CN107236827A - Transmissible gastro-enteritis virus detection kit and detection method - Google Patents
Transmissible gastro-enteritis virus detection kit and detection method Download PDFInfo
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Abstract
The invention belongs to technical field of molecular biological detection, and in particular to a kind of transmissible gastro-enteritis virus detection kit and detection method.For prior art lack it is a kind of it is simple to operate, with low cost, be swift in response and the problem of result accurately detects the method for transmissible gastro-enteritis virus, the present invention provides a kind of transmissible gastro-enteritis virus detection kit and detection method.The kit includes：Detect just primers F 3, reversely peripheral primer B3, cross primer CPR, forward direction probe DF5F and the reverse probe DF5B to the periphery of transmissible gastro-enteritis virus.The present invention is using nucleic acid constant-temperature amplification principle detection transmissible gastro-enteritis virus, and detection method is sensitive reliable, simple to operate, saves manpower, time and financial cost, has great importance.
Description
Technical field
The invention belongs to Animal diseases technical field of molecular biological detection, and in particular to a kind of transmissible gastroenteritis of swine disease
Malicious detection kit and detection method.
Background technology
Transmissible gastroenteritis of swine (transmissible gastroenteritis of swine, TGE) is infected by pig
One kind caused by property marcy agent (transmissible gastroenteritis virus of swine, TGEV) is to vomit
Tell, watery diarrhea, dehydration are the highly contagious disease of principal character, the U.S. confirms the cause of disease for 1945 first, and the world is moved
Thing health organization (OIE) is classified as the infectious disease that must be examined in B class diseases.Since China reports first to 1950s,
Current China most area has the sick happening and prevelence, and the autumn and winter are the sick high-incidence season, and the pig of various different cultivars and week old is all
TGEV disease morbidities can be infected, wherein with death rate highest, up to 100% after piglet morbidity in 2 week old；Piglets more than 5 week old
Infect the death rate after transmissible gastroenteritis relatively low, but the growth in piglet later stage can be influenceed, reduce the efficiency of feed utilization of piglet, be weight
One of pig virus diarrhoea disease wanted, serious harm is brought to pig industry.However, the cause of disease and Porcine epidemic diarrhea virus
(PEDV) clinical symptoms, caused by other cause of diseases such as porcine rotavirus (RV), respiratory and enteric coronavirus (PRCV) are similar, only lead to
Accurate Diagnosis can not be made by crossing epidemiology and clinical symptoms, and effective laboratory diagnostic method the disease occurs and popular
Control serve vital effect.
TGEV belongs to coronaviridae coronavirus genus, and its genome is big single-stranded underlying stock RNA, its core of N gene codes
Capsid protein, virus replication, transcription and it is pathogenic in terms of play an important roll, and gene structure is highly conserved, in vaccine
Using and molecule diagnosis in terms of have larger value.
Test in laboratory TGEV method is a lot, although these common detection methods technically comparative maturity, at present
Remain in some problems：The separation identification of virus is to confirm one of most accurate etiological diagnosis method of the cause of disease, general most
Number infectious disease is established a capital really to be identified by the separation of cause of disease.But the method short time consumption length (7~10 days), and there is cost
The shortcomings of high, operating process is complicated, it is difficult to meet ageing required by clinical diagnosis, is mainly used in scientific research.Electronic Speculum
Technology for detection TGEV is also one of conventional etiological diagnosis method, but due to the hat such as TGEV and PEDV PRCVs
Shape morphology of virus is similar, only by electron microscopic observation can not antidiastole, immunoelectron microscopic method can definitely detect clinical sample with more sensitive
TGEV in product or cell culture, and virus serology identification can be provided.However, either conventional Electronic Speculum detection technique is still
, all there is complex operation in immuno-electron microscope detection technique, cost is high, the shortcomings of requiring high to instrument and equipment.Also it can be neutralized with serum
The immunoassay TGEV such as experiment, EUSA, immunofluorescence technique and immunoperoxidase assay, specifically
The quality of property antibody is the primary factor of the immunology detection degree of accuracy, and PRCV and TGEV antigenicity has certain correlation,
Easily there is very much false positive reaction, be one of the significant drawbacks that the technology is applied to detection TGEV.Conventional RT-PCR method is current
The TGEV detection methods that Yi Kong centers, scientific research institution generally select, can remove the process of virus purification culture from, meanwhile, detection
The all more conventional virus purification of sensitivity and the degree of accuracy and amynologic diagnostic method are high, but this method requires higher to instrument and equipment,
Complex operation, amplified production easily causes Aerosol Pollution, is not suitable for promoting in basic unit, multiple PCR method also has identical
Defect.Though the problem of quantitative fluorescent PCR can solve Aerosol Pollution, the general testing agency's difficulty or ease of expensive instrument and equipment are held
By.
LAMP method is another molecular detecting method, and this method is cheap, quick, sensitivity and specificity are high, and because
Its constant-temperature amplification does not need the expensive devices such as PCR instrument, and can judge amplification by observing the presence or absence of amplification accessory substance.But
Be, there are some researches show by expand accessory substance observe result there is no electrophoresis sensitive, and if uncap electrophoresis observation result general
Greatly increase the possibility of Aerosol Pollution, and the false positive damage ratio Standard PCR/RT- of aerosol of LAMP amplified productions institute primer
PCR is more serious, therefore, and how effectively detection LAMP amplified production is the big factor for restricting technology development.Gene core
Chip technology is also a developing direction of future disease quick diagnosis, but the technology is also immature at present.
Therefore, still it is used for detecting TGEV without a kind of sensitive detection method of simple to operate, with low cost, Detection results.
Cross primer isothermal amplification technology (Crossing Priming Amplification, CPA) is a kind of nucleus
Sour isothermal amplification technique (the patent No.：200810134583.1).The technology is using a kind of strand displacement archaeal dna polymerase at 62 DEG C or so
Constant temperature under insulation 1 hour can specifically, fast and efficiently complete nucleic acid amplification reaction.The technology and some quick inspections
Survey technology combines the (such as Rapid detection test strip [patent No.：200610003429.1], disposable full closed target nucleic amplifier
Device for fast detecting [the patent No.：200610109620.4] etc.), a platform can be provided for live rapid molecular detection, grasped
Make simple, it is only necessary to which a water-bath or metal bath can complete nucleic acid amplification, its amplified production is with closed detection of nucleic acids test paper
Bar carries out result judgement, and testing result naked eyes are visible, and can be prevented effectively from Aerosol Pollution.It is easy, quick, accurate, sensitive,
Food safety detection, infectiousness Pathogen test, the monitoring of daily cause of disease, customs inspection quarantine, the strategy of emerging public health cause
The fields such as property technological reserve are widely used.
The content of the invention
The technical problem to be solved in the present invention is：Prior art lacks a kind of detection pig simple to operate, with low cost and passed
The method of metachromia marcy agent.
The present invention solve technical problem technical scheme be：There is provided a kind of transmissible gastro-enteritis virus detection kit and
Detection method.
The invention provides a kind of transmissible gastro-enteritis virus detection kit, including：Detect transmissible gastroenteritis of swine
Just primers F 3, reversely peripheral primer B3, cross primer CPR, forward direction probe DF5F and the reverse probe DF5B to the periphery of virus.
Wherein, in above-mentioned transmissible gastro-enteritis virus detection kit, the described just nucleotides of primers F 3 to the periphery
Sequence is at least five continuous nucleotide in nucleotide sequence shown in SEQ ID NO.1.
Wherein, in above-mentioned transmissible gastro-enteritis virus detection kit, described reverse peripheral primer B3 nucleotides
Sequence is at least five continuous nucleotide in nucleotide sequence shown in SEQ ID NO.2.
Wherein, in above-mentioned transmissible gastro-enteritis virus detection kit, described cross primer CPR nucleotide sequence
At least five continuous nucleotide in the nucleotide sequence shown in SEQ ID NO.3.
Wherein, in above-mentioned transmissible gastro-enteritis virus detection kit, described positive probe DF5F nucleotides sequence
At least five continuous nucleotide in nucleotide sequence shown in SEQ ID NO.4 is classified as, 5 ' ends are marked with FAM groups.
Wherein, in above-mentioned transmissible gastro-enteritis virus detection kit, described reverse probe DF5B nucleotides sequence
At least five continuous nucleotide in nucleotide sequence shown in SEQ ID NO.5 is classified as, 5 ' ends are marked with Biotin groups.
Wherein, in above-mentioned transmissible gastro-enteritis virus detection kit, the nucleic acid constant-temperature amplification reaction solution composition bag
Include：Just to the periphery 3 0.1~0.6 μm of ol of primers F in reaction solution in every 16 μ L, reversely periphery primer 0.1~0.6 μm of ol of B3, is handed over
Pitch primer CPR 0.4~1.6 μm of ol, positive 0.2~1 μm of ol of probe DF5F, reverse probe DF5B 0.2~1 μm of ol, MgSO4
0~6mmol, dNTP solution 0.1~0.6mmol, Betaine 0~1.5mol, GspF enzyme 8U/ μ L 4~12U, 10 × GspF
The μ L of 2 μ L, AMV 0.02~0.5U, 25 × RNA Secure of Buffer 0.8, surplus is distilled water.
It is preferred that, in above-mentioned transmissible gastro-enteritis virus detection kit, the nucleic acid constant-temperature amplification reaction solution composition
Including：Just to the periphery 3 0.1~0.3 μm of ol of primers F in reaction solution in every 16 μ L, reverse periphery primer 0.1~0.2 μm of ol of B3,
Cross primer CPR 0.9~1.0 μm of ol, positive 0.3~0.4 μm of ol of probe DF5F, reverse 0.7~0.8 μm of ol of probe DF5B,
MgSO4 1~3mmol, dNTP solution 0.3~0.5mmol, Betaine 0.9~1.0mol, GspF enzyme 8U/ μ L 9~10U, 10
The μ L of 2 μ L, AMV 0.1~0.2U, 25 × RNA Secure of × GspF Buffer 0.8, surplus is distilled water.
It is furthermore preferred that in above-mentioned transmissible gastro-enteritis virus detection kit, the nucleic acid constant-temperature amplification reaction solution group
Into including：Just to the periphery 3 0.26 μm of ol of primers F in reaction solution in every 16 μ L, reverse periphery primer 0.1 μm of ol of B3, intersection is drawn
Thing CPR 1.0 μm of ol, positive 0.4 μm of ol of probe DF5F, reverse 0.8 μm of ol of probe DF5B, MgSO4 2mmol, dNTP solution
0.4mmol, Betaine 1.0mol, GspF enzyme 8U/ μ L 10U, 10 × GspF Buffer 2 μ L, AMV 0.2U, 25 × RNA
The μ L of Secure 0.8, surplus is distilled water.
It is furthermore preferred that in above-mentioned transmissible gastro-enteritis virus detection kit, described 10 × GspF Buffer contain
Molar concentration is 500mM Tris-HCl (8.5,25 DEG C of pH), 300mM KCl, 300mM (NH4)SO4With 1%Triton X-
100。
Further, in above-mentioned transmissible gastro-enteritis virus detection kit, in addition to positive control and negative control；
The positive control be the DNA fragmentation containing transmissible gastro-enteritis virus N genes, sequence be SEQ ID NO.6 shown in, it is described
Negative control is aseptic double-distilled water.
Present invention also offers a kind of method that virus is detected using above-mentioned transmissible gastro-enteritis virus detection kit,
Comprise the following steps：
A, with RNA extracts kits extract RNA from sample to be detected；
B, the RNA of extraction mixed with transmissible gastro-enteritis virus detection kit, the amplified reaction at 58~65 DEG C
30~90 minutes, with compareing contrast sentence read result after 15min.
Wherein, in the method for above-mentioned use transmissible gastro-enteritis virus detection kit detection virus, described in step a
Sample to be detected include one kind in pig lymphonodi mesenterici, jejunum, small intestine or intestinal contents, excrement or lung.
Wherein, in the method for above-mentioned use transmissible gastro-enteritis virus detection kit detection virus, step b is 63
Amplified reaction 60 minutes at DEG C.
Beneficial effects of the present invention are：
(1) compared to traditional Physiology and biochemistry detection method, cross primer nucleic acid constant-temperature amplification technology is applied to directly to be checked
Target gene is expanded in sample, it is only necessary to which a constant-temperature amplification can just detect that transmissible gastro-enteritis virus whether there is, significantly
Improve efficiency to have saved the time, proliferation time is only the 1/3~1/2 of conventional RT-PCR；
(2) amplified production observes result by visualizing nucleic acid membrane chromatographic Rapid detection test strip, compared to traditional PCR
TRAP, saves the cumbersome operation sequences such as deallocation glue, electrophoresis, gel imaging, and do not contact the nucleic acid dye of potential carcinogenic risk
Material, meanwhile, test strip is placed in full closed target nucleic amplifier fast testing device, can be prevented effectively from Aerosol Pollution
Caused false sun detection；
(3) sample detection of high flux and small throughput can be met simultaneously；
(4) the invention provides a kind of real-time detection to transmissible gastro-enteritis virus and inspection and quarantining for import/export
New technology, to improve the immune prevention and control level of China transmissible gastroenteritis of swine disease, introduce a fine variety, routine monitoring, disease purification, disease are examined
Survey etc. is significant.
Brief description of the drawings
Fig. 1 show TGEV-PCR cell toxicants RT-PCR amplifications in embodiment 5；
Fig. 2 show TGEV-PCR plasmids RT-PCR amplifications in embodiment 5.
Embodiment
The invention provides a kind of transmissible gastro-enteritis virus detection kit, including：Detect transmissible gastroenteritis of swine
Just primers F 3, reversely peripheral primer B3, cross primer CPR, forward direction probe DF5F and the reverse probe DF5B to the periphery of virus.
In the kit, there are two peripheral primers, two detection probes and a cross primer.In kit of the present invention
5 oligonucleotide sequences rely on the strand displacement characteristic of the high activity of GspF enzymes so that strand displacement DNA synthesis constantly self amplification
Circulation.
The cross primer isothermal amplification reactions of kit of the present invention have 3 stages, first, and cross primer site is drawn
Enter, then self cyclic amplification of nucleic acid fragment, the generation of last amplified production at a constant temperature.Because two probes are marked respectively
There are a FAM groups and Biotin groups, specific amplification products are marked simultaneous with biotin and FAM, so that product can be
It is positive and is detected in nucleic acid test strip.
Heretofore described intersection amplimer refers to the main primer for intersecting amplification, wherein 5 ' end end sequences
It is identical with FAM label probe sequences.
Peripheral primer refers to the short chain primer for being located at intersection amplimer rear, and it is poly- in strand displacement Bst DNA that it, which is acted on,
In the presence of synthase or GspF archaeal dna polymerases, the extended chain of amplified production is peeled off from template.
Probe primer is detection primer, refers to the short chain primer marked with biotin or haptens FAM, its effect is to make expansion
Increase production thing with mark, for detection.
Haptens FAM refers to the chemical substance for detection primer, the combination of they and corresponding antibody specificity, is used for
Detect amplified production.
In order to more fully explain the implementation of the present invention, there is provided for detecting transmissible gastro-enteritis virus
The embodiment of CPA kits.These embodiments are only to explain, rather than limitation the scope of protection of present invention.Wherein
Raw material used is common commercially available.
Embodiment 1 prepares transmissible gastro-enteritis virus DNA
Comprise the following steps：
(1) enter performing PCR amplification using two periphery primer SEQ ID.1 and SEQ ID.2 and obtain target gene；
(2) pcr amplification product that step (1) is obtained is purified；
(3) amplified production after purification for obtaining step (2) in time and is built by plasmid transfection and complete contains mesh
Genes of SEQ ID NO.6 shown in plasmid sequence；
(4) plasmid extracted is quantified and is diluted to 106Copy/20 DEG C of μ L, ﹣ is preserved.
Wherein, in step (2), using TaKaRa Mini BEST Agarose Gel DNA glue reclaim kits pair
Pcr amplification product is purified；Using TaKaRa pMDTM 18-T Vector Cloning plasmid transfections in step (3)
Kit.
