccine Bartha-K61 strain, are provided by Guangxi Center for Animal Disease Control ＆ Prevention.
1st, the design of primer
With reference to PEDV classics strain CV777 strain (AF353511) for delivering in GenBank, variant CHGD-01 strain
(JX261936) and vaccine strain CV777 strain (GU372744), 3 pairs of specific primers (table 1) are designed.
1 primer information of table
In table 1, PEDV-ORF3 primer is street strain's (classical strain and variant) and vaccine strain universal primer, it is contemplated that amplification
ORF3 genetic fragment is 198bp, is 148bp in vaccine strain in street strain；PEDV-SJ primer is classical strain and vaccine strain specificity
Primer, it is contemplated that amplification S genetic fragment 429bp；PEDV-SB primer is variant specific primer, it is contemplated that amplification S genetic fragment
274bp.Therefore, using primer PEDV-ORF3/PEDV-SJ/PEDV-SB, at the same amplify 198bp and 429bp fragment person be through
Allusion quotation strain is while amplify 148bp and 429bp fragment person for vaccine strain while amplifying 198bp and 274bp fragment person for variation
Strain.
2nd, prepared by sample
Take PEDV vaccine CV777 strain, wild poisons allusion quotation strain, variant respectively, extract according to RNA extracts kit description total
RNA, obtains cDNA using 6 aggressiveness of random primer and the reverse transcription of Prime Script II RTase enzyme.With cDNA as template, point
Not Ying Yong 3 couples of specific primers PEDV-ORF3, PEDV-SJ, PEDV-SB enter performing PCR amplification.The test kit recovery of application glue reclaim,
Purified pcr product, connection pMD18-T carrier, conversion CH5 α competent cell, coated plate, the positive bacterium colony Zengjing Granule of screening, use
Plasmid extraction kit extracts plasmid, and performing PCR of going forward side by side identification, enzyme action identification, sequencing identification are corresponding to confirm successfully to construct
Recombiant plasmid standard substance.P-PEDV-ORF3Y, p- is respectively designated as the gene constructed plasmid of ORF3 in vaccine strain, street strain
PEDV-ORF3J/B, for the gene constructed plasmid of S classical strain/vaccine strain, variant be respectively designated as p-PEDV-SJ/Y,
p-PEDV-SB.
With reference to RNA/DNA extracts kit description, extract respectively FMDV, CSFV, PRRSV, TGEV, PRoV RNA and
The DNA of PCV2, PRV.
3rd, test kit is prepared
According to result of study, detection kit is assembled with convenient use.Test kit contains following reagent：Reverse transcription RT reacts
Liquid, PCR reactant liquor, sterilizing distilled water, wherein：
The every 15 μ L of pipe of reverse transcription RT reactant liquor, including：5 × Prime Scrip II buffer 4.0 μ L, dNTP are (each
10mM) 1.0 μ L, 6 aggressiveness of random primer (50pmol/ μ L, purchased from TaKaRa company), PrimeScriptTM II1st Strand
1.5 μ L of cDNA Synthesis Kit test kit, 0.5 μ L, RNase H- type reverse transcription of RNase inhibitor (40U/ μ L)
(200U/ μ L) 1.0 μ L, sterilize 7 μ L of distilled water；
The every 45 μ L of pipe of PCR reactant liquor, including：2 × Taq PCR MasterMix, 25 μ L, PEDV-ORF3 upstream and downstream primer
(25pmol/ μ L) each 1 μ L, PEDV-SJ upstream and downstream primer (25pmol/ μ L) each 0.75 μ L, PEDV-SB upstream and downstream primer
(25pmol/ μ L) each 1.25 μ L, sterilize 14 μ L of distilled water.
4th, multiple RT-PCR program and condition
Optimized, determining the reaction condition of multiple RT-PCR, two step programs is expanded including reverse transcription and PCR.
20 μ L reverse transcription systems are：5 × Prime Scrip II buffer, 4.0 1.0 μ L of μ L, dNTP (each 10mM), with
6 aggressiveness of power traction thing (50pmol/ μ L) 1.5 μ L, 0.5 μ L, RNase H- type reverse transcription (200u/ of RNase inhibitor (40u/ μ L)
μ L) 1.0 μ L, 5.0 μ L of template total serum IgE, sterilize 7.0 μ L of distilled water；Reverse transcription program is：30 DEG C of 10min, 48 DEG C of 45min, 95
℃5min.
50 μ L PCR reaction systems are：2 × Taq PCR MasterMix, 25 μ L, PEDV-ORF3 upstream and downstream primer
(25pmol/ μ L) each 1 μ L, PEDV-SJ upstream and downstream primer (25pmol/ μ L) each 0.75 μ L, PEDV-SB upstream and downstream primer
(25pmol/ μ L) each 5 μ L of 1.25 μ L, cDNA, sterilizing distilled water is mended to 50 μ L；Response procedures are：94℃2min；94 DEG C of 30s,
56.2 DEG C of 30s, 72 DEG C of 45s, 40 circulations；72℃10min.
5. experimental applications of the present invention
The test kit of aforementioned preparation and the optimum reaction condition for determining is applied, with the various combination of 4 kinds of plasmids, and
The DNA of the cDNA of FMDV, CSFV, PRRSV, TGEV, PRoV and PCV2, PRV carries out multiplex PCR as template, detects which is special
Property.As shown in figure 1, only plasmid reaction assumes the positive, and FMDV, CSFV, PRRSV, TGEV, PRoV, PCV2, PRV are the moon
Property, without cross reaction.As a result show, using Porcine epidemic diarrhea virus virulent strain of the present invention and the multiple RT-PCR of vaccine strain
It is stronger special that detection kit and its detection method differentiate that detection PEDV open country poisons allusion quotation strain, variant and vaccine strain have
Property.
