CN106191313A - A kind of six kinds of pig breeding dysfunction virus liquid phase chip agent boxes - Google Patents
A kind of six kinds of pig breeding dysfunction virus liquid phase chip agent boxes Download PDFInfo
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- CN106191313A CN106191313A CN201610578516.3A CN201610578516A CN106191313A CN 106191313 A CN106191313 A CN 106191313A CN 201610578516 A CN201610578516 A CN 201610578516A CN 106191313 A CN106191313 A CN 106191313A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Abstract
The invention provides a kind of six kinds of pig breeding dysfunction virus liquid phase chip agent boxes, it contains specific forward primer probe, specific forward primer probe sequence as shown in SEQ ID NO:1 ~ 6；Sequence possibly together with specific downstream primer probe and specific downstream primer probe is shown in SEQ ID NO:7 ~ 12, and the 5 ' ends at any one of specific Down Stream primed probe add one section of biotin；Possibly together with specificity microsphere, the sequence of specificity microsphere is as shown in SEQ ID NO:13 ~ 18.Present invention achieves and 6 kinds of pig breeding dysfunction viruses can be carried out liquid-phase chip detection in a reaction system simultaneously, there is high speed, high flux, high specific and accuracy high.
Description
Technical field
The invention belongs to field of biological genes, relate to a kind of liquid phase chip reagent box, a kind of six boars are numerous
Grow obstacle virus liquid phase chip reagent box.
Background technology
Boar is the source of whole pig farm production line, and the health status of boar has been largely fixed whole pig farm swinery
Health status, for the needs of pig breed, since the nineties in 20th century, boar circulation volume of trade is increased sharply, and we send out from raising pigs
While reaching country's excellent boar gene of introduction and advanced yard feeding pattern, the pressure of control and prevention of disease is the most increasing.In recent years, I
The sow of state's many large-scale pig farms is affected by Reproduction Disorder the most to some extent, causes destructiveness to pig industry
Strike.Pig breeding dysfunction disease is to miscarry with in-pig, produces stillborn fetus, mummy tire, the weak son of debility, deformity
Tire, few young and sterile disease for principal character, the cause of disease is more complicated, can be divided into congenital, functional, trophism, mechanicalness
With disease, wherein with infectious disease especially viral disease harm maximum, often in large-area endemicity and popularity
Infect, cause the miscarriage of substantial amounts of in-pig, stillborn fetus and newborn piglet dead.Owing to the cause of disease causing breeding difficulty is complicated, permitted
Many diseases Symptoms clinically is inconspicuous, and even a lot of disease symptomses are similar and are difficult to differentiate, the control causing this disease is difficult
Spend the biggest.
The pathogenesis causing Reproduction Disorder has a lot, and virus infection is Etiological, mainly includes swine fever
Virus (CSFV), Pseudorabies virus (PRV), reproductive and respiratory syndrome virus (PRRSV), circovurus type 2 (PCV-2), parvovirus
(PPV), encephalitis b virus (JEV) etc..At present, to the detection of pig breeding dysfunction disease mainly with nosetiology, serology and point
Sub-biology is main, although the separation and Culture of virus is the goldstandard of etiological diagnosis, but owing to its cycle is longer, operation is complicated
It is restricted when detection uses；Although serology and molecular biology have the features such as rapid sensitive, but in the high pass of cause of disease
Amount context of detection Shortcomings.Therefore, actual production and Clinical detection just require to have high flux, response speed fast, quick
Perception is high, reproducible, detection method that accuracy is high, liquid-phase chip technology conform exactly to above some.
Liquid-phase chip technology, as a kind of high-throughout detection technique, is that the mid-90 in 20th century is by U.S. Luminex
Development of company gets up.This technology collecting type cell technology, fluorescence-encoded micro-beads, laser, Digital Signal Processing and traditional life
Change technology, in one, is a kind of multi-functional analysis platform, this technology platform be can guarantee information quality, be provided that again relatively
High-throughout a new generation molecular detection technology platform.In recent years, the most liquid-phase chip technology is used for key biological molecule,
As antigen, antibody, cytokine, the expression of phosphorylated protein or level of activation change, DNA sequencing, snp analysis, gene mutation
Study with expression, screening anticancer medicine etc., and progressively for clinical diagnosis.Developed tumor markers, anaphylactogen examination,
Autoimmune disease, cytokine etc. diagnosis microsphere, and calendar year 2001 be approved by the fda in the United States for clinic carry out autoimmune disease,
The diagnosis of human leucocyte antigen typing, this is also the biochip being uniquely approved by the fda in the United States for clinical diagnosis, opening of it
Send out and application is considered as the impressive progress that biochip field is developed.It can realize carrying out multi objective determining simultaneously
Property, quantitative analysis.There is high flux, highly sensitive, reproducible, the feature of saving sample.
