CN108048584A - The brucellar probe of RAA Fluorometric assays and kit - Google Patents
The brucellar probe of RAA Fluorometric assays and kit Download PDFInfo
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Abstract
The present invention discloses a kind of detection brucella RAA Fluorometric assay kits, and detection kit includes following components：The general reaction reagent of RAA basic fluorescences, reaction buffer, probe and primer mixture, negative quality-control product, positive quality control product and critical positive quality control product.The probe is the terminal sequence of 5 ' terminal sequences/fluorescent reporter group //idSp//quenching group/3 '；This kit can directly detect brucella DNA, be used especially for the relatively low sample of detection brucella DNA content；This kit carries out isothermal duplication detection at 30-42 DEG C, and 5-20min completes detection.Kit is easy to operate, and cooperation miniature instrument, easy to carry has the advantages that quick, sensitive, accurate, reproducible；It is suitble to base and Site Detection, the good application prospect of tool.
Description
Technical field
The invention belongs to molecular Biological Detection fields, and in particular to a kind of detection brucella RAA Fluorometric assays
Probe, primer and kit.
Background technology
Brucellosis (brucellosis) abbreviation cloth disease, also known as " Mediterranean fruit fly ", " Malta fever ", " ripple latent heat "
Deng being by domestic animals such as a kind of brucella (Brucella) infected cattle, sheep, pigs, pass through respiratory tract, alimentary canal, skin and life
Infecting both domestic animals and human hyperinfection disease caused by growing system etc..
Brucella is a kind of Gram-negative bacteria, its main feature is that not moving, almost without pod membrane, (smooth type has micro- pod
Film), thermophilic oxygen bacterium, oxidizing ferment, catalase-positive can reduce nitrate, cytozoicus, the interior survival of the carcass that can be in.It is wrapped altogether
Include six kinds：Brucella bovis (bacillus abortus), sheep type brucella (Maltese brucella), pig cloth
Lu Shi bacillus, dog brucella, pack rat brucella and Brucella ovis etc..Cloth disease principal pathogenetic is non-in the Middle East
Continent, Latin America, the several areas in the Central Asia and Mediterranean periphery, global brucella patient is up to 500,000 or so, epidemic situation
It is on the rise, agriculture district is propagated to even to the trend of Urban Sprawl from pastoral area, half pastoral area.After this bacterium intrusion human body, with lymph
Lymph node is reached, is swallowed by phagocyte, if cloth bacterium is not killed by phagocyte, then bacterium is in intracellular growth and breeding, formation office
Portion's primary lesion.This stage someone is known as lymph source property nomadic phase, is equivalent to incubation period, and incubation period is 7-60 days, and averagely two
In week, small number of patients was up to several months or 1 year or more.Bacterium amount reproduction in phagocyte causes phagocyte to rupture, with big
Amount bacterium enters lymph and blood circulation forms bacteremia.Bacterium is swallowed again by hemophagocyte in blood, and with
Blood flow is brought to whole body, and breeding, forms multiple venereal disease in monokaryon-mononuclear phagocyte system of liver, spleen, lymph node, marrow etc.
Stove causes clinically not only have bacteremia, septicemia, but also has the performance of toxaemia.After the regular period, focus of infection
Bacterial growth breeding enters blood again, causes palindromia.Histopathological lesions are extensive.Clinical manifestation also just variation.It is so anti-
Become chronic infection again, chronic phase is invaded more and backbone and large joint, shows as long-term fever (including low-heat), hidrosis, arthralgia
Suspects and the signs such as simultaneous or liver, spleen, lymph node and testis enlargement.Epidemic situation after eighties of last century China experience is very popular twice
Downward trend, but the beginning of this century are begun with, epidemic situation has rebound momentum again.At the beginning of 2007, have 25 provinces, cities and autonomous regions' hairs
Brucellosis between the existing human world and poultry.According to statistics China by the number of brucella infection potential risk up to 3.5 hundred million, often
About 30,000 people of year neopathy number of cases.Cloth disease is China's one of infectious disease of number of the infected rapid development in recent years.At present《Middle Chinese
People republic law on the prevention and control of infectious diseases》Brucellosis is classified as Category B notifiable disease, it is more known " non-with us
Allusion quotation ", " swine flu ", " anthrax ", " AIDS ", " rabies ", " hepatitis B " etc. belong to a kind of infectious disease together.
