CN106636474A - Primer group, kit and method for detecting six mouse viruses by multiple immunity fluorescence - Google Patents

Primer group, kit and method for detecting six mouse viruses by multiple immunity fluorescence Download PDF

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CN106636474A
CN106636474A CN201710044633.6A CN201710044633A CN106636474A CN 106636474 A CN106636474 A CN 106636474A CN 201710044633 A CN201710044633 A CN 201710044633A CN 106636474 A CN106636474 A CN 106636474A
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primers
mnv
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CN106636474B (en
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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 a primer group, a kit and a method for detecting six mouse viruses by multiple immunity fluorescence. The primer group is designed by the aid of multiple RT-PCR (reverse transcription-polymerase chain reaction) technologies and Luminex liquid chip technologies and can simultaneously detect six high-infection viruses such as TMEV, MHV, MNV, Reo-3, MVM and MPV of mice. By the aid of the primer group, target amplification products are acquired through specific PCR, the amplification products, fluorescence encoding microspheres and streptavidin-phycoerythrin are hybridized, and different types of viruses are distinguished when a mean fluorescence intensity value is read by a Luminex detector. The method has the advantages of rapidness, high efficiency, high specificity and sensitivity, good repeatability and the like and can be applied to quality monitoring, epidemiological investigation and early warning of test mice. The primer group is good in flexibility, and varieties detecting the viruses can be increased and decreased as required on the basis.

Description

The primer sets of Multiple immunizations fluoroscopic examination six kinds of virus of mouse, kit and method
Technical field
The invention belongs to field of biological detection, more particularly, to drawing for six kinds of viruses of Multiple immunizations fluoroscopic examination mouse Thing group, kit and method.
Background technology
The detection of animal used as test microbial quality is the important indicator of evaluation experimental animal quality, and microorganism infection is not only to reality Test animal to work the mischief in itself, potential interference is also resulted in research work.In experimental animal infection disease, viral disease is accounted for There is important position, it was found that experiment mice virus infection is more in recessive sense in the monitoring of daily Bioexperiment animal pathogenic Dye, without obvious clinical symptoms, disease diagnosis are relatively difficult；And zoogenetic infection virus often shows as persistent infection, Once there is virus infection than more difficult to remove, needing could be from by detecting and eliminating (test-and-removal) method for facility Contaminated facility thoroughly eradicates virus, therefore, animal used as test method for detecting virus rapidly and efficiently is set up, timely and accurately send out Existing pathogen infection, control and removing epidemic situation, for improving, Quality of Experimental Animals is extremely important.
Experiment mice up to ten several, SPF specified in China's animal used as test standard GB/T 14922.2-2011 of virus Level mouse need to exclude 11 kinds of viruses.Mouse encephalomyelitis virus (Theiler ' s murine in the present invention Encephalomyelitis virus, TMEV), MHV (Murine hepatitis virus, MHV), mouse exhale The lonely virus type III (Reovirus Type 3, Reo-3) of intestines and minute parvovirus of mice MVM strains (Minute virus of mice, MVM) be national standard need exclude cause of disease.Mouse norovirus (Murine Norovirus, MNV) and minute parvovirus of mice MPV strains (Mouse parvovirus, MPV) are the virus of newfound infection experiment mouse, and both viruses are external experiments One essential items for inspection of animal health monitoring, although not yet MNV and MPV are listed in detection in China's animal used as test national standard Mesh, but some animals used as test produce or all it are classified as examination project using unit and some CRO companies.TMEV、MHV、 MNV, MVM, MPV are the higher virus of infection rate in current experiment mice, are resisted in the report experiment mice 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 can not produce normal resisting Precursor reactant.Antigen context of detection conventional viral isolation and identification method was not only complicated but also loaded down with trivial details, was unfavorable for routine testing.Traditional detection Technology can not meet Quality of Experimental Animals detection demand, the molecular biological testing detection virus tool based on PCR The features such as having quick, sensitive, special, is the conventional detection method for clinically detecting and diagnosing the illness at present.But regular-PCR method Needs to be uncapped and carry out gel electrophoresis analysis to product, and method of operating not enough simplifies, and easily produce Aerosol Pollution, cause Existing false positive.Because of pinpoint accuracy, high sensitivity is that virus infection at present is examined the advantages of pollution is few to Real-Time Fluorescent Quantitative PCR Technique Disconnected important means.But real-time fluorescence PCR technology is limited by fluorescent species and instrument itself, at most can only be to 5 targets Detected, and the difficulty of Success in Experiment is greatly, cost is also of a relatively high.
