CN106636474B - Six kinds of viral primer sets of Multiple immunizations fluoroscopic examination mouse, kit and method - Google Patents

Six kinds of viral primer sets of Multiple immunizations fluoroscopic examination mouse, kit and method Download PDF

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CN106636474B
CN106636474B CN201710044633.6A CN201710044633A CN106636474B CN 106636474 B CN106636474 B CN 106636474B CN 201710044633 A CN201710044633 A CN 201710044633A CN 106636474 B CN106636474 B CN 106636474B
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袁文
张钰
郭鹏举
黄韧
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Guangdong Laboratory Animals Monitoring Institute
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Abstract

The invention discloses six kinds of viral primer sets of Multiple immunizations fluoroscopic examination mouse, kit and methods.Multiple RT round pcrs and Luminex liquid-phase chip technologies are combined by the present invention, mouse TMEV, MHV, MNV, Reo 3, the primer sets of this six kinds high infection rate virus of MVM, MPV can be detected simultaneously by devising, target amplification product is obtained by specific PCR using the primer sets, then amplified production, fluorescence-encoded micro-beads and Streptavidin phycoerythrin are hybridized, when reading MFI values by Luminex detectors, so as to differentiate different types of virus.This method have rapidly and efficiently, high specificity, high sensitivity, the advantageous effects such as reproducible, can be applied to quality-monitoring, epidemiology survey and the early warning of experiment mice.Flexibility of the present invention is good, can add and subtract the species of detection virus on this basis as needed.

Description

Six kinds of viral primer sets of Multiple immunizations fluoroscopic examination mouse, kit and method
Technical field
The invention belongs to field of biological detection, draw more particularly, to six kinds of Multiple immunizations fluoroscopic examination mouse is viral Object group, kit and method.
Background technology
The detection of experimental animal microbial quality is the important indicator of evaluation experimental animal quality, and microorganism infection is not only to reality It tests animal to damage in itself, potential interference is also resulted in research work.In experimental animal infection disease, viral disease accounts for There is important position, it was found that the infection of experiment mice virus is mostly in recessive sense in the monitoring of daily Bioexperiment animal pathogenic Dye, without apparent clinical symptoms, disease diagnosis is relatively difficult；And zoogenetic infection virus often shows as persistent infection, Once facility is in the presence of virus infection than more difficult to remove, it is necessary to could be from by detecting and eliminating (test-and-removal) method Contaminated facility thoroughly eradicates virus, therefore, establishes experimental animal method for detecting virus rapidly and efficiently, timely and accurately sends out Existing pathogen infection, control and removing epidemic situation, it is extremely important for improving Quality of Experimental Animals.
Experiment mice virus up to ten is several, SPF specified in China experimental animal standard GB/T 14922.2-2011 Grade mouse need to exclude 11 kinds of viruses.Mouse encephalomyelitis virus (Theiler ' s murine in the present invention Encephalomyelitis virus, TMEV), mouse hepatitis virus (Murine hepatitis virus, MHV), mouse exhale MVM plants of the lonely viral type III (Reovirus Type 3, Reo-3) of intestines and minute parvovirus of mice (Minute virus of mice, MVM it is) that national standard needs the cause of disease excluded.Mouse norovirus (Murine Norovirus, MNV) and minute parvovirus of mice MPV plants (Mouse parvovirus, MPV) are the virus of newfound infection experiment mouse, both viruses are external experiments One essential items for inspection of animal health monitoring, although MNV and MPV are not yet included in detection in China's experimental animal national standard Mesh, but some experimental animals produce or all it are classified as examination project using unit and some CRO companies.TMEV、MHV、 MNV, MVM, MPV are the virus that infection rate is higher in current experiment mice, the report experiment mice moderate resistance such as Wang Cui pretty young woman in 2014 Body positive rate be respectively 15.1% (n=325), 19.9% (n=971), 42.2% (n=329), 12.0% (n=326) and 1.3% (n=390).
