CN106755589A - Five kinds of primer sets of pathogen of rat, kit and Multiple immunizations fluorescence analysis method are detected simultaneously - Google Patents

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

Info

Publication number
CN106755589A
CN106755589A CN201710044031.0A CN201710044031A CN106755589A CN 106755589 A CN106755589 A CN 106755589A CN 201710044031 A CN201710044031 A CN 201710044031A CN 106755589 A CN106755589 A CN 106755589A
Authority
CN
China
Prior art keywords
kinds
pathogen
original
tmev
rtv
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201710044031.0A
Other languages
Chinese (zh)
Other versions
CN106755589B (en
Inventor
黄韧
袁文
郭鹏举
张钰
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangdong Laboratory Animals Monitoring Institute
Original Assignee
Guangdong Laboratory Animals Monitoring Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangdong Laboratory Animals Monitoring Institute filed Critical Guangdong Laboratory Animals Monitoring Institute
Publication of CN106755589A publication Critical patent/CN106755589A/en
Application granted granted Critical
Publication of CN106755589B publication Critical patent/CN106755589B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Abstract

The invention discloses five kinds of primer sets of pathogen of detection rat, kit and Multiple immunizations fluorescence analysis method simultaneously.Be combined for multiple PCR technique and Luminex liquid-phase chip technologies by the present invention, and devising can be while detects rat RTV, TMEV, RCV, PVM, M.pulmonisThis five kinds of primer sets 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.The method have rapidly and efficiently, the beneficial effect such as high specificity, sensitivity is high, reproducible, can be applied to quality-monitoring, epidemiology survey and the early warning of experimental rat.Flexibility of the present invention is good, and the species of detection cause of disease can be on this basis added and subtracted as needed.

Description

Five kinds of primer sets of pathogen of rat, kit and Multiple immunizations fluorescence are detected simultaneously Analysis method
Technical field
The invention belongs to field of biological detection, more particularly, to five kinds of primer sets of pathogen of detection rat, examination 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 Test animal to cause harm in itself, potential interference is also resulted in research work.It is in subclinical infection more experimental rat pathogenic microorganism, does not have There are obvious clinical symptoms, disease diagnosis are relatively difficult.And zoogenetic infection pathogen often shows as persistent infection, facility Once exist pathogenic infection than it is more difficult to remove, it is necessary to pass through to detect and eliminate (test-and-removal) method could be from receiving The thorough eradication of pathogens of facility of pollution, therefore, experimental animal pathogen detection method rapidly and efficiently is set up, timely and accurately Find the cause of disease infection, controls and remove epidemic situation, extremely important for improving Quality of Experimental Animals.
SPF grades of rat specified in China experimental animal standard GB/T 14922.2-2011 need to exclude 21 kinds of micro- lifes of cause of disease Thing.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 Standard needs the cause of disease for excluding.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 conventional environment under raise rat in RTV, TMEV 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 listed in detection project in family's standard, some experimental animals produce or use unit, Yi Jiyi A little CRO companies are all classified as it examination project.
At present, Serology test such as enzyme linked immunological of the domestic Diagnosis of Viral Infections method primarily directed to antibody test Adsorption test (ELISA), immuno-enzymatic test (IEA) and immunofluorescent test (IFA) etc., but be may not apply in these methods The detection of immunologic hypofunction or immunodeficient rats, because they can not produce 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 can not meet experiment Animal quality detection demand, the molecular biological testing detection virus based on PCR has quick, sensitive, special etc. Feature, is the conventional detection method for clinically detecting and diagnosing the illness at present.But regular-PCR method needs to uncap to carry out product Gel electrophoresis analysis, operating method not enough simplifies, and easily produces Aerosol Pollution, causes false positive occur.Real-time fluorescence Quantitative PCR technique pollutes the important means that the advantages of lacking is current Diagnosis of Viral Infections because of pinpoint accuracy, high sensitivity.But it is real When Fluorescence PCR assay limited by fluorescent species and instrument itself, 5 targets can only at most be detected, and test into Greatly, cost is also of a relatively high for the difficulty of work(.
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 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 detection rat five kinds of pathogen of 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：
Rat five kinds of primer sets of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis are detected simultaneously, including 6 pairs are drawn Thing, 6 pairs of primers are formed by carrying out modification to 6 pairs of original primers；One be modified in each pair original primers Arm is connected between 5 ' ends of original primers pass through with 3 ' ends of corresponding tag sequences, and 5 ' end additions of another original primers are biological Element mark；
Described 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’；
Described 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 trims in the middle of 12~18 primers of atom between described.
Preferably, arm between arm is six ethylene glycol between described.
Rat five kinds of kits of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis are detected simultaneously, it is characterised in that： The kit contains and detect simultaneously five kinds of primer sets 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, described independent fluorescence coding microball contains complementary with tag sequences in above-mentioned primer The anti-tag sequences of pairing.
Further, mentioned reagent box is also containing SA-PE 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, Five kinds of nucleic acid samples of pathogen of M.pulmonis；The negative control for do not contain RTV, TMEV, RCV, PVM, Five kinds of nucleic acid samples of pathogen of M.pulmonis.
The Multiple immunizations fluorescence analysis side of rat five kinds of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis is detected simultaneously Method, comprises the following steps：
1) pathogen RNA or DNA are extracted from sample, as template, while setting blank, negative control, mixing Positive control；The blank is without template；The negative control be with do not contain RTV, TMEV, RCV, PVM, Five kinds of nucleic acid samples of pathogen of M.pulmonis are template；It is described mixing positive control be with simultaneously contain RTV, TMEV, Five kinds of nucleic acid samples of pathogen of RCV, PVM, M.pulmonis are template；
2) add the nucleic acid of internal standard MS2 as Quality Control in the sample, RT-PCR is carried out with the primer sets in above-mentioned kit Amplification；
3) 6 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 different pathogens MFI values；
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；
The above 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 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 8.
Further, step 2) in, the response procedures of RT-PCR amplifications are：48~52 DEG C of 25~35min of reverse transcription；95℃ Predegeneration 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 10min eventually.
Further, step 3) in, 100 μ L reaction systems of hybridization contain：6 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 beneficial effects of the invention are as follows：
Be combined for multiple PCR technique and Luminex liquid-phase chip technologies by the present invention, and devising one kind can be while detects big Mouse RTV, TMEV, RCV, PVM, M.pulmonis this five kinds of primer sets of pathogen, are obtained using the primer sets by specific PCR Target amplification product, is then hybridized amplified production, fluorescence-encoded micro-beads and SA-PE, is passed through When Luminex detectors read MFI values, so as to differentiate different types of pathogen.The method have rapidly and efficiently, specificity The beneficial effect such as by force, sensitivity is high, reproducible, can be applied to quality-monitoring, epidemiology survey and the early stage of experimental rat Early warning.It is specific as follows：
1) experimental animal common detection methods are unitem detections at present, 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 detection project can flexibly be increased according to increasing for cause of disease；Sample consumption is few simultaneously, 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 stronger.
3) liquid-phase chip utilizes biotin-avidin signal amplifying system, and its affinity is up to 1015L/moL, than simple Affinity of antibody is high by 104More than times, make testing result it is sensitiveer, by environmental disturbances are smaller, stability is high；The inspection of the inventive method Survey remolding sensitivity regular-PCR 1~2 order of magnitude high.
4) Luminex liquid-phase chip technologies overcome piece membrane DNA chip in macromolecular due to being reacted in the solution using microballoon Kinetics is influenceed by surface tension, three-dimensional effect etc. during detection, the repeatability of sample detection is substantially increased, is detected Reliable results stabilization；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 cause of disease can be on this basis added and subtracted as needed.
Brief description of the drawings
Fig. 1：5 kinds of RT-PCR electrophoretograms 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, M：DL500DNA marker】；
Fig. 2：5 kinds of multi-fluorescence immunoassay method test experience result figures of rat pathogen are detected simultaneously；
Fig. 3：The multi-fluorescence immunoassay method of 5 kinds of rat pathogen of detection detects specificity experiments result figure simultaneously；
Fig. 4：5 kinds of multi-fluorescence immunoassay method detection sensitivity experimental result pictures of rat pathogen are detected simultaneously；
Fig. 5：5 kinds of multi-fluorescence immunoassay method clinical sample test experience results of rat pathogen are 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 does not describe in detail be this area routine operation.
Embodiment 1, while detect rat five kinds of primer sets of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis
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 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：AF125582) and MS2 coating egg White gene order (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 No more 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 is selected between 100~250bp, so advantageously reduced 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 The ability that sequence forms primer dimer is analyzed, and have selected tag sequences most suitable with original primers sequence.
Due in multiplex PCR system multiple primer 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 SA-PE Also very strong fluorescence signal can be inspired after effect, negative background's fluorescence very high can be thus produced, the judgement of result is influenceed, Even result in test failure.Therefore design of primers is key of the invention, is also the base for carrying out Luminex liquid-phase chip detections Plinth.
Inventor have passed through substantial amounts of experiment 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 it is immune detect simultaneously rat RTV, TMEV, RCV, PVM, Preferably, the preferred primer sets are by following original primers to further modification for the specificity of M.pulmonis pathogen and sensitivity 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 made a distinction to rat RTV, TMEV, RCV, PVM, M.pulmonis pathogen, thus by above-mentioned primer make into The modification of one step, is required with meeting corresponding operation.In 5 ' the corresponding tag sequences of end connection of the original sense primer of each bar, its Connected by arm 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 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 bar are also added with biotin labeling.
Embodiment 2, while detect rat five kinds of kits of pathogen of 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) 6 kinds of fluorescence-encoded micro-beads for including anti-tag sequences for encoding different iridescent, the anti-tag sequences Row can correspondingly with multi-fluorescence immunoassay primer in tag sequence complementary pairings；6 kinds of microballoons are purchased from luminex companies, It is MTAG-A034, MTAG- that wherein RTV, TMEV, RCV, PVM, M.pulmonis and IC distinguish corresponding fluorescence-encoded micro-beads number A046, MTAG-A028, MTAG-019, MTAG-029 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 To contain five kinds of nucleic acid samples of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis simultaneously；The negative control be without There are five kinds of nucleic acid samples of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis.
Embodiment 3, while detect rat five kinds of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis multi-fluorescence exempt from The foundation of epidemic disease analysis method
(1) nucleic acid extraction
Extract instrument (Tiangeng company) automatically with nucleic acid and extract five kinds of cause of diseases of RTV, TMEV, RCV, PVM, M.pulmonis respectively The RNA or DNA of body, as the template of multi-fluorescence immunoassay detection method.Wherein internal standard MS2 standard items are added to each sample In, the overall process of the synchronous extraction for participating in sample nucleic, sample-adding, RT-PCR amplifications and signal detection.
(2) preparation of plasmid standard
RN or DNA with five kinds of pathogen of RTV, TMEV, RCV, PVM, M.pulmonis as masterplate, respectively with embodiment 1 Corresponding original primers amplified production are carried out agarose respectively and coagulated to carrying out RT-PCR amplifications in designed original primers group Gel electrophoresis detection, purpose fragment is reclaimed with glue reclaim kit, the purpose fragment of recovery is connected into pMD-19T carriers, and turn 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.Extract plasmid with plasmid extraction kit, micro ultraviolet specrophotometer determine concentration with Purity, is calculated according to the following equation copy number.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 is tested every time using the OneStep RT-PCR Kit of Qiangen companies Positive control, negative control and blank be set simultaneously, wherein positive control with the tissue containing above-mentioned five kinds of pathogen or Used as positive control template, wherein negative control is not containing above-mentioned five kinds of pathogen nucleic acid samples for the nucleic acid that cell culture is extracted Used as negative control template, blank is and is not added with template pair product (can be intact animal tissue or normal cell culture) According to (No Template Control, NTC), i.e., replace template with water 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 predegeneration 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 10min eventually.
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 6 kinds of microballoons for being coated with special anti-tag sequences, wherein anti-tag sequences 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 difference is 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。
Abundant resuspended fluorescence-encoded micro-beads working solution, each sample well and control wells add the μ L of microballoon working solution 20, then 5 μ L PCR primers are added in sample well, 5 μ L PCR positive controls, negative control and blank are also separately added into 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 50 above-mentioned reaction solutions of μ L detected, 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 3 times of MFI values of MFI values more than or equal to blank of sample, 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
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 The scorching virus (LCMV) of film, 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, and each sample adds internal standard MS2 as Quality Control, Negative control and blank are set simultaneously, and 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 RTV, TMEV, RCV, PVM, M.pulmonis is the positive, and in the absence of friendship Fork reaction, other pathogens are feminine gender, illustrate that the inventive method 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, use The multi-fluorescence immunoassay method of above-mentioned foundation 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 RTV, The lowest detection of TMEV, RCV, PVM, M.pulmonis is limited to 1 × 102copies/μL。
The detection of embodiment 6, clinical sample
Sample to be tested is 24 parts of rat samples (numbering is 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 positive control, negative control and blank are set, 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 2,5,7,8,11,12,13,22,23 is negative sample；Sample 9, 10th, 20 is RTV nucleic acid positive samples；Sample 3,4 is TMEV nucleic acid positive samples；Sample 16,17,18,19 is positive 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 passes through sequencing analysis, as a result unanimously.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment Limitation, it is other it is any without departing from Spirit Essence of the 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>Five kinds of primer sets of pathogen of rat, kit and Multiple immunizations fluorescence analysis method are detected simultaneously
<130>
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
ggaaacttag agcagacatc g 21
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
tccacagaga acaaccatcg 20
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
cgttggaaaa gcacctctca c 21
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
tcggagtcgg ttgacttaga t 21
<210> 5
<211> 23
<212> DNA
<213>Artificial sequence
<400> 5
tggmatcctc aagaagacca ctt 23
<210> 6
<211> 23
<212> DNA
<213>Artificial sequence
<400> 6
atgccmgaaa accargagta atg 23
<210> 7
<211> 19
<212> DNA
<213>Artificial sequence
<400> 7
gctggcattc cacataacc 19
<210> 8
<211> 19
<212> DNA
<213>Artificial sequence
<400> 8
cccacaaggt acacggaga 19
<210> 9
<211> 22
<212> DNA
<213>Artificial sequence
<400> 9
ggaaatgccc taagtatgac gg 22
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence
<400> 10
cggataacgc ttgcacccta 20
<210> 11
<211> 19
<212> DNA
<213>Artificial sequence
<400> 11
cgcagaatcg caaatacac 19
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<400> 12
taacaataag ctcgcagtcg 20
<210> 13
<211> 24
<212> DNA
<213>Artificial sequence
<400> 13
acttatttct tcactactat atca 24
<210> 14
<211> 24
<212> DNA
<213>Artificial sequence
<400> 14
ttaaacaatc tactattcaa tcac 24
<210> 15
<211> 24
<212> DNA
<213>Artificial sequence
<400> 15
cacttaattc attctaaatc tatc 24
<210> 16
<211> 24
<212> DNA
<213>Artificial sequence
<400> 16
atactttaca aacaaataac acac 24
<210> 17
<211> 24
<212> DNA
<213>Artificial sequence
<400> 17
tactacttct ataactcact taaa 24
<210> 18
<211> 24
<212> DNA
<213>Artificial sequence
<400> 18
attaaacaac tcttaactac acaa 24