Embodiment 2 prepares the kit reaction solution of detection transmissible gastro-enteritis virus
Comprise the following steps：
(1) RNA extracts reagents：Purchased from the commercialization column method Total RNA Mini Kits of in the market；
(2) transmissible gastro-enteritis virus nucleic acid constant-temperature amplification reaction solution：Just to the periphery primers F 3 (SEQ ID NO.1)
0.2 μm of ol, reverse periphery primer B3 (SEQ ID NO.2) 0.1 μm of ol, positive 0.4 μm of ol of probe DF5F (SEQ ID NO.3),
Positive probe DF5F (SEQ ID NO.4 5 ' end linkage flag group FAM groups) 0.8 μm of ol, cross primer CPR (SEQ ID
NO.5 5 ' end linkage flag group Biotin groups) 1 μm of ol10 × Buffer, MgSO42mmol, dNTP solution 0.4mmol,
Betaine 1mol, GspF enzyme 10U, AMV enzymes 0.2U, 25 × RNA Secure and aseptic double-distilled water composition, overall reaction liquid product
For 16 μ L.
(3) transmissible gastro-enteritis virus DNA (SEQ ID NO.6) prepared by embodiment 1 is used as positive control, nothing
Bacterium distilled water is used as negative control.
Wherein, all primer and probes transfer to qualified genome company to synthesize；GspF enzymes and 10 × GspF
Buffer：Purchased from NEB companies；AMV：It is required that 65 DEG C of active reverse transcriptases；DNTP, RNA Secure and aseptic double-distilled water are equal
For ordinary commercial products.
Embodiment 3 detects transmissible gastro-enteritis virus with the kit of embodiment 2
Comprise the following steps：
(1) with column method Total RNA Mini Kits from sample to be detected (lymphonodi mesenterici, intestines and interior
Tolerant, excrement) middle extraction RNA；
(2) sample RNA is taken as template to be added in the PCR pipe equipped with reaction solution, in 63 DEG C of amplified reactions 60 minutes, its
Middle sample RNA is 1 with the ratio for redissolving buffer solution:4, sample addition is not less than 4 μ L；The positive is separately added into control PCR pipe
Contrast template and negative control template；
(3) PCR pipe after warm bath is placed into disposable nucleic acid detection apparatus and detected, interpretation knot after 15 minutes
Really.When containing transmissible gastro-enteritis virus nucleic acid in sample, take on a red color band in the detection line of test strips；Note no matter why
Sample, control line all should be red stripes；
(4) other two batches kit is taken to repeat experiment 2 times, altogether 3 clinical sample detection experiments, as a result such as table 1 below
It is shown.
Wherein, " +++ " represents strong positive, and "+" represents positive, and "-" represents negative.
The different lot number kit testing results of table 1
It can be seen that by embodiment 3：The kit testing result of 3 batches illustrates kit of the present invention without significant difference
Testing result between different batches has comparativity, with good repeatability.The detection method of the present invention is reproducible, and only
Need to complete within 2 hours, greatly shorten detection time, while only needing a people to complete operation, save manpower and detection
Cost.
The kit of the present invention of embodiment 4 detects the selectivity of transmissible gastro-enteritis virus
According to method detection Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (RV), the CSFV of embodiment 3
(CSFV), high pathogenic pig blue-ear disease malicious (HP-PRRSV), PRV (PRV), porcine circovirus 2 type (PCV2), the positive
Reference substance, testing result is as shown in table 2 below.
Wherein, " +++ " represents strong positive, and "+" represents positive, and "-" represents negative.
The kit of the present invention of table 2 detects the result of different virus
As shown in Table 2：Kit detection transmissible gastro-enteritis virus nucleic acid of the present invention has very strong selectivity.
Embodiment 5 detects the Sensitivity comparison with PCR assay methods using kit of the present invention
Comprise the following steps：
(1) magnificent strain (H strain) the cell toxicant physiological saline of weak TGEV will be caused to make 10 times of gradient dilutions successively, so
Amplification after nucleic acid, detection are extracted according to the method for embodiment 3 afterwards, to determine that kit of the present invention is used to detect pig transmissible stomach and intestine
The sensitivity of scorching viral nucleic acid.Amplified reaction terminates, and takes reaction tube in detection means, closes device, stands 15min observation knots
Really, positive findings is shown as at CT lines being red stripes, and negative findings shows at only C lines to be red stripes, with present invention examination
Agent box detection the minimum of transmissible gastro-enteritis virus can detect 74TCID50/mL。
By the TGEV positive control plasmids of preparation with 10 times of gradient dilutions are made successively with physiological saline, then according to embodiment
3 method is expanded to various concentrations plasmid, detected, to determine that kit of the present invention is used to detect transmissible gastroenteritis of swine disease
The test limit of malicious positive plasmid.Amplified reaction terminates, and takes reaction tube in detection means, closes device, stands 15min observation knots
Really, positive findings is shown as at CT lines being red stripes, and negative findings shows at only C lines to be red stripes, with present invention examination
Agent box detection the minimum of transmissible gastro-enteritis virus can detect 45copies/ μ L.
(2) RT-PCR method：
Amplification primers：The just primers F 3 and reversely peripheral primer B3 to the periphery expanded from CPA
RT~PCR reactions are 20 μ l systems:
Amplification program is：50℃30min
Amplified sample：For magnificent strain (H strain) the cell toxicant nucleic acid of the weak TGEV of the cause of above-mentioned different dilution factors and difference
Concentration positive plasmid.
RT-PCR products take 10 μ l in 40min under 2% agarose gel electrophoresis, 100V voltages, pass through gel imaging system
Observation, RT-PCR method is minimum can to detect 740TCID50/ mL (Fig. 1), 450copies/ μ L (Fig. 2).
The comparing result of the inventive method and PCR method is as shown in table 3 below.
Wherein, " +++ " represents strong positive, and "+" represents positive, and "-" represents negative.
Table 3 uses distinct methods testing result
It was found from the result of embodiment 5：The method sensitivity of the present invention can be examined apparently higher than the susceptibility of RT-PCR method
Measure the lower sample of transmissible gastro-enteritis virus content.
The kit reaction solution concentration of embodiment 6 gropes experiment
Comprise the following steps：
(1) transmissible gastro-enteritis virus nucleic acid constant-temperature amplification reaction solution is prepared according to the method for embodiment 2, according to reaction
The concentration difference of each composition is divided into tri- groups of A, B, C in liquid, and every group of reaction solution concentration is as shown in table 4 below.
The kit of the differential responses liquid concentration of table 4
| Group A | Group B | Group C | |
| Just primers F 3 to the periphery | 0.2μmol | 0.3μmol | 0.1μmol |
| Reverse periphery primer B3 | 0.1μmol | 0.2μmol | 0.1μmol |
| Cross primer CPR | 1μmol | 1μmol | 0.9μmol |
| Positive probe DF5F | 0.4μmol | 0.4μmol | 0.3μmol |
| Reverse probe DF5B | 0.8μmol | 0.8μmol | 0.7μmol |
| MgSO4 | 2mmol | 3mmol | 1mmol |
| DNTP solution | 0.4mmol | 0.5mmol | 0.3mmol |
| Betaine | 1mol | 1mol | 0.9mol |
| GspF enzyme 8U/ μ L | 10U | 10U | 9U |
| 10×GspF Buffer | 2μL | 2μL | 2μL |
| AMV | 0.2U | 0.2U | 0.1U |
| 25×RNA Secure | 0.8μL | 0.8μL | 0.8μL |
| Aseptic double-distilled water | Supply to 16 μ L | Supply to 16 μ L | Supply to 16 μ L |
(2) magnificent strain (H strain) the cell toxicant physiological saline of weak TGEV will be caused to make 10 times of gradient dilutions successively, pressed
Method according to embodiment 3 extracts nucleic acid, respectively with being detected after above-mentioned group of A, group B, group C system amplification, to determine examination of the present invention
Optimal system and the sensitivity of suboptimum system detection transmissible gastro-enteritis virus nucleic acid in agent box.Amplified reaction terminates,
Reaction tube is taken in detection means, device is closed, 15min observation results are stood, it is red that positive findings, which is shown as at CT lines,
Band, negative findings shows at only C lines to be red stripes, and detailed results see the table below 5.
Wherein, wherein, " +++ " represents strong positive, and "+" represents positive, and " +/- " expression weakly positive, "-" represents negative.
Table 5 uses the kit sensitivity analysis of different compositions
From the result of table 5, group A systems (the optimal system of kit i.e. of the present invention) detection transmissible gastro-enteritis virus
It is minimum to detect 74TCID50The minimum detectable limit of/mL, group B and group C system detection transmissible gastro-enteritis virus is compared with group
A is poor, is 740TCID50/mL。
From above-mentioned experiment, transmissible gastro-enteritis virus is detected using nucleic acid constant-temperature amplification the invention provides one kind
Kit, different reaction conditions is optimized, such as primer and probe concentration, Mg2+Concentration, reaction temperature etc. it is excellent
Change, and the present invention is combined with nucleic acid detection test strip detecting system, establish transmissible gastro-enteritis virus nucleic acid constant-temperature
The method for expanding qualitative detection.The sensitivity of the kit can detect 45copies/ μ L in each reaction system, can meet
The requirement of quick detection transmissible gastro-enteritis virus, has great importance.
SEQUENCE LISTING
<110>Sichuan, which is received, compares bio tech ltd;Yousida Biological Technology Co., Ltd., Hangzhou
<120>Transmissible gastro-enteritis virus detection kit and detection method
<130> A170587
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>The just nucleotide sequence of primers F 3 to the periphery
<400> 1
caatgggagc agtgccaag 19
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>Reverse periphery primer B3 nucleotide sequence
<400> 2
taatctgctg aaggaattgt tc 22
<210> 3
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223>Cross primer CPR nucleotide sequence
<400> 3
attggctgaa tgtgttcctg gcaagtggta tttgtgtgtg a 41
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Positive probe DF5F nucleotide sequence
<400> 4
attggctgaa tgtgttcc 18
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>Reverse probe DF5B nucleotide sequence
<400> 5
tctgtgtcta gcattttgtt tg 22
<210> 6
<211> 182
<212> DNA
<213> Artificial Sequence
<220>
<223>The nucleotide sequence of transmissible gastro-enteritis virus DNA
<400> 6
caatgggagc agtgccaagc attacccaca attggctgaa tgtgttccat ctgtgtctag 60
cattttgttt ggaagctatt ggacttcaaa ggaagatggc gaccagatag aagtcacgtt 120
cacacacaaa taccacttgc caaaggatga tcctaaaact gaacaattcc ttcagcagat 180
ta 182
Claims (10)
1. transmissible gastro-enteritis virus detection kit, it is characterised in that including following component：Detect transmissible gastroenteritis of swine
Just primers F 3, reversely peripheral primer B3, cross primer CPR, forward direction probe DF5F and the reverse probe DF5B to the periphery of virus.
2. transmissible gastro-enteritis virus detection kit according to claim 1, it is characterised in that meet it is following at least
One：
Described at least five that just nucleotides sequence of primers F 3 is classified as in nucleotide sequence shown in SEQ ID NO.1 to the periphery connects
Continuous nucleotides；
At least five that described reverse peripheral primer B3 nucleotides sequence is classified as in nucleotide sequence shown in SEQ ID NO.2 connects
Continuous nucleotides；
Described cross primer CPR nucleotides sequence is classified as at least five continuous kernel in nucleotide sequence shown in SEQ ID NO.3
Thuja acid；
At least five that described positive probe DF5F nucleotides sequence is classified as in nucleotide sequence shown in SEQ ID NO.4 is continuous
Nucleotides；
At least five that described reverse probe DF5B nucleotides sequence is classified as in nucleotide sequence shown in SEQ ID NO.5 is continuous
Nucleotides.
3. transmissible gastro-enteritis virus detection kit according to claim 1 or 2, it is characterised in that：It is described just
FAM groups are marked with to 5 ' ends of probe DF5F sequences, 5 ' ends of the reverse probe DF5B sequences are marked with Biotin groups.
4. the transmissible gastro-enteritis virus detection kit according to any one of claims 1 to 3, it is characterised in that：Institute
Stating nucleic acid constant-temperature amplification reaction solution composition includes：Just to the periphery 3 0.1~0.6 μm of ol of primers F in reaction solution in every 16 μ L, reversely
Peripheral 0.1~0.6 μm of ol of primer B3, cross primer CPR 0.4~1.6 μm of ol, positive 0.2~1 μm of ol of probe DF5F, reversely
Probe DF5B 0.2~1 μm of ol, MgSO4 0~6mmol, dNTP solution 0~1.5mol of 0.1~0.6mmol, Betaine,
2 μ L, AMV 0.02~0.5U, 25 × RNA Secure of GspF enzyme 8U/ μ L 4~12U, 10 × GspF Buffer 0.8 μ L, it is remaining
Measure as distilled water.
5. transmissible gastro-enteritis virus detection kit according to claim 4, it is characterised in that：The nucleic acid constant-temperature
Amplification reaction solution composition includes：Just to the periphery 3 0.1~0.3 μm of ol of primers F in reaction solution in every 16 μ L, reverse periphery primer B3
0.1~0.2 μm of ol, cross primer CPR 0.9~1.0 μm of ol, positive 0.3~0.4 μm of ol of probe DF5F, reverse probe DF5B
0.7~0.8 μm of ol, MgSO4 1~3mmol, dNTP solution 0.3~0.5mmol, Betaine 0.9~1.0mol, GspF enzyme
The μ L of 2 μ L, AMV 0.1~0.2U, 25 × RNA Secure of 8U/ μ L 9~10U, 10 × GspF Buffer 0.8, surplus is double
Steam water.
6. transmissible gastro-enteritis virus detection kit according to claim 5, it is characterised in that：The nucleic acid constant-temperature
Amplification reaction solution composition includes：Just to the periphery 3 0.26 μm of ol of primers F in reaction solution in every 16 μ L, reverse periphery primer B3 0.1
μm ol, cross primer CPR 1.0 μm of ol, positive 0.4 μm of ol of probe DF5F, reverse probe DF5B 0.8 μm of ol, MgSO4
2mmol, dNTP solution 0.4mmol, Betaine 1.0mol, GspF enzyme 8U/ μ L 10U, 10 × GspF Buffer 2 μ L, AMV
The μ L of 0.2U, 25 × RNA Secure 0.8, surplus is distilled water.
7. transmissible gastro-enteritis virus detection kit according to claim 6, it is characterised in that：Described 10 ×
GspF Buffer contain molar concentration for 500mM Tris-HCl (8.5,25 DEG C of pH), 300mM KCl, 300mM (NH4)SO4With
1%Triton X-100.
8. the method that usage right requires the transmissible gastro-enteritis virus detection kit detection virus described in 1~7 any one,
It is characterised in that it includes following steps：
A, with RNA extracts kits extract RNA from sample to be detected；
B, the RNA of extraction mixed with transmissible gastro-enteritis virus detection kit, at 58~65 DEG C amplified reaction 30~
With compareing contrast sentence read result after 90min, 15min.
9. the method for transmissible gastro-enteritis virus detection kit detection virus according to claim 8, its feature exists
In：Sample to be detected described in step a is including in pig lymphonodi mesenterici, jejunum, small intestine or intestinal contents, excrement or lung
One kind.
10. the method for transmissible gastro-enteritis virus detection kit detection virus according to claim 8 or claim 9, it is special
Levy and be：Step b is amplified reaction 60 minutes at 63 DEG C.
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| CN201710675424.1A CN107236827B (en) | 2017-08-09 | 2017-08-09 | Kit and method for detecting transmissible gastroenteritis virus of swine |
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| CN110438249A (en) * | 2019-04-29 | 2019-11-12 | 浙江省疾病预防控制中心 | A kind of gondii nucleic acid constant-temperature amplification detection kit and application method |
| CN112725530A (en) * | 2021-01-19 | 2021-04-30 | 中国计量大学 | Cross primer amplification primer group for detecting bean pod mottle virus, kit and application |
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| CN107236827B (en) | 2020-11-20 |
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Effective date of registration: 20220118 Address after: No.204, Jinchuan Road, Jintang County, Chengdu, Sichuan 610400 Patentee after: CHENGDU NABII BIOTECHNOLOGY CO.,LTD. Patentee after: Hangzhou yousida Biotechnology Co., Ltd Address before: 620800 Sichuan Nabi Biotechnology Co., Ltd., Guanyin District, Pengshan County, Meishan City, Sichuan Province Patentee before: SICHUAN NABII BIOTECHNOLOGY CO.,LTD. Patentee before: Hangzhou yousida Biotechnology Co., Ltd |