At present, domestic Diagnosis of Viral Infections method is primarily directed to the Serology test such as enzyme linked immunological of antibody test Adsorption test (ELISA), immuno-enzymatic test (IEA) and immunofluorescent test (IFA) etc., but may not apply in these methods The detection of immunologic hypofunction or immunodeficient mouse such as SCID mice and nude mouse etc. because they cannot generate it is normal anti- Precursor reactant.Antigen context of detection conventional viral isolation and identification method was not only complicated but also cumbersome, was unfavorable for routine testing.Traditional detection Technology has been unable to meet Quality of Experimental Animals detection demand, the molecular biological testing detection virus tool based on PCR There is the features such as quick, sensitive, special, be the common detection method of current clinically detect and diagnose disease.But regular-PCR method It needs to uncap and gel electrophoresis analysis is carried out to product, operating method not enough simplifies, and easily generates Aerosol Pollution, causes Existing false positive.Real-Time Fluorescent Quantitative PCR Technique is because of pinpoint accuracy, and high sensitivity, it is that current virus infection is examined to pollute the advantages that few Disconnected important means.But real-time fluorescence PCR technology is limited be subject to fluorescent species and instrument itself, at most can only be to 5 targets It is detected, and the difficulty of Success in Experiment is very big, cost is also relatively high.
Luminex liquid-phase chip technologies are a kind of new multi-fluorescence immunoassay sides risen the 1990s Method, the technology is organically whole and fluorescence-encoded micro-beads technologies, laser analysis technology, Flow Cytometry, high-speed digital signal The multinomial technology such as treatment technology, computer algorithm.Compared with traditional clinical testing procedure, major advantage is can be with Independent assortment, high throughput, high sensitivity, reproducible, detection range is wide, reaction speed is fast, inexpensive, easy to operate, can Detection albumen can detect nucleic acid etc. again.The present invention using Luminex liquid-phase chip technologies establish it is a kind of detect simultaneously mouse TMEV, The detection method of the virus of this six kinds of high infection rates of MHV, MNV, Reo-3, MVM, MPV, the technology can be applied to experiment mice Quality-monitoring, epidemiology survey and early warning.
The content of the invention
It is an object of the invention to provide Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV diseases Primer, kit and the method for poison.
The technical solution used in the present invention is：
For the primer sets of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, including 7 To primer, 7 pairs of primers are by 7 pairs of original primers modify；It is described to be modified in each pair original primers Arm connects between 5 ' ends of one original primers pass through with 3 ' ends of corresponding tag sequences, 5 ' end additions of another original primers Biotin labeling；
The original primers are to as follows：
The original sense primers of TMEV：5 '-GATCTAAGTYAACCGACTCC-3 ',
The original anti-sense primers of TMEV：5 '-GAAGGAAGGGGCAACACATA-3 ',
The original sense primers of MHV：5 '-TGGMATCCTCAAGAAGACCACTT-3 ',
The original anti-sense primers of MHV：5 '-ATGCCMGAAAACCARGAGTAATG-3 ',
The original sense primers of MNV：5 '-TCTGTYCTGCGCTGGGTGC-3 ',
The original anti-sense primers of MNV：5 '-GCTGCGCCATCACTCATCC-3 ',
The original sense primers of Reo-3：5 '-CCTCGCCTACGTGAAGAAGT-3 ',
The original anti-sense primers of Reo-3：5 '-CATCATCGAGTCCCTGGGTG-3 ',
The original sense primers of MVM：5 '-TTGTTCCACCACCACTAAATG-3 ',
The original anti-sense primers of MVM：5 '-GGTTTGTGTTCAAGATCTAGTTC-3 ',
The original sense primers of MPV：5 '-CACCAGCAACAGACAACCAA-3 ',
The original anti-sense primers of MPV：5 '-TGAATGCGTTGAGTGTTCTC-3 ',
The original sense primers of internal standard MS2：5 '-CGCAGAATCGCAAATACAC-3 ',
The original anti-sense primers of internal standard MS2：5’-TAACAATAAGCTCGCAGTCG-3’；
The tag sequences are as follows：
The tag sequences of TMEV are：5 '-TTAAACAATCTACTATTCAATCAC-3 ',
The tag sequences of MHV are：5 '-CACTTAATTCATTCTAAATCTATC-3 ',
The tag sequences of MNV are：5 '-TACTTCTTTACTACAATTTACAAC-3 ',
The tag sequences of Reo3 are：5 '-CACTACACATTTATCATAACAAAT-3 ',
The tag sequences of MVM are：5 '-TACTACTTCTATAACTCACTTAAA-3 ',
The tag sequences of MPV are：5 '-ACTTATTTCTTCACTACTATATCA-3 ',
The tag sequences of internal standard MS2 are：5’-ATTAAACAACTCTTAACTACACAA-3’.
Further, arm is trim among the primer of 12~18 atoms between described.
Preferably, arm arm between six ethylene glycol between described.
