CN106755589B - The primer sets, kit and Multiple immunizations fluorescence analysis method of five kinds of pathogen of rat are detected simultaneously - Google Patents

The primer sets, kit and Multiple immunizations fluorescence analysis method of five kinds of pathogen of rat are detected simultaneously Download PDF

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CN106755589B
CN106755589B CN201710044031.0A CN201710044031A CN106755589B CN 106755589 B CN106755589 B CN 106755589B CN 201710044031 A CN201710044031 A CN 201710044031A CN 106755589 B CN106755589 B CN 106755589B
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黄韧
袁文
郭鹏举
张钰
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Guangdong Laboratory Animals Monitoring Institute
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Abstract

The invention discloses the primer sets, kit and Multiple immunizations fluorescence analysis method for detecting five kinds of pathogen of rat simultaneously.Multiple PCR technique and Luminex liquid-phase chip technologies are combined by the present invention, and rat RTV, TMEV, RCV, PVM, M. can be detected simultaneously by devisingpulmonisThe primer sets of this five kinds of pathogen, 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 pathogen.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 experimental rat.Flexibility of the present invention is good, can add and subtract the species of detection cause of disease on this basis as needed.

Description

The primer sets, kit and Multiple immunizations fluorescence of five kinds of pathogen of rat are detected simultaneously Analysis method
Technical field
The invention belongs to field of biological detection, more particularly, to primer sets, the examination for detecting five kinds of pathogen of rat simultaneously Agent box and Multiple immunizations fluorescence analysis 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.Experimental rat pathogenic microorganism in subclinical infection, does not have mostly There are apparent clinical symptoms, disease diagnosis is relatively difficult.And zoogenetic infection pathogen often shows as persistent infection, facility Once there are pathogenic infection than it is more difficult to remove, it is necessary to by detect and eliminate (test-and-removal) method could from by The facility of pollution thoroughly eradicates pathogen, therefore, establishes experimental animal pathogen detection method rapidly and efficiently, timely and accurately Find the cause of disease infection, control and removing epidemic situation, extremely important for improving Quality of Experimental Animals.
SPF grades of rats specified in China experimental animal standard GB/T 14922.2-2011 need to exclude 21 kinds of micro- lifes of cause of disease Object.Rat coronaviruse/sialodacryoadenitis virus (Rat coronavirus, RCV) in the present invention, mouse pneumonia virus (Pneumonia virus of mice, PVM), mycoplasma pulmonis (Mycoplasma pulmonis, M.pulmonis) are countries The cause of disease that standard needs exclude.Rat Taylor virus (Rat theilovirus, RTV) and mouse encephalomyelitis virus (Theiler ' s murine encephalomyelitis virus, TMEV) is the virus of newfound infection experiment rat, We investigate find RTV, TMEV in the rat raised under conventional environment infection rate be respectively 37.6% (n=101) and 24.8% (n=101).Both viruses are an essential items for inspection of external experimental animal health monitoring, experimental animal state of China Although RTV and TMEV are not yet included in detection project in family's standard, some experimental animals produce or using unit, Yi Jiyi A little CRO companies are all classified as it examination project.
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 rats, because they cannot generate normal antibody response.Antigen context of detection Conventional viral isolation and identification method was not only complicated but also cumbersome, was unfavorable for routine testing.Traditional detection technique has been unable to meet experiment Animal quality detection demand, the molecular biological testing detection virus based on PCR have quick, sensitive, special etc. Feature is the common detection method of current clinically detect and diagnose disease.But regular-PCR method needs, which are uncapped, carries out product Gel electrophoresis analysis, operating method not enough simplifies, and easily generates Aerosol Pollution, causes false positive occur.Real-time fluorescence Quantitative PCR technique pollutes the important means that the advantages that few is current Diagnosis of Viral Infections because of pinpoint accuracy, high sensitivity.It is but real When Fluorescence PCR assay limited be subject to fluorescent species and instrument itself, 5 targets can only be at most detected, and test into The difficulty of work(is very big, and 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 rat RTV, The Multiple immunizations fluorescence analysis method of five kinds of pathogen of TMEV, RCV, PVM, M.pulmonis, can be applied to the matter of experimental rat Amount monitoring, epidemiology survey and early warning.
The content of the invention
It is an object of the invention to provide detect five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis simultaneously Primer sets, kit and Multiple immunizations fluorescence analysis method.