Claims (10)

1. rat RTV, TMEV, RCV, PVM, M. are detected simultaneouslypulmonisFive kinds of primer sets of pathogen, including 6 pairs of primers, 6 pairs of primers are formed by carrying out modification to 6 pairs of original primers；One be modified in each pair original primers is original Arm is connected between 5 ' ends of primer pass through with 3 ' ends of corresponding tag sequences, another 5 ' end addition biotin marks of original primers Note；
Described 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 ',
M. pulmonisOriginal sense primer：5 '-GGAAATGCCCTAAGTATGACGG-3 ',
M. pulmonisOriginal anti-sense primer：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’；
Described 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 ',
M. pulmonisTag sequences be：5 '-TACTACTTCTATAACTCACTTAAA-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 be repaiied in the middle of 12 ~ 18 primers of atom Jewelry.
3. primer sets according to claim 2, it is characterised in that：Described arm is arm between six ethylene glycol.
4. rat RTV, TMEV, RCV, PVM, M. are detected simultaneouslypulmonisFive kinds of kits of pathogen, it is characterised in that： The kit contains the fluorescence-encoded micro-beads of the different iridescent of primer sets described in claim 1,6 kinds of codings, and described is only Vertical fluorescence-encoded micro-beads contain 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 RTV, TMEV, RCV, PVM, M.pulmonisFive kinds of nucleic acid samples of pathogen；The negative control for do not contain RTV, TMEV、RCV、PVM、M. pulmonisFive kinds of nucleic acid samples of pathogen.
6. rat RTV, TMEV, RCV, PVM, M. are detected simultaneouslypulmonisFive kinds of Multiple immunizations fluorescence analysis sides of pathogen Method, comprises the following steps：
1）Pathogen RNA or DNA are extracted from sample, as template, while setting blank, negative control, the mixing positive Control；The blank is without template；The negative control is not contain RTV, TMEV, RCV, PVM, M.pulmonisFive kinds of nucleic acid samples of pathogen are template；It is described mixing positive control be with simultaneously contain RTV, TMEV, RCV, PVM、M. pulmonisFive kinds of nucleic acid samples of pathogen 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 6 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 pathogens；
5）Result judges：When MFI value >=3.0 of the MFI values/blank of sample, the positive is judged to, is otherwise judged to feminine gender；
The above 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 μ L reaction systems of RT-PCR amplifications contain Have：Each pair primer mixed liquor in 5 × buffer4 μ L, dNTP0.8 μ L, Enzyme mix0.8 μ L, primer sets（10µM）Each 0.4 μ L, the μ L of template 4, without the μ L of RNase water 8.
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 predegeneration 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 10min eventually.
9. method according to claim 6, it is characterised in that：Step 3）In, 100 μ L reaction systems of hybridization contain：6 kinds Encode the μ L of fluorescence-encoded micro-beads 20, the μ L of SA-PE 75, the μ L of amplified production 5 of different iridescent.
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.
CN201710044031.0A 2017-01-10 2017-01-19 The primer sets, kit and Multiple immunizations fluorescence analysis method of five kinds of pathogen of rat are detected simultaneously Active CN106755589B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710018758 2017-01-10
CN2017100187581 2017-01-10

Publications (2)

Publication Number Publication Date
CN106755589A true CN106755589A (en) 2017-05-31
CN106755589B CN106755589B (en) 2018-05-25

Family

ID=58945029

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710044031.0A Active CN106755589B (en) 2017-01-10 2017-01-19 The primer sets, kit and Multiple immunizations fluorescence analysis method of five kinds of pathogen of rat are detected simultaneously

Country Status (1)

Country Link
CN (1) CN106755589B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107312873A (en) * 2017-06-28 2017-11-03 广东省实验动物监测所 A kind of multiple liquid phase genetic chip detection primer, kit and method of 5 kinds of Respiratory Tract of Mice cause of diseases of quick differentiation
CN108004338A (en) * 2017-12-22 2018-05-08 杭州医学院 Detect the Primer composition and application and the product and detection SPF mouse pathogen methods using it of SPF mouse pathogens

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105331741A (en) * 2015-11-13 2016-02-17 广东省实验动物监测所 HRM detection method and primer for quick identification of mouse encephalomyelitis virus and rat Theiler virus
CN106191311A (en) * 2016-07-19 2016-12-07 广东省实验动物监测所 A kind of quick detection Cavia porcellus LCMV, SV, PVM, Reo 3 virus multiple liquid phase method for gene chip and reagent

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105331741A (en) * 2015-11-13 2016-02-17 广东省实验动物监测所 HRM detection method and primer for quick identification of mouse encephalomyelitis virus and rat Theiler virus
CN106191311A (en) * 2016-07-19 2016-12-07 广东省实验动物监测所 A kind of quick detection Cavia porcellus LCMV, SV, PVM, Reo 3 virus multiple liquid phase method for gene chip and reagent