Multiplex PCR will be carried out as template after 10 times of 4 kinds of recombiant plasmid equal-volume mixing being serially diluted, as a result as Fig. 2,
The detection lower limit of three pairs of primers is 2.62 × 103Copy/μ L.Show using Porcine epidemic diarrhea virus virulent strain of the present invention and
The multiple RT-PCR detection kit of vaccine strain and its detection method differentiate detection PEDV open country poisons allusion quotation strain, variant and vaccine
Strain has stronger sensitivity.
With final concentration of 2.62 × 1064 kinds of plasmid mixtures of copy/μ L carry out multiplex PCR for template.As a result as Fig. 3,
Repeat for 5 times the equal purpose fragment that can amplify uniformity is tested, show using Porcine epidemic diarrhea virus virulent strain of the present invention
Differentiate the wild poisons allusion quotation strain of detection PEDV, variant and epidemic disease with the multiple RT-PCR detection kit of vaccine strain and its detection method
Seedling strain has preferably repeatability.
Claims (4)
1. Porcine epidemic diarrhea virus virulent strain and vaccine strain multiple RT-PCR detection primer group, it is characterised in that including three couples of spies
Specific primer, is primer 1 and 2, primer 3 and 4, primer 5 and 6 respectively, and they are respectively provided with sequence table SEQ .ID.No.1 extremely
The base sequence of SEQ.ID.No.6.
2. multiple RT-PCR detection primer group according to claim 1, it is characterised in that：The primer 1, primer 2, primer
3rd, primer 4, primer 5, the mol ratio of primer 6 are 1: 1: 1: 1: 1: 1.
3. a kind of Porcine epidemic diarrhea virus virulent strain and vaccine strain multiple RT-PCR detection kit, it is characterised in that the reagent
Box contains following reagent：Reverse transcription RT reactant liquor, PCR reactant liquor, sterilizing distilled water, the PCR reactant liquor includes three pairs specifically
Property primer, is primer 1 and 2, primer 3 and 4, primer 5 and 6 respectively, and they are respectively provided with sequence table SEQ .ID.No.1 extremely
The base sequence of SEQ.ID.No.6.
4. multiple RT-PCR detection kit according to claim 3, it is characterised in that：The reverse transcription RT reactant liquor is every
15 μ L of pipe, including：5 × Prime Scrip II buffer 4.0 μ L, dNTP 1.0 μ L contain each dNTP 10mM, random primer 6
Aggressiveness 50pmol/ μ L, 1.5 μ L, RNase inhibitor 40U/ μ L, 0.5 μ L, RNase H- type reverse transcription 200U/ μ L, 1.0 μ L,
Sterilizing 7 μ L of distilled water；The every 45 μ L of pipe of the PCR reactant liquor, including：2 × Taq PCR MasterMix, 25 μ L, primer 1 and 2
The each 1 μ L of 25pmol/ μ L, primer 3 and each 0.75 μ L of 4 25pmol/ μ L, primer 5 and each 1.25 μ L of 6 25pmol/ μ L, sterilizing is double to steam
14 μ L of water.
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Cited By (4)
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| CN106947832A (en) * | 2017-03-30 | 2017-07-14 | 河南省农业科学院 | The primer and probe of Porcine epidemic diarrhea virus quantitative fluorescent PCR |
| CN107557494A (en) * | 2017-09-26 | 2018-01-09 | 华中农业大学 | Differentiate the primer of detection diarrhea of pigs coronavirus and multiple RT PCR methods |
| CN108315489A (en) * | 2018-04-18 | 2018-07-24 | 白玲 | A kind of primer sets and kit for transmissible gastro-enteritis virus now separation strains and vaccine strain antidiastole |
| CN114015815A (en) * | 2021-12-17 | 2022-02-08 | 广西壮族自治区动物疫病预防控制中心 | Microdroplet digital PCR kit for swine atypical pestivirus and detection method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106947832A (en) * | 2017-03-30 | 2017-07-14 | 河南省农业科学院 | The primer and probe of Porcine epidemic diarrhea virus quantitative fluorescent PCR |
| CN107557494A (en) * | 2017-09-26 | 2018-01-09 | 华中农业大学 | Differentiate the primer of detection diarrhea of pigs coronavirus and multiple RT PCR methods |
| CN108315489A (en) * | 2018-04-18 | 2018-07-24 | 白玲 | A kind of primer sets and kit for transmissible gastro-enteritis virus now separation strains and vaccine strain antidiastole |
| CN114015815A (en) * | 2021-12-17 | 2022-02-08 | 广西壮族自治区动物疫病预防控制中心 | Microdroplet digital PCR kit for swine atypical pestivirus and detection method thereof |
| CN114015815B (en) * | 2021-12-17 | 2024-04-30 | 广西壮族自治区动物疫病预防控制中心 | Microdroplet digital PCR kit for swine atypical pestivirus and detection method thereof |
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