Luminex liquid-phase chip technologies are a kind of new multi-fluorescence immunoassay sides that the nineties in 20th century rises 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, its major advantage is can be with Independent assortment, high flux, sensitivity is high, 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 set up it is a kind of detect simultaneously mouse TMEV, The viral detection method of MHV, MNV, Reo-3, MVM, MPV this six kinds of high infection rates, the technology can be applicable 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 is sick The primer of poison, kit and method.
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 formed by carrying out modification to 7 pairs of original primers；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；
Described original primers are to as follows：
The original upstream primers of TMEV：5 '-GATCTAAGTYAACCGACTCC-3 ',
The original downstream primers of TMEV：5 '-GAAGGAAGGGGCAACACATA-3 ',
The original upstream primers of MHV：5 '-TGGMATCCTCAAGAAGACCACTT-3 ',
The original downstream primers of MHV：5 '-ATGCCMGAAAACCARGAGTAATG-3 ',
The original upstream primers of MNV：5 '-TCTGTYCTGCGCTGGGTGC-3 ',
The original downstream primers of MNV：5 '-GCTGCGCCATCACTCATCC-3 ',
The original upstream primers of Reo-3：5 '-CCTCGCCTACGTGAAGAAGT-3 ',
The original downstream primers of Reo-3：5 '-CATCATCGAGTCCCTGGGTG-3 ',
The original upstream primers of MVM：5 '-TTGTTCCACCACCACTAAATG-3 ',
The original downstream primers of MVM：5 '-GGTTTGTGTTCAAGATCTAGTTC-3 ',
The original upstream primers of MPV：5 '-CACCAGCAACAGACAACCAA-3 ',
The original downstream primers of MPV：5 '-TGAATGCGTTGAGTGTTCTC-3 ',
The original upstream primer of internal standard MS2：5 '-CGCAGAATCGCAAATACAC-3 ',
The original downstream primer of internal standard MS2：5’-TAACAATAAGCTCGCAGTCG-3’；
Described 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 in the middle of the primer of 12～18 atoms between described.
Preferably, arm is arm between six ethylene glycol between described.
For the kit of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, its feature It is：The kit contains above-mentioned viral for Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV Primer sets, the fluorescence-encoded micro-beads of the different iridescent of 7 kinds of codings, described independent fluorescence coding microball contains and above-mentioned primer The anti-tag sequences of middle tag sequences complementary pairing.
Further, mentioned reagent box, also containing SA-PE compound, RT-PCR amplifing reagents, interior Mark MS2 standard items, mixing positive control, negative control, the mixing positive control is for simultaneously containing TMEV, MHV, MNV, Reo- 3rd, the nucleic acid samples of six kinds of virus of MVM, MPV；The negative control is not contain TMEV, MHV, MNV, Reo-3, MVM, MPV six Plant the nucleic acid samples of virus.
A kind of method of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, including it is as follows Step：
1) viral RNA or DNA are extracted from sample, as template, while arranging blank, negative control, mixing sun Property control, the blank is without template；The mixing positive control is to contain TMEV, MHV, MNV, Reo- simultaneously 3rd, the nucleic acid samples of six kinds of virus of MVM, MPV are template；The negative control be with do not contain TMEV, MHV, MNV, Reo-3, The nucleic acid samples of six kinds of virus of MVM, MPV are template；
2) add the nucleic acid of internal standard MS2 as Quality Control in the sample, with the primer sets in above-mentioned kit RT-PCR is carried out Amplification；
3) 7 kinds in amplified production, mentioned reagent box are encoded into the fluorescence-encoded micro-beads of different iridescent, strepto- is affine Element-phycoerythrin is hybridized；
4) after hybridization terminates, hybrid product is tested and analyzed by Luminex systems, reads the MFI of different virus Value；
5) result judges：When MFI value >=3.0 of the MFI values/blank of sample, the positive is judged to, is otherwise judged to the moon Property；
Said method is used for the diagnosis and treatment of non-diseases.
Further, step 2) in, 20 μ L reaction systems of RT-PCR amplifications contain：5×buffer4μL、dNTP0.8μ The each 0.4 μ L of each pair primer mixed liquor (10 μM), the μ L of template 4 in L, Enzyme mix0.8 μ L, primer sets, without the μ L of RNase water 7.6.
Further, step 2) in, the response procedures of RT-PCR amplifications are：48～52 DEG C of 25～35min of reverse transcription；95℃ Denaturation 15min；94 DEG C of denaturation 30s, 60 DEG C of annealing 90s, 72 DEG C of extension 30s；Circulation 35 times；72 DEG C extend eventually 10min.
Further, step 3) in, 100 μ L reaction systems of hybridization contain：7 kinds encode the fluorescence-encoded of different iridescent The μ L of microballoon 20, the μ L of SA-PE 75, the μ L of amplified production 5.
Further, step 3) in, the response procedures of hybridization are：43～47 DEG C of 20～30min of reaction.
The invention has the beneficial effects as follows：
The present invention combines multiple PCR technique and Luminex liquid-phase chip technologies, and devising can be while detects mouse The primer sets of the high infection rate viruses of this six kinds of TMEV, MHV, MNV, Reo-3, MVM, MPV.Carry out first with the primer sets multiple PCR obtains target amplification product, is then hybridized amplified production, fluorescence-encoded micro-beads and SA-PE, When reading MFI values by Luminex detectors, so as to differentiate different types of virus.The method have rapidly and efficiently, specificity The beneficial effect such as by force, sensitivity is high, reproducible, can be applicable to quality-monitoring, epidemiology survey and the early stage of experiment mice Early warning.It is specific as follows：
1) at present animal used as test common detection methods are unitem detections, it is impossible to the various cause of diseases of one-time detection, with biography System detection method is compared, the inventive method realize to same sample in various different molecules of interest while detect, reality Existing high flux detection, and flexibly increase detection project according to can increasing for cause of disease；Simultaneously sample consumption is few, simple to operate, Quickly, testing cost can be substantially reduced.
2) PCR primer is captured by specific microsphere probe, better than traditional multiple detection method PCR primer fragment length Result judgement is carried out, detection specificity is higher.
3) Luminex liquid-phase chips utilize biotin-avidin signal amplifying system, its affinity to be up to 1015L/moL, It is higher than simple affinity of antibody by 104More than times, make testing result it is sensitiveer, by environmental disturbances are less, 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 reacting in the solution using microballoon During detection kinetics is affected by surface tension, three-dimensional effect etc., substantially increased the repeatability of sample detection, 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, and the species of detection virus can be on this basis added and subtracted 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, M：DL500DNAmarker】；
Fig. 2：The multi-fluorescence immune analysis method test experience result figure of 6 kinds of Murine Virus is detected simultaneously；
Fig. 3：Simultaneously the multi-fluorescence immune analysis method of 6 kinds of Murine Virus of detection detects specificity experiments result figure；
Fig. 4：The multi-fluorescence immune analysis method detection sensitivity experimental result picture of 6 kinds of Murine Virus is detected simultaneously；
Fig. 5：The multi-fluorescence immune analysis 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 adopts conventional method.Embodiment In the method that do not describe in detail be this area routine operation.
Embodiment 1, for Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus primer Group
According to goal of the invention, a large amount of sequences are analyzed and are compared, filter 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 CaM 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：NC001630) and MS2 envelope protein gene sequence (GenBank accession number： NC001417, as internal standard, internal control, abbreviation IC).
Primer is designed according to nucleotide sequence, and is carried out primer secondary structure and is formed the ability of primer dimer from each other Evaluated.The present invention follows following design principle when primer is designed：Primer length is 18～25bp, between upstream and downstream primer Less than 5bp, make G+C contents between 40%～60% as far as possible, primer itself there can not be continuous 4 base complementrities, keep away as far as possible Exempt to form dimeric structure, hairpin structure, the clip size of amplification selects between 100～250bp, so to advantageously reduce Steric effect during Luminex microballoon hybridization reactions, is more beneficial for the carrying out of follow-up hybridization reaction；Meanwhile, inventor is also to tag Sequence forms the ability of primer dimer and is analyzed, and have selected tag sequences most suitable with 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, biotinylated primer is formed after dimer, in SA-PE Also very strong fluorescence signal can be inspired after effect, very high negative background's fluorescence can be thus produced, the judgement of result is affected, Even result in test failure.Therefore design of primers is the key of the present invention, is also the base for carrying out Luminex liquid-phase chip detections Plinth.
