CN108950083A - The multiplex RT-PCR method of GETV, PEDV, TGEV, PDCoV and PoRV are detected simultaneously - Google Patents
The multiplex RT-PCR method of GETV, PEDV, TGEV, PDCoV and PoRV are detected simultaneously Download PDFInfo
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Abstract
The invention discloses a kind of for detecting his virus (GETV), the multiple RT-PCR primer sets of Porcine epidemic diarrhea virus (PEDV), transmissible gastro-enteritis virus (TGEV), pig Delta coronavirus (PDCoV) and pig A rotavirus (PoRV) of pig lid simultaneously, and the nucleotide sequence of the multiple RT-PCR primer sets is as shown in SEQ ID NO:1~SEQ ID NO:10.The invention also discloses a kind of multiple RT-PCR detection methods for once detecting GETV, PEDV, TGEV, PDCoV and PoRV directly from sample using multiple RT-PCR primer sets.Compared with existing conventional RT-PCR, the detection method high specificity, high sensitivity can be realized while identify five kinds of viruses of GETV, PEDV, TGEV, PDCoV and PoRV, and testing result is accurate, and detection efficiency is high.
Description
Technical field
The invention belongs to RT-PCR detection technique fields, and in particular to one kind be used for and meanwhile detect GETV, PEDV, TGEV,
The multiplex RT-PCR method of PDCoV and PoRV.
Background technique
He belongs to Togaviridae alphavirus at viral (Getah virus, GETV) to pig lid, is a kind of Zoonosis worm
Matchmaker's virus, can the media such as tissue/organ, secretion and excreta through mosquito matchmaker, aerosol and infected animals carry out horizontal biography
It broadcasts, can also be by animal placenta vertical transmission, and human poultry infection and certain animals is caused to fall ill.Antibody neutralization test has confirmed
GETV antibody positive rate is up to 20% or more in human serum sample, and is classified as Amphixenosis accordingly, although so far still
No virus infection people and the report for leading to clinical onset.
Porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea virus, PEDV), transmissible gastroenteritis of swine
Viral (Transmissible gastroenteritis virus, TGEV) and pig Delta coronavirus (Porcine
Deltacoronavirus, PDCoV) it is coronavirus genus member, and can cause the enteron aisle acute, highly infectious of pig
Disease.Especially nearest Ohio State Univ-Columbus USA and Dutch Utrecht university research personnel prove that pig coronavirus can be felt
Human cell is contaminated, therefore human health may be threatened.
Pig A rotavirus (porcinerota virus, PoRV) causes a variety of newborn and young animals and the abdomen of people
It rushes down.Research shows that rotavirus carries out propagation and gene rearrangement between different plant species, and the virus of gene rearrangement can cause people
Diarrhea.
Since GETV, PEDV, TGEV, PDCoV and PoRV have obvious or potential threat, especially its sense to population health
Dye spectrum is than wide, therefore, carries out above-mentioned 5 kinds of viral strict inspections with important to animal derived product especially food
Public health meaning.GETV, PEDV, TGEV, PDCoV and PoRV traditional detection method mainly has virus to be separately cultured and serum
Method, but it is laborious time-consuming and be difficult to effective district partitivirus type.
Summary of the invention
The object of the present invention is to provide one kind, and GETV, PEDV, TGEV, PDCoV and PoRV are directly once detected from sample
Multiple RT-PCR detection method.Compared with existing RT-PCR detection method, detection method of the invention removes high specificity, spirit
Sensitivity is high outer, additionally it is possible to realize while identify five kinds of viruses of GETV, PEDV, TGEV, PDCoV and PoRV, time and labour saving is laborsaving, inspection
Survey it is high-efficient, effectively to detect and identify GETV, PEDV, TGEV, PDCoV and PoRV in animal derived product especially food
Five kinds of viruses provide a kind of economic, quickly, special, sensitive method.