The technical solution used in the present invention is：
The primer sets of five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis are detected simultaneously, are drawn including 6 Duis Object, 6 pairs of primers are by 6 pairs of original primers modify；One be modified in each pair original primers Arm connects between 5 ' ends of original primers pass through with 3 ' ends of corresponding tag sequences, 5 ' end addition biologies of another original primers Element mark；
The original primers are to as follows：
The original sense primers of RTV：5 '-GGAAACTTAGAGCAGACATCG-3 ',
The original anti-sense primers of RTV：5 '-TCCACAGAGAACAACCATCG-3 ',
The original sense primers of TMEV：5 '-CGTTGGAAAAGCACCTCTCAC-3 ',
The original anti-sense primers of TMEV：5 '-TCGGAGTCGGTTGACTTAGAT-3 ',
The original sense primers of RCV：5 '-TGGMATCCTCAAGAAGACCACTT-3 ',
The original anti-sense primers of RCV：5 '-ATGCCMGAAAACCARGAGTAATG-3 ',
The original sense primers of PVM：5 '-GCTGGCATTCCACATAACC-3 ',
The original anti-sense primers of PVM：5 '-CCCACAAGGTACACGGAGA-3 ',
The original sense primers of M.pulmonis：5 '-GGAAATGCCCTAAGTATGACGG-3 ',
The original anti-sense primers of M.pulmonis：5 '-CGGATAACGCTTGCACCCTA-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 RTV are：5 '-ACTTATTTCTTCACTACTATATCA-3 ',
The tag sequences of TMEV are：5 '-TTAAACAATCTACTATTCAATCAC-3 ',
The tag sequences of RCV are：5 '-CACTTAATTCATTCTAAATCTATC-3 ',
The tag sequences of PVM are：5 '-ATACTTTACAAACAAATAACACAC-3 ',
The tag sequences of M.pulmonis are：5 '-TACTACTTCTATAACTCACTTAAA-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.
The kit of five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis is detected simultaneously, it is characterised in that： The kit contains while detects the primer sets of five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis, 6 kinds of volumes The fluorescence-encoded micro-beads of the different iridescent of code, the independent fluorescence coding microball contain complementary with tag sequences in above-mentioned primer The anti-tag sequences of pairing.
Further, mentioned reagent box also contains streptavidin-phycoerythrin compound, RT-PCR amplifing reagents, interior Mark MS2 standard items, mixing positive control, negative control, the mixing positive control for simultaneously containing RTV, TMEV, RCV, PVM, The nucleic acid samples of five kinds of pathogen of M.pulmonis；The negative control for do not contain RTV, TMEV, RCV, PVM, The nucleic acid samples of five kinds of pathogen of M.pulmonis.
The Multiple immunizations fluorescence analysis side of five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis is detected simultaneously Method includes the following steps：
1) pathogen RNA or DNA are extracted from sample, as template, while blank control, negative control, mixing are set Positive control；The blank control is not add template；The negative control be with do not contain RTV, TMEV, RCV, PVM, The nucleic acid samples of five kinds of pathogen of M.pulmonis are template；It is described mixing positive control be with simultaneously contain RTV, TMEV, The nucleic acid samples of five kinds of pathogen of RCV, PVM, M.pulmonis 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 6 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 different pathogens MFI values；
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 8 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：6 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 devising one kind can detect greatly simultaneously The primer sets of this five kinds of pathogen of mouse RTV, TMEV, RCV, PVM, M.pulmonis are obtained using the primer sets by specific PCR Then amplified production, fluorescence-encoded micro-beads and streptavidin-phycoerythrin are hybridized, passed through by target amplification product When Luminex detectors read MFI values, so as to differentiate different types of pathogen.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 experimental rat 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) liquid-phase chip utilizes biotin-avidin signal amplifying system, and affinity is up to 1015L/moL, than simple Affinity of antibody is high by 104Times or more, make testing result it is sensitiveer, by environmental disturbances are smaller, stability is high；The inspection of the method for the present invention Survey high 1~2 order of magnitude of remolding sensitivity 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 cause of disease on this basis as needed.
Description of the drawings
Fig. 1：The RT-PCR electrophoretograms of 5 kinds of rat pathogen are detected simultaneously【1：RTV, 2：TMEV, 3：RCV, 4：PVM, 5： M.pulmonis, 6：IC, 7：Mixing positive control template, 8：Blank control, M：DL500DNA marker】；
Fig. 2：The multi-fluorescence immunoassay method test experience result figure of 5 kinds of rat pathogen is detected simultaneously；
Fig. 3：The multi-fluorescence immunoassay method detection specificity experiments result figure of 5 kinds of rat pathogen of detection simultaneously；
Fig. 4：The multi-fluorescence immunoassay method detection sensitivity experimental result picture of 5 kinds of rat pathogen is detected simultaneously；
Fig. 5：The multi-fluorescence immunoassay method clinical sample test experience result of 5 kinds of rat pathogen is detected simultaneously Figure.
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 while the primer sets for detecting five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis
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 RTV：EU542581), 5 ' UTR gene orders (the GenBank accession number of TMEV： X56019), N gene orders (the GenBank accession number of RCV：AF088984), (GenBank is logged in the NS1 gene orders of PVM Number：AY743910), 16sRNA gene orders (the GenBank accession number of M.pulmonis：) and the coating egg of MS2 AF125582 White gene order (GenBank accession number：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 rat RTV, TMEV, RCV, PVM, The specificity of M.pulmonis pathogen and sensitivity are best, the preferred primer sets by following original primers to further modifying and Into.
The base sequence of original primers pair is as follows：
The original sense primers of RTV：5’-GGAAACTTAGAGCAGACATCG-3’(SEQ ID NO：1),
The original anti-sense primers of RTV：5’-TCCACAGAGAACAACCATCG-3’(SEQ ID NO：2),
The original sense primers of TMEV：5’-CGTTGGAAAAGCACCTCTCAC-3’(SEQ ID NO：3),
The original anti-sense primers of TMEV：5’-TCGGAGTCGGTTGACTTAGAT-3’(SEQ ID NO：4),
The original sense primers of RCV：5’-TGGMATCCTCAAGAAGACCACTT-3’(SEQ ID NO：5),
The original anti-sense primers of RCV：5’-ATGCCMGAAAACCARGAGTAATG-3’(SEQ ID NO：6),
The original sense primers of PVM：5’-GCTGGCATTCCACATAACC-3’(SEQ ID NO：7),
The original anti-sense primers of PVM：5’-CCCACAAGGTACACGGAGA-3’(SEQ ID NO：8),
The original sense primers of M.pulmonis：5’-GGAAATGCCCTAAGTATGACGG-3’(SEQ ID NO：9),
The original anti-sense primers of M.pulmonis：5’-CGGATAACGCTTGCACCCTA-3’(SEQ ID NO：10),
The original sense primers of internal standard MS2：5’-CGCAGAATCGCAAATACAC-3’(SEQ ID NO：11),
The original anti-sense primers of internal standard MS2：5’-TAACAATAAGCTCGCAGTCG-3’(SEQ ID NO：12)；
In order to be distinguished to rat RTV, TMEV, RCV, PVM, M.pulmonis pathogen, thus by above-mentioned primer make 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 RTV are：5’-ACTTATTTCTTCACTACTATATCA-3’(SEQ ID NO：13)；
The tag sequences of TMEV are：5’-TTAAACAATCTACTATTCAATCAC-3’(SEQ ID NO：14)；
The tag sequences of RCV are：5’-CACTTAATTCATTCTAAATCTATC-3’(SEQ ID NO：15)；
The tag sequences of PVM are：5’-ATACTTTACAAACAAATAACACAC-3’(SEQ ID NO：16)；
The tag sequences of M.pulmonis are：5’-TACTACTTCTATAACTCACTTAAA-3’(SEQ ID NO：17)；
The tag sequences of internal standard MS2 are：5’-ATTAAACAACTCTTAACTACACAA-3’(SEQ ID NO：18).
In addition, 5 ' ends of each original anti-sense primer of item are also added with biotin labeling.
Embodiment 2 while the kit for detecting five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis
Kit includes following components：
1) primer of rat RTV, TMEV, RCV, PVM, M.pulmonis pathogen is detected while designed by embodiment 1 Group；
2) fluorescence-encoded micro-beads for including anti-tag sequences of the different iridescent of 6 kinds of codings, the anti-tag sequences Row can correspondingly with the tag sequence complementary pairings in multi-fluorescence immunoassay primer；6 kinds of microballoons are purchased from luminex companies, Wherein the corresponding fluorescence-encoded micro-beads number of RTV, TMEV, RCV, PVM, M.pulmonis and IC are MTAG-A034, MTAG- A046, MTAG-A028, MTAG-019, MTAG-029 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 the nucleic acid samples simultaneously containing five kinds of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis；The negative control be without There are the nucleic acid samples of five kinds of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis.
It embodiment 3 while detects the multi-fluorescences of five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis and exempts from The foundation of epidemic disease analysis method
(1) nucleic acid extraction
It extracts instrument (Tiangeng company) automatically with nucleic acid and extracts five kinds of cause of diseases of RTV, TMEV, RCV, PVM, M.pulmonis respectively The RNA or DNA of body, the template as multi-fluorescence immunoassay detection method.Wherein internal standard MS2 standard items are added to each sample In, the synchronous extraction for participating in sample nucleic, sample-adding, RT-PCR is expanded and the overall process of signal detection.
(2) preparation of plasmid standard
Using the RN or DNA of five kinds of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis as masterplate, respectively with embodiment 1 Amplified production is carried out agarose and coagulated by corresponding original primers respectively to carrying out RT-PCR amplifications in designed original primers group Gel electrophoresis detect, and target fragment is recycled with plastic recovery kit, and the target fragment of recycling is connected to pMD-19T carriers, and is turned Change into DH5 α competent cells, with the LB agar plate screening positive clones containing Amp, positive colony bacterium is identified with PCR, and To positive recombinant plasmid sequence verification.With plasmid extraction kit extract plasmid, micro ultraviolet specrophotometer measured concentration with Copy number is calculated according to the following equation in purity.Copy number (copies/ μ L)=6.022 × 1023(copies/moL)×DNA Concentration (g/ μ L)/mass M W (g/moL).Wherein, MW=DNA bases number (bp) × 660daltons/bp, DNA base number=load Body series number+insetion sequence base number.
(3) multiplex RT-PCR amplification
With the primer sets designed by embodiment 1, respectively to RTV, TMEV, RCV, PVM, M.pulmonis and MS2 (internal standard) Multiplex RT-PCR amplification is carried out, multiple RT-PCR reagent uses the OneStep RT-PCR Kit of Qiangen companies, tests every time Positive control, negative control and blank control be set simultaneously, wherein positive control with contain the tissue of above-mentioned five kinds of pathogen or The nucleic acid of cell culture extraction is as positive control template, and wherein negative control is not to contain above-mentioned five kinds of pathogen nucleic acid samples Product (can be intact animal tissue or normal cell culture) are used as negative control template, and blank control is to be not added with template pair According to (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 6 kinds of microballoons for being coated with special anti-tag sequences, wherein anti-tag sequences It correspondingly can mutually be recruited with the tag sequences on five kinds of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis and internal standard MS2 primers It is right.6 kinds of microballoons are purchased from luminex companies, and specific RTV, TMEV, RCV, PVM, M.pulmonis and IC are corresponding glimmering Pumped FIR laser microballoon number is MTAG-A034, MTAG-A046, MTAG-A028, MTAG-019, MTAG-029 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.During the MFI values of detector five kinds of pathogen of reading, different types of pathogen 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
It is thin with RTV, TMEV, RCV, PVM, M.pulmonis, mouse reovirus type III (Reo-3) and mouse respectively Small virus MVM plants (MVM), mouse norovirus (MNV), minute parvovirus of mice MPV plants (MPV), lymphatic choroid plexus brain Film scorching viral (LCMV), sendai virus (SV), 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 The RNA or DNA of malicious (MCMV) carry out multi-fluorescence immunoassay detection as template, each sample addition internal standard MS2 as Quality Control, Negative control and blank control are set simultaneously, 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 RTV, TMEV, RCV, PVM, M.pulmonis are the positive, and there is no hand over Fork reaction, other pathogens are feminine gender, illustrate that the method for the present invention and kit specificity are good.
Embodiment 5, sensitivity test
By the plasmid standard Easy of five pathogen of RTV, TMEV, RCV, PVM, M.pulmonis of preparation Dilution (Takara companies) does 10 times and is serially diluted, and obtains 1 × 107~1 × 100Copies/ μ L series standard templates are used The multi-fluorescence immunoassay method of above-mentioned foundation 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 RTV, The lowest detection of TMEV, RCV, PVM, M.pulmonis are limited to 1 × 102copies/μL。
The detection of embodiment 6, clinical sample
Sample to be tested is 24 parts of rat samples (number 1-24) that In Guangdong Province is collected, and is extracted in Rats Organs and Tissues and caecum Tolerant RNA/DNA, each sample adds internal standard MS2 as Quality Control, while sets positive control, negative control and blank control, 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 2,5,7,8,11,12,13,22,23 is negative sample；Sample 9, 10th, 20 be RTV nucleic acid positive samples；Sample 3,4 is TMEV nucleic acid positive samples；Sample 16,17,18,19 is positive for RCV nucleic acid Sample；Sample 15,21,24 is PVM nucleic acid positive samples；Sample 1,6,14 is M.pulmonis nucleic acid positive samples.Positive sample This 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>The primer sets, kit and Multiple immunizations fluorescence analysis method of five kinds of pathogen of rat are detected simultaneously
<130>
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<170> PatentIn version 3.5
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Claims (10)

1. detect the primer sets of five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis simultaneously, including 6 pairs of primers, 6 pairs of primers are by 6 pairs of original primers modify；Described be modified in each pair original primers one original Arm connects between 5 ' ends of primer pass through with 3 ' ends of corresponding tag sequences, 5 ' end addition biotin marks of another original primers Note；
The original primers are to as follows：
The original sense primers of RTV：5 '-GGAAACTTAGAGCAGACATCG-3 ',
The original anti-sense primers of RTV：5 '-TCCACAGAGAACAACCATCG-3 ',
The original sense primers of TMEV：5 '-CGTTGGAAAAGCACCTCTCAC-3 ',
The original anti-sense primers of TMEV：5 '-TCGGAGTCGGTTGACTTAGAT-3 ',
The original sense primers of RCV：5 '-TGGMATCCTCAAGAAGACCACTT-3 ',
The original anti-sense primers of RCV：5 '-ATGCCMGAAAACCARGAGTAATG-3 ',
The original sense primers of PVM：5 '-GCTGGCATTCCACATAACC-3 ',
The original anti-sense primers of PVM：5 '-CCCACAAGGTACACGGAGA-3 ',
The original sense primers of M.pulmonis：5 '-GGAAATGCCCTAAGTATGACGG-3 ',
The original anti-sense primers of M.pulmonis：5 '-CGGATAACGCTTGCACCCTA-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：
Tag sequences in the original upstream and downstream primers of RTV are：5 '-ACTTATTTCTTCACTACTATATCA-3 ',
Tag sequences in the original upstream and downstream primers of TMEV are：5 '-TTAAACAATCTACTATTCAATCAC-3 ',
Tag sequences in the original upstream and downstream primers of RCV are：5 '-CACTTAATTCATTCTAAATCTATC-3 ',
Tag sequences in the original upstream and downstream primers of PVM are：5 '-ATACTTTACAAACAAATAACACAC-3 ',
Tag sequences in the original upstream and downstream primers of M.pulmonis are：5 '-TACTACTTCTATAACTCACTTAAA-3 ',
Tag sequences in the original upstream and downstream primers of internal standard MS2 are：5’-ATTAAACAACTCTTAACTACACAA-3’.
2. primer sets according to claim 1, it is characterised in that：Described arm is repaiied among the primer for 12~18 atoms Jewelry.
3. primer sets according to claim 2, it is characterised in that：Described arm arm between six ethylene glycol.
4. the kit of five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis is detected simultaneously, it is characterised in that：Institute It states kit and contains primer sets described in claim 1, the fluorescence-encoded micro-beads of the different iridescent of 6 kinds of codings, the 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 The nucleic acid samples of five kinds of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis；The negative control for do not contain RTV, TMEV, The nucleic acid samples of five kinds of pathogen of RCV, PVM, M.pulmonis.
6. the Multiple immunizations fluorescence analysis side of five kinds of pathogen of rat RTV, TMEV, RCV, PVM, M.pulmonis is detected simultaneously Method includes the following steps：
1) pathogen RNA or DNA are extracted from sample, as template, while sets blank control, negative control, mixing positive Control；The blank control is not add template；The negative control be with do not contain RTV, TMEV, RCV, PVM, The nucleic acid samples of five kinds of pathogen of M.pulmonis are template；It is described mixing positive control be with simultaneously contain RTV, TMEV, The nucleic acid samples of five kinds of pathogen of RCV, PVM, M.pulmonis 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 6 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 pathogens；
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 × buffer4 μ L, dNTP0.8 μ L, Enzyme mix0.8 μ L, each 0.4 μ L of 10 μM of each pair primer mixed liquor in primer sets, 4 μ L of template, without 8 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：6 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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