Inventor have passed through substantial amounts of test and improvement to designed primer, just overcomes above-mentioned multiple PCR primer and sets The difficult point of meter, preferably goes out following primer sets, its be used for multi-fluorescence immunity detect simultaneously mouse TMEV, MHV, MNV, Reo-3, Preferably, the preferred primer sets are formed by following original primers to further modification for the specificity of MVM, MPV virus and sensitivity.
The base sequence of original primers pair is as follows：
The original upstream primers of TMEV：5’-GATCTAAGTYAACCGACTCC-3’(SEQ ID NO：1),
The original downstream primers of TMEV：5’-GAAGGAAGGGGCAACACATA-3’(SEQ ID NO：2),
The original upstream primers of MHV：5’-TGGMATCCTCAAGAAGACCACTT-3’(SEQ ID NO：3),
The original downstream primers of MHV：5’-ATGCCMGAAAACCARGAGTAATG-3’(SEQ ID NO：4),
The original upstream primers of MNV：5’-TCTGTYCTGCGCTGGGTGC-3’(SEQ ID NO：5),
The original downstream primers of MNV：5’-GCTGCGCCATCACTCATCC-3’(SEQ ID NO：6),
The original upstream primers of Reo-3：5’-CCTCGCCTACGTGAAGAAGT-3’(SEQ ID NO：7),
The original downstream primers of Reo-3：5’-CATCATCGAGTCCCTGGGTG-3’(SEQ ID NO：8),
The original upstream primers of MVM：5’-TTGTTCCACCACCACTAAATG-3’(SEQ ID NO：9),
The original downstream primers of MVM：5’-GGTTTGTGTTCAAGATCTAGTTC-3’(SEQ ID NO：10),
The original upstream primers of MPV：5’-CACCAGCAACAGACAACCAA-3’(SEQ ID NO：11),
The original downstream primers of MPV：5’-TGAATGCGTTGAGTGTTCTC-3’(SEQ ID NO：12),
The original upstream primer of internal standard MS2：5’-CGCAGAATCGCAAATACAC-3’(SEQ ID NO：13),
The original downstream primer of internal standard MS2：5’-TAACAATAAGCTCGCAGTCG-3’(SEQ ID NO：14)；
In order to make a distinction to mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, thus above-mentioned original primers are made into The modification of one step, is required with meeting corresponding operation.In the corresponding tag sequences of 5 ' end connections of the original upstream primer of each bar, its By arm connection between six ethylene glycol between 3 ' ends of middle tag sequences and 5 ' ends of primer sequence.
The tag sequences for being 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 the original downstream primer of each bar are also added with biotin labeling.
Embodiment 2, for Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus reagent 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 7 kinds of coding difference iridescent, the anti-tag sequences Row can correspondingly with multi-fluorescence immunoassay primer in tag sequence complementary pairings, 7 kinds of microballoons are purchased from Luminex companies, It is MTAG-A046, MTAG- that wherein TMEV, MHV, MNV, Reo-3, MVM, MPV and MS2 distinguish corresponding fluorescence-encoded micro-beads number A028, MTAG-A015, MTAG-042, MTAG-029, MTAG-034 and MTAG-036；
3) SA-PE 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 the nucleic acid samples of six kinds of virus of TMEV, MHV, MNV, Reo-3, MVM, MPV；The negative control is not contain The nucleic acid samples of six kinds of virus 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 Set up
(1) nucleic acid extraction
Instrument (Tiangeng company) is extracted automatically with nucleic acid extracts six kinds of viruses of TMEV, MHV, MNV, Reo-3, MVM, MPV respectively RNA or DNA, as the template of 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
RNA or DNA with six kinds of viruses of TMEV, MHV, MNV, Reo-3, MVM, MPV as masterplate, respectively with the institute of embodiment 1 Amplified production is carried out respectively Ago-Gel to carrying out RT-PCR amplifications for corresponding original primers in the original primers group of design Electrophoresis detection, with glue reclaim kit purpose fragment is reclaimed, and the purpose fragment of recovery is connected to into pMD-19T carriers, and is converted Into DH5 α competent cells. with the LB agar plate screening positive clones containing Amp, positive colony bacterium is identified with PCR, and it is right Positive recombinant plasmid sequence verification.Extract plasmid with plasmid extraction kit, micro ultraviolet specrophotometer determine 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 Degree (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, TMEV, MHV, MNV, Reo-3, MVM, MPV and MS2 (internal standard) are entered respectively Row multiplex RT-PCR amplification, multiple RT-PCR reagent is tested every time same using the OneStep RT-PCRKit of Qiangen companies When positive control, negative control and blank are set, wherein positive control is with the tissue containing above-mentioned six kinds of virus or cell , used as positive control template, wherein negative control (can not contain above-mentioned six kinds of viral nucleic acid samples for the nucleic acid that culture is extracted To be intact animal tissue or normal cell culture) used as negative control template, blank is and is not added with Template Controls (No Template Control, NTC), i.e., with water replace template in the reaction.