To realize goal of the invention, The technical solution adopted by the invention is as follows:
Present invention firstly provides one kind for simultaneously detect pig lid he virus (GETV), Porcine epidemic diarrhea virus (PEDV),
Transmissible gastro-enteritis virus (TGEV), pig Delta coronavirus (PDCoV) and pig A rotavirus (PoRV) it is multiple
RT-PCR primer group, the multiple RT-PCR primer sets are the primer for being sensed by his virus of pig lid, detection pig epidemic
The primer of diarrhea virus, the primer for detecting transmissible gastro-enteritis virus, the primer for detecting pig Delta coronavirus and detection pig A
The primer of rotavirus forms, wherein
For detecting the nucleotide sequence of his viral primer of pig lid are as follows:
Upstream primer P1:5'-GGTGGCAGGTTCACAATCC-3'(SEQ ID NO:1),
Downstream primer P2:5'-TTCTTCTGTTCCTTCTGGGGT-3'(SEQ ID NO:2)；
For detecting the nucleotide sequence of the primer of Porcine epidemic diarrhea virus are as follows:
Upstream primer P3:5'-CGTGGTGGGTTTGGTTGAT-3'(SEQ ID NO:3),
Downstream primer P4:5'-CGGTGACAAGTGAAGCACAGAT-3'(SEQ ID NO:4)；
For detecting the nucleotide sequence of the primer of transmissible gastro-enteritis virus are as follows:
Upstream primer P5:5'-CAAACATCACGAGGTCTTGCTAC-3'(SEQ ID NO:5),
Downstream primer P6:5'-CAAAGTCAACTGGACATCT-3'(SEQ ID NO:6)；
For detecting the nucleotide sequence of the primer of pig Delta coronavirus are as follows:
Upstream primer P7:5'-CCAGCAACCACTCGTGTTACTT-3'(SEQ ID NO:7),
Downstream primer P8:5'-TCAGCCATACCCGTCTTCTC-3'(SEQ ID NO:8)；
For detecting the nucleotide sequence of the primer of pig A rotavirus are as follows:
Upstream primer P9:5'-TCACAACGAAATGGGATAGC-3'(SEQ ID NO:9),
Downstream primer P10:5'-GCAAGCACAGATTCACAAAC-3'(SEQ ID NO:10)；
The detection is not for the purpose of medical diagnosis on disease and/or treatment.
Above-mentioned multiple RT-PCR primer sets can be used in his virus, Porcine epidemic diarrhea virus, pig of preparation detection pig lid
Infectious gastroenteritis virus, pig Delta coronavirus and pig A rotavirus reagent.
The present invention also provides a kind of kits containing above-mentioned multiple RT-PCR primer sets.The kit can be used for together
When detection pig lid his virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pig Delta coronavirus and pig A groups
Rotavirus, the application of the kit is not for the purpose of medical diagnosis on disease and/or treatment.
It is used for the present invention provides one kind while detecting his virus, Porcine epidemic diarrhea virus, pig transmissible stomach and intestine of pig lid
Scorching viral, pig Delta coronavirus and pig A rotavirus multiplex RT-PCR method, comprising the following steps:
Step 1: synthesize it is above-mentioned for and meanwhile to detect pig lid his virus, Porcine epidemic diarrhea virus, transmissible gastroenteritis of swine sick
Malicious, pig Delta coronavirus and pig A rotavirus multiple RT-PCR primer sets；
Step 2: extracting the total serum IgE of sample to be tested, then carry out reverse transcription by template of the total serum IgE of extraction, obtain cDNA；
Step 3: cDNA being obtained as template using reverse transcription, the multiple RT-PCR primer sets for using step 1 to synthesize carry out PCR for primer
Amplified reaction；
Step 4: analysis pcr amplification product, result judgement；
The multiplex RT-PCR method is not for the purpose of medical diagnosis on disease and/or treatment.
According to above-mentioned multiplex RT-PCR method, it is preferable that the reaction system of reverse transcription described in step (2) are as follows: 5 ×
4 μ L of buffer, 1 μ L of 10mmol/L dNTPs, 20 μm of 0.5 μ L of ol/L pd (N) 6 (random hexamers), 20 μm of ol/L
0.5 μ L of Oligo (dT), 0.5 μ L of 40U/ μ L RNase inhibitor, 0.5 μ L and RNA template 13 of 200U/ μ L reverse transcriptase (M-MLV)
μL；The reaction condition of the reverse transcription are as follows: 42 DEG C of 1h, 95 DEG C of 10min.
According to above-mentioned multiplex RT-PCR method, it is preferable that the reaction system of pcr amplification reaction described in step (3) are as follows:
2 × Es Taq MasterMix, 25 μ L, 2 μ L of mix primer, 5 μ L of cDNA template, ddH218 μ L of O, wherein the mix primer
The primer of his virus of to be by initial concentration the be detection pig lid of 20 μm of ol/L, the primer for detecting Porcine epidemic diarrhea virus, inspection
It surveys the primer of transmissible gastro-enteritis virus, detect the primer of pig Delta coronavirus and drawing for detection pig A rotavirus
The ratio of 30:10:3:2:2 is uniformly mixed and obtains object by volume；The reaction condition of the pcr amplification reaction are as follows: initial denaturation
95 DEG C of 5min, 94 DEG C of 30s, 57 DEG C of 80s, 72 DEG C of 60s, totally 35 recycle；Last 72 DEG C of extensions 10min.
According to above-mentioned multiplex RT-PCR method, it is preferable that analysis pcr amplification product described in step (4) is expanded PCR
Increase production object and carry out agarose gel electrophoresis analysis, type viral in sample is determined according to electrophoresis result.
Compared with prior art, the positive beneficial effect that the present invention obtains are as follows:
(1) present invention refers to the Capsid gene order of the pig lid his viral (GETV) logged in GenBank, pig epidemic diarrhea
Viral the E protein coding gene sequence of (PEDV), the S protein coding gene sequence of transmissible gastro-enteritis virus (TGEV), pig
The N protein coding gene sequence of Delta coronavirus (PDCoV) and the VP6 encoding histone base of pig A rotavirus (PoRV)
Because of sequence, five pairs of specific primers are designed；Five pairs of primer specificities are strong, and amplification efficiency and high sensitivity are homologous each other
Property it is low, can be realized while identifying five kinds of viruses of GETV, PEDV, TGEV, PDCoV and PoRV, and lactation can be caused young with other
Pig common diarrhoea virus, swine fever virus (CSFV), encephalitis B virus (JEV) and porcine reproductive and respiratory syndrome virus (PRRSV) nothing
Cross reaction, it is not significant to same type different batches sample detection result difference, to GETV, PEDV, TGEV, PDCoV and PoRV
Five kinds of hybrid virus nucleic acid (volume ratio 1:1:1:1:1) maximum dilution multiples and limit of identification are respectively 10-2With 85.72ng/ μ
L.Therefore, the present invention provides a kind of cost for effectively detection and identification five kinds of viruses of GETV, PEDV, TGEV, PDCoV and PoRV
Low, quick, special, accurate method.
(2) compared with the regular-PCR for successively expanding single virus respectively, multiple RT-PCR detection method of the present invention has spy
It is anisotropic by force and detector efficiency it is high, while detecting and the advantages of 5 kinds of different virus can be identified.
Detailed description of the invention
Fig. 1 is the specific test knot of PEDV, PoRV, TGEV, PDCoV and GETV of multiplex RT-PCR method of the present invention
Fruit；Wherein, M M.DL2000Marker, swimming lane 1 represent the pure of five kinds of virocytes of PEDV, TGEV, PoRV, PDCoV and GETV
Culture mix, swimming lane 2-10 respectively represent PEDV, PoRV, TGEV, PDCoV, GETV, PRRSV (porcine reproductive and respiratory syndrome
Virus), CSFV (swine fever virus), JEV (japanese encephalitis virus) and negative control.
Fig. 2 is the result of sequence alignment；Wherein, (a) is the E sequence alignment result of PEDV, (b) is the S sequence ratio of TGEV
Pair as a result, (c) be PoRV VP6 sequence alignment result, (d) be PDCoV N sequence alignment result, (e) be GETV Capsid
Sequence alignment result；(a) in: CV777 is PEDV strain, and HN148 represents the E segment of the PEDV of amplification 964bp；(b) in: P115
For TGEV strain, HN394 represents the S segment of the TGEV of amplification 507bp；(c) in: 2010/WH-a VP6 is PoRV strain,
HN1007 represents the VP6 segment of the PoRV of amplification 698bp；(d) in: USA/IIIinois121/2014 is PDCoV strain,
HN513 represents the N segment of the PDCoV of amplification 410bp；(e) in: Alphavirus M1 is GETV strain, and HN513 represents amplification
The Capsid segment of the GETV of 189bp.
Fig. 3 is the test result for carrying out sensitivity Detection to PEDV using single RT-PCR method of the invention；
M is M.DL2000Marker；Swimming lane 1~6 is respectively the 10 of PEDV positive cDNA-1Dilution, 10-2Dilution, 10-3Dilution,
10-4Dilution, 10-5Dilution, 10-6Dilution.
Fig. 4 is the test result for carrying out sensitivity Detection to PoRV using single RT-PCR method of the invention；
M is M.DL2000Marker；Swimming lane 1~6 is respectively the 10 of PoRV positive cDNA-1Dilution, 10-2Dilution, 10-3Dilution,
10-4Dilution, 10-5Dilution, 10-6Dilution.
Fig. 5 is the test result for carrying out sensitivity Detection to TGEV, PDCoV using single RT-PCR method of the invention；
M is M.DL2000Marker；Swimming lane 1~6 is respectively the 10 of TGEV positive cDNA-1Dilution, 10-2Dilution, 10-3Dilution,
10-4Dilution, 10-5Dilution, 10-6Dilution；Swimming lane 7~12 is respectively the 10 of PDCoV positive cDNA-1Dilution, 10-2Dilution, 10-3It is dilute
It releases, 10-4Dilution, 10-5Dilution, 10-6Dilution.
Fig. 6 is the test result for carrying out sensitivity Detection to GETV using single RT-PCR method of the invention；
M is M.DL2000Marker；Swimming lane 1~6 is respectively the 10 of GETV positive cDNA-1Dilution, 10-2Dilution, 10-3Dilution,
10-4Dilution, 10-5Dilution, 10-6Dilution；Detecting maximum dilution multiple is 10-6。
Fig. 7 is multiplex RT-PCR method of the present invention to PEDV, PoRV, TGEV, the test of PDCoV and GETV sensitivity Detection
As a result；
M is M.DL2000Marker；Swimming lane 1~6 is respectively PEDV, PoRV, TGEV, PDCoV and GETV mixing positive cDNA
10-1Dilution, 10-2Dilution, 10-3Dilution, 10-4Dilution, 10-5Dilution, 10-6Dilution.
Fig. 8 is the repetitive test result using multiplex RT-PCR method of the present invention；
M is M.DL2000Marker；Swimming lane 1~3 is respectively third day, the 6th day, the 9th day PEDV, PoRV, TGEV,
The mixing pure culture of PDCoV and GETV, swimming lane 4 are negative control.
Specific embodiment
Below by way of specific embodiment, invention is further described in detail, but does not limit the scope of the invention.?
Under without departing from the spirit and scope of the present invention, can with the details and forms of the technical scheme of the invention are modified or replaced, but
These modifications or substitutions each fall within protection scope of the present invention.Unless otherwise specified, raw material used in embodiment, chemical reagent
It is conventional commercial commodity, the conventional means that technological means used is well known to the skilled person.
Test strain and reagent:
(1) strain: his virus (GETV), porcine reproductive and respiratory syndrome virus (Porcine Reproductive of pig lid is tested
And Respiratory Syndrome Virus, PRRSV), swine fever virus (Classical Swine Fever Virus,
CSFV) and Japanese B encephalitis virus (Japanese Encephalitis Virus, JEV) Reference strains, by Henan agriculture
Sparetime university learns poultry diease research institute and saves；Pig Delta coronavirus (PDCoV) strain is by Agricultural University Of He'nan's Henan Province's animal foodstuff
The present of security concern laboratory；The weak poison of CV777 plants of Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus magnificent strain, pig A groups
NX plants of rotavirus purchases are in market diarrhea triple vaccine.
(2) reagent: agarose (Sigma) is purchased from Sangon Biotech (Shanghai) Co., Ltd.；DEPC
(Amresco), TRIzon Reagent extraction agent and 2 × EsTaqMasterMix have purchased from Beijing health for century biotechnology
Limit company；Source of mouse reverse transcriptase (M-MLV), RNase inhibitor, dNTP and DL1000DNA Marker are purchased from precious bioengineering
(Dalian) Co., Ltd；It is raw that SanPrep pillar DNA plastic recovery kit and a small amount of extraction agent boxes of Plasmid DNA are purchased from raw work
Object engineering (Shanghai) limited liability company, other reagents are domestic or Import Analysis is pure.
Embodiment 1: design of primers and synthesis
(1) for detecting the design of his viral primer of pig lid:
Formation and survive most important and height of the Capsid protein coding gene to virion in pig lid his viral (GETV)
It is conservative, therefore selecting Capsid gene is the target gene for detecting his virus of pig lid.Genbank database is compared using DNAMAN 6.0
In all Capsid refer to gene order, find out opposite conservative fragments.The conservative fragments of the Capsid gene found are inputted
In 5.0 software of Primer Premier, with upstream and downstream primer length 19bp and 60 DEG C and 58.3 DEG C of 21bp, Tm value, G+C content
57.9% and 47.6%, it avoids primer itself from forming hairpin structure, reduces formation dimer between primer, reduces non-specific expand
Increasing etc. is parameter, the upstream and downstream primer sequence of design amplification Capsid gene.Then by the primer sequence of acquisition using in NCBI
BLAST online software is verified, and optimal primer sequence (being shown in Table 1) is obtained.
(2) for detecting the design of the primer of Porcine epidemic diarrhea virus:
E protein plays an important role and protects in the self assembly and Budding process of virus in Porcine epidemic diarrhea virus (PEDV)
It keeps, therefore selecting E protein encoding gene is the target gene for detecting Porcine epidemic diarrhea virus.It is compared using DNAMAN 6.0
All E proteins refer to gene order in Genbank database, find out opposite conservative fragments.By the conservative of the E protein gene found
Segment inputs in 5.0 software of Primer Premier, with upstream and downstream primer length 19bp and 58 DEG C and 60.3 DEG C of 22bp, Tm value,
G+C content 52.6% and 50% avoids primer itself from forming hairpin structure, reduces formation dimer between primer, reduces non-spy
Specific amplification etc. is parameter, the upstream and downstream primer sequence of design amplification E protein gene.Then the primer sequence of acquisition is utilized
BLAST online software is verified in NCBI, obtains optimal primer sequence (being shown in Table 1).
(3) for detecting the design of the primer of transmissible gastro-enteritis virus:
S protein is high glycosylation albumen in transmissible gastro-enteritis virus (TGEV), generates neutralizing antibody simultaneously in induction body
The various aspects such as immunoprotection, cell fusion and tissue tropism are provided and plays an important role and guards relatively, therefore S protein is selected to encode base
Because detecting the target gene of transmissible gastro-enteritis virus.All S eggs in Genbank database are compared using DNAMAN 6.0
White ginseng examines gene order, finds out opposite conservative fragments.The conservative fragments of the S protein gene found are inputted into Primer Premier
In 5.0 softwares, with upstream and downstream primer length 23bp and 60.3 DEG C and 54 DEG C of 19bp, Tm value, G+C content 47.8% and 42.1%,
It avoids primer itself from forming hairpin structure, reduce formation dimer between primer, reduction non-specific amplification etc. as parameter, design
Expand the upstream and downstream primer sequence of S protein gene.Then the primer sequence of acquisition is carried out using BLAST online software in NCBI
Verifying, obtains optimal primer sequence (being shown in Table 1).
(4) for detecting the design of the primer of pig Delta coronavirus:
N protein is a kind of multi-functional phosphorylation structural proteins in pig Delta coronavirus (PDCoV), and structure has conservative
And species specificity, therefore selecting N protein encoding gene is the target gene for detecting pig Delta coronavirus.Utilize DNAMAN6.0
It compares all N proteins in Genbank database and refers to gene order, find out opposite conservative fragments.By the N protein gene found
Conservative fragments input in 5.0 software of Primer Premier, with upstream and downstream primer length 22bp and 60.3 DEG C of 20bp, Tm value and
62 DEG C, G+C content 50% and 55%, avoid primer itself formed hairpin structure, reduce primer between formed dimer, reduce it is non-
Specific amplification etc. is parameter, the upstream and downstream primer sequence of design amplification N protein gene.Then the primer sequence of acquisition is utilized
BLAST online software is verified in NCBI, and obtaining optimal primer sequence is (being shown in Table 1).
(5) for detecting the design of the primer of pig A rotavirus:
VP6 albumen is strongly immunogenic and highly conserved in pig A rotavirus (PoRV), therefore selects VP6 protein coding gene for inspection
Survey the target gene of pig A rotavirus.All S proteins in Genbank database, which are compared, using DNAMAN 6.0 refers to gene
Sequence finds out opposite conservative fragments.The conservative fragments of the VP6 protein gene found are inputted into 5.0 software of Primer Premier
In, with upstream and downstream primer length 20bp and 58 DEG C and 58 DEG C of 20bp, Tm value, G+C content 45% and 45%, avoid primer from figure
At hairpin structure, formation dimer between primer, reduction non-specific amplification etc. are reduced as parameter, design amplification VP6 albumen base
The upstream and downstream primer sequence of cause.Then the primer sequence of acquisition is verified using BLAST online software in NCBI, is obtained most
Good primer sequence (being shown in Table 1).
Table 1 is used to detect the primer of GETV, PEDV, TGEV, PDCoV and PoRV
It is above-mentioned for detect pig lid his virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pig A groups take turns
The primer of shape virus and pig Delta coronavirus is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Embodiment 2: nucleic acid extraction
According to TRIzon Reagent extraction agent operation instructions nucleic acid extraction is carried out to virus respectively, obtains each virus
RNA.
Embodiment 3:RT-PCR amplification
Respectively using the RNA of the virus of extraction as template, reverse transcription becomes cDNA.The reverse transcription system are as follows: 5 × buffer4 μ L,
1 μ L of 10mmol/L dNTPs, 20 μm of 0.5 μ L of ol/L pd (N) 6 (random hexamers), 20 μm of ol/L Oligo (dT) 0.5
μ L, 0.5 μ L of 40U/ μ L RNase inhibitor, 0.5 μ L and RNA template of 200U/ μ L reverse transcriptase (M-MLV), 13 μ L；The reversion
The reaction condition of record are as follows: 42 DEG C of 1h, 95 DEG C of 10min.
Using cDNA as template, pcr amplification reaction is carried out.The reaction system of the pcr amplification reaction are as follows: 2 × Es Taq
25 μ L of MasterMix, 2 μ L of mix primer, 5 μ L of cDNA template, ddH218 μ L of O, wherein the mix primer is will be initial dense
Primer, the detection pig transmissible that degree is the primer of his virus of the detection pig lid of 20 μm of ol/L, detects Porcine epidemic diarrhea virus
The primer of the primer of marcy agent, the primer for detecting pig Delta coronavirus and detection pig A rotavirus is by volume
The ratio of 30:10:3:2:2, which is uniformly mixed, to be obtained.The reaction condition of the pcr amplification reaction are as follows: 95 DEG C of 5min of initial denaturation；94
DEG C 30s, 57 DEG C of 80s, 72 DEG C of 60s, totally 35 circulations；Last 72 DEG C of extensions 10min.2% Ago-Gel of pcr amplification product
Carry out electrophoresis detection analysis.
Embodiment 4: multiple RT-PCR specific test
In order to verify the present invention be used for while detecting pig lid his virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus,
The specificity of the multiple RT-PCR primer sets of pig A rotavirus and pig Delta coronavirus is designed by the invention more
Weight RT-PCR primer group respectively to PEDV, TGEV, PoRV, PDCoV, GETV, PRRSV (porcine reproductive and respiratory syndrome virus),
CSFV (swine fever virus) and JEV (japanese encephalitis virus) carries out RT-PCR amplification, while also using multiple RT-PCR primer sets pair
The mixture of five kinds of virocyte pure cultures of PEDV, TGEV, PoRV, PDCoV and GETV carries out RT-PCR amplification.Concrete operations
Steps are as follows:
(1) PEDV, TGEV, PoRV, PDCoV, GETV, PRRSV are extracted respectively according to method for extracting nucleic acid as described in example 2
The RNA of (porcine reproductive and respiratory syndrome virus), CSFV (swine fever virus) and JEV (japanese encephalitis virus), then respectively with
The RNA of PEDV, TGEV, PoRV, PDCoV, GETV, PRRSV, CSFV and JEV are template, according to RT-PCR described in embodiment 3
Amplification method carries out RT-PCR amplified reaction using multiple RT-PCR primer sets of the invention, and amplified production carries out Ago-Gel
Electrophoretic analysis, the result is shown in Figure 1.DNA recycling is carried out to the electrophoretic band of above-mentioned each RT-PCR amplified production, through purifying after recycling
Sequencing and comparison are carried out after processing.By taking the RNA of PEDV is the RT-PCR amplified reaction of template as an example, amplified production is returned
It receives, the concrete operations of sequencing and comparison are as follows: use DNA plastic recovery kit to using the RNA of PEDV as the RT-PCR of template
Amplified production is recycled (operating procedure is referring to DNA plastic recovery kit specification), is connect after purification with pMD18-T carrier,
Then Transformed E .coli DH5 α competent cell, is screened through Amp, and PCR is accredited as positive bacterium solution and send raw work bioengineering
(Shanghai) limited liability company is sequenced；Each target fragment chooses 3 positive colony samples, and each sample repeats to be sequenced
3 times；The partial sequence for measuring the PEDV announced in sequence and GenBak is compared.TGEV, PoRV, PDCoV and GETV
The sequencing and comparison method of amplified production are similar with PEDV.Sequencing and the result of comparison are shown in Fig. 2.
(2) according to method for extracting nucleic acid as described in example 2 to five kinds of viruses of PEDV, TGEV, PoRV, PDCoV and GETV
The mixture of cell pure culture carries out Total RNAs extraction, then using the total serum IgE of extraction as template, according to described in embodiment 3
RT-PCR amplification method carries out RT-PCR amplification using multiple RT-PCR primer sets of the invention, and it is solidifying that amplified production carries out agarose
Gel electrophoresis analysis, the result is shown in Figure 1.
By Fig. 1 and Fig. 2 it is found that it is template that only PEDV, TGEV, PoRV, PDCoV and GETV, which have one or more cell cultures,
When have specific band amplification, and amplify come specific band be consistent with target fragment；And with PRRSV, CSFV and JEV
It is expanded when cell culture is template without specific band.Thus illustrate design for simultaneously detect pig lid he virus, pig flow
Row diarrhea virus, transmissible gastro-enteritis virus, pig A rotavirus and the multiple RT-PCR of pig Delta coronavirus draw
Object group has good specificity.
Embodiment 5: primer sensitivity test
(1) single RT-PCR sensitivity tests
This five kinds of viral RNA of PEDV, TGEV, PoRV, PDCoV and GETV are extracted respectively according to method as described in example 2, are pressed
Five kinds of viral RNA reverse transcriptions are become into cDNA according to the method that embodiment 3 is recorded；Detected respectively using microplate reader PEDV, TGEV,
Then the cDNA nucleic acid content of PoRV, PDCoV and GETV adjust the nucleic acid concentration of five kinds of virus cDNA to same concentration
(8572ng/μL).Then 10 times of continuous doubling dilutions are carried out to five kinds adjusted viral cDNA respectively and (10 is arranged altogether-1、
10-2、10-3、10-4、10-5、10-6Six dilutions).Then by five kinds of viral cDNA of the same dilution by volume 1:
1:1:1:1 mixing, obtains 10-1、10-2、10-3、10-4、10-5、10-6The mixutre genome of six dilutions.It is dilute with six respectively
The mixutre genome for degree of releasing is template, carries out the single RT-PCR amplification (method and the basic phase of embodiment 3 of single RT-PCR amplification
Together, the difference is that, the primer used in the reaction system of pcr amplification reaction is single primer), separately verify the present invention
Detect pig lid he virus primer, detect Porcine epidemic diarrhea virus primer, detect transmissible gastro-enteritis virus primer,
The sensibility of the primer of pig A rotavirus and the primer of detection pig Delta coronavirus is detected, amplified production carries out electrophoresis
Detection.As the result is shown (Fig. 3~Fig. 6), PEDV, PoRV, TGEV, PDCoV and GETV detect maximum dilution multiple point to electrophoresis detection
It Wei 10-3、10-4、10-5、10-6With 10-6, i.e., when mixing nucleic acid extension rate is greater than 10-3, can be obtained effective amplification.
(2) multiple RT-PCR sensitivity tests
This five kinds of viral RNA of PEDV, TGEV, PoRV, PDCoV and GETV are extracted respectively according to method as described in example 2, are pressed
Five kinds of viral RNA reverse transcriptions are become into cDNA according to the method that embodiment 3 is recorded；Detected respectively using microplate reader PEDV, TGEV,
Then the cDNA nucleic acid content of PoRV, PDCoV and GETV adjust the nucleic acid concentration of five kinds of virus cDNA to same concentration
(8572ng/μL).Then 10 times of continuous doubling dilutions are carried out to five kinds adjusted viral cDNA respectively and (10 is arranged altogether-1、
10-2、10-3、10-4、10-5、10-6Six dilutions).Then by five kinds of viral cDNA of the same dilution by volume 1:
1:1:1:1 mixing, obtains 10-1、10-2、10-3、10-4、10-5、10-6Six dilution mixutre genomes.It is diluted respectively with six
The mixutre genome of degree is template, carries out RT-PCR amplification (the amplification side RT-PCR using multiple RT-PCR primer sets of the invention
Method is with embodiment 3), amplified production carries out electrophoresis detection.Electrophoresis detection as the result is shown (Fig. 7), GETV, PEDV, TGEV, PoRV and
Five kinds of hybrid virus nucleic acid (volume ratio 1:1:1:1:1) maximum dilution multiples of PDCoV and limit of identification are respectively 10-2With
85.72ng/μL。
Embodiment 6: multiple RT-PCR repetitive test
Third day after five kinds of viral samples detect for the first time, the 6th day and the 9th day, five kinds of diseases are extracted with same procedure respectively
The nucleic acid of malicious pure culture carries out RT-PCR amplification using RT-PCR amplification method described in embodiment 3, and amplified production carries out electricity
Swimming detection.Electrophoresis result (Fig. 8) display, 3 batches, which repeat test, can amplify uniform purpose band, show this hair
Bright multiplex RT-PCR method repeatability is good.
Embodiment 7: while detecting his virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pig A groups of pig lid
The multiplex RT-PCR method of rotavirus and pig Delta coronavirus
With the multiple RT-PCR detection method of the invention established to 93 portions of fresh meats, chitling tissue and the excrement sample in 3 city of Henan
Product are detected, and are compared with conventional RT-PCR testing result, as the result is shown (table 2), have 41 parts in 93 parts of test samples
The positive is 32 parts containing single virus, is 9 parts containing hybrid virus, and positive rate is up to 44.09%；Contain single virus
In 32 parts, 3 parts are GETV, and 17 parts are PEDV, and 9 parts are TGEV, and 2 parts are PoRV, and 1 part is PDCoV；9 parts containing hybrid virus
In, 1 part of sample contains PEDV, TGEV and PoRV simultaneously, and 5 parts of samples contain PEDV and PoRV simultaneously, and 3 parts of samples contain simultaneously
PEDV and GETV.It is compared with conventional RT-PCR testing result it is found that two methods concordance rate is 100%.
The testing result of the multiple RT-PCR of the present invention of table 2 and conventional RT-PCR detection method to 93 parts of target samples
Above embodiments are only to illustrate implementer's case of the invention rather than limit, although referring to preferred embodiment pair
The present invention is described in detail, still, all within the spirits and principles of the present invention, made any modification, equivalent replacement,
Improve etc., it should all be included in the protection scope of the present invention.
Sequence table
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Claims (5)
1. one kind is used for while detecting his virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pig Delta of pig lid
The multiple RT-PCR primer sets of coronavirus and pig A rotavirus, which is characterized in that the multiple RT-PCR primer sets be by
For detecting the primer of his virus of pig lid, the primer for detecting Porcine epidemic diarrhea virus, detecting transmissible gastro-enteritis virus
Primer, the primer for detecting pig Delta coronavirus and the primer composition for detecting pig A rotavirus, wherein
For detecting the nucleotide sequence of his viral primer of pig lid are as follows:
Upstream primer P1:5'-GGTGGCAGGTTCACAATCC-3'(SEQ ID NO:1),
Downstream primer P2:5'-TTCTTCTGTTCCTTCTGGGGT-3'(SEQ ID NO:2)；
For detecting the nucleotide sequence of the primer of Porcine epidemic diarrhea virus are as follows:
Upstream primer P3:5'-CGTGGTGGGTTTGGTTGAT-3'(SEQ ID NO:3),
Downstream primer P4:5'-CGGTGACAAGTGAAGCACAGAT-3'(SEQ ID NO:4)；
For detecting the nucleotide sequence of the primer of transmissible gastro-enteritis virus are as follows:
Upstream primer P5:5'-CAAACATCACGAGGTCTTGCTAC-3'(SEQ ID NO:5),
Downstream primer P6:5'-CAAAGTCAACTGGACATCT-3'(SEQ ID NO:6)；
For detecting the nucleotide sequence of the primer of pig Delta coronavirus are as follows:
Upstream primer P7:5'- CCAGCAACCACTCGTGTTACTT-3'(SEQ ID NO:7),
Downstream primer P8:5'- TCAGCCATACCCGTCTTCTC-3'(SEQ ID NO:8)；
For detecting the nucleotide sequence of the primer of pig A rotavirus are as follows:
Upstream primer P9:5'-TCACAACGAAATGGGATAGC-3'(SEQ ID NO:9),
Downstream primer P10:5'-GCAAGCACAGATTCACAAAC-3'(SEQ ID NO:10)；
The detection is not for the purpose of medical diagnosis on disease and/or treatment.
2. multiple RT-PCR primer sets described in claim 1 are in his virus, Porcine epidemic diarrhea virus, pig of preparation detection pig lid
Application in the reagent of infectious gastroenteritis virus, pig Delta coronavirus and pig A rotavirus.
3. containing the kit of multiple RT-PCR primer sets described in claim 1.
4. kit as claimed in claim 3 is sick in detection pig lid his virus, Porcine epidemic diarrhea virus, transmissible gastroenteritis of swine
Application in poison, pig Delta coronavirus and pig A rotavirus, the application are not with medical diagnosis on disease and/or treatment
Purpose.
5. one kind is used for while detecting his virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pig Delta of pig lid
The multiplex RT-PCR method of coronavirus and pig A rotavirus, which comprises the following steps:
Step 1: synthesizing multiple RT-PCR primer sets described in claim 1；
Step 2: extracting the total serum IgE of sample to be tested, then carry out reverse transcription by template of the total serum IgE of extraction, obtain cDNA；
Step 3: cDNA being obtained as template using reverse transcription, the multiple RT-PCR primer sets for using step 1 to synthesize carry out PCR for primer
Amplified reaction；
Step 4: the product that analysis pcr amplification reaction obtains, result judgement；
The multiplex RT-PCR method is not for the purpose of medical diagnosis on disease and/or treatment.
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| CN109554508A (en) * | 2019-01-31 | 2019-04-02 | 福建省农业科学院畜牧兽医研究所 | For identifying the detection primer and probe of anomaly PEDV and TGEV |
| CN109652596A (en) * | 2019-01-30 | 2019-04-19 | 珠海出入境检验检疫局检验检疫技术中心 | Triple real-time RT-PCR primer sets, kit and the method for PEDV, TGEV and PoRV |
| CN109868331A (en) * | 2019-01-25 | 2019-06-11 | 长江大学 | The dual RT-Nested PCR primer and detection method of universal Porcine epidemic diarrhea virus and pig fourth type coronavirus and application |
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