It the Center for Disease Control (center for disease control, CDC) therefore is also considered as it and can be made
Chemical and biological weapons, and it is defined as " B class bioterrorisms medicament ".It can be seen that cloth disease has huge potential threat people
Health, it is a serious public health problem, and serious economic loss and higher Global Health is triggered to bear
Load, prevention cloth disease, which is propagated, becomes China and global common mission.
At present, brucellar traditional sensing techniques mainly include pathogen separation technology, cultural character identification technology, blood
It is clear to learn the technologies such as detection technique, immunology detection technique and molecular biology.
Brucellosis pathogeny detection is to be separately cultured brucella from the blood or tissue of infection, using admittedly
Body culture medium such as serum glucose agar, tryptose soya agar culture medium is cultivated, and testing principle is by cloth Lu Shi
Thalli morphology, dyeing characteristic, colonial morphology, growth characteristics, biochemical reaction feature and the brucella Anti-TNF-α of bacillus
The methods of body agglutination test, is differentiated.The method method is brucellosis diagnosis " goldstandard ", and cost is relatively low, tool
Difinite quality and quantitative analysis feature.But exist simultaneously separation rate than it is relatively low, it is easy pollution, take it is longer the problems such as, and separate cloth
Lu Shi bacillus, there are bio-safety risk, thus limits the use of this method to environment and staff.
Serum inspection is a kind of medically common detection means, including various agglutinating reactions, complement fixation reaction, precipitation
Reaction etc..
Test tube agglutination (SAT) is a kind of serology agglutinating reaction of classics, it refers to that particulate antigen is for example red thin
Born of the same parents, bacterium etc. with corresponding antibody under the conditions of existing for suitable electrolyte, it is macroscopic through being condensed into after a certain period of time
Agglutinator.SAT is the legal experiment of China's cloth disease detection, and advantage is SAT detection serum immune globulin M (IgM) sensibility
Higher, easy to operate, it is easy to judge, specificity is poor when having higher diagnostic value, but being used alone, and cannot distinguish between
Artificial immunity and natural infection.And diagnostic level is not achieved in the antibody titer of part infection animal, thus also easily cause mistaken diagnosis
With fail to pinpoint a disease in diagnosis.
Plate agglutination test (PAT) nineteen thirty-nine Huddleson has carried out plate agglutination test first, because its detection speed compared with
Soon, in early 20th century, PAT is quarantine and a kind of easy detection method of the quick primary dcreening operation of epidemiology in crowd between poultry.It is used
Antigen is that highly concentrated bacterium solution is placed in high strong brine, is made of crystal violet and malachite green dye dyeing thalline, is tested with SAT
It is higher compared to sensibility, and specificity is higher, but also may occur in which nonspecific agglutination.
Rose bengal precipitation test is to be specifically used to diagnosis brucellosis, it is 3.8 ± 0.2 highly concentrated bacterium solutions of pH, warp
Standard serum mark is simultaneously formed with brave red dye (tetrachlorotetraiodofluorescein sodium's salt) dyeing thalline.It is mainly characterized by activating
There is specificity to be up to 96% up to 97.1% and sensibility for IgG, this method, and can remove nonspecific agglutination element in serum, as a result
Reliably, method is simple, of low cost, is particularly suitable for the large area quarantine and epidemiology survey of cloth disease.But it cannot reflect
Other artificial immunity and natural infection.
Precipitation reaction is mainly used for the qualitative detection of antigen or antibody.Its principle refers to soluble antigen and corresponding antibodies
In the presence of having electrolyte, the visible precipitate object phenomenon that is formed by proper proportion.The precipitation of phenomenon design is real accordingly
Testing mainly includes flocculation test, the precipitation test in ring precipitation test and gel.