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 denaturations 15min；94 DEG C of denaturation 30s, 60 DEG C of annealing 90s, 72 DEG C of extension 30s；Circulation 35 times；72 DEG C extend eventually 10min.
PCR primer enters row agarose gel electrophoresis analysis, and its electrophoretogram is as shown in Figure 1, it is seen that amplify purpose band.
(4) hybridize
Multiple RT-PCR product, fluorescence-encoded micro-beads, Streptavidin phycoerythrin (SA-PE) are hybridized, including it is 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 six kinds of cause of diseases of TMEV, MHV, MNV, Reo-3, MVM, MPV and tag sequences on internal standard MS2 primer Pairing (SEQ ID NO：15～21).7 kinds of microballoons are purchased from Luminex companies, specific TMEV, MHV, MNV, Reo-3, MVM, MPV and MS2 distinguish corresponding fluorescence-encoded micro-beads number be 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 into 10 μ g/ μ with 1 × Tm Hybrdization Buffer L。
Abundant resuspended fluorescence-encoded micro-beads working solution, each sample well and control wells add the μ L of microballoon working solution 20, subsequently 5 μ L PCR primers are added in sample well, 5 μ L PCR positive controls, negative control and blank are also separately added in control wells Product, is eventually adding the SA-PE working solutions of 75 μ L, fully mixes, 45 DEG C of incubation 25min in metal heater.
According to the explanation of the detectors of Luminex 200 by hybridization after the above-mentioned reactant liquors of 50 μ L detected, as a result such as Fig. 2 It is shown.During the MFI values of detector six kinds of viruses of reading, 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, i.e., the MFI values of sample/ The positive is judged to during MFI value >=3.0 of blank, feminine gender is otherwise judged to.
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 strains (KRV), rat parvovirus H-1 strains (H-1) and mouse giant cell disease Used as template, each sample adds internal standard MS2 as Quality Control to the RNA or DNA of malicious (MCMV), while setting negative control and blank are right According to the multi-fluorescence immunoassay analysis set up with embodiment 3 are detected.
Experimental result as shown in figure 3, IC control set up, according to criterion：The MFI values of the MFI values/blank of sample The positive is judged to when >=3.0, feminine gender is otherwise judged to, only TMEV, MHV, MNV, Reo-3, MVM, MPV are the positive, and there is no friendship Fork reaction, other viruses are feminine gender, illustrate that the inventive method and kit specificity are good.
Embodiment 5, sensitivity test
The plasmid standard of 6 virus for preparing is done into 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 set up with embodiment 3 Method is detected.
The multi-fluorescence immunoassay sensitivity technique result of table 1
Multi-fluorescence immunoassay detection sensitivity experimental result as shown in table 1 and Fig. 4, test result indicate that TMEV, The lowest detection of MHV, MNV and MVM is 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 (numbering 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 it is right to arrange positive control, negative control and blank According to the multi-fluorescence immunoassay analysis set up using upper embodiment 3 are detected.
Experimental result as shown in figure 5, IC control set up, according to criterion：The MFI values of the MFI values/blank of sample The positive is judged to when >=3.0, feminine gender is otherwise judged to, 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 is 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 passes through sequencing analysis, as a result unanimously.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment Limit, other any Spirit Essences without departing from the present invention and the change, modification, replacement made under principle, 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>The primer sets of Multiple immunizations fluoroscopic examination six kinds of virus of mouse, kit and method
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<170> PatentIn version 3.5
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Claims (10)

1. the primer sets of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus, including 7 pairs are used for Primer, 7 pairs of primers are formed by carrying out modification to 7 pairs of original primers；It is described be modified in each pair original primers one Arm connects between 5 ' ends of bar original primers pass through with 3 ' ends of corresponding tag sequences, 5 ' end addition lifes of another original primers Thing element mark；
Described original primers are to as follows：
The original upstream primers of TMEV：5 '-GATCTAAGTYAACCGACTCC-3 ',
The original downstream primers of TMEV：5 '-GAAGGAAGGGGCAACACATA-3 ',
The original upstream primers of MHV：5 '-TGGMATCCTCAAGAAGACCACTT-3 ',
The original downstream primers of MHV：5 '-ATGCCMGAAAACCARGAGTAATG-3 ',
The original upstream primers of MNV：5 '-TCTGTYCTGCGCTGGGTGC-3 ',
The original downstream primers of MNV：5 '-GCTGCGCCATCACTCATCC-3 ',
The original upstream primers of Reo-3：5 '-CCTCGCCTACGTGAAGAAGT-3 ',
The original downstream primers of Reo-3：5 '-CATCATCGAGTCCCTGGGTG-3 ',
The original upstream primers of MVM：5 '-TTGTTCCACCACCACTAAATG-3 ',
The original downstream primers of MVM：5 '-GGTTTGTGTTCAAGATCTAGTTC-3 ',
The original upstream primers of MPV：5 '-CACCAGCAACAGACAACCAA-3 ',
The original downstream primers of MPV：5 '-TGAATGCGTTGAGTGTTCTC-3 ',
The original upstream primer of internal standard MS2：5 '-CGCAGAATCGCAAATACAC-3 ',
The original downstream primer of internal standard MS2：5’-TAACAATAAGCTCGCAGTCG-3’；
Described 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：Described arm is to repair in the middle of the primer of 12～18 atoms Jewelry.
3. primer sets according to claim 2, it is characterised in that：Described arm is arm between six ethylene glycol.
4. the kit of Multiple immunizations fluoroscopic examination mouse TMEV, MHV, MNV, Reo-3, MVM, MPV virus is used for, and its feature exists In：The kit contains the fluorescence-encoded micro-beads of the different iridescent of primer sets described in claim 1,7 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 SA-PE compound, RT-PCR amplifing reagents, internal standard MS2 standard items, mixing positive control, negative control, the mixing positive control is for while contain There are the nucleic acid samples of six kinds of virus of TMEV, MHV, MNV, Reo-3, MVM, MPV；The negative control for do not contain TMEV, MHV, The nucleic acid samples of six kinds of virus 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, walks including following Suddenly：
1）Viral RNA or DNA are extracted from sample, as template, while it is right to arrange blank, negative control, the mixing positive According to the blank is without template；It is described mixing positive control be with simultaneously contain TMEV, MHV, MNV, Reo-3, The nucleic acid samples of six kinds of virus of MVM, MPV are template；The negative control be with do not contain TMEV, MHV, MNV, Reo-3, MVM, The nucleic acid samples of six kinds of viruses of MPV are template；
2）Add the nucleic acid of internal standard MS2 as Quality Control in the sample, carried out with the primer sets in kit described in claim 4 RT-PCR is expanded；
3）By fluorescence-encoded micro-beads, the strepto- of the different iridescent of 7 kinds of codings in kit described in amplified production, claim 4 Avidin-phycoerythrin is hybridized；
4）After hybridization terminates, hybrid product is tested and analyzed by Luminex systems, read the MFI values of different virus；
5）As a result judge：When MFI value >=3.0 of the MFI values/blank of sample, the positive is judged to, is otherwise judged to feminine gender；
Said method is used for the diagnosis and treatment of non-diseases.
7. method according to claim 6, it is characterised in that：Step 2）In, 20 L reaction systems of RT-PCR amplifications contain Have：Each pair primer mixed liquor in 5 × buffer4 L, dNTP0.8 L, Enzyme mix0.8 L, primer sets（10µM）Each 0.4 L, the L of template 4, without the L of RNase water 7.6.
8. method according to claim 6, it is characterised in that：Step 2）In, the response procedures of RT-PCR amplifications are：48～ 52 DEG C of 25～35min of reverse transcription；95 DEG C of denaturations 15min；94 DEG C of denaturation 30s, 60 DEG C of annealing 90s, 72 DEG C of extension 30s；Circulation 35 times；72 DEG C extend eventually 10min.
9. method according to claim 6, it is characterised in that：Step 3）In, 100 L reaction systems of hybridization contain：7 kinds The L of fluorescence-encoded micro-beads 20, the L of SA-PE 75, the L of amplified production 5 of the different iridescent of coding.
10. method according to claim 6, it is characterised in that：Step 3）In, the response procedures of hybridization are：43～47 DEG C 20～30min of reaction.
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