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DANIELA HOFLER等: "A Bead-Based Multiplex Assay for the Detection of DNA Viruses Infecting Laboratory Rodents", 《PLOS ONE》 *
WEN YUAN等: "Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler’s murine encephalomyelitis virus and rat theilovirus", 《JOURNAL OF VIROLOGICAL METHODS》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107312873A (en) * 2017-06-28 2017-11-03 广东省实验动物监测所 A kind of multiple liquid phase genetic chip detection primer, kit and method of 5 kinds of Respiratory Tract of Mice cause of diseases of quick differentiation
CN107312873B (en) * 2017-06-28 2018-07-24 广东省实验动物监测所 A kind of multiple liquid phase genetic chip detection primer, kit and method of 5 kinds of Respiratory Tract of Mice cause of diseases of quick differentiation
CN108004338A (en) * 2017-12-22 2018-05-08 杭州医学院 Detect the Primer composition and application and the product and detection SPF mouse pathogen methods using it of SPF mouse pathogens
CN108004338B (en) * 2017-12-22 2021-03-12 杭州医学院 Primer composition for detecting SPF mouse pathogenic bacteria, application and product using primer composition and method for detecting SPF mouse pathogenic bacteria

Also Published As

Publication number Publication date
CN106755589B (en) 2018-05-25

Similar Documents

Publication Publication Date Title
Reslova et al. xMAP technology: applications in detection of pathogens
CN104017908B (en) The rapid gene type identification and analysis of human papilloma virus and device thereof
CN106399517B (en) Nucleic acid detection technology combining multi-cross constant-temperature amplification with gold nano biosensing
CN106957927A (en) African swine fever fluorescence PCR detection reagent, African swine fever fluorescence PCR detection reagent kit and its application
CN106544434B (en) Method for detecting Listeria monocytogenes by combining multi-cross amplification with gold nano biosensing
Li et al. Rapid detection of Brucella spp. and elimination of carryover using multiple cross displacement amplification coupled with nanoparticles-based lateral flow biosensor
CN106282416B (en) A kind of the multi-fluorescence immunoassay primer, kit and method of 5 kinds of fowl immunosupress cause of diseases of quick differentiation
CN111518948B (en) Method for detecting SARS-CoV-2 by reverse transcription multi-cross displacement amplification combined with nano biological sensing
KR102493424B1 (en) A method of detecting rna
CN106636474B (en) Six kinds of viral primer sets of Multiple immunizations fluoroscopic examination mouse, kit and method
Barclay et al. Hydrogel particles improve detection of SARS-CoV-2 RNA from multiple sample types
CN106755589B (en) The primer sets, kit and Multiple immunizations fluorescence analysis method of five kinds of pathogen of rat are detected simultaneously
CN106636454A (en) Real-time fluorescence multi-RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for simultaneously detecting human coronaviruses 229E, OC43, NL63 and HKU1
CN108350511A (en) Improved composition and method for detecting virus
CN113186341B (en) CRISPR-mediated one-step constant-temperature amplification SARS-CoV-2 detection method
CN101177714A (en) Monocyte hyperplasia Listeria PCR detecting method adding amplified interior label
CN107326098B (en) Multiplex fluorescence immunoassay method and reagent for rapidly distinguishing rabbit plague virus, sendai virus and rabbit rotavirus
CN106011313B (en) A kind of the multi-fluorescence immunoassay method and reagent of quick differentiation ILTV, IBV, MG and MS
CN103773894B (en) For detecting the dual probe determination method of HCV
CN105603081B (en) Non-diagnosis-purpose qualitative and quantitative detection method for intestinal microorganisms
CN106319091B (en) The multi-fluorescence immunoassay method and kit of a kind of quick differentiation canine distemper virus, canine parvovirus and rabies viruses
CN106086241B (en) A kind of primer, kit and the method for the multi-fluorescence immunoassay of 4 kinds of fowl respiratory pathogens of quick differentiation
CN106544447B (en) A kind of Multiple immunizations fluorescence analysis primer, kit and method for detecting chicken Marek&#39;s disease virus and chicken infectious anemia virus
KR20220048385A (en) Reverse transcriptase nested polymerase chain reaction for the detection of Aichivirus-A from water environment and positive control for confirmation of the false positive reaction
CN106191312B (en) A kind of quick multi-fluorescence immunoassay method and reagent for distinguishing AIV, NDV, MG and MS

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant