CN101701265A - Method for detecting infectious bovine rhinotracheitis virus and application thereof - Google Patents
Method for detecting infectious bovine rhinotracheitis virus and application thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting infectious bovine rhinotracheitis virus. Internal amplification control (IAC) markers indicating the false negative are added to a polymerase chain reaction (PCR) system; an IAC fragment is built through the rearrangement of the base, the design of the primers and the PCR gene amplification with the bypass method; the added concentration of an internal maker template is 10<2> MuL in each reaction process, the added concentration of the Mg2+ is 2.0 mmol/L, the added concentration of the each dNTP is 0.3 mmol/L, the added concentration of each primer is 0.5 mmol/L, and the added concentration of a probe is 0.2 mmol/L; the primers are amplified at 50 DEG C for 2min in 1 cycle, 95 DEG C for 5min in 1 cycle, 95 DEG C for 15s in 45 cycles and 60 DEG C for 60s in 45 cycles. Therefore, the existence of inhibitory components or the occurrence of false negative indication can be accurately showed. The method is 10-100 times more sensitive than the conventional PCR method and the virus isolation test method and can be widely used for detecting the infectious bovine rhinotracheitis virus.
Description
Invention field
The invention belongs to field of virus detection, be specifically related to the detection method of viral infectious, particularly relate to detection method and the Application Areas of a kind of infectious bovine rhinotrachetis virus.
Background technology
(Infectious bovine rhinotracheitis is a kind of viral infectious of the ox that caused by bovid herpesvirus 1 type (Bovineherpes virus 1) IBR) to infectious bovine rhinotrachetis, and the bovid herpesvirus 1 type is called for short BHV-1.The serious clinical symptom such as respiratory tract infection, conjunctivitis, encephalitis, the decline of giving milk, metritis, enteritis and miscarriage of the normal appearance of morbidity ox.Widely distributed in this disease world wide, the serosurvey of China part provinces and regions also shows the existence of this disease.OIE (OIE) classifies IBR as the category-B animal epidemic, and also clear and definite regulation in China's animal and plant quarantine law is imported and exported Niu Bixu and detected IBR, so that control the generation of this disease and popular.
At present, the detection method that BHV-1 is commonly used has virus to separate and Serum Antibody Detection, and Serum Antibody Detection is unfavorable for the diagnosis of BHV-1 acute infection or early infection; The virus separation exists detection time long, and sensitivity is lower, and has many drawbacks such as cytotoxicity of sample, is unfavorable for the operation of viral separation test.Along with developing rapidly of Protocols in Molecular Biology, the application of fluorescent PCR detection technique has improved detection efficiency and detection sensitivity greatly.But owing to there is the not principal component of large amount of complex in the sample, and in the sample preparation process, the residual of some materials can influence pcr amplification, makes to react the sensitivity reduction that records even cause false negative result, detect for fluorescent PCR, can't correctly determine the goal gene copy number.
By retrieval, do not see also that both at home and abroad related detection is highly sensitive, specificity is good, and is easy to operate and can indicate and calibrate false negative result, improves the report of fluorescent quantitative PCR detection method of the detection BHV-1 of accuracy rate.Therefore; explore and research detection sensitivity height; specificity is good; easy to operate and can indicate and proofread and correct the method for false negative result; for promptly and accurately effectively detecting BHV-1, protection livestock industry develops in a healthy way, promotes the agricultural products in China outlet and protects the aspects such as favorable image of China in international trade all significant.
Summary of the invention
Highly sensitive at not seeing related detection at present, specificity is good, and is easy to operate and can indicate and proofread and correct false negative result, improves the fluorescence quantifying PCR method of the detection BHV-1 of accuracy rate.The present invention is by primer and probe design, use the bypass method pcr amplification, obtained mark template in the BHV-1 fluorescent quantitative PCR, and addition and the reaction conditions of internally marking template are optimized, set up the fluorescence quantitative PCR detection system that contains internal standard substance, thereby realized the sensitivity to BHV-1, efficient, quick, accurately detection.
The object of the present invention is to provide a kind of detection sensitivity height, specificity is good, the test period is short, and can indicate and proofread and correct false negative result, guarantees the BHV-1 detection method of PCR detection accuracy.
The invention provides a kind of infectious bovine rhinotrachetis virus conclusive evidence and accurate quantitative method for quick.
Technical scheme of the present invention: by having set up the detection method of a kind of infectious bovine rhinotrachetis virus, employing adds the false-negative amplification interior label of indication (internal amplificationcontrol in the PCR reaction system, IAC), by base rearrangement, design of primers and bypass method pcr amplification, made up an IAC fragment, through condition optimizing, successful structure contain in target quantitative fluorescent PCR reaction system, can show existence that suppresses composition or the generation of indicating false negative result, realization is implemented monitoring to process of the test, guarantees to detect quality.
The present invention specifically provides a kind of infectious bovine rhinotrachetis virus detection method, and concrete steps are as follows:
1. the design of primer and probe is with synthetic:
Design amplifies Auele Specific Primer Ip2, Ir2 and the fluorescent probe ITpro of 141bp according to the BHV-1gB gene order, and template makes up primer I p1 and Ir1, and wherein Ip1, Ir1 are positioned at Ip2, the Ir2 outside, amplify the fragment that length is 787bp.Probe I Tpro sequence is reset, and design primer I c1 and Ic2 utilize the base mutation principle, by the joint sequence of artificial interpolation, and mark probe I Cpro in making up.
2. the preparation of target dna template:
BHV-1 is bred through cell cultures, get supernatant liquor after the freeze thawing and extract viral DNA with the DNAZo1 extracting solution; Seminal fluid carries out nucleic acid extraction with DNAZol after using Sephacryl S-400 gel-filtration.
With the viral DNA is template, is amplimer with Ip1, Ir1, and reaction conditions is: 95 ℃ of sex change 5min; 96 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 2min, 35 circulations; Extend 10min at 72 ℃ at last.Amplified production reclaims through gel-purified, is connected on the pMD18-T carrier, transforms DH5 α competent cell, extracts plasmid, through enzyme cut, after PCR and order-checking identify, called after pMD18-IT is called for short IT.
3. the design of bypass method pcr amplification and interior mark probe:
With plasmid IT is template, adopts primer I p1, Ic1 and Ic2, Ir1 to carry out pcr amplification for the pairing primer respectively, and the amplification condition of Ip1, Ic1 is: 95 ℃ of 5min; 96 ℃ of 1min, 56 ℃ of 45s, 72 ℃ of 45s, 35 circulations; 72 ℃ of 10min; The amplification condition of Ic2, Ir1 is: 95 ℃ of 5min; 96 ℃ of 1min, 62 ℃ of 45s, 72 ℃ of 45s, 35 circulations; 72 ℃ of 10min.Amplified production is called after Ip1c1, Ic2r1 respectively.
With after Ip1c1, the Ic2r1 balanced mix as template, carry out pcr amplification with primer I p1 and Ir1, reaction conditions is: 95 ℃ of sex change 5min; 96 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 2min, 35 circulations; Extend 10min at 72 ℃ at last.Product purification after the amplification is reclaimed and is connected on the pMD18-T carrier, transform DH5 α competent cell, extract plasmid, through enzyme cut, PCR and order-checking identify correct after, called after pMD18-IC is called for short IC.
4. the foundation of substance fluorescent PCR amplification system:
With IT and IC is template, is that primer and probe increase with Ip1, Ir1, ITpro and Ip1, Ir1, ICpro respectively, and dNTP concentration is 0.15～3.0mmol/L, and primer concentration is 0.2～0.6mmol/L, and concentration and probe concentration is 0.1～0.4mmol/L, Mg
2+Concentration is 1.0～4.0mmol/L, and the Taq archaeal dna polymerase is 1.25～2.5u, and Tm is 59～61 ℃, and loop parameter is 40,45 and 50, other parameter constants when optimizing.
5. being template with the IT and the IC mixture of different concns respectively, is that primer and probe carry out the fluorescent PCR coamplification with Ip1, Ir1, ITpro and ICpro, determines the interpolation concentration of IC template in the coamplification reaction system.
Among the present invention, be numbered BHV-1gB gene order design primer and the fluorescent probe of AJ004801.1 in the reference gene storehouse, the long 135305bp of this BHV-1 gene order, those of ordinary skills can be known by the public's channel.
Among the present invention, be template with the BHV-1 gene, amplify the fragment that length is 787bp with primer I p1 and Ir1, this fragment cloning obtains To Template pMD18-IT to the pMD-18T carrier, is called for short IT.
Among the present invention, be template, amplify the fluorescent PCR amplified fragments that length is 141bp with primer I p2 and Ir2 with IT.
Among the present invention, be template with IT, amplifying length respectively with primer I p1, Ic1 and Ic2, Ir1 is 227bp and 588bp fragment, called after Ip1c1 and Ic2r1.
Among the present invention, be template with Ip1c1, Ic2r1 equal amount of mixture, carry out pcr amplification with primer I p1 and Ir1 and go out the fragment that length is 787bp that this fragment cloning is to the pMD-18T carrier, mark template pMD18-IC is called for short IC in obtaining.
Simultaneously, the present invention fully takes into account, and when IC and IT two templates increase simultaneously, can compete enzyme, primer and probe, amplification is exerted an influence, to the reaction conditions of coamplification system with detect lower bound and carried out optimizing research.Get 10
6～10
1The IT of copy/μ L concentration and IC mixture are that primer and probe carry out the fluorescent PCR coamplification with Ip1, Ir1, ITpro and ICpro, and through optimizing, interior mark template interpolation concentration is each reaction 10 in the coamplification system
2Copy, Mg
2+2.0mmol/L, each 0.3mmol/L of dNTP, each 0.5mmol/L of primer, each 0.2mmol/L of probe, amplification condition are 50 ℃, 2min, 1 circulation; 95 ℃, 5min, 1 circulation; 95 ℃ of 15s, 60 ℃ of 60s, 45 circulations.
Further, the present invention is to the specificity of infectious bovine rhinotrachetis virus detection method and can repeat conclusive evidence, and has set up typical curve.
The nucleic acid samples such as BVDV, BLV, BTV, PRV, MDV that increased respectively with amplification system provided by the invention, sample does not all have positive amplification curve, shows that this method has specificity preferably.
Use amplification system provided by the invention, with 10
2The IC template of copy/reaction is respectively with 10
5, 10
2With 10
1The IT of copy/reaction carries out repeatedly repeated augmentation detection, and the C t value standard deviation and the variation coefficient that different tests obtains are analyzed, and the Ct value variation coefficient that different tests obtains is 0.38～2.8, shows that this method has better repeatability.
Further, the present invention passes through 10
6～10
1Adding concentration in the IT of copy/reaction density and the system is 10
2The IC coamplification of copy has been set up BHV-1 fluorescent PCR coamplification examination criteria curve.
By implementing the concrete summary of the invention of the present invention, can reach following beneficial effect.
1. detection time is short: traditional isolation of virus at least will be more than 7d to the detection time of BHV-1, and this invention comprises that sample pre-treatments arrives the acquisition detected result within 2h detection time, at least also will shorten 30min than the PCR detection method of routine.
2. detection sensitivity height: this method can detect the recombinant plasmid and the 0.01TCID of 10 copies
50Virus particle, the conventional PCR of remolding sensitivity and viral separate high 10～100 times.
3. specificity is good: by correlated virus nucleic acid samples such as BVDV, BLV, BTV, PRV, MDV and BHV-1 are detected, have only the BHV-1 test positive.
4. good reproducibility: detect by the virus of different titers being carried out three repeatability, the Ct value variation coefficient that different tests obtains is 0.38～2.8%, all within 10%.
5. stability is high: detect by the repeatability to sample, all obtain the consistence result.
6. pollute few: detect with regular-PCR and compare, entire reaction is carried out in the pipe of same sealing, has reduced the pollution that electrophoresis step causes.
7. can monitor in real time and detection by quantitative:, realize the real-time monitoring of entire reaction course and result's detection by quantitative by the reception of instrument to producing fluorescent signal in each amplification.
8. can indicate the generation of false negative result:,, illustrate that this detected result is a false negative result if sample and interior mark template all do not have amplification curve because of the interior mark template in this reaction system increases along with reaction starts.
9. flow process is simple: strong operability, be easy to grasp, and as long as possess molecular biology rudimentary knowledge, can finely finish just need not good special training.
10. cost is low: the reagent that uses is the molecular biology common agents, and low price is easy to buying, and consumption is few.
11. interior mark stencil design is reasonable: reset and the bypass method pcr amplification by base, interior mark template two terminal sequences and the target gene sequences that make up are in full accord, at the probe joint bit sequence change has taken place only, make it and to combine with interior mark probe, the character of interior segmental base sequence of mark and goal gene is also identical substantially, with combine with a pair of primer, therefore in mark fragment and goal gene same amplification efficiency is arranged, guaranteed the carrying out of coamplification.
12. the addition of interior mark template is reasonable: the mark template is 10 in adding
2The recombinant plasmid of individual copy, this concentration can reach the monitoring to reaction process, does not influence the augmentation detection to the To Template quantitative fluorescent PCR again.
13. practical value height: realize process of the test is implemented monitoring, effectively solved the false negative result that exists in the traditional detection method, can carry out quality monitoring, guarantee the accuracy of detected result to the laboratory.
14. effect is good: detect with this system with this inventive method 40 parts of ox seminal fluid and 10 parts of nose of an ox chamber swabs, all obtain expected results to clinical collection.
15. reference is big: the foundation of this method, for correlated virus inspection and quarantine Study on Technology provides reference preferably.And has the reference of guidance to the development of standardization for the standard detection reagent.
16. have a extensive future: this method can be widely used in inspection and quarantining for import/export department, animal and veterinary department and culture unit, for effective prevention and control of eqpidemic disease significant.
Description of drawings
Figure 1 shows that the BHV-1PCR amplification, wherein, 1,2 be primer I p1 and Ir1 to BHV-1PCR amplified production (787bp), M is DL 2000marker.
Figure 2 shows that recombinant plasmid pcr amplification result, wherein, 1 be primer I p1 and Ir1 to recombinant plasmid pcr amplification product (787bp), M is DL2000marker.
Figure 3 shows that the recombinant plasmid enzyme cuts qualification result, wherein, 1,2 fragment that produces for the reorganization plasmid enzyme restriction, M is DL2000marker.
Figure 4 shows that Ip1c1 and Ic2r1PCR amplification, wherein, 1,2 is primer I p1 and the Ic1 amplification (227bp) to IT, and 3,4 is primer I c2 and the Ir1 amplification (588bp) to IT, and M is DL 2000marker.
Figure 5 shows that primer, the relative template position of probe.
Figure 6 shows that IT and IC template fluorescent PCR amplification partial sequence synoptic diagram.
Figure 7 shows that ITpro (figure A) and ICpro (figure B) fluorescent quantitative PCR curve, wherein, 1 is IT (figure A) and IC (figure B); 2 is IC (figure A) and IT (figure B); The result shows that probe has specificity preferably at each self-template.
Figure 8 shows that 10
2The IT coamplification curve of the IC of copy/reaction and serial gradient dilution, wherein, 1-6 is 10
6-10
1The IT of copy/reaction (schemes a) and corresponding IC (figure b) amplification curve.
Figure 9 shows that the coamplification typical curve.
Figure 10 shows that the replica test of coamplification system, wherein, 1-3 is respectively 10
5, 10
2, 10
1IT of copy/reaction (figure A) and corresponding IC (figure B) amplification curve.
Embodiment
Below, for embodiment the present invention is described, still, the present invention is not limited to following embodiment.
The virus of selecting for use among the present invention, sample and bacterial classification:
BHV-1Barta Nu/67 causes low virulent strain and MDBK ox kidney passage cell available from China Veterinery Drug Inspection Office, and samples such as ox seminal fluid, swab and serum pick up from certain cattle farm.
Bovine viral diarrhea virus (BVDV), bovine leukemia virus (BLV), blue tongue virus (BTV), foot and mouth disease virus (FMDV), pseudorabies virus (PRV), Marek's disease virus nucleic acid samples such as (MDV) are preserved by this laboratory, and those of ordinary skills also can obtain.
The main agents of selecting for use among the present invention:
It is hundred Imtech's products that DNA Zol (DP3002), plasmid prepare test kit (DP1001); PMD18-T carrier (D101A), gel-purified reclaim test kit (DV805A), DH5 α competent cell, Taq archaeal dna polymerase, primer, TaqMan probe etc. and are jewellery biotechnology (Dalian) company limited product.
The key instrument of selecting for use among the present invention:
Inverted microscope (Leica DMI 6000B), micro sample adding appliance (Eppendorf), high speed frozen centrifugation (HITACHI CF16RX), fluorescent PCR instrument (ABI7300), PCR instrument (Biometra), electrophoresis apparatus (Bio-RAD MODEL200), digital image analyzer (Alpha innotech corporation IS-2200), Lambda35 ultraviolet spectrophotometer etc.
All reagent selected for use among the present invention and instrument all are well known selecting for use, but do not limit enforcement of the present invention, and other reagent more well known in the art and equipment are all applicable to the enforcement of the following embodiment of the present invention.
Be numbered BHV-1gB gene order design primer and the fluorescent probe of AJ004801.1 among the present invention in the reference gene storehouse, those of ordinary skills can be known by the public's channel.
Embodiment one: the detection of infectious bovine rhinotrachetis virus
1. the design of primer and probe is with synthetic:
Design amplifies Auele Specific Primer Ip2, Ir2 and the fluorescent probe ITpro of 141bp according to the BHV-1gB gene order, and template makes up primer I p1 and Ir1, and wherein Ip1, Ir1 are positioned at Ip2, the Ir2 outside, amplify the fragment that length is 787bp.Probe I Tpro sequence is reset, and design primer I c1 and Ic2 utilize the base mutation principle, by the joint sequence of artificial interpolation, and mark probe I Cpro in making up.
2. the preparation of target dna template:
BHV-1 is bred through cell cultures, get supernatant liquor after the freeze thawing and extract viral DNA with the DNAZo1 extracting solution; Seminal fluid carries out nucleic acid extraction with DNAZol after using Sephacryl S-400 gel-filtration.
With the viral DNA is template, is amplimer with Ip1 and Ir1, and reaction conditions is: 95 ℃ of sex change 5min; 96 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 2min, 35 circulations; Extend 10min at 72 ℃ at last.Amplified production reclaims through gel-purified, is connected on the pMD18-T carrier, transforms DH5 α competent cell, extracts plasmid, through enzyme cut, after PCR and order-checking identify, called after pMD18-IT (being called for short IT).
3. the design of bypass method pcr amplification and interior mark probe:
With plasmid IT is template, adopts primer I p1, Ic1 and Ic2, Ir1 to carry out pcr amplification for the pairing primer respectively, and the amplification condition of Ip1, Ic1 is: 95 ℃ of 5min; 96 ℃ of 1min, 56 ℃ of 45s, 72 ℃ of 45s, 35 circulations; 72 ℃ of 10min; The amplification condition of Ic2, Ir1 is: 95 ℃ of 5min; 96 ℃ of 1min, 62 ℃ of 45s, 72 ℃ of 45s, 35 circulations; 72 ℃ of 10min.Amplified production is called after Ip1c1, Ic2r1 respectively.
With after Ip1c1 and the Ic2r1 balanced mix as template, carry out pcr amplification with primer I p1 and Ir1, reaction conditions is: 95 ℃ of sex change 5min; 96 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 2min, 35 circulations; Extend 10min at 72 ℃ at last.Product purification after the amplification is reclaimed and is connected on the pMD18-T carrier, transform DH5 α competent cell, extract plasmid, through enzyme cut, PCR and order-checking identify correct after, called after pMD18-IC (being called for short IC).
4. the foundation of substance fluorescent PCR amplification system:
Being template with IT and IC respectively, is that primer and probe increase with Ip1, Ir1, ITpro and Ip1, Ir1, ICpro respectively, and dNTP concentration is 0.15～3.0mmol/L, and primer concentration is 0.2～0.6mmol/L, and concentration and probe concentration is 0.1～0.4mmol/L, Mg
2+Concentration is 1.0～4.0mmol/L, and the Taq archaeal dna polymerase is 1.25～2.5u, and Tm is 59～61 ℃, and loop parameter is 40,45 and 50, other parameter constants when optimizing.
5. being template with the IT and the IC mixture of different concns respectively, is that primer and probe carry out the fluorescent PCR coamplification with Ip1, Ir1, ITpro and ICpro, determines the interpolation concentration of IC template in the coamplification reaction system.
Further, the present invention gets 10 respectively
6～10
1The IT of copy/μ L concentration and IC mixture are that primer and probe carry out the fluorescent PCR coamplification with Ip1, Ir1, ITpro and ICpro, and through optimizing, interior mark template interpolation concentration is each reaction 10 in the coamplification system
2Copy, Mg
2+2.0mmol/L, each 0.3mmol/L of dNTP, each 0.5mmol/L of primer, each 0.2mmol/L of probe, amplification condition are 50 ℃, 2min, 1 circulation; 95 ℃, 5min, 1 circulation; 95 ℃ of 15s, 60 ℃ of 60s, 45 circulations.
Primer code name among the present invention, sequence, position see Table 1.Wherein, be numbered BHV-1gB gene order design primer and the fluorescent probe of AJ004801.1 in the reference gene storehouse, the long 135305bp of this BHV-1 gene order, those of ordinary skills can be known by the public's channel.
Table 1: primer code name, sequence, position and few nucleotide
Embodiment two: the acquisition of To Template
BHV-1 is bred through cell cultures, get supernatant liquor after the freeze thawing and extract viral DNA with the DNAZol extracting solution, seminal fluid carries out nucleic acid extraction with DNAZol after using Sephacryl S-400 gel-filtration.
With the viral DNA is template, is that amplimer carries out pcr amplification with Ip1, Ir1,20 μ L reaction systems, wherein: Mg
2+1.5mmol/L, each 0.25mmol/L of dNTP, each 1mmol/L of primer, Taq archaeal dna polymerase 1.25u, template 5 μ L.Amplification condition is: 95 ℃ of sex change 5min; 96 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 2min, 35 circulations; Extend 10min at 72 ℃ at last.Amplified production 1% agarose electrophoresis, and gel-purified recovery are connected on the pMD18-T carrier, transform DH5 α competent cell, and the extraction plasmid carries out PCR, enzyme is cut and check order evaluation.
1. the pcr amplification of recombinant plasmid is identified: after feminine gender moves slow 10 times of dilutions of recombinant plasmid during the screening electrophoresis, getting 1 μ L is template, add 2.0 μ L, 10 * PCR damping fluid, 2 μ L dNTP (2.5mM) mixtures, 0.25 μ L Taq archaeal dna polymerase (5u/ μ L), each 2 μ L of upstream and downstream primer (10pmoL), moisturizing to 20 μ L volume carries out pcr amplification, amplification condition is the same, and 1% agarose gel electrophoresis detects.
2. the enzyme of recombinant plasmid is cut evaluation: add 10 * restriction enzyme damping fluid (buffer E), 1 μ L in the 0.5mL pipe, each 0.5 μ L of BamHI and Hind III enzyme (12u/ μ L), BSA (10mg/mL) 0.25 μ L, plasmid DNA 4 μ L, aqua sterilisa mend to 10 μ L, centrifugal mixing, put 37 ℃ of water-bath digestion 2h, 65 ℃ of water-bath 10min deactivation restriction endonucleases add 2 μ L6 * last sample buffer, and 1% agarose gel electrophoresis detects.
3. the order-checking of recombinant plasmid: pcr amplification, enzyme are cut evaluation be the male plasmid clone, incubated overnight is proposed the positive plasmid order-checking.
Agarose gel electrophoresis shows, is template with the full gene DNA of BHV-1, and Ip1 and Ir1 are primer, amplifies the 787bp fragment that size conforms to, and recombinant plasmid is cut evaluation through primer I p1 and Ir1 pcr amplification and enzyme and all obtained the 787bp fragment, and order-checking is identified all correct.Through identifying that correct recombinant plasmid is To Template, called after pMD18-IT is called for short IT.The result is referring to accompanying drawing 1,2,3.
Embodiment three: the structure of interior mark template
With plasmid IT is template, adopts primer I p1, Ic1 and Ic2, Ir1 to carry out pcr amplification, 20 μ L reaction systems, wherein Mg for the pairing primer respectively
2+1.5mmol/L, each 0.25mmol/L of dNTP, each 1mmol/L of primer, Taq enzyme 1.25u, template 5 μ l.Ip1, Ic1 amplification condition are: 95 ℃ of 5min; 96 ℃ of 1min, 56 ℃ of 45s, 72 ℃ of 45s, 35 circulations; 72 ℃ of 10min.Ic2, Ir1 amplification condition are: 95 ℃ of 5min; 96 ℃ of 1min, 62 ℃ of 45s, 72 ℃ of 45s, 35 circulations; 72 ℃ of 10min.Amplify the fragment of size respectively for 227bp and 588bp, and called after Ip1c1 and Ic2r1, referring to accompanying drawing 4.
With after Ip1c1, the Ic2r1 balanced mix as template, carry out pcr amplification with primer I p1 and Ir1, reaction system and amplification condition are with embodiment one, two.Product purification after the amplification is reclaimed and is cloned into the pMD18-T carrier, transform DH5 α competent cells, extract plasmid with embodiment one, two, through enzyme cut, PCR and order-checking identify correct after, called after pMD18-IC is called for short IC.Mark probe sequence in Ic1 and Ic2 splicing part are.
Each primer, the relative template position of probe and interior mark template design of graphics are referring to accompanying drawing 5.IT, IC fluorescent PCR amplification partial sequence is as follows, can be referring to accompanying drawing 6.
IT?sequence：
1 GCTCGCGGAG?CTGGAGGTGA?T
G?TTCTGGAGGA
61?CCGCGAGTTC?TTGCCGCTAG?AAGTGTACAC?GCGCGCCGAG?CTCGCCGACA?CGGGTCTGCT
121CGACTACAGC?GAGATACAGC?G
IC?sequence：
1 GCTCGCGGAG?CTGGAGGTGA?T
G?TTCTGGAGGA
61?CCGCGAGTTC?TTGCCGCTAG?AAGTGTACAC?GCGCGCCGAG?CTCGCCGACA?CGGGTCTGCT
121?CGACTACAGC?GAGATACAGC?G
Embodiment four: the foundation of substance fluorescent PCR amplification system
Being template with IT and IC respectively, is that primer and probe increase with Ip1, Ir1, ITpro and Ip1, Ir1, ICpro respectively, 25 μ L reaction system, wherein Mg
2+1.5mmol/L, each 0.2mmol/L of dNTP, each 0.4mmol/L of primer, probe 0.2mmol/L, Taq archaeal dna polymerase 1.25u, template 1 μ L, amplification condition is 50 ℃, 2min, 1 circulation; 95 ℃, 5min, 1 circulation; 95 ℃ of 15s, 60 ℃ of 60s, 45 circulations.The result shows that probe has specificity preferably at each self-template, and amplification curve is referring to accompanying drawing 7.
Embodiment five: coamplification detects the mensuration of lower bound
When IC and IT two templates increase simultaneously, can compete enzyme, primer and probe, amplification is exerted an influence, should restudy the reaction conditions of coamplification system and detect lower bound.
Get 10 respectively
6～10
1The IT of copy/μ L concentration and IC mixture are that primer and probe carry out the fluorescent PCR coamplification with Ip1, Ir1, ITpro and ICpro.Because IC and IT increase in same reaction, it is also identical to detect primer, and the two can be at war with to substrate, primer, enzyme etc., and therefore, the IC fragment of selection suitable concentration is added the fluorescence quantitative PCR detection system to and wanted to closing.Through optimizing, mark template interpolation concentration is each reaction 10 in selecting in the coamplification system of the present invention
2Copy, Mg
2+2.0mmol/L, each 0.3mmol/L of dNTP, each 0.5mmol/L of primer, each 0.2mmol/L of probe, all the other conditions are with embodiment four, and amplification curve is referring to accompanying drawing 8.
Embodiment six: the foundation of typical curve
Get 10
6～10
1The IT of copy/μ L concentration and IC mixture are that primer and probe carry out the quantitative fluorescent PCR coamplification with Ip1, Ir1, ITpro and ICpro, and through optimizing, it is each reaction 10 that the interior mark of the final selection of coamplification system template is added concentration
2Copy, wherein Mg
2+2.0mmol/L, each 0.3mmol/L of dNTP, each 0.5mmol/L of primer, each 0.2mmol/L of probe, amplification condition are 50 ℃, 2min, 1 circulation; 95 ℃, 5min, 1 circulation; 95 ℃ of 15s, 60 ℃ of 60s, 45 circulations.Amplification curve with IT is formulated typical curve, referring to accompanying drawing 9.From the typical curve that obtains as can be seen, in the concentration range of being surveyed, the concentration of standard substance presents tangible linear dependence with corresponding Ct value and concerns that the slope of its regression curve is-3.42, and intercept is 35.61, coefficient R
2Be 0.9997, this coefficient is greater than 0.95, and each dilution amplification point all rationally is distributed on the typical curve, shows that the typical curve that this research obtains is good.
Embodiment seven: the specific detection of method
With reaction system among the embodiment six and the amplification condition nucleic acid samples such as BVDV, BLV, BTV, PRV, MDV that increased respectively, sample is all negative, and illustration method has specificity preferably.
Embodiment eight: repeatability detects
With the reaction system among the embodiment six and amplification condition respectively to 10
5, 10
2Carry out repeatedly repeated augmentation detection with the IT of 10 copy/reactions, the Ct value variation coefficient that different tests obtains is 0.38～2.8, all in 10%, shows that this method has good repeatability and stability, and amplification curve is referring to accompanying drawing 10.
Embodiment nine: the remolding sensitivity of method
1. with the comparison of viral separation method
Get 10
3-10
-4TCI D
50The BHV-1 of/50 μ L detects with the fluorescence quantifying PCR method that the present invention sets up respectively, fluorescent PCR is S type amplification curve to occur, Ct＜40, and negative control and blank all do not have amplification curve and Ct value, but corresponding interior mark template has S type amplification curve as positive criterion.The virus separation is kept liquid with the MEM that contains 2% foetal calf serum and is replaced above-mentioned MEM to carry out 10 times of serial dilutions, and each extent of dilution is respectively got 50uL and carried out the virus separation.Above-mentioned viral sample separation is inoculated 8 holes, 96 hole individual layer MDBK cells, and each gradient is inoculated 8 holes, and 37 ℃ of absorption 1h add and keep liquid 150uL/ hole, and 37 absorption 1h add the 150ul/ hole and keep liquid, put CO
2Incubator 7d, with highly diluted multiple that CPE occurs as the isolating sensitivity of virus.This fluorescence PCR method can detect 0.01TCID
50Viral level, and viral separation method has only 3 holes CPE to occur for the cell hole of the viral level of 0.1TCID50, this fluorescence PCR method is higher at least more than 10 times than viral separation method sensitivity, the results are shown in Table 2.
2. the target fluorescent PCR detects relatively with regular-PCR and in not containing
The DNA that above-mentioned series of diluted samples is extracted is that primer carries out the regular-PCR amplification with Ip1 and Ir1, reaction system and amplification condition are with implementing one, two, the reaction product detected through gel electrophoresis, amplify the specific fragment that length is 787bp, with the high dilution that is positive as detection sensitivity.The interior target fluorescent PCR method of setting up with embodiment four that do not contain detects simultaneously.The result show this contain in target fluorescence PCR method detection sensitivity highly sensitive 100 times than regular-PCR, this sensitivity with do not contain in the sensitivity of target fluorescent PCR suitable.
Mark fluorescence PCR method and viral separation test are relatively in the table 2:BHV1
Embodiment ten: to the detection of clinical sample
In increasing negative MEM, bovine serum, fresh bovine seminal fluid and frozen ox seminal fluid, adds the marker method fluorescent PCR of setting up through the present invention the virus of known quantity respectively, detect with this marker method fluorescence PCR method, the result shows that mark fluorescence PCR method can be about the 0.01TCID50/ reaction to the detection sensitivity of above-mentioned sample in this.40 parts of ox seminal fluid (wherein 8 parts have been accredited as the positive) and 10 parts of nose of an ox chamber swabs (wherein 3 parts have been accredited as the positive) to clinical collection detect with this system.There are the sample and the interior mark template of 3 parts of ox seminal fluid (comprising 1 part of positive) all not to have amplification curve, show to exist in the seminal fluid and suppress composition.Amplification again after nucleic acid is further purified, interior mark template and positive all amplify curve.
Sequence table
Organization?Applicant
＜110〉OrganizationName: the Application Project of Inspection and Quarantine Center of Xinjiang Entry-Exit Inspection and Quarantin
＜120〉Title: detection method of a kind of infectious bovine rhinotrachetis virus and application
＜130〉AppFileReference: detection method of a kind of infectious bovine rhinotrachetis virus and application
<140>CurrentAppNumber：
Sequence
<213>OrganismName：Bovine?herpesvirus1
<400>PreSequenceString：
cggtctcctt?tgccttcggc?aacgagagcg?agccggtgga?gggccagctc?ggcgaggaca 60
acgagctgct?gccgggccgc?gagctcgtgg?agccctgcac?cgccaaccac?aagcgctact 120
tccgctttgg?cgcggactac?gtgtactacg?agaactacgc?gtacgtgcgg?cgggtcccgc 180
tcgcggagct?ggaggtgatc?agcacctttg?tggacctaaa?cctcacggtt?ctggaggacc 240
gcgagttctt?gccgctagaa?gtgtacacgc?gcgccgagct?cgccgacacg?ggtctgctcg 300
actacagcga?gatacagcgc?cgcaaccagc?tgcacgagct?ccggttctac?gacattgacc 360
gcgtggtcaa?gacggacggc?aatatggcca?tcatgcgagg?gctcgccaac?ttctttcagg 420
gcctgggcgc?cgtcgggcag?gcggtgggca?cggtggtgct?gggcgccgcg?ggtgccgcgc 480
tctcgaccgt?gtcgggcatc?gcctcgttta?ttgcgaaccc?gttcggcgcg?ctggccacgg 540
ggctgctggt?gctcgccggg?ctggtggccg?ctttcctggc?gtaccggtac?atttcccgcc 600
tccgcagcaa?ccccatgaag?gcgctgtacc?cgatcaccac?gcgcgcgctc?aaggacgacg 660
cccggggcgc?aaccgccccg?ggcgaggaag?aggaggagtt?tgacgcggcc?aaactggagc 720
aggcccgcga?gatgatcaag?tatatgtcgc?tcgtgtcagc?ggtcgagcgg?caagagcaca 780
aggcgaa 787
<212>Type：DNA
<211>Length：787
SequenceName：IT
SequenceDescription：
Sequence
<213>OrganismName：
<400>PreSequenceStr?ing：
cggtctcctt?tgccttcggc?aacgagagcg?agccggtgga?gggccagctc?ggcgaggaca 60
acgagctgct?gccgggccgc?gagctcgtgg?agccctgcac?cgccaaccac?aagcgctact 120
tccgctttgg?cgcggactac?gtgtactacg?agaactacgc?gtacgtgcgg?cgggtcccgc 180
tcgcggagct?ggaggtgatt?ccacccttgc?aatcctcgtc?gtctagagtt?ctggaggacc 240
gcgagttctt?gccgctagaa?gtgtacacgc?gcgccgagct?cgccgacacg?ggtctgctcg 300
actacagcga?gatacagcgc?cgcaaccagc?tgcacgagct?ccggttctac?gacattgacc 360
gcgtggtcaa?gacggacggc?aatatggcca?tcatgcgagg?gctcgccaac?ttctttcagg 420
gcctgggcgc?cgtcgggcag?gcggtgggca?cggtggtgct?gggcgccgcg?ggtgccgcgc 480
tctcgaccgt?gtcgggcatc?gcctcgttta?ttgcgaaccc?gttcggcgcg?ctggccacgg 540
ggctgctggt?gctcgccggg?ctggtggccg?ctttcctggc?gtaccggtac?atttcccgcc 600
tccgcagcaa?ccccatgaag?gcgctgtacc?cgatcaccac?gcgcgcgctc?aaggacgacg 660
cccggggcgc?aaccgccccg?ggcgaggaag?aggaggagtt?tgacgcggcc?aaactggagc 720
aggcccgcga?gatgatcaag?tatatgtcgc?tcgtgtcagc?ggtcgagcgg?caagagcaca 780
aggcgaa 787
<212>Type：DNA
<211>Length：787
SequenceName：IC
SequenceDescription：
Sequence
<213>OrganismName：
<400>PreSequenceString：
cagcaccttt?gtggacctaa?acctcacggt?tctggaggac?cgcgagttct?tgccgctaga 60
agtgtacacg?cgcgccgagc?tcgccgacac?gggtctgctc?gactacagcg?agatacagcg 120
ccgcaaccag?ctgcacgagc?tccggttcta?cgacattgac?cgcgtggtca?agacggacgg 180
caatatggcc?atcatgcgag?ggctcgccaa?cttctttcag?ggcctgggcg?ccgtcgggca 240
ggcggtgggc?acggtggtgc?tgggcgccgc?gggtgccgcg?ctctcgaccg?tgtcgggcat 300
cgcctcgttt?attgcgaacc?cgttcggcgc?gctggccacg?gggctgctgg?tgctcgccgg 360
gctggtggcc?gctttcctgg?cgtaccggta?catttcccgc?ctccgcagca?accccatgaa 420
ggcgctgtac?ccgatcacca?cgcgcgcgct?caaggacgac?gcccggggcg?caaccgcccc 480
gggcgaggaa?gaggaggagt?ttgacgcggc?caaactggag?caggcccgcg?agatgatcaa 540
gtatatgtcg?ctcgtgtcag?cggtcgagcg?gcaagagcac?aaggcgaa 588
<212>Type ：DNA
<211>Length：588
SequenceName：Ic2r1
SequenceDescription：
Sequence
<213>OrganismName：
<400>PreSequenceString：
cggtctcctt?tgccttcggc?aacgagagcg?agccggtgga?gggccagctc?ggcgaggaca 60
acgagctgct?gccgggccgc?gagctcgtgg?agccctgcac?cgccaaccac?aagcgctact 120
tccgctttgg?cgcggactac?gtgtactacg?agaactacgc?gtacgtgcgg?cgggtcccgc 180
tcgcggagct?ggaggtgatt?ccacccttgc?aatcctcgtc?gtctaga 227
<212>Type：DNA
<211>Length：227
SequenceName：Ip1c1
SequenceDescription：
Claims (3)
1. an infectious bovine rhinotrachetis virus detection method is characterized in that, described detection method concrete steps are as follows:
(1) amplify Auele Specific Primer Ip2, Ir2 and the fluorescent probe ITpro of 141bp according to BHV-1 gB gene order design, template makes up primer I p1 and Ir1, and wherein Ip1, Ir1 are positioned at Ip2, the Ir2 outside, amplify the fragment that length is 787bp; Probe I Tpro sequence is reset, design primer I c1 and Ic2, by the joint sequence of artificial interpolation, mark probe I Cpro in making up;
(2) BHV-1 is bred through cell cultures, get supernatant liquor after the freeze thawing and extract viral DNA with the DNAZo1 extracting solution; Seminal fluid carries out nucleic acid extraction with DNAZo1 after using Sephacryl S-400 gel-filtration; With the viral DNA is template, is amplimer with Ip1 and Ir1, and reaction conditions is: 95 ℃ of sex change 5min; 96 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 2min, 35 circulations; Extend 10min at 72 ℃ at last; Amplified production reclaims through gel-purified, is connected on the pMD18-T carrier, transforms DH5 α competent cell, extracts plasmid, cuts, after PCR and order-checking identify, makes up plasmid IT through enzyme;
(3) be template with plasmid IT, adopt primer I p1, Ic1 and Ic2, Ir1 to carry out pcr amplification for the pairing primer respectively, the amplification condition of Ip1, Ic1 is: 95 ℃ of 5min; 96 ℃ of 1min, 56 ℃ of 45s, 72 ℃ of 45s, 35 circulations; 72 ℃ of 10min; The amplification condition of Ic2, Ir1 is: 95 ℃ of 5min; 96 ℃ of 1min, 62 ℃ of 45s, 72 ℃ of 45s, 35 circulations; 72 ℃ of 10min; With after amplified production Ip1c1, the Ic2r1 balanced mix as template, carry out pcr amplification with primer I p1 and Ir1, reaction conditions is: 95 ℃ of sex change 5min; 96 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 2min, 35 circulations; Extend 10min at 72 ℃ at last; Product purification after the amplification is reclaimed and is connected on the pMD18-T carrier, transform DH5 α competent cell, extract plasmid, through enzyme cut, PCR and order-checking identify, makes up plasmid IC;
(4) be template with plasmid IT and IC respectively, be that primer and probe increase with Ip1, Ir1, ITpro and Ip1, Ir1, ICpro respectively, dNTP concentration is 0.15～3.0mmol/L, and primer concentration is 0.2～0.6mmol/L, concentration and probe concentration is 0.1～0.4mmol/L, Mg
2+Concentration is 1.0～4.0mmol/L, and the Taq archaeal dna polymerase is 1.25～2.5u, and Tm is 59～61 ℃, and loop parameter is 40,45 and 50;
(5) being template with the IT and the IC mixture of different concns respectively, is that primer and probe carry out the fluorescent PCR coamplification with Ip1, Ir1, ITpro and ICpro, determines the interpolation concentration of IC template in the coamplification reaction system.
2. according to claim 1 infectious bovine rhinotrachetis virus detection method, it is characterized in that in the described coamplification detection architecture, it is 10 that interior mark template is added concentration
2Copy, Mg
2+2.0mmol/L, each 0.3mmol/L of dNTP, each 0.5mmol/L of primer, each 0.2mmol/L of probe, Taq archaeal dna polymerase 1.5u.
3. according to claim 1 infectious bovine rhinotrachetis virus detection method, it is characterized in that described coamplification detection architecture amplification condition is 50 ℃, 2min, 1 circulation; 95 ℃, 5min, 1 circulation; 95 ℃ of 15s, 60 ℃ of 60s, 45 circulations.
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