Pig breeding dysfunction sexually transmitted disease (STD) poison is that a class is widely present and endangers serious virus, rarely has the most both at home and abroad and uses liquid phase core
The report that this viroid is detected and identifies by sheet method.
Summary of the invention
For above-mentioned technical problem of the prior art, the invention provides a kind of six kinds of pig breeding dysfunction virus liquid phase cores
Sheet test kit, described this six kinds of pig breeding dysfunction virus liquid phase chip agent boxes solve and detect six boars in prior art
The method workload of breeding difficulty virus is big, cost is high, and time-consumingly long technical problem.
The invention provides a kind of six kinds of pig breeding dysfunction virus liquid phase chip agent boxes, it contains:
1) specific forward primer probe, the sequence such as SEQ ID NO:1 of described specific forward primer probe
～shown in 6；
2) specific downstream primer probe, the sequence of described specific downstream primer probe is SEQ ID NO:7
～shown in 12, the 5 ' ends at any one of specific Down Stream primed probe add one section of biotin；
3) specificity microsphere, the sequence of described specificity microsphere is as shown in SEQ ID NO:13～18.
Further, any one specific upstream primed probe has one section of ATG label complementary mutually with microsphere also
With 12 C separately.
Further, in the sequence of any one of specificity microsphere and corresponding specific upstream primed probe
TAG label is mutually complementary.
Further, hybridization night, Streptavidin (SAPE) are also comprised.
Further, multiplex PCR Mix 1, multiplex PCR Mix 2 and a kind of six kinds of pig breeding dysfunction virus liquids are also comprised
Phase chip agent box operation instructions.
Operation principle of the present invention is: collect PPV Viral structural protein VP2 gene in GenBank, the ORF2 opening of PCV is read
Frame sequence, the gB gene of PRV, the NSP2 gene of PRRSV, the raq gene sequence of CSFV and the M gene of JEV, utilize
The primer5.0 equimolecular biological software sequence analysis and comparison to collecting, filters out the special conserved sequence of said gene also
Design primer and probe, add the preceding paragraph TAG sequence at specific upstream primer 5 ' end, and centre is believed as termination with 12 carbon bridges
Number, at specificity downstream primer 5 ' end plus biotin (Biotin)；Select with its TAG label mutually complementation microsphere (with
Anti-TAG sequence)；Remove to expand sample to be checked with specific probe primer, then take the PCR primer expanded and hybridize with microsphere；
Upper machine, the specific probe carrying microsphere passes through flow cytometer under the power that sheath fluid flows, and machine sends twice simultaneously
Laser, red laser by distinguishing the numbering of microsphere to the identification of coding microball, and green laser is used for identifying downstream primer 5 '
The biotin of link is quantitative as reporter gene, finally draws a MFI value (using 300 as threshold value).
The present invention is a kind of high-throughout detection technique for six kinds of pig breeding dysfunction virus liquid phase chip agent boxes, collects
Low cytometric analysis, fluorescence-encoded micro-beads, laser, Digital Signal Processing and traditional biochemical technology, in one, are that one is many
The analysis platform of function.Liquid-phase chip technology accuracy and specificity are high, are 100% with the coincidence rate of regular-PCR, liquid phase simultaneously
The chip technology detection cycle is short, and this test kit can complete the screening to six kinds of pig breeding dysfunction viruses in 3-4 hour；With commonly
PCR detection is compared, and it is short that this test kit not only detects the time, and avoids the EB harm to people, highly sensitive, and flux is big, minimum
Detectable concentration can reach 102Copies/ μ L, can complete the detection of 6 kinds of pig breeding dysfunction viruses simultaneously.
This test kit uses multiple PCR products to be combined with specificity microsphere as probe, improves the specificity of detection, logical
Overflow-type cell instrument and laser scanner technique, finally provide a MFI value, qualitative can not only can also carry out sample relatively
Quantitatively.Use this test kit that detection, Epidemiological study and the risk assessment of clinical six kinds of pig breeding dysfunctions virus are had weight
The using value wanted.
This research and utilization Luminex distinctive xTAG technology, foundation can carry out level mirror to six kinds of pig breeding dysfunction viruses
Fixed liquid-phase chip technology, the detection for pig breeding dysfunction virus provides a kind of high-throughout new method with identifying, in early days
Quickly determine pathogen.For clinical economics, Epidemiological study all by significant.
The present invention compares with prior art, and its technological progress is significant.The test kit using the present invention carries out pig six kinds
During the detection of pig breeding dysfunction virus liquid-phase chip technology, there is the advantages such as quick, high flux, sensitivity height, high specificity, greatly
Shorten experimental period, opened up the application prospect of this test kit.
Accompanying drawing explanation
Fig. 1 is the schematic diagram that liquid-phase chip instrument reads data.
Fig. 2 is to expand pig parvoviral, porcine circovirus 2 type and PRV (Pseudorabies virus) with the triple PCR of this experimental design
The result increased.
Fig. 3 is the result that swine fever virus, pig blue-ear disease and encephalitis b virus are expanded by the triple PCR of this experimental design.
Fig. 4 is to utilize traditional PCR method to pig parvoviral, porcine circovirus 2 type and PRV (Pseudorabies virus) 8 dilution ladder
The amplification of degree, display minimal detectable concentration is 500pg/ μ L.
Fig. 5 is the checking liquid-phase chip minimal detectable concentration to porcine circovirus 2 type, when detectable concentration is diluted to
During 102copies/ μ L, the MFI that liquid-phase chip shows is 528.
Fig. 6 is specific probe and the schematic diagram of microsphere coupling and principle.
Fig. 7 is when microsphere passes through flow cytometer under the promotion of flowing sheath fluid, is taken microsphere surface by two bundle laser
The information of band is scanned and reads.
Fig. 8 be specific PCR amplified production under suitable hybridization temperature, carry out coupling with specificity microsphere.
Fig. 9 is to go checking to be clinically separated known AIV, NDV, TGEV, SIV, PEDV viral nucleic acid sample with this test kit
(our unit's preservation), the genomic DNA (extract RNA and be converted into cDNA) of sample expands as PCR amplification template PCR system
Detect in the liquid-phase chip detection system set up after increasing.Result display MFI value is below 300.
Figure 10 is the partial results using this test kit that 388 parts of clinical samples carry out liquid-phase chip detection.
Detailed description of the invention
The following example is further intended to describe invention rather than limit invention by any way, without departing substantially from the present invention
Spirit and principle under, any change that ability made for the present invention or those of ordinary skill easily realize falls within this
Within the bright interest field that awaits the reply.
Embodiment 1 test kit design principle
The present invention, first according to the difference of gene, collects PPV Viral structural protein VP2 gene, the ORF2 of PCV in GenBank
Open reading frame sequence, the gB gene of PRV, the NSP2 gene of PRRSV, the raq gene sequence of CSFV and the M gene of JEV, profit
With the primer5.0 equimolecular biological software sequence analysis and comparison to collecting, filter out the special conserved sequence of said gene
And design primer SEQ ID NO:1～12；Ensureing that six boar breedings are hindered by primer specificity and the premise without dimer impact
Hindering virus to divide into two groups of multiplex PCR systems (being preserved strain by this laboratory, refer to table 1), first group is PPV, PCV2With
PRV, is shown in Fig. 2；Second group is CSFV, PRRS and JEV, sees Fig. 3；The preceding paragraph TAG sequence is added at specific upstream primer 5 ' end, in
Between with 12 carbon bridges as termination signal, at specificity downstream primer 5 ' end plus biological (Biotin)；Select to mark with its TAG
Sign microsphere (with Anti-TAG sequence, SEQ ID 13～18) complementary mutually to go to expand sample to be checked with specific probe primer,
Then take the PCR primer after amplification and hybridize (see Fig. 8) with microsphere；Mixed liquor after hybridization is put in liquid-phase chip instrument and reads
Number, the specific probe carrying microsphere passes through flow cytometer under the power that sheath fluid flows, and machine sends twice laser simultaneously,
Red laser by distinguishing the numbering of microsphere to the identification of coding microball, and green laser is used for identifying what downstream primer 5 ' linked
Biotin as reporter gene quantitatively (see Fig. 1), finally draws a MFI value (using 300 as threshold value).
1 test kit of table sets up the strain information that multiple PCR method is used
Embodiment 2 test kit forms
Multiplex PCR reagent: PCR system 25 μ L
(comprising multiplex PCR Mix 12.5 μ L, each 0.5 μ L of primer, ddH2O of PPV, PCV-2 and PRV mends to 25 μ in combination 1
L) (such as SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:3, SEQ ID NO:9
Shown in), combination 2 (comprise multiplex PCR Mix 12.5 μ L, each 0.5 μ L of primer of CSFV, PRRSV and JEV (such as SEQ ID NO:
4, shown in SEQ ID NO:10, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:6, SEQ ID NO:12), reverse transcription
Enzyme 1 μ L, ddH2O mend to 25 μ L)
Liquid-phase chip reagent: reaction system 90 μ L, the reaction microsphere 1 μ L, 1 × Tm that wherein comprise each coding mend to 20 μ L
(as shown in SEQ ID NO:13～18), 70 μ LSAPE diluents (69.3 μ L 1 × Tm and 0.7 μ LSAPE).
Embodiment 3 test kit using method
One, the extraction of nucleic acid and multiplex PCR
To single six kinds of virocyte cultures of PPV, PCV2, PRV, CSFV, PRRSV and JEV, PPV, PCV2, PRV,
CSFV, PRRSV and JEV six kinds of virus mixture (isopyknic PPV, PCV2, PRV, CSFV, PRRSV and JEV cell culture
Mixing), and process clinical sample supernatant in unknown virus nucleic acid use MagPure Viral RNA Kit for
MagMax ExPRVess nucleic acid extraction kit (Mei Ji company) extracts nucleic acid, after nucleic acid extraction completes, micro-with Nano-drop
Amount spectrophotometer carries out concentration mensuration to it.Sample adds 5 μ L Proteinase K Solution, 5 μ L magnetic beads, 90 μ L in 96 orifice plate A holes
Lysate and 50 μ L measuring samples or comparisons；Adding 150 μ L washing liquids in the B of hole, in C and D of hole, each 150 μ L that add remove saline solution,
Hole H adds 50 μ L sterilizing deionized waters；96 orifice plates and eluting bar installing sample and reagent are put in instrument, start journey
Sequence；After EP (end of program), take out eluting bar, stick sealed membrane, be directly used in detection, or put-70 DEG C of preservations.
Reaction system is 25 μ L, 23 μ L multiplex PCR reagent (combination 1,2) and 2 μ L nucleic acid to be checked.
Operating procedure: after being diluted according to above-mentioned requirements by sample nucleic acid to be checked, be separately added into 2 combinations in test kit many
In weight PCR.
Combine 1 reaction condition: 94 DEG C of denaturations 5min；94 DEG C of 30S, 56 DEG C of 30S, 72 DEG C of 45S, circulate 30 times；72℃
10min。
Combine 2 reaction conditions: 94 DEG C of denaturations 5min；94 DEG C of 30S, 52 DEG C of 30S, 72 DEG C of 45S, circulate 30 times；72℃
10min。
Two, hybridization
The most each coding microball participates in reaction (i.e. 1 μ L) with 2500 microspheres, complements to 20 μ L with 1 × Tm；
2. add the product after 2 μ L above-mentioned PCR amplifications, be adjusted to final volume 25 μ L with ddH2O；
3. with 1 × Tm hybridization buffer SAPE is diluted to 8 μ g/ml, and adds 70 μ L to each reacting hole, softly mix
Even；
4. put into PCR instrument, use 40 DEG C of hybridization 30min (see Fig. 8).
Three, liquid-phase chip instrument reads result
First machine should be carried out before upper machine, and laser is calibrated and verifies；Take liquid-phase chip reagent 90 μ L, add
Enter hybridization solution n μ L (2≤n≤5) to be checked, mend to final volume 95 μ L with ddH2O；Liquid-phase chip instrument is every to 100 by 2 bundle laser
Plant coding microball and carry out laser scanning surface (see Fig. 7), read 100 microsphere surface fluorescence values and then take median, finally obtain MFI
Value (with 300 as threshold value, be positive more than 300, be judged to feminine gender less than 300).
Embodiment 4 test kit specific test
Extract known AIV, NDV, TGEV, SIV, PEDV, PCV2Viral nucleic acid sample (corresponding sample 1-6, diagnostic center
Preserve), after genomic DNA (extract RNA and be converted into cDNA) the 2 μ L of sample expand as PCR amplification template PCR system
Detect in the liquid-phase chip detection system set up, set negative and positive comparison simultaneously
Hybridization condition: 40 DEG C, 30min；
Liquid-phase chip reaction condition: the copper coin in liquid-phase chip instrument is heated to 40 DEG C in advance, the premixed liquid hybridized should
Put rapidly to liquid-phase chip instrument, click on " Run " and run.
Liquid-phase chip instrument result shows only PCV2Have MFI value (2055), for positive findings, and control group A IV, NDV,
The MFI value of TGEV, SIV, PEDV is below 300, for negative findings (see Fig. 9).
Result: high specificity
Embodiment 5 test kit sensitivity tests
The DNA of extraction porcine circovirus 2 type, and carry out the mensuration of DNA concentration, its DNA concentration is 70.4ng/ μ L.To carry
The DNA taken carries out 10 times of dilutions, altogether 7 titres of dilution, its concentration be respectively 7.04ng/ μ L, 704pg/ μ L, 70.4pg/ μ L,
7.04pg/ μ L, 704fg/ μ L, 70.4fg/ μ L, 7.04fg/ μ L multiplexed PCR amplification hybridizes with corresponding microsphere, then after completing
Reading data by liquid-phase chip instrument, result shows that MFI value is less than threshold value 300 as DNA concentration 704fg/ μ L, so showing examination
Agent box is 7.04pg/ μ L (result is shown in Fig. 5) to porcine circovirus 2 type nucleic acid minimal detectable concentration.By above-mentioned 8 dilution gradients
DNA goes to run PCR and carries out sensitivity contrast, and result shows do not have band when DNA is 7.04pg/ μ L (the 7th hole), refers to Fig. 4.
Result: sensitivity is up to 7.04pg/ μ L
The detection of embodiment 6 Real-time fluorescent PCR detects with liquid-phase chip
With reference to the method for embodiment 3, to from 2 pig farms of Shanghai International Automobile City Tourist Festival, 6 pig farms of Pudong and Shanghai certain pig farm bright is altogether
Count 388 parts of 9 pig farms sow cord blood sample and carry out detection and chip method inspection with Real-time fluorescence PCR method
Survey (as shown in Figure 10).
Result: Real-time fluorescent PCR result of the test and the result repetitive rate 2325/2328=of liquid-phase chip detection
99.87%.Refer to table 2.
The detailed table of table 2 clinical sample testing result
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the model that the application appended claims is limited equally
Enclose.
Claims (5)
1. one kind six kinds pig breeding dysfunction virus liquid phase chip agent box chip agent boxes, it is characterised in that it contains:
1) specific forward primer probe, sequence such as SEQ ID NO:1 ~ 6 institute of described specific forward primer probe
Show；
2) specific downstream primer probe, the sequence of described specific downstream primer probe is SEQ ID NO:7 ~ 12 institutes
Showing, the 5 ' ends at any one of specific Down Stream primed probe add one section of biotin；
3) specificity microsphere, the sequence of described specificity microsphere is as shown in SEQ ID NO:13 ~ 18.
A kind of six kinds of pig breeding dysfunction virus liquid phase chip agent boxes the most according to claim 1, it is characterised in that: arbitrarily
One specific upstream primed probe has one section of ATG label complementary mutually with microsphere with 12 C separately.
A kind of six kinds of pig breeding dysfunction virus liquid phase chip agent boxes the most according to claim 1, it is characterised in that: arbitrarily
The sequence of one described specificity microsphere is mutually complementary with TAG label in corresponding specific upstream primed probe.
A kind of six kinds of pig breeding dysfunction virus liquid phase chip agent boxes the most according to claim 1, it is characterised in that: comprise
1 × Tm hybridizes night, Streptavidin.
A kind of six kinds of pig breeding dysfunction virus liquid phase chip agent boxes the most according to claim 1, it is characterised in that: comprise
Multiplex PCR Mix 1, multiplex PCR Mix 2, six kinds of pig breeding dysfunction virus liquid phase chip agent box operation instructions.
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| CN113684309A (en) * | 2021-07-06 | 2021-11-23 | 浙江省动物疫病预防控制中心 | 7 primer probe and kit for detecting viruses related to porcine reproductive disorder diseases based on liquid chip technology and application of primer probe and kit |
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| CN113684309A (en) * | 2021-07-06 | 2021-11-23 | 浙江省动物疫病预防控制中心 | 7 primer probe and kit for detecting viruses related to porcine reproductive disorder diseases based on liquid chip technology and application of primer probe and kit |
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