Complement fixation test (CFT) (CFT)：Since 1909 establish complement fixation test (CFT) technology, it has been widely applied.Its
Principle is that complement can be combined with any antigen antibody complex, but cannot be combined with individual antigen or antibody.Experiment needs two
System (suspect system and instruction system) and complement, CFT methods be considered as in current serological test technology the most accurate it
One, and be used widely.Document Nielsen K.Diagnosis of brucellosis by serology. [J]
.Veterinary Microbiology,2002,90(1–4):447-459. reports think that CFT needs a large amount of different reagents, a large amount of
Various control group, every time experiment need substantial amounts of titration, and conclusion also has subjectivity, and cannot differentiate and manually exempt from
Epidemic disease and natural infection, experiment condition is complicated harsh, in view of these shortcomings, CFT methods are unfavorable for promoting to base, should not be at the scene
It carries out.
In recent years, the immunological method for applying to brucella bacterium clinical detection mainly has:Rose bengal precipitation test
(RBPT), tube agglutination test (SAT), complement fixation test (CFT) (CFT), newborn ring test (MRT), enzyme-linked immunosorbent assay
(ELISA) and colloidal gold immunity chromatography (GICA) etc..Immunological method is simple and practicable, but susceptibility is low, and specificity is not high.
Molecular biology method has many advantages, such as high sensitivity, high specificity, and main technology has PCR
(abbreviation PCR) and loop-mediated isothermal amplification technique.
PCR (abbreviation PCR) was founded in 1985, had become the conventional skill of the pathogenic microorganism examination at present
Art has the characteristics that high specificity, high sensitivity, simplicity, quick, relatively low to the purity requirement of sample, in the base of PCR method
On plinth, some new molecular biology methods are employed for brucellar detection therewith.Document
Hekmatimoghaddam S,Sadeh M, Khalili M B,et al.Comparison of PCR,Wright
agglutination test and blood culture for diagnosis of brucellosis in
suspected patients[J].Pakistan Journal of Biological Sciences Pjbs,2013, 16
(22):1589 compare PCR, the 3 kinds of detection method sensitivity of tube agglutination and blood culture, specificity, positive predictive value and feminine gender
Predicted value, PCR are higher than the efficiency of blood culture, and inspection result is consistent with tube agglutination.Patent " 201610617030.6 cloth Lu Shi
Detection method, kit and its application of bacterium " is reported, and using PCR methods, cloth Lu Shi is obtained from serum specimen with centrifugal column method
Bacterium DNA obtains sample to be tested；Pcr amplification reaction；Reaction result is detected using fluorescence quantitative PCR instrument, during fluorescence signal collection
It is set as that Taqman fluorescence probe BKV-FP 5 ' holds the corresponding fluorescein of fluorophor, fluorescence signal, minimum inspection is collected at 60 DEG C
Go out to can reach 1000copies/mL；This method is sensitive, special, recall rate is high, but this method is to the special of instrument requirements and personnel
The requirement of industry attainment is high, and needs certain hardware supported, high to the DNA purity requirements of extraction when detection time 1.5-2 is small.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, LAMP) is by day
This scholar Notomi T et al. design 4 specific primers for 6 regions of target gene, in strand displacement archaeal dna polymerase (Bst
DNA polymerase) effect under, 60-65 DEG C of constant-temperature amplification, its amplification efficiency can reach 10 within 15-60 minutes9~1010A quantity
Grade.Liu Q etc. report the LAMP method that brucellosis is detected using 6 specific primers.The method design design is complicated,
False positive occurs high.
There is more universal pathogeny detection technology with reference to the traditional detection method of above analysis brucella, although cost
It is low, but have the verification and measurement ratio low, time-consuming and the deficiencies of professional technician is needed to operate simultaneously；Immunological method have sensitivity it is low,
It is not easy to detect in initial infection, there are problems that missing inspection, be not easy to be that experimentation needs animal real the reason for popularization
It tests, experimental period is long, of high cost, cumbersome；Molecular Biological Detection technology can meet the detection need of sensitive specificity
It asks, but technology such as round pcr is needed using complicated instrument, well-equipped laboratory and professional operator at present,
Although LAMP technology need not be expensive PCR instrument, multipair primer and very high to target-gene sequence requirement, and false sun need to be designed
Property occur it is high the shortcomings of.