For the kit of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, feature It is：The kit contains above-mentioned for Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus Primer sets, the fluorescence-encoded micro-beads of 7 kinds of different iridescent of coding, the independent fluorescence coding microball contains and above-mentioned primer The anti-tag sequences of middle tag sequences complementary pairing.
Further, mentioned reagent box also contains streptavidin-phycoerythrin compound, RT-PCR amplifing reagents, interior MS2 standard items, mixing positive control, negative control are marked, the mixing positive control is to contain TMEV, MHV, MNV, Reo- simultaneously 3rd, six kinds of viral nucleic acid samples of MVM, MPV；The negative control is not contain TMEV, MHV, MNV, Reo-3, MVM, MPV six The nucleic acid samples of kind virus.
A kind of method of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, including as follows Step：
1) viral RNA or DNA are extracted from sample, as template, while blank control, negative control, mixing sun are set Property control, the blank control is does not add template；The mixing positive control is to contain TMEV, MHV, MNV, Reo- simultaneously 3rd, six kinds of viral nucleic acid samples of MVM, MPV are template；The negative control be with do not contain TMEV, MHV, MNV, Reo-3, Six kinds of viral nucleic acid samples of MVM, MPV are template；
2) nucleic acid of internal standard MS2 is added in the sample as Quality Control, and RT-PCR is carried out with the primer sets in above-mentioned kit Amplification；
3) it is fluorescence-encoded micro-beads, the strepto- of the different iridescent of 7 kinds of codings in amplified production, mentioned reagent box is affine Element-phycoerythrin is hybridized；
4) after hybridizing, analysis is detected hybrid product by Luminex systems, reads the MFI of different virus Value；
5) result judges：When the MFI values >=3.0 of MFI values/blank control of sample, the positive is judged to, is otherwise judged to the moon Property；
The above method is used for the diagnose and treat of non-disease.
Further, in step 2), 20 μ L reaction systems of RT-PCR amplifications contain：5×buffer4μL、dNTP0.8μ L, Enzyme mix0.8 μ L, each 0.4 μ L of each pair primer mixed liquor (10 μM), 4 μ L of template in primer sets, without 7.6 μ L of RNase water.
Further, in step 2), the response procedures of RT-PCR amplifications are：48~52 DEG C of 25~35min of reverse transcription；95℃ Pre-degeneration 15min；94 DEG C of denaturation 30s, 60 DEG C of annealing 90s, 72 DEG C of extension 30s；Xun Huan 35 times；72 DEG C extend 10min eventually.
Further, in step 3), 100 μ L reaction systems of hybridization contain：7 kinds encode the fluorescence-encoded of different iridescent 20 μ L of microballoon, 75 μ L of streptavidin-phycoerythrin, 5 μ L of amplified production.
Further, in step 3), the response procedures of hybridization are：43~47 DEG C of 20~30min of reaction.
The beneficial effects of the invention are as follows：
Multiple PCR technique and Luminex liquid-phase chip technologies are combined by the present invention, and mouse can be detected simultaneously by devising The primer sets of this six kinds high infection rate virus of TMEV, MHV, MNV, Reo-3, MVM, MPV.It is carried out first with the primer sets multiple PCR obtains target amplification product, then hybridizes amplified production, fluorescence-encoded micro-beads and streptavidin-phycoerythrin, When reading MFI values by Luminex detectors, so as to differentiate different types of virus.This method have rapidly and efficiently, specificity By force, the advantageous effects such as high sensitivity, reproducible can be applied to quality-monitoring, epidemiology survey and the early stage of experiment mice Early warning.It is specific as follows：
1) at present experimental animal common detection methods be unitem detection, can not a variety of cause of diseases of one-time detection, with biography System detection method is compared, and the method for the present invention is realized is carried out at the same time detection to a variety of different molecules of interest in same sample, real Existing high-throughput detection, and can detection project flexibly be increased according to increasing for cause of disease；Sample dosage is few simultaneously, easy to operate, Quickly, testing cost can be substantially reduced.
2) PCR product is captured by specific microsphere probe, better than traditional multiple detection method PCR product fragment length Result judgement is carried out, detection specificity is stronger.
3) Luminex liquid-phase chips utilize biotin-avidin signal amplifying system, and affinity is up to 1015L/moL, It is higher than simple affinity of antibody by 104Times or more, make testing result it is sensitiveer, by environmental disturbances are smaller, stability is high；The present invention The detection sensitivity of method 1~2 order of magnitude higher than regular-PCR.
4) Luminex liquid-phase chip technologies overcome piece membrane DNA chip in macromolecular due to being reacted in the solution using microballoon Kinetics is influenced by surface tension, three-dimensional effect etc. during detection, substantially increases the repeatability of sample detection, is detected Reliable results are stablized；The repeatability of detection can often reach more than 90%, and the range of linearity is also very wide.
5) flexibility of the present invention is good, can add and subtract the species of detection virus on this basis as needed.
Description of the drawings
Fig. 1：The RT-PCR electrophoretograms of 6 kinds of Murine Virus are detected simultaneously【1：TMEV, 2：MHV, 3：MNV, 4：Reo-3,5： MVM, 6：MPV, 7：IC, 8：Mixing positive control template, 9：Blank control, M：DL500DNAmarker】；
Fig. 2：The multi-fluorescence immunoassay method test experience result figure of 6 kinds of Murine Virus is detected simultaneously；
Fig. 3：The multi-fluorescence immunoassay method detection specificity experiments result figure of 6 kinds of Murine Virus of detection simultaneously；
Fig. 4：The multi-fluorescence immunoassay method detection sensitivity experimental result picture of 6 kinds of Murine Virus is detected simultaneously；
Fig. 5：The multi-fluorescence immunoassay method clinical sample test experience result figure of 6 kinds of Murine Virus is detected simultaneously.
Specific embodiment
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.In following embodiments The reagent raw material is commercially available common raw material in addition to especially source is indicated, and the preparation of reagent uses conventional method.Embodiment In the method that is not described in detail be this field routine operation.
Embodiment 1, the primer for Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus Group
According to goal of the invention, analysis comparison is carried out to a large amount of sequences, filters out the specific and conserved sequence of following viruses： 5 ' UTR gene orders (the GenBank accession number of TMEV：X56019), N gene orders (the GenBank accession number of MHV： FJ647223), ORF1~ORF2 calmodulin binding domain CaMs gene order (GenBank accession number of MNV：AY228235), the S1 bases of Reo-3 Because of sequence (GenBank accession number：HM159619), VP2 gene orders (the GenBank accession number of MVM：NC001510), MPV VP2 gene orders (GenBank accession number：) and envelope protein gene sequence (the GenBank accession number of MS2 NC001630： NC001417, as internal standard, internal control, abbreviation IC).
Primer is designed according to nucleotide sequence, and carries out primer secondary structure and forms the ability of primer dimer from each other It is evaluated.The present invention follows following design principle when designing primer：Primer length is 18~25bp, between upstream and downstream primer No more than 5bp, make G+C contents as far as possible between 40%~60%, primer itself there cannot be continuous 4 base complementrities, keep away as far as possible Exempt to form dimeric structure, hairpin structure, the clip size of amplification is selected between 100~250bp, so advantageously reduced Steric effect during Luminex microballoon hybridization reactions is more advantageous to the progress of follow-up hybridization reaction；Meanwhile inventor is also to tag The ability that sequence forms primer dimer is analyzed, and has been selected and the most suitable tag sequences of original primers sequence.
Due in multiplex PCR system multiple primers and template in same reaction tube, it is easy to cause to interfere with each other, and And be easy to that dimer can be formed between primer, after biotinylated primer forms dimer, in streptavidin-phycoerythrin Also very strong fluorescence signal can be inspired after effect, very high negative background's fluorescence can be thus generated, influence the judgement of result, Even result in test failure.Therefore design of primers is the key that the present invention and the base for carrying out Luminex liquid-phase chip detections Plinth.
Inventor have passed through designed primer substantial amounts of experiment and improve, and just overcomes above-mentioned multiple PCR primer and sets The difficult point of meter preferably goes out following primer sets, be used for multi-fluorescence be immunized while detect mouse TMEV, MHV, MNV, Reo-3, The specificity of MVM, MPV virus and sensitivity are best, and the preferred primer sets are by following original primers to further modifying.
The base sequence of original primers pair is as follows：
The original sense primers of TMEV：5’-GATCTAAGTYAACCGACTCC-3’(SEQ ID NO：1),
The original anti-sense primers of TMEV：5’-GAAGGAAGGGGCAACACATA-3’(SEQ ID NO：2),
The original sense primers of MHV：5’-TGGMATCCTCAAGAAGACCACTT-3’(SEQ ID NO：3),
The original anti-sense primers of MHV：5’-ATGCCMGAAAACCARGAGTAATG-3’(SEQ ID NO：4),
The original sense primers of MNV：5’-TCTGTYCTGCGCTGGGTGC-3’(SEQ ID NO：5),
The original anti-sense primers of MNV：5’-GCTGCGCCATCACTCATCC-3’(SEQ ID NO：6),
The original sense primers of Reo-3：5’-CCTCGCCTACGTGAAGAAGT-3’(SEQ ID NO：7),
The original anti-sense primers of Reo-3：5’-CATCATCGAGTCCCTGGGTG-3’(SEQ ID NO：8),
The original sense primers of MVM：5’-TTGTTCCACCACCACTAAATG-3’(SEQ ID NO：9),
The original anti-sense primers of MVM：5’-GGTTTGTGTTCAAGATCTAGTTC-3’(SEQ ID NO：10),
The original sense primers of MPV：5’-CACCAGCAACAGACAACCAA-3’(SEQ ID NO：11),
The original anti-sense primers of MPV：5’-TGAATGCGTTGAGTGTTCTC-3’(SEQ ID NO：12),
The original sense primers of internal standard MS2：5’-CGCAGAATCGCAAATACAC-3’(SEQ ID NO：13),
The original anti-sense primers of internal standard MS2：5’-TAACAATAAGCTCGCAGTCG-3’(SEQ ID NO：14)；
In order to be distinguished to mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, thus above-mentioned original primers are made into The modification of one step, to meet corresponding operation requirement.Corresponding tag sequences are connected at 5 ' ends of the original sense primer of each item, It is connected between 3 ' ends of middle tag sequences and 5 ' ends of primer sequence by arm between six ethylene glycol.
The tag sequences connected are as follows：
The tag sequences of TMEV are：5’-TTAAACAATCTACTATTCAATCAC-3’(SEQ ID NO：15),
The tag sequences of MHV are：5’-CACTTAATTCATTCTAAATCTATC-3’(SEQ ID NO：16),
The tag sequences of MNV are：5’-TACTTCTTTACTACAATTTACAAC-3’(SEQ ID NO：17),
The tag sequences of Reo3 are：5’-CACTACACATTTATCATAACAAAT-3’(SEQ ID NO：18),
The tag sequences of MVM are：5’-TACTACTTCTATAACTCACTTAAA-3’(SEQ ID NO：19),
The tag sequences of MPV are：5’-ACTTATTTCTTCACTACTATATCA-3’(SEQ ID NO：20),
The tag sequences of internal standard MS2 are：5’-ATTAAACAACTCTTAACTACACAA-3’(SEQ ID NO：21).
In addition, 5 ' ends of each original anti-sense primer of item are also added with biotin labeling.
Embodiment 2, the reagent for Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus Box
Kit includes following components：
1) it is sick for Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV designed by embodiment 1 The primer sets of poison；
2) fluorescence-encoded micro-beads for including anti-tag sequences of the different iridescent of 7 kinds of codings, the anti-tag sequences Row can be correspondingly with the tag sequence complementary pairings in multi-fluorescence immunoassay primer, and 7 kinds of microballoons are purchased from Luminex companies, Wherein the corresponding fluorescence-encoded micro-beads number of TMEV, MHV, MNV, Reo-3, MVM, MPV and MS2 are MTAG-A046, MTAG- A028, MTAG-A015, MTAG-042, MTAG-029, MTAG-034 and MTAG-036；
3) streptavidin-phycoerythrin compound, RT-PCR amplifing reagents, internal standard MS2 standard items, mixing are positive right According to, negative control；The RT-PCR amplifing reagents contain 5 × buffer, dNTP, Enzyme mix；The mixing positive control For simultaneously containing six kinds of viral nucleic acid samples of TMEV, MHV, MNV, Reo-3, MVM, MPV；The negative control is not contain Six kinds of viral nucleic acid samples of TMEV, MHV, MNV, Reo-3, MVM, MPV.
Embodiment 3, a kind of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV viral methods It establishes
(1) nucleic acid extraction
It extracts instrument (Tiangeng company) automatically with nucleic acid and extracts six kinds of viruses of TMEV, MHV, MNV, Reo-3, MVM, MPV respectively RNA or DNA, the template as multiplex PCR.Wherein internal standard MS2 standard items are added in each sample, synchronous to participate in sample nucleic Extraction, sample-adding, RT-PCR amplification and signal detection overall process.
(2) preparation of plasmid standard
Using TMEV, MHV, MNV, Reo-3, MVM, MPV six kinds of viral RNA or DNA as masterplate, respectively with 1 institute of embodiment Amplified production is carried out Ago-Gel by corresponding original primers respectively to carrying out RT-PCR amplifications in the original primers group of design Electrophoresis detection recycles target fragment with plastic recovery kit, the target fragment of recycling is connected to pMD-19T carriers, and is converted LB agar plate screening positive clones of the containing Amp into DH5 α competent cells identifies positive colony bacterium with PCR, and right Positive recombinant plasmid sequence verification.With plasmid extraction kit extract plasmid, micro ultraviolet specrophotometer measured concentration with it is pure Degree, is calculated according to the following equation copy number.Copy number (copies/ μ L)=6.022 × 1023(copies/moL) × DNA is dense Spend (g/ μ L)/mass M W (g/moL).Wherein, MW=DNA bases number (bp) × 660daltons/bp, DNA base number=carrier Series number+insetion sequence base number.
(3) multiplex RT-PCR amplification
With the primer sets designed by embodiment 1, respectively to TMEV, MHV, MNV, Reo-3, MVM, MPV and MS2 (internal standard) into Row multiplex RT-PCR amplification, multiple RT-PCR reagent use the OneStep RT-PCRKit of Qiangen companies, and experiment is same every time When positive control, negative control and blank control be set, wherein positive control is to contain above-mentioned six kinds viral tissues or cell For the nucleic acid of culture extraction as positive control template, wherein negative control (can not contain above-mentioned six kinds of viral nucleic acid samples To be intact animal tissue or normal cell culture) as negative control template, blank control is to be not added with Template Controls (No Template Control, NTC), i.e., in the reaction with water come instead of template.
RT-PCR amplification reaction systems are as follows：
The response procedures of amplification are：50 DEG C of reverse transcription 30min；95 DEG C of pre-degeneration 15min；94 DEG C of denaturation 30s, 60 DEG C of annealing 90s, 72 DEG C of extension 30s；Xun Huan 35 times；72 DEG C extend 10min eventually.
PCR product is analyzed into row agarose gel electrophoresis, and electrophoretogram is as shown in Figure 1, it is seen that amplifies purpose band.
(4) hybridize
Multiple RT-PCR product, fluorescence-encoded micro-beads, Streptavidin phycoerythrin (SA-PE) are hybridized, including following Step：
The fluorescence-encoded micro-beads are 7 kinds of microballoons for being coated with special anti-tag sequences respectively, wherein anti-tag Sequence can be correspondingly complementary with the tag sequences on six kinds of cause of diseases of TMEV, MHV, MNV, Reo-3, MVM, MPV and internal standard MS2 primers Match (SEQ ID NO：15~21).7 kinds of microballoons are purchased from Luminex companies, specific TMEV, MHV, MNV, Reo-3, MVM, The corresponding fluorescence-encoded micro-beads number of MPV and MS2 for MTAG-A046, MTAG-A028, MTAG-A015, MTAG-042, MTAG-029, MTAG-034 and MTAG-036.
The preparation of fluorescence-encoded micro-beads working solution：By 2500/μ L fluorescence-encoded micro-beads with 1.1 × Tm Hybrdization Buffer are diluted to 1 μ L and about contain 125/kind fluorescence-encoded micro-beads.
It is prepared by SA-PE working solutions：1mg/mL SA-PE are diluted to 10 μ g/ μ with 1 × Tm Hybrdization Buffer L。
Fluorescence-encoded micro-beads working solution is fully resuspended, each sample well and control wells add in 20 μ L of microballoon working solution, then 5 μ L PCR products are added in sample well, 5 μ L PCR positive controls, negative control and blank control are also separately added into control wells Product is eventually adding the SA-PE working solutions of 75 μ L, abundant mixing, 45 DEG C of incubation 25min in metal heater.
The 50 above-mentioned reaction solutions of μ L after hybridization are detected by the explanation according to 200 detectors of Luminex, as a result such as Fig. 2 It is shown.When detector reads six kinds of viral MFI values, different types of virus can be substantially offered an explanation.
As a result criterion：When the MFI values of sample are more than or equal to 3 times of MFI values of blank control, i.e., the MFI values of sample/ The positive is judged to during MFI values of blank control >=3.0, is otherwise judged to feminine gender.
Embodiment 4, specific test
Respectively with TMEV, MHV, MNV, Reo-3, MVM, MPV, lymphocytic choriomeningitis virus (LCMV), celestial being Platform virus (SV), mouse pneumonia virus (PVM), mouse hemorrhagic fever viruse (HV), mouse pox virus (Ect), polyomavirus (Poly), Mouse adenovirus (Mad), rat parvovirus KRV plants (KRV), rat parvovirus H-1 plants (H-1) and mouse giant cell disease For the RNA or DNA of malicious (MCMV) as template, each sample adds internal standard MS2 as Quality Control, while sets negative control and blank pair According to the multi-fluorescence immunoassay analysis established with embodiment 3 is detected.
Experimental result is as shown in figure 3, IC controls are set up, according to criterion：The MFI values of MFI values/blank control of sample The positive is judged to when >=3.0, is otherwise judged to feminine gender, only TMEV, MHV, MNV, Reo-3, MVM, MPV are the positive, and there is no hand over Fork reaction, other viruses are feminine gender, illustrate that the method for the present invention and kit specificity are good.
Embodiment 5, sensitivity test
6 viral plasmid standards of preparation are done 10 times with Easy dilution (Takara companies) to be serially diluted, Obtain 1 × 107~1 × 100Copies/ μ L series standard templates, the multi-fluorescence immune detection analysis side established with embodiment 3 Method is detected.
1 multi-fluorescence immunoassay sensitivity technique result of table
Multi-fluorescence immunoassay detection sensitivity experimental result as shown in table 1 and Fig. 4, the experimental results showed that TMEV, The lowest detection of MHV, MNV and MVM are limited to 1 × 102The lowest detection of copies/ μ L, Reo-3 and MPV is limited to 1 × 103copies/μL。
The detection of embodiment 6, clinical sample
Sample to be tested is 24 parts of mouse samples (number is 1~24) that In Guangdong Province is collected, and extracts mice organs and caecum The RNA/DNA of content, each sample adds internal standard MS2 as Quality Control, while sets positive control, negative control and blank pair According to the multi-fluorescence immunoassay analysis established using upper embodiment 3 is detected.
Experimental result is as shown in figure 5, IC controls are set up, according to criterion：The MFI values of MFI values/blank control of sample The positive is judged to when >=3.0, is otherwise judged to feminine gender, as a result：Sample 1,2,3,19 is negative sample；Sample 14,15,17 is TMEV cores Sour positive sample；Sample 4,5,6,7,8 is MHV nucleic acid positive samples；Sample 11,18,20,23 is MNV nucleic acid positive samples；Sample Originally 12,13,24 be Reo-3 nucleic acid positive samples；Sample 9,16 is MVM nucleic acid positive samples；Sample 10,21,22 is MPV nucleic acid Positive sample.Positive sample is by sequencing analysis, as a result unanimously.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Experimental Animals Supervising Station, Guangdong Prov.
<120>Six kinds of viral primer sets of Multiple immunizations fluoroscopic examination mouse, kit and method
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Claims (10)

1. for the primer sets of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, including 7 pairs Primer, 7 pairs of primers are by 7 pairs of original primers modify；It is described be modified in each pair original primers one Arm connects between 5 ' ends of the original sense primer of item pass through with 3 ' ends of corresponding tag sequences, and the 5 ' of another original anti-sense primer End addition biotin labeling；
The original primers are to as follows：
The original sense primers of TMEV：5 '-GATCTAAGTYAACCGACTCC-3 ',
The original anti-sense primers of TMEV：5 '-GAAGGAAGGGGCAACACATA-3 ',
The original sense primers of MHV：5 '-TGGMATCCTCAAGAAGACCACTT-3 ',
The original anti-sense primers of MHV：5 '-ATGCCMGAAAACCARGAGTAATG-3 ',
The original sense primers of MNV：5 '-TCTGTYCTGCGCTGGGTGC-3 ',
The original anti-sense primers of MNV：5 '-GCTGCGCCATCACTCATCC-3 ',
The original sense primers of Reo-3：5 '-CCTCGCCTACGTGAAGAAGT-3 ',
The original anti-sense primers of Reo-3：5 '-CATCATCGAGTCCCTGGGTG-3 ',
The original sense primers of MVM：5 '-TTGTTCCACCACCACTAAATG-3 ',
The original anti-sense primers of MVM：5 '-GGTTTGTGTTCAAGATCTAGTTC-3 ',
The original sense primers of MPV：5 '-CACCAGCAACAGACAACCAA-3 ',
The original anti-sense primers of MPV：5 '-TGAATGCGTTGAGTGTTCTC-3 ',
The original sense primers of internal standard MS2：5 '-CGCAGAATCGCAAATACAC-3 ',
The original anti-sense primers of internal standard MS2：5’-TAACAATAAGCTCGCAGTCG-3’；
The tag sequences are as follows：
The tag sequences of TMEV are：5 '-TTAAACAATCTACTATTCAATCAC-3 ',
The tag sequences of MHV are：5 '-CACTTAATTCATTCTAAATCTATC-3 ',
The tag sequences of MNV are：5 '-TACTTCTTTACTACAATTTACAAC-3 ',
The tag sequences of Reo3 are：5 '-CACTACACATTTATCATAACAAAT-3 ',
The tag sequences of MVM are：5 '-TACTACTTCTATAACTCACTTAAA-3 ',
The tag sequences of MPV are：5 '-ACTTATTTCTTCACTACTATATCA-3 ',
The tag sequences of internal standard MS2 are：5’-ATTAAACAACTCTTAACTACACAA-3’.
2. primer sets according to claim 1, it is characterised in that：Among primer of the described arm for 12~18 carbon atoms Trim.
3. primer sets according to claim 2, it is characterised in that：Described arm arm between six ethylene glycol.
4. for the kit of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, feature exists In：The kit contains primer sets described in claim 1, the fluorescence-encoded micro-beads of the different iridescent of 7 kinds of codings, described Independent fluorescence coding microball contains the anti-tag sequences with tag sequences complementary pairing in claim 1 primer.
5. kit according to claim 4, it is characterised in that：Also containing streptavidin-phycoerythrin compound, RT-PCR amplifing reagents, internal standard MS2 standard items, mixing positive control, negative control, the mixing positive control is to contain simultaneously Six kinds of viral nucleic acid samples of TMEV, MHV, MNV, Reo-3, MVM, MPV；The negative control for do not contain TMEV, MHV, Six kinds of viral nucleic acid samples of MNV, Reo-3, MVM, MPV.
6. a kind of method of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, including walking as follows Suddenly：
1) viral RNA or DNA are extracted from sample, as template, while sets blank control, negative control, mixing positive right According to the blank control is not add template；It is described mixing positive control be with simultaneously contain TMEV, MHV, MNV, Reo-3, Six kinds of viral nucleic acid samples of MVM, MPV are template；The negative control be with do not contain TMEV, MHV, MNV, Reo-3, MVM, Six kinds of viral nucleic acid samples of MPV are template；
2) nucleic acid of internal standard MS2 is added in the sample as Quality Control, and the primer sets described in claim 4 in kit carry out RT-PCR is expanded；
3) by fluorescence-encoded micro-beads, the strepto- of the different iridescent of 7 kinds of codings described in amplified production, claim 4 in kit Avidin-phycoerythrin is hybridized；
4) after hybridizing, analysis is detected hybrid product by Luminex systems, reads the MFI values of different virus；
5) result judges：When the MFI values >=3.0 of MFI values/blank control of sample, the positive is judged to, is otherwise judged to feminine gender；On State diagnose and treat of the method for non-disease.
7. according to the method described in claim 6, it is characterized in that：In step 2), 20 μ L reaction systems of RT-PCR amplifications contain Have：5 × buffer, 4 μ L, dNTP 0.8 μ L, 0.8 μ L of Enzyme mix, each 0.4 μ of 10 μM of each pair primer mixed liquor in primer sets L, 4 μ L of template, without 7.6 μ L of RNase water.
8. according to the method described in claim 6, it is characterized in that：In step 2), the response procedures of RT-PCR amplifications are：48~ 52 DEG C of 25~35min of reverse transcription；95 DEG C of pre-degeneration 15min；94 DEG C of denaturation 30s, 60 DEG C of annealing 90s, 72 DEG C of extension 30s；Xun Huan 35 times；72 DEG C extend 10min eventually.
9. according to the method described in claim 6, it is characterized in that：In step 3), 100 μ L reaction systems of hybridization contain：7 kinds Encode 20 μ L of fluorescence-encoded micro-beads, 75 μ L of streptavidin-phycoerythrin, 5 μ L of amplified production of different iridescent.
10. according to the method described in claim 6, it is characterized in that：In step 3), the response procedures of hybridization are：43~47 DEG C React 20~30min.
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