CN106167837A - The real-time fluorescence quantitative PCR detection kit of bovine viral diarrhoea bovine diarrhoea virus and primer special thereof and probe - Google Patents
The real-time fluorescence quantitative PCR detection kit of bovine viral diarrhoea bovine diarrhoea virus and primer special thereof and probe Download PDFInfo
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Abstract
The invention discloses a kind of for detecting I type and the real-time fluorescence quantitative PCR detection kit of II type bovine viral diarrhoea bovine diarrhoea virus and primer special thereof and TaqMan probe.Utilize that the test kit of the present invention is easy and simple to handle to BVDV detection, high specificity, highly sensitive, reproducible, cannot be only used for I type, II type bovine viral diarrhoea bovine diarrhoea virus and the discriminating of swine fever virus, more can realize I type and the accurate quantitative analysis of II type bovine viral diarrhoea bovine diarrhoea virus, can be used for the field such as raw material and semi-finished product detection, dairy product quality inspection in production of vaccine.
Description
Technical field
The invention belongs to the veterinary animal Pathogen test in technical field of biological, particularly to one for I type
With II type Bovine Viral Diarrhea-Mucosal Disease Virus carry out qualitative and quantitative analysis real-time fluorescence quantitative PCR detection kit and
Primer special and TaqMan probe.
Background technology
Bovine viral diarrhoea-mucosal disease
Bovine viral diarrhoea-mucosal disease (Bovine viral diarrhea-mucosal disease, BVD-MD), letter
Claim bovine viral diarrhoea or mucosa sick, Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) cause, take place mostly in cattle
A kind of infectious disease acute, hot, it faces to examine and is characterized as mucosa inflammation, erosion, necrosis, diarrhoea and Pregnant cows miscarriage.2008
The Ministry of Agriculture of China promulgated announces No. 96 and bovine viral diarrhea is listed in three class epidemic diseases.
Bovine Viral Diarrhea-Mucosal Disease Virus
The rounded granule of Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) particle, diameter is about 50-80nm, has cyst membrane, table
There is fine lug structure in face.According to producing cytopathic situation, BVDV is divided into 2 types, non-cell pathogenicity type (NCP) and cause cell
Pathotype (CP), its genome length respectively may be about 12.5Kb and 12.3Kb.BVDV genomic nucleic acids is that single-stranded positive RNA divides
Son, including 5 ' UTR districts, three, ORF and 3 ' NRT district part.According to 5 ' UTR region sequences, BVDV is divided into two kinds of genotype, i.e. I type
And II type (BVDV-II) (BVDV-I), Epidemiological study finds that BVDV-I type is popular the most extensive, but along with II type BVDV disease
The incoming China of poison, II type BVDV virus is the most increasingly paid close attention to by more people.BVDV and the nucleotide of swine fever virus (CSFV)
Sequence and amino acid sequence homology are the highest, respectively may be about 66% and 85%.BVDV virus in a lot of cell cultures all
Can breeding, the cell culture such as such as tire Ren Bovis seu Bubali, testis, spleen, fetal lamb testis, Ren sus domestica, current Ren Bovis seu Bubali secondary cells strain is by extensively
Application.
The popularity of Bovine Viral Diarrhea-Mucosal Disease Virus
At present, bovine viral diarrhoea-mucosal disease (BVD-MD) is distributed in all over the world, from 20th century eight, the nineties with
Coming, at Bei Meizhou, the U.S., the infection rate of Canada BVD-MD reach 50%-85%；European, French, German, through serology
Survey result display BVD-MD infection rate be 76%, the sickness rate of Sweden between 10%-80%, the infection rate of Finland beef cattle
Not less than 50%, Greece's positive rate is 1.3%-14%, and Lithuania's positive rate is 70%-100%, Switzerland and the average sun of Poland
Property rate is respectively 12.5% and 86%；In South America, Brazil, Venezuela, Peru, New Zealand and the sun of Australia BVD-MD
Property rate is respectively 15.1%-56%, 36%-37%, 50%-96% and 89%.
China found BVDV first in 1980, and more than 20 province, city in the whole nation detect BVDV the most successively afterwards.Villous themeda
The multiple spring etc. acquires 576 parts of samples from Henan Province, including having adopted 395 parts of blood serum samples and 6 cattle farms from 5 steer ration fields
Having adopted 181 parts of milk samples, these 576 parts of samples carry out the detection of BVDV antibody, result shows, beef cattle blood serum sample and milch cow
The average BVDV positive rate of milk sample is respectively 31.39% and 58.60% (Henan bovine viral diarrhoea mucosal disease epidemiology
Investigation, the villous themeda multiple spring etc., China's Veterinary Journal, 2012,48 (5): 3-6).Between 2006-2008, when Yang get Sheng etc. spends 3 years
Between 9 regional 56 in Fujian Province are raised scattered the Niu Jinhang serosurvey of family and 15 large-scale cattle farms, result BVDV is average
Positive rate be up to 93.1% (serosurvey of Fujian Province's bovine viral diarrhea, Yang get Sheng etc., China's animal quarantine, 2008,
25 (12): 36-37).These researchs show, BVDV detects not only to judging that milch cow is the most ill significant, or milch cow
Give milk the important leverage of safety.
The prevention of bovine viral diarrhoea-mucosal disease
Because of being widely current of bovine viral diarrhoea-mucosal disease (BVD-MD), vaccination is to prevent this pathogenetic effectively
Means, the weak poison epidemic disease being made mainly cultivated by the current vaccine producing upper use with the cell culture of BVDV research strain
Seedling or inactivated vaccine.It was verified that BVDV inactivated vaccine and attenuated vaccine can effectively protect homology or antigenic difference relatively
Little strain, but to different antigenic BVDV strains, then cannot protect completely.Meyers etc. have evaluated BVDV I type inactivation epidemic disease
The immune effect that BVDV II type is infected by Seedling, result BVDV inactivated vaccine can only cross protection allos hypotype to a certain extent
(Meyers G, Tantz N, Dubovi EJ, Dubovi, Heinz-J ü rgen is T.1991.Viral in BVDV infection
cytopathogenicity correlated with integration of Ubiquitin-coding sequences
[J] .Virol:180:602～616).Additionally, due to BVDV attenuated vaccine exists causes uterine infection, immunosuppressant and external source
The risk that virus is polluted, therefore, the use of BVDV attenuated vaccine still suffers from dispute.Certainly, undeniable BVDV attenuated vaccine exists
To a certain extent or safety.
The diagnosis of bovine viral diarrhoea-mucosal disease
The Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) generally existed, and the pathogenesis of complexity, cause cattle disease
The preventing and treating difficulty of virus diarrhea-mucosal disease (BVD-MD).This disease-specific is not the most shown clinical in China's BVD-MD case
Symptom, great majority are inapparent infection, such as persistent infection, immunologic tolerance etc..And inapparent infection generally gets the brush-off in pasture, because of
This does not obtain enough monitoring.Use vaccine to there is again the problem that effect is undesirable, safety is poor, cause BVD-MD
At China's ever more popular.
BVDV detects
At present, for the detection of BVDV virus, the most commonly used method has regular-PCR detection technique, fluorescent quantitation
PCR detection technique and RPA detection technique, its have a problem in that regular-PCR detection technique because detecting only with pair of primers, right
The strong adaptability of aim sequence, causes the virus that nucleic acid sequence homology is high specific band also occur, thus causes false positive,
And RPA detection technique sensitivity fluorescence quantitative PCR detection technique to be less than, such as RPA patent documentation CN201510292386.2
Detection sensitivity is only 2TCID50, and the detection sensitivity of quantitative PCR patent documentation CN201410658803.6 is up to
0.32TCID50But, because the primer of different fluorescence quantifying PCR method designs is different with the site of probe sequence, carrying out BVDV
During Viral diagnosis, its sensitivity is the most identical, as sea day antiperspirant " differentiates GSFV Yu BVDV1 dual real-time fluorescence quantifying PCR method
Foundation " document detect viral susceptibility be only 102Copies/uL, and the foundation of the method is just for I BVDV virus
Without regard to II type BVDV.Owing to the nucleotide sequence difference of I type and II type BVDV virus is bigger, find a good site
The detection simultaneously carrying out I type and II type BVDV is extremely difficult, although patent documentation CN201410658803.6 sets up detection method
Reproducible, period standard deviation is only up to 0.6, but the BVDV strain used because of this patent is C24V strain, this poison
Strain belongs to I type BVDV, is not directed to II type BVDV strain in the document, and whether the method can detect II BVDV strain also needs
Further confirm that.Further, BVDV detection also faces the interference of swine fever virus (CSFV), because of swine fever virus (CSFV) and
BVDV is the member of same genus, and homology is high, has the strongest serological cross reaction, by agar gel diffusion test, immunity
All there is certain cross reaction in fluorescent technique, elisa, virus neutralization tests and Standard PCR technology, very
Difficult by both thoroughly differentiations, therefore there is false positive in BVDV detection.
Correlation technique
Real-Time Fluorescent Quantitative PCR Technique (Quantitative Real-time PCR, qPCR) be 1996 by the U.S.
Applied Biosystems company releases, and this technology refers to add fluorophor in PCR reaction system, utilizes fluorescence to believe
Whole PCR process is monitored in real time in number accumulation, finally by standard curve unknown template carried out quantitative analysis (Heid CA,
Stevens J,Livak KJ,Williams PM.Real time quantitative PCR.Genome Res.1996Oct；
6(10):986-94.).Real-Time Fluorescent Quantitative PCR Technique is a kind of relative quantification technology detecting viral genome, with
Regular-PCR technology is compared, and adds two ends with fluorescently-labeled oligonucleotide probe, has highly sensitive, specificity
By force, reproducible, quantitative advantage the most accurately, it is possible to use multiple fluorophor realizes the detection of several genes, and available in
Between oligonucleotide probe realize the similarity discriminating compared with high gene.At present, the method is widely used in examining of human infectious disease
Disconnected and cause of disease is quantitatively and the detection of animal pathogen, the inspection and quarantine of animal products, the field such as qualification of biological product
(Jozefczuk J,Adjaye J.Quantitative real-time PCR-based analysis of gene
expression.Methods Enzymol.2011；500:99-109.)
Summary of the invention
First purpose of the present invention is to provide for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV)
Carry out primer and the TaqMan probe of real-time fluorescence quantitative PCR detection, and realize Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV)
Discriminating with swine fever virus (CSFV).
It is provided by the present invention for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus are carried out real time fluorescent quantitative
The primer of PCR detection is: the nucleotide sequence of forward primer (BVDV-F) is as shown in SED ID NO:1 in sequence table, and downstream is drawn
The nucleotide sequence of thing (BVDV-R) is as shown in SEQ ID NO:2 in sequence table.
The primer sequence derived by above-mentioned primer falls within present invention.Described derived sequence refers at SEQ ID NO:
On the basis of 1 and/or SEQ ID NO:2 through one to ten base replacement, lack or add the primer sequence obtained.
It is provided by the present invention for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus are carried out real time fluorescent quantitative
The TaqMan probe of PCR detection is: SED ID NO:3 institute in the nucleotide sequence such as sequence table of TaqMan probe (BVDV-P)
Show；Described probe is through fluorescently-labeled, and its 5 ' end is marked with reporter fluorescence group, and 3 ' ends are marked with quenching fluorescence group.
Present invention is fallen within by the derived sequence of above-mentioned TaqMan probe sequence.Described derived sequence refers to
On the basis of SEQID NO:3,5 ' ends and/or 3 ' ends in sequence add, reduce the sequence that one or more base obtains.
It is described for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus are carried out real-time fluorescence quantitative PCR detection
5 ' end reporter fluorescence groups of TaqMan probe (BVDV-P) are ROX, and 3 ' end fluorescent quenching groups are BHQ2.
For preventing PCR to be extended when expanding, phosphatizing treatment is held in the 3 ' of described TaqMan probe.
Second object of the present invention is to provide for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV)
Carry out the test kit of real-time fluorescence quantitative PCR detection.
Real-time fluorescence quantitative PCR detection kit provided by the present invention, including above-mentioned for I type and II type bovine viral
Property diarrhoea-bovine diarrhoea virus carries out primer and the TaqMan probe of real-time fluorescence quantitative PCR detection.
Specifically, described test kit includes the following reagent for 25 μ L real-time fluorescence quantitative PCR reaction systems: in real time
Fluorescent quantitation one-step method PCR reactant liquor 2 × One Step RT-PCR Buffer III 12.5 μ L (being purchased from TakaRa company),
TaKaRa Ex Taq HS 0.5 μ L (being purchased from TakaRa company), PrimeScript RT Enzyme Mix II 0.5 μ L (purchases
In TakaRa company), BVDV-F (20 μMs) 0.5 μ L, BVDV-R (20 μMs) 0.5 μ L, BVDV-P (10 μMs) 1 μ L, RNA-free
H2O 7.5μL。
For convenience of detection, also including positive control and negative control in described test kit, described positive control is bovine viral
Property diarrhoea-bovine diarrhoea virus I type and II type geneome RNA, described negative control be without I type and II type bovine viral diarrhoea-
The reaction system of bovine diarrhoea virus, such as H2O (distilled water, sterile deionized water etc.).
Third object of the present invention is to provide described primer, probe or test kit to I type and II type bovine viral abdomen
Rush down-bovine diarrhoea virus (BVDV) carries out the detection of non-diseases diagnostic purpose or carrying out non-to BVDV with swine fever virus (CSFV)
The application of the discriminating of medical diagnosis on disease purpose.
One of this application is a kind of above-mentioned real-time fluorescence quantitative PCR detection kit and Real-Time Fluorescent Quantitative PCR Technique
I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) are carried out the side of the qualitative and quantitative analysis of non-diseases diagnostic purpose
Method.The detection of this real-time fluorescence quantitative PCR comprises the following steps:
1) Criterion curve: choose 5 ' UTR region genes of I type and II type bovine viral diarrhoea bovine diarrhoea virus
Specific and conserved sequence (I type virus holds 131-606 bit base from 5 ') builds sequence as standard substance, and design is to I type and II
Type bovine viral diarrhoea bovine diarrhoea virus carries out the standard substance primer of real-time fluorescence quantitative PCR detection, builds and carries I type and II type
The recombiant plasmid pCR TOPO-BVDV standard of 5 ' UTR region detection genes of bovine viral diarrhoea bovine diarrhoea virus
Plasmid, to identifying that correct recombiant plasmid is transcribed, prepares RNA standard substance, will carry I type and II type bovine viral abdomen
The RNA that the recombiant plasmid pCR TOPO-BVDV standard plasmid of 5 ' the UTR detection genes rushing down bovine diarrhoea virus transcribes
Product is as standard substance, and 10 times of gradient dilutions become 1 × 10 respectively8、1×107、1×106、1×105、1×104、1×103、1×
102、1×101Copy (copies)/μ L, using the standard substance of variable concentrations as template, in above-mentioned primer and TaqMan probe
Real-time fluorescence quantitative PCR detection is carried out, after detection terminates, with the concentration Log value (X-axis) of each standard substance to its corresponding Ct under guiding
Value (Y-axis) mapping, draws standard curve；
2) extract testing sample geneome RNA, with extract geneome RNA as template, at above-mentioned primer and TaqMan
Real-time fluorescence quantitative PCR detection is carried out under the guiding of probe；
3) realize I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus with the change of the CT value obtained or fluorescence signal
(BVDV) qualitative detection, occurs that " S " type amplification curve shows in testing sample containing I type and II type bovine viral diarrhoea-viscous
Film disease virus (BVDV), further according to intensity and the step 1 of fluorescence signal) in standard curve, draw in testing sample contained I type
Copy number with II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV), it is achieved detection by quantitative.
The two of application are the discriminating of a kind of Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) and swine fever virus (CSFV),
Also include after above-mentioned steps:
4) result judges:
The real-time fluorescence quantitative PCR detection method of the present invention only BVDV sample is had amplification curve and to CSFV sample without expanding
Increasing curve, the present invention makes full use of the difference of both gene orders, and utilizes and cannot expand on 2 bases of probe sequence difference
Principle, filter out preferable site at three (from 5 ' end 192,193,206 sites), can with BVDV-F, BVDV-R and BVDV-P
Amplification I type and II type BVDV simultaneously, if PCR amplification occurs that " S " type curve of standard can confirm that it is I type and II type BVDV disease
Poison, cannot expand RT (" S " type curve do not occur) because having the difference of 3 bases with RT, thus realize mirror
Other Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) and the purpose of swine fever virus (CSFV).
Concrete decision method: if there is " S " type amplification curve in 35 circulations, then confirm as the BVDV positive and (contain in sample
Have I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus), more than 38 circulations " S " type amplification curve does not occurs, then confirm as
BVDV is negative (not containing I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus in sample), is judged between 35-38 circulation
Suspicious, need to heavily examine.
In the above-mentioned methods, described step 2) in testing sample can be to pick up from tire cattle or calf for production of vaccine
Raw material serum, vaccine semi-finished product, it is also possible to produced milk by milch cow, to its detection for non-diagnostic purpose.By this type of is treated
In test sample product, the detection by quantitative of BVDV virus is to realize the monitoring to vaccine raw material and semi-finished product etc., and provides visitor for follow-up disposal
See data.
Described step 1) and step 2) in 25 μ L real-time fluorescence quantitative PCR reaction systems comprise the steps that template 2 μ L, in real time
Fluorescent quantitation one-step method PCR reactant liquor 2 × One Step RT-PCR Buffer III 12.5 μ L (being purchased from TakaRa company),
TaKaRa Ex Taq HS 0.5 μ L (being purchased from TakaRa company), PrimeScript RT Enzyme Mix II 0.5 μ L (purchases
In TakaRa company), BVDV-F (20 μMs) 0.5 μ L, BVDV-R (20 μMs) 0.5 μ L, BVDV-P (10 μMs) 1 μ L, RNA-free
H2O 7.5μL.Primer is diluted to 20ng/ μ L, and in reaction system, final dosage is 10ng.TaqMan probe is diluted to 10ng/ μ L,
In reaction system, final dosage is 10ng.
Described step 1) and step 2) in real-time fluorescence quantitative PCR reaction condition can be: first 52 DEG C of 15min, 95 DEG C
2min；Then 94 DEG C of 5s, 58 DEG C of 30s, 45 circulations.Fluorescence signal detection is carried out at the end of the annealing of each circulation.
The invention provides a kind of real-time fluorescence for detecting I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus fixed
Amount PCR detection kit and primer special, TaqMan probe.The present invention can be to I type and II type bovine viral diarrhoea-mucosal disease
Virus implements quickly detection, can be the quality monitoring in vaccine production process and rationalization joins Seedling (as vaccine antigen is prepared in assessment
Exact level, provide data basis for vaccine antigen content) strong foundation is provided, it is ensured that the safety of vaccination and rationally
Property, the production to Bovine Viral Diarrhea-Mucosal Disease Virus I type and II type vaccine has directive function；Can also be to cow producing milk
Quality is monitored, and provides objective data, to ensure the safety of milk and goods thereof for follow-up disposal.The reagent of the present invention
Box and detection method is easy and simple to handle, high specificity, highly sensitive, reproducible, cannot be only used for I type and II type bovine viral abdomen
Rush down-bovine diarrhoea virus and the discriminating of swine fever virus, more can realize I type and the standard of II type Bovine Viral Diarrhea-Mucosal Disease Virus
The amount of determination, (includes clinical pathological material of disease or training in clinical diagnosis by the detection at I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus
Support I type and the accurate detection of II type Bovine Viral Diarrhea-Mucosal Disease Virus in thing) and production of vaccine and milk product produce (non-
The application of diagnostic purpose) in play a significant role, have a extensive future.
Below in conjunction with specific embodiment, the present invention is described in further details.
Accompanying drawing explanation
Fig. 1 is that present invention Real-Time Fluorescent Quantitative PCR Technique is to I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus
(BVDV) technology path of qualitative and quantitative analysis is carried out
Fig. 2 is that the present invention is fixed for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) carry out real-time fluorescence
The amplification curve of the standard substance of amount PCR detection
Fig. 3 is that the present invention is fixed for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) carry out real-time fluorescence
The standard curve of amount PCR detection
Fig. 4 is I type and the real-time fluorescence quantitative PCR detection method of II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV)
Specific detection result
Fig. 5 is I type and the real-time fluorescence quantitative PCR detection method of II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV)
Sensitivity technique result
Fig. 6 is I type and the real-time fluorescence quantitative PCR detection method of II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV)
Repeated testing result
Detailed description of the invention
In following embodiment, method therefor is conventional method if no special instructions, and concrete steps can be found in:
" Molecular Cloning:A Laboratory Manual " (Sambrook, J., Russell, David W.,
Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring
Harbor)。
Described percent concentration is mass/mass (W/W, unit g/100g) percent concentration, matter if no special instructions
Amount/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, Unit/mL/100mL) percent concentration.
The acquirement approach of the various biomaterials described in embodiment is only to provide a kind of approach of acquisition of testing to reach
To specifically disclosed purpose, the restriction to biological material source of the present invention should not become.It is true that used biomaterial
Source is widely, any keeps on the right side of the law and the biomaterial that can obtain of moral ethics can be according to carrying in embodiment
Show that replacement uses.
The primer is synthesized by Beijing Hua Da gene company limited；Probe used is synthesized by TAKARA genome company.
Embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and concrete
Operating process, embodiment will assist in and understands the present invention, but protection scope of the present invention is not limited to following embodiment.
Biological genome is an index the most objective of direct reaction biology essential information, contained by different virus
Genomic information is different, genome base can be utilized mutual by different virus taxises to different groups by genomic information
Recruit to principle, a large amount of amplifications of specific site gene order can be realized, thus make naked eyes visible by certain method.
As it is shown in figure 1, the present invention devises a pair specific primer based on above ultimate principle and an oligonucleotide is visited
Pin, utilizes the principle of base pair complementarity, establishes a kind of specific detection I type and II type bovine viral diarrhoea-mucosal disease is sick
The real-time fluorescence quantitative PCR detection method of poison (BVDV).
Embodiment 1, design Real-Time Fluorescent Quantitative PCR Technique detection I type and II type bovine viral diarrhoea bovine diarrhoea virus
Primer and TaqMan probe
Owing to the nucleotide sequence difference of I type and II type BVDV virus is bigger, finds a good site and carry out I simultaneously
The detection of type and II type BVDV is crucial.From the nucleic acid database GenBank of NCBI (http: //
Www.ncbi.nlm.nih.gov) retrieval obtains bovine viral diarrhoea bovine diarrhoea virus I type and 5 ' UTR region genes of II type
(BVDV I type No. GenBank: JN704144, its complete genome sequence as shown in SED ID NO:4 in sequence table, BVDV II type
No. GenBank: FJ431191.1, its 5 ' end gene order as shown in SED ID NO:5 in sequence table) sequence, use DNA Man
After software is compared, according to primer, TaqMan probe design principle, choose bovine viral diarrhoea bovine diarrhoea virus I type and II
Preferred specificity is also protected by 5 ' the UTR region genes (selecting this gene to be because between BVDV difference strain the most conservative) of type
(this detection sequence is that I type bovine viral diarrhoea bovine diarrhoea virus holds 131-606 position alkali from 5 ' as detection sequence to keep sequence
Base, SED ID NO:6 in sequence table), by Primer Express 6.0 software design to bovine viral diarrhoea bovine diarrhoea virus I
Type and II type carry out primer and the TaqMan probe of real-time fluorescence quantitative PCR detection, and sequence is as follows:
BVDV-F (forward primer): 5 '-AGCAACAGTGGTGAGTTCGTTGGA-3 ' (SED ID NO:1 in sequence table)
BVDV-R (downstream primer): 5 '-GCCCTCGTCCACGTGGC/TATCTCG-3 ' (SED ID NO:2 in sequence table)
BVDV-P (TaqMan fluorescent probe): 5 '-ROX-AGTACAGGGTAGTCGTCAA/GTGGTTCG-BHQ2-3 ' (sequence
SED ID NO:3 in list).
The reporter fluorescence group of BVDV-P is ROX, and fluorescent quenching group is BHQ2.
For preventing PCR to be extended when expanding, phosphatizing treatment is held in the 3 ' of described probe.
Embodiment 2, by primer and the TaqMan probe of the present invention, I type and II type bovine viral diarrhoea bovine diarrhoea virus are entered
Row real-time fluorescence quantitative PCR detects
One, the geneome RNA of testing sample is extracted
(it is used for obtaining standard substance and acquisition to I type and II type bovine viral diarrhoea bovine diarrhoea virus cell culture respectively
Positive control) and testing sample extraction geneome RNA, concrete grammar comprises the following steps (AxyprepTMBody Fluid
Viral DNA/RNA Miniprep Kit, AXYGEN company):
(1) AXYGEN test kit prepares: by test kit description, is pre-configured with the isopropanol containing 1% glacial acetic acid and in examination
Agent Buffer W1A and Buffer W2 adds the dehydrated alcohol of prescribed concentration.
(2) adding the measuring samples of 200uL in 1.5mL centrifuge tube, and add 200uL Buffer V-L, whirlpool shakes
After mixing, stand 5min.
(3) adding 75uL Buffer V-N in the 1.5mL sample and reagent mixing centrifuge tube of step (2), whirlpool concussion is mixed
Even, 12000g is centrifuged 5min.
(4) supernatant is transferred in 2mL centrifuge tube (providing in test kit), adds 300uL isopropanol (1% glacial acetic acid), on
Lower inversion 6-8 time, mix homogeneously.
(5) being placed in preparing pipe in 2mL centrifuge tube in (providing in test kit), the mixed liquor taking step (4) moves into preparation
Guan Zhong, 6000g are centrifuged 1min.
(6) abandoning filtrate, put back into preparing pipe to 2mL centrifuge tube, add 500uL Buffer W1A, room temperature stands 1min.
12000g is centrifuged 1min.
(7) abandoning filtrate, put back into preparing pipe to 2mL centrifuge tube, add 800uL Buffer W2,12000g is centrifuged 1min.
(8) putting back into preparing pipe in 2mL centrifuge tube, 12000g is centrifuged 1min.
(9) it is placed in preparing pipe in the 1.5mL centrifuge tube (providing in test kit) of cleaning, adds 40uL preparing periosteum central authorities
Water without enzyme, room temperature stands 1min.12000g is centrifuged 1min eluted rna.
The RNA extracted from testing sample is as detection sample；From I type and II type bovine viral diarrhoea bovine diarrhoea virus cell
The RNA extracted in cultivation is as positive control sample；Method according to following two will be from I type and II type bovine viral diarrhoea mucosal disease
The RNA that virocyte extracts in cultivating prepares standard substance.
Two, the foundation of the real-time fluorescence quantitative PCR standard curve of I type and II type bovine viral diarrhoea bovine diarrhoea virus
1, PCR amplification I type and II type bovine viral diarrhoea bovine diarrhoea virus pcr amplification primer beyond the region of objective existence enclose 5 ' UTR region
Gene
The geneome RNA from I type and II type bovine viral diarrhoea bovine diarrhoea virus extracting step one carries out PCR expansion
Increase, obtain the purpose fragment of 5 ' UTR region genes of I type and II type bovine viral diarrhoea bovine diarrhoea virus, 25 μ L reaction systems
As follows:
Table 1 I type and the PCR amplification system of II type bovine viral diarrhoea bovine diarrhoea virus 5 ' UTR region gene
2, prepared by standard substance
The I type that purification is reclaimed and the 5 ' UTR region of the II type bovine viral diarrhoea bovine diarrhoea virus mesh containing detection gene
Fragment be cloned in pCR TOPO carrier (purchased from Invitrogen company), be built into recombiant plasmid, and screen positive restructuring
Plasmid send Hua Da genome company to check order, and checking plasmid construction knows no success.Sequencing result show to obtain sequence correct carry I
The recombiant plasmid of 5 ' UTR region genes of type and II type bovine viral diarrhoea bovine diarrhoea virus, named pCR TOPO-BVDV
Standard plasmid, to identifying that correct recombiant plasmid uses the T7 transcript reagent box of TAKARA to transcribe, is prepared into
To RNA standard substance.
3, the foundation of real-time fluorescence quantitative PCR standard curve
Recombiant plasmid to 5 ' UTR genes of the check order correct I type that carries and II type bovine viral diarrhoea bovine diarrhoea virus
The RNA product that pCR TOPO-BVDV standard plasmid transcribes, measures concentration with Nanodrop, and calculates each standard substance
Copy number, according to 10 times of gradients, standard substance are diluted to 1 × 108、1×107、1×106、1×105、1×104、1×103、1
×102、1×101Copies/ μ L, each sample does 2 repetitions, using the standard substance of variable concentrations as template, at primer and
Real-time fluorescence quantitative PCR detection is carried out under the guiding of TaqMan probe.
The reaction system (25 μ L) of real-time fluorescence quantitative PCR is as follows:
The real-time fluorescence quantitative PCR reactant of 5 ' UTR genes of table 2 I type and II type bovine viral diarrhoea bovine diarrhoea virus
System
| Reagent name | Volume (μ L) |
| 2 × One Step RT-PCR Buffer III (purchased from TaKaRa company) | 12.5 |
| TaKaRa Ex Taq HS (purchased from TakaRa company) | 0.5 |
| PrimeScript RT Enzyme Mix II (purchased from TakaRa company) | 0.5 |
| BVDV-F(20μM) | 0.5 |
| BVDV-R(20μM) | 0.5 |
| BVDV-P(10μM) | 1.0 |
| DNA profiling | 2.0 |
| RNA-free H2O | 7.5 |
The reaction condition of real-time fluorescence quantitative PCR is (quantitative PCR apparatus, model C FX96, purchased from U.S. Bole): reverse transcription
Condition: 52 DEG C of 15min, 95 DEG C of 2min；PCR amplification condition: 94 DEG C of 5s, 58 DEG C of 30s, 45 circulations.
The real-time fluorescence quantitative PCR amplification curve of standard substance is as in figure 2 it is shown, standard substance amplification curve is smooth serpentine
Curve (positive), in Fig. 2, the standard concentration of eight groups of lines correspondences from left to right is respectively 1 × 108、1×107、1×106、1×
105、1×104、1×103、1×102、1×101copies/μL.After detection terminates, with the concentration Log value (X-axis) of each standard substance
Mapping its corresponding Ct value (Y-axis), draw standard curve, standard curve is as it is shown on figure 3, correlation coefficient is respectively R2=1.000,
Error is less, and standard curve can be used, standard curve the linear equation obtained is: y=-3.295x+42.139.
4, the real-time fluorescence quantitative PCR of I type in vaccine raw material serum and II type bovine viral diarrhoea bovine diarrhoea virus is examined
Survey
I type that the present embodiment is set up by the present invention and the real time fluorescent quantitative of II type bovine viral diarrhoea bovine diarrhoea virus
The RNA (detection sample) that 12 parts of collected vaccines raw material serum (testing sample) are extracted by PCR detection method detects, to go out
The RNA that the I type lived and II type bovine viral diarrhoea bovine diarrhoea virus cell culture extract is positive control sample, without enzyme water to be
Negative control sample, according to real-time fluorescence quantitative PCR testing result, sick to I type in sample and II type bovine viral diarrhoea mucosal disease
Poison judges, and carries out the copy number of virus quantitatively.
Result is as shown in table 3, has 4 parts (not have I type and II type bovine viral abdomen for feminine gender in the 12 parts of detection samples detected
Rush down bovine diarrhoea virus), 8 parts of serum are judged to the positive (there is I type and II type bovine viral diarrhoea bovine diarrhoea virus), and period is equal
Less than 35, and its copy number is more than 101Copy, it is not possible to raw as I type and II type bovine viral diarrhoea bovine diarrhoea virus vaccine
The raw material (not allowing in raw material containing BVDV virus) produced.Visible use the inventive method can monitor in vaccine raw material serum
Virus levels, Effective selection goes out acceptable material.
Table 3 sample real-time fluorescence quantitative PCR testing result
| Sample | Quantitative result (period) | Quantitative result (copies/ μ L) | Result of determination |
| Positive control | 26.18 | 5.17E+04 | Set up |
| Blood serum sample 1 | 32.29 | 2.56E+02 | Positive |
| Blood serum sample 2 | 0 | 0 | Negative |
| Blood serum sample 3 | 33.64 | 8.90E+01 | Positive |
| Blood serum sample 4 | 31.54 | 4.65E+02 | Positive |
| Blood serum sample 5 | 0 | 0 | Negative |
| Blood serum sample 6 | 32.85 | 1.65E+02 | Positive |
| Blood serum sample 7 | 34.99 | 1.52E+01 | Positive |
| Blood serum sample 8 | 34.97 | 3.11E+01 | Positive |
| Blood serum sample 9 | 34.94 | 3.19E+01 | Positive |
| Blood serum sample 10 | 39.54 | 8.45E-01 | Negative |
| Blood serum sample 11 | 28.10 | 7.01E+03 | Positive |
| Blood serum sample 12 | 0 | 0 | Negative |
| Negative control | 0 | 0 | Set up |
Embodiment 3, specificity experiments
Utilize AxyGen test kit that DNA/RNA carries out extracting simultaneously to I type and II type cattle according to method described in embodiment 2
Viral diarrhea bovine diarrhoea virus (BVDV), swine fever virus (CSFV) extract RNA, to porcine circovirus 2 type (PCV2), nose trachea
Scorching virus (IBRV) extracts DNA, and I type and II type bovine viral diarrhoea bovine diarrhoea virus cell culture with inactivation extract simultaneously
RNA be positive control, with without enzyme water as negative control, carry out the most glimmering under the guiding of primer of the present invention and TaqMan probe
Fluorescent Quantitative PCR detects, and PCR reaction system and reaction condition are with reference to embodiment 2, to verify the specificity of the method.
As shown in Figure 4, only there is specific " S " type amplification curve to testing result in BVDV, and result is positive, and other sample is not
Specific " S " type amplification curve occur, result is negative.Testing result shows can specifically detect I by the method for the present invention
Type and II type bovine viral diarrhoea bovine diarrhoea virus.
Embodiment 4, sensitivity experiment
Described in embodiment 2, the pCR of 5 ' UTR genes of bovine viral diarrhoea bovine diarrhoea virus I type and II type will be carried
TOPO-BVDV standard plasmid standard substance are transcribed RNA product and are diluted to 1 × 10 respectively according to 10 times of gradients8、1×
107、1×106、1×105、1×104、1×103、1×102、1×101Copies/ μ L, using the standard substance of variable concentrations as mould
Plate, carries out real-time fluorescence quantitative PCR detection, PCR reaction system and reaction under the guiding of primer of the present invention and TaqMan probe
Condition is with reference to embodiment 2, to verify the sensitivity of the method.
Testing result is as it is shown in figure 5, show I type of the present invention and the real-time fluorescence of II type bovine viral diarrhoea bovine diarrhoea virus
Quantitative PCR detecting method can detect to 1 × 101Copies/ μ L, with other fluorescent quantitative PCR detection method (Hai Han, mirror
The foundation of other GSFV Yu BVDV1 dual real-time fluorescence quantifying PCR method, Agricultural University of the Inner Mongol's Master's thesis) compare, sensitivity
Want high 10 times.
Embodiment 5, repeated experiment
Described in embodiment 2, using 8 gradients of quantitative approach standard substance of having diluted as template, each gradient is entered
Row 3 repetition, carries out real-time fluorescence quantitative PCR detection, PCR reaction system under the guidance of primer of the present invention and TaqMan probe
And reaction condition is with reference to embodiment 2, to verify the repeatability of the method.
Testing result is as shown in Fig. 6 and Biao 4, and the amplification curve of each Concentraton gradient is relatively gathered as seen from the figure, circulation
Number, without obvious gap, shows the real-time fluorescence quantitative PCR detection method of I type of the present invention and II type bovine viral diarrhea virus
Preferably, period standard deviation difference only up to 0.59, with other fluorescent quantitation (CN201410658803.6) PCR for repeatability
The repeatability of detection method highest standard deviation 0.6 is quite.
The real time fluorescent quantitative repeatability of 5 ' UTR genes of table 4 bovine viral diarrhoea bovine diarrhoea virus I type and II type is tested
Confirmatory test result
| Sample message | Repeat 1 | Repeat 2 | Repeat 3 | Standard deviation |
| Standard substance | Period | Period | Period | Period |
| 1.00E+08 | 13.43 | 13.38 | 13.36 | 0.03 |
| 1.00E+07 | 16.75 | 16.78 | 16.55 | 0.13 |
| 1.00E+06 | 19.95 | 20.20 | 20.19 | 0.14 |
| 1.00E+05 | 23.24 | 23.41 | 23.55 | 0.16 |
| 1.00E+04 | 26.66 | 26.83 | 26.75 | 0.08 |
| 1.00E+03 | 29.91 | 29.82 | 29.84 | 0.05 |
| 1.00E+02 | 32.85 | 32.71 | 32.44 | 0.21 |
| 1.00E+01 | 0 | 36.54 | 37.37 | 0.59 |
Embodiment 6, detection bovine viral diarrhoea bovine diarrhoea virus I type and the real-time fluorescence quantitative PCR detectable of II type
Box
Based on embodiment 1 and 2, real-time fluorescence quantitative PCR detection kit provided by the present invention, including described for right
I type and II type bovine viral diarrhoea bovine diarrhoea virus carry out the primer (BVDV-F and BVDV-R) of real-time fluorescence quantitative PCR detection
With TaqMan probe (BVDV-P).
Specifically, this test kit includes the following reagent for 25 μ L real-time fluorescence quantitative PCR reaction systems: the most glimmering
Light quantitative one-step method PCR reactant liquor 2 × One Step RT-PCR Buffer III 12.5 μ L (being purchased from TakaRa company),
TaKaRa Ex Taq HS 0.5 μ L (being purchased from TakaRa company), PrimeScript RT Enzyme Mix II 0.5 μ L (purchases
In TakaRa company), BVDV-F (20 μMs) 0.5 μ L, BVDV-R (20 μMs) 0.5 μ L, BVDV-P (10 μMs) 1 μ L, RNA-free
H2O 7.5μL。
For convenience of detection, may also include positive control and negative control in test kit, described positive control is bovine viral
Diarrhoea-bovine diarrhoea virus I type and II type geneome RNA, described negative control is without Bovine Viral Diarrhea-Mucosal Disease Virus I
Type and the reaction system of II type, such as H2O (distilled water, sterile deionized water etc.).
In test kit, the use of each reagent refers to the content of embodiment 2.
To the real-time fluorescence quantitative PCR detection of antigen in vaccine semi-finished product in embodiment 7, vaccine production process
The present embodiment is to vaccine antigen I type in semi-finished product sample in BVDV production of vaccine and II type bovine viral diarrhoea mucosa
Sick viral level detects.Detection method is same as in Example 2, and measuring samples is BVDV vaccine semi-finished product 1-14.
Testing result such as table 5:
5 14 parts of BVDV semi-finished product sample detection results of table
| Sample number into spectrum | Period | Copy number | Sample number into spectrum | Period | Copy number |
| Vaccine sample 1 | 22.60 | 1.06E+06 | Vaccine sample 2 | 20.08 | 5.01E+06 |
| Vaccine sample 3 | 23.38 | 5.89E+05 | Vaccine sample 4 | 22.07 | 1.09E+06 |
| Vaccine sample 5 | 21.57 | 2.33E+06 | Vaccine sample 6 | 19.53 | 7.65E+06 |
| Vaccine sample 7 | 30.46 | 2.79E+03 | Vaccine sample 8 | 31.44 | 8.28E+02 |
| Vaccine sample 9 | 21.06 | 3.42E+06 | Vaccine sample 10 | 20.76 | 2.98E+06 |
| Vaccine sample 11 | 20.79 | 4.17E+06 | Vaccine sample 12 | 18.51 | 1.67E+07 |
| Vaccine sample 13 | 28.80 | 9.80E+03 | Vaccine sample 14 | 19.81 | 6.18E+06 |
According to this detection by quantitative result, copy number reaches 105Semi-finished product Seedling can carry out the preparation of finished product Seedling, and copy number
7,8, No. 13 low sample virus content are not enough, it is impossible to carry out the preparation of finished product Seedling.
To I type and the real-time fluorescence quantitative PCR of II type bovine viral diarrhoea bovine diarrhoea virus in embodiment 8, cow producing milk
Detection
In the present embodiment sample given milk to milch cow, I type and II type bovine viral diarrhoea bovine diarrhoea virus content are examined
Survey.Detection method is same as in Example 2, and measuring samples is cattle farm output milk.
Testing result such as table 6:
The BVDV Viral diagnosis result of 6 10 parts of milk samples of table
| Sample number into spectrum | Period | Copy number | Sample number into spectrum | Period | Copy number |
| Milk sample 1 | 32.18 | 5.04E+02 | Milk sample 2 | 0 | 0 |
| Milk sample 3 | 33.83 | 1.40E+02 | Milk sample 4 | 34.44 | 8.74E+01 |
| Milk sample 5 | 0 | 0 | Milk sample 6 | 0 | 0 |
| Milk sample 7 | 0 | 0 | Milk sample 8 | 0 | 0 |
| Milk sample 9 | 0 | 0 | Milk sample 10 | 40.05 | 2.92E+00 |
According to this detection by quantitative result, in 10 parts of milk samples, 3 parts of samples are the BVDV detection positive, respectively 1,3, and 4
Number, remain 7 parts and be feminine gender (period is feminine gender more than 38, therefore sample 10 is also judged to feminine gender).According to this detection, for the positive
Output milk need to be further processed, and can set up accordingly and review archives.
Claims (10)
1. for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus carried out real-time fluorescence quantitative PCR detection primer or
TaqMan probe, according to bovine viral diarrhoea bovine diarrhoea virus I type and the specific and conserved sequence of 5 ' UTR region genes of II type
Obtaining as detection sequential design, the base of this detection sequence is as shown in SED ID NO:6 in sequence table.
The most according to claim 1 fixed for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus being carried out real-time fluorescence
The primer of amount PCR detection, it is characterised in that SED ID NO:1 in the nucleotide sequence such as sequence table of forward primer (BVDV-F)
Shown in, the nucleotide sequence of downstream primer (BVDV-R) is as shown in SEQ ID NO:2 in sequence table.
The most according to claim 1 fixed for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus being carried out real-time fluorescence
The TaqMan probe of amount PCR detection, SED ID NO:3 institute in the nucleotide sequence such as sequence table of this TaqMan probe (BVDV-P)
Show；Described probe is through fluorescently-labeled, and its 5 ' end is marked with reporter fluorescence group, and 3 ' ends are marked with quenching fluorescence group.
TaqMan probe the most according to claim 3, it is characterised in that: described reporter fluorescence group is ROX, fluorescent quenching
Group is BHQ2.
5. for I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus being carried out the test kit of real-time fluorescence quantitative PCR detection,
Including the TaqMan probe described in the primer described in claim 1 or 2 and claim 1 or 3 or 4.
Test kit the most according to claim 5, it is characterised in that: described test kit includes following for 25 μ L real-time fluorescences
The reagent of quantitative PCR reaction system: real time fluorescent quantitative one-step method PCR reactant liquor 2 × One Step RT-PCR Buffer
III 12.5 μ L (is purchased from TakaRa company), TaKaRa Ex Taq HS 0.5 μ L (being purchased from TakaRa company), PrimeScript
RT Enzyme Mix II 0.5 μ L (being purchased from TakaRa company), BVDV-F (20 μMs) 0.5 μ L, BVDV-R (20 μMs) 0.5 μ L,
BVDV-P (10 μMs) 1 μ L, RNA-free H2O 7.5μL。
7. according to the test kit described in claim 4 or 5, it is characterised in that: described test kit may also include standard substance, the positive
Comparison and negative control, described standard substance are containing I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus pcr amplification product fragment
Plasmid transcription RNA product, described positive control is I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus geneome RNA, institute
Stating negative control is without Bovine Viral Diarrhea-Mucosal Disease Virus I type and the reaction system of II type, such as H2O is (distilled water, aseptic
Deionized water etc.).
8. one kind with the arbitrary described real-time fluorescence quantitative PCR detection kit of claim 5-7 to I type and II type bovine viral
The method that diarrhoea-bovine diarrhoea virus (BVDV) carries out non-diseases diagnostic purpose qualitative and quantitative analysis, comprises the following steps:
1) Criterion curve: build the recombiant plasmid pCR TOPO-BVDV standard carrying described detection gene
Plasmid, to identifying that correct recombiant plasmid is transcribed, prepares RNA standard substance, standard substance is diluted to 1 × 108、1
×107、1×106、1×105、1×104、1×103、1×102、1×101Copy (copies)/μ L, with the standard of variable concentrations
Product, as template, are carried out under the guiding of TaqMan probe described in primer described in claim 1 or 2 and claim 1 or 3 or 4
Real-time fluorescence quantitative PCR detects, and after detection terminates, makees its corresponding Ct value (Y-axis) with the concentration Log value (X-axis) of each standard substance
Figure, draws standard curve；
2) extract testing sample geneome RNA, with extract geneome RNA as template, at primer described in claim 1 or 2
Real-time fluorescence quantitative PCR detection is carried out under guiding with TaqMan probe described in claim 1 or 3 or 4；
3) with the change of the CT value obtained or fluorescence signal, I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) are entered
Row qualitative detection, occurs that " S " type amplification curve shows in testing sample containing Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) I
Type or II type；Intensity and step 1 further according to fluorescence signal) in standard curve, draw in testing sample contained I type or II type
The copy number of Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV), it is achieved detection by quantitative；
Described step 1) and step 2) in 25 μ L real-time fluorescence quantitative PCR reaction systems comprise the steps that template 2 μ L, real-time fluorescence
Quantitatively one-step method PCR reactant liquor 2 × One Step RT-PCR Buffer III 12.5 μ L (being purchased from TakaRa company),
TaKaRa Ex Taq HS 0.5 μ L (being purchased from TakaRa company), PrimeScript RT Enzyme Mix II 0.5 μ L (purchases
In TakaRa company), BVDV-F (20 μMs) 0.5 μ L, BVDV-R (20 μMs) 0.5 μ L, BVDV-P (10 μMs) 1 μ L, RNA-free
H2O 7.5μL；Described step 1) and step 2) in real-time fluorescence quantitative PCR reaction condition can be: first 52 DEG C of 15min, 95 DEG C
2min；Then 94 DEG C of 5s, 58 DEG C of 30s, 45 circulations.
9. the method differentiating I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus (BVDV) and swine fever virus (CSFV),
Also include after claim 8 step:
4) result judges:
If " S " type amplification curve occurring in 35 circulations, then confirm as BVDV positive (containing I type or II type bovine viral in sample
Property diarrhoea-bovine diarrhoea virus), 38 circulations are above there is not " S " type amplification curve, then confirm as BVDV negative (in sample not
Containing I type and II type Bovine Viral Diarrhea-Mucosal Disease Virus), it is judged to suspicious between 35-38 circulation, need to heavily examine.
Method the most according to claim 8 or claim 9, it is characterised in that: described step 2) in testing sample include as epidemic disease
The blood serum sample of Seedling raw materials for production, semi-finished product vaccine sample or cattle farm milk sample.
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