The content of the invention
In order to solve in prior art detection method time-consuming and laborious, long time period and efficiency is low, need professional and
The problems such as expensive instrument, false positive rate is high, an embodiment of the present invention provides a kind of for detecting drawing for Brucellosis nucleic acid
Object, fluorescence probe and kit.The kit and detection method of the present invention using RAA technologies, overcome more than the shortcomings that, tool
Have short detection time, high sensitivity, high specificity, it is easy to operate the features such as.It need to only be reacted 5~20 minutes at 39 DEG C
Analysis result.Have the characteristics that it is quick, sensitive, easy to operate, to future prevention brucellosis propagate have it is very important
Meaning has larger application prospect.The technical solution is as follows：
First, the present invention discloses a kind of brucellar probe of RAA Fluorometric assays, and the probe is：
The terminal sequence of 5 ' terminal sequences/fluorescent reporter group //idSp//quenching group/3 '；Wherein：
As shown in SEQ ID NO.1, sequence information is the nucleotide sequence of the 5 ' terminal sequence
CTTTATGATGGCAAGGGCAAGGTGGAAGAT；
As shown in SEQ ID NO.2, sequence information is the nucleotide sequence of the 3 ' terminal sequence
TGCGCCTTCTGGCGAC。
Therefore, entire probe sequence can also be expressed as：5 '-CTTTATGATGGCAAGGGCAAGGTGGAAGAT/ fluorescence
Reporter group //idSp//quenching group/TGCGCCTTCTGGCGAC-3 '.
Wherein, the idSp is 1 base room；The fluorescent reporter group can select：FAM、HEX、TET、JOE、
Any one of VIC, ROX, Cy3 or Cy5, the quenching group can select：TAMRA、Eclipse、BHQ1、BHQ2、 BHQ3
Or any one of DABCYL.
5’-CTTTATGATGGCAAGGGCAAGGTGGAAGA/i6FAMdT//THF//iBHQdT/GCGCCTTC
TGGCGAC-3 ' is probe specifically used in an embodiment of the present invention, and design is characterized in that：The fluorescence probe is repaiied
Between adoring within the probe, fluorescent reporter group is modified on the position from 5' ends base number 30bp, quenching group modification is from 3' ends
On the position of base number 16bp, empty 1 base positions, residual for modifying tetrahydrofuran between fluorescent reporter group and quenching group
Base；
Another aspect of the present invention is to disclose a kind of brucella RAA Fluorometric assay kits, including above
The probe and following primer：
Upstream primer sequence is as shown in SEQ ID NO.3 in sequence table：
5’-CTCGGTTGCCAATATCAATGCGATCAAGTC-3’；
Downstream primer sequence is as shown in SEQ ID NO.4 in sequence table：
5’-CTTGCCTTTCAGGTCTGCGACCGATTTGATG-3’；
A kind of brucella RAA Fluorometric assay kits described in for technical solution above, the present invention is to probe
It is not limited with storage concentration of the primer in kit, for the accuracy of experiment, under normal circumstances, the probe makes
In reaction system when the final concentration of 0.02-0.05mM of used time in the reaction system, the sense primer and anti-sense primer use
In final concentration of 0.05-0.1mM.
A kind of brucella RAA Fluorometric assay kits described in for technical solution above, the kit is also
Including the general reaction reagent of reagent RAA basic fluorescences and reaction buffer.Wherein：The general reaction examination of the RAA basic fluorescences
Agent is the freeze-dried powder by frozen drying, is provided by Jiangsu Qi Tian genes bio tech ltd；It is that commodity article No. is
The basic reaction unit of F00001.Those skilled in the art can also select it according to the principle of RAA Fluorometric assay methods
He can be as the product of the general reaction reagent of RAA basic fluorescences as replacement.
A kind of brucella RAA Fluorometric assay kits described in for technical solution above, the reaction are delayed
Fliud flushing is made of following reagent：Its storage concentration in kit is not limited at this, for the accuracy of experiment, one
As in the case of, in the reaction buffer concentration of each component be Tris-HCl Buffer of 450mM, 260mM
The PEG 10000 of MgAc and 20% W/V.
A kind of brucella RAA Fluorometric assay kits described in for technical solution above, the kit is also
Including following reagent：Negative quality-control product, positive quality control product and/or critical positive quality control product；
It is brucellar genomic DNA fragment that the positive quality control product, which contains, at this to its storage in kit
Concentration does not limit, and for the accuracy of experiment, under normal circumstances, the concentration of positive quality control product is 1.0 × 104IU/
mL；Any one concentration of this concentration can also be selected above as positive quality control product.
The critical positive quality control product is containing brucellar genomic DNA fragment, at this to it in kit
Storage concentration does not limit, for the accuracy of experiment, under normal circumstances, the concentration of critical positive quality control product for 1.0 ×
103IU/mL；
The feminine gender quality-control product is the reagent for not containing brucellar genomic fragment, is typically chosen and does not contain Bu Lu
The DNA plasmid of the genomic fragment of family name bacillus.
Brucellar method is detected using mentioned reagent box, is included the following steps：
(1) DNA of sample to be tested is extracted；DNA is extracted by normal method, the certified products that may be employed has sale on the market extracts
Kit extracts the DNA of sample to be tested, such as Xi'an it is grand, QIAGEN kits, Tiangeng kit, can also be used from kinetonucleus
Sour extraction apparatus carries out DNA extractions；
(2) Buffer is reacted to prepare：47 μ L reaction buffers are taken, add in the mixture of 2 μ L probes and primer, it is fully mixed
；Prepared 49 μ L reagents are added in the general reaction reagent freeze-dried powder of RAA basic fluorescences, it is made fully to dissolve and mix
；Extracted 1 μ L samples DNA is added as template, total volume is 50 μ L；
(3) it is put into detection FAM fluorescence detection equipments and carries out real-time RAA fluorescence methods reaction；Real-time RAA fluorescence methods reaction item
Part is：39 DEG C of reaction 20min；
(4) interpretation of result：
FAM fluorescence detection equipments detect that signal substantially increases within 20min, show amplification judging as the positive；
FAM fluorescent instruments signal is determined as feminine gender without increase within 20 min.
Description of the drawings
The detection amplification figure of Fig. 1 embodiment of the present invention 1.
The testing result figure of Fig. 2 embodiment of the present invention 2.
The testing result figure of Fig. 3 embodiment of the present invention 3.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to embodiment party of the present invention
Formula is described in further detail.But present disclosure is not limited thereto.
Primed probe and DNA plasmid are closed by Sangon Biotech (Shanghai) Co., Ltd. in following embodiment
Into；Embodiment 1
The embodiment of the present invention provides a kind of detection brucella RAA Fluorometric assays kit and its detection method,
In, brucellar specific conservative's gene is selected as target detection gene, by American National Biotechnology Information
The heart (NCBI) (http://www.ncbi.nlm.nih.gov), brucella bcsp31 gene orders are obtained, and are carried out more
Sequence alignment, and one section of conserved sequence is therefrom selected, the sequence is as shown in SEQ ID NO.6 in sequence table：
TGGCTCGGTTGCCAATATCAATGCGATCAAGTCGGGCGCTCTGGAGTCCGGCTTTACGC
AGTCAGACGTTGCCTATTGGGCCTATAACGGCACCGGCCTTTATGATGGCAAGGGCAAG
GTGGAAGATTTGCGCCTTCTGGCGACGCTTTACCCGGAAACGATCCATATCGTTGCGCGT
AAGGATGCAAACATCAAATCGGTCGCAGACCTGAAAGGCAAGCG；According to more than sequence student on commission's work bioengineering (on
Sea) limited company's progress synthetic DNA material, plasmid size 222bp；
According to the design that RAA technologies primer and probe design principle carry out, by screening and evaluating, finally determine：
Upstream primer sequence 5 '-CTCGGTTGCCAATATCAATGCGATCAAGTC-3 '；
Downstream primer sequence 5 '-CTTGCCTTTCAGGTCTGCGACCGATTTGATG-3 '；
Probe sequence and method of modifying are：
5’-CTTTATGATGGCAAGGGCAAGGTGGAAGA/i6FAMdT//THF //iBHQdT/
GCGCCTTCTGGCGAC-3 ', such as SEQ ID NO.5.
Wherein, FAM is fluorescent reporter group, and BHQ is fluorescent quenching group, and THF is tetrahydrofuran residue, method of modifying
For FAM：6-Carboxyfluorescein；THF:tetrahydrofuran；BHQ:black hole quencher；
phosphate: 3’phosphate to block elongation。
Table 1 is the relevant information table of primer and probe
2 kit forms of the present invention of table
By 5ul 1010The working standard of different gradients is made in IU/mL brucella DNA plasmids, is respectively：
Working standard 1, contains 1.0 × 107The non-infectious DNA fragmentation of the brucellar bcsp31 genes of IU/mL.
Working standard 2, contains 1.0 × 106The non-infectious DNA fragmentation of the brucellar bcsp31 genes of IU/mL.
Working standard 3, contains 1.0 × 105The non-infectious DNA fragmentation of the brucellar bcsp31 genes of IU/mL.
Working standard 4, contains 1.0 × 104The non-infectious DNA fragmentation of the brucellar bcsp31 genes of IU/mL.
Working standard 5, contains 1.0 × 103The non-infectious DNA fragmentation of the brucellar bcsp31 genes of IU/mL.
Working standard 6, contains 1.0 × 102The non-infectious DNA fragmentation of the brucellar bcsp31 genes of IU/mL.
Buffer is reacted to prepare：By 376 μ L reaction buffers are drawn, the mixture of 16 μ L probes and primer is added in, fully
It is mixed；It draws the 49 μ L reagents of buffer solution after being mixed to be added separately in the general reaction reagent pipe of 8 RAA basic fluorescences, makes jelly
Dry powder is fully dissolved and is mixed；
1 μ L feminine genders quality-control product, standard work product 6, standard work product 5, standard work are separately added into 8 prepared pipes
Works 4, standard work product 3, standard work product 2, standard work product 1 are template, and each reaction tube is fully mixed, each
Reaction tube total volume is 50 μ L；
Detecting instrument uses the RAA-F1620 fluorescence detectors that Jiangsu Qi Tian genes bio tech ltd provides；
Instrument is arranged to：39 DEG C of reaction temperature, 20 minutes reaction time.
The reaction tube being mixed is put into RAA-F1620 fluorescence detectors, is reacted 20 minutes at 39 DEG C.
Testing result is as shown in Figure 1.The results show substantially has amplification in most fast 2 minutes, all standard work in 10 minutes
Product have amplification, repeat above example, can obtain identical amplification fluorescent signal, reproducible.
Embodiment 2
Primed probe is same as Example 1.
Table 3 is the kit of embodiment 2
Samples sources and DNA extractions
Sample is provided by Liaoning international travel health care center, is separated brucella in pig, sheep tissue samples
The DNA of extraction is cultivated, the DNA extracted is saved backup at -80 DEG C.
Buffer is reacted to prepare：Three reaction tubes are taken, respectively by following operation, 27 μ L reaction buffers is drawn, adds in 2 μ L
The mixture of probe and primer, is fully mixed；It is general anti-that 49 μ L reagents of buffer solution after being mixed are added to RAA basic fluorescences
It answers in Reagent Tube, freeze-dried powder is made fully to dissolve and be mixed；
1 μ L feminine genders quality-control product is separately added into 3 prepared pipes, the Brucella suis sample that 1 μ L have been extracted
The sheep brucella sample DNA that DNA, 1 μ L have been extracted is template, and each reaction tube is fully mixed, and each reaction tube is total
Volume is 50 μ L；
Detecting instrument uses the RAA-F1620 fluorescence detectors that Jiangsu Qi Tian genes bio tech ltd provides；
Instrument is arranged to：39 DEG C of reaction temperature, 20 minutes reaction time.
The reaction tube being mixed is put into RAA-F1620 fluorescence detectors, is reacted 20 minutes at 39 DEG C.
Testing result is as shown in Figure 2.The results show substantially has amplification in 3 minutes, repeats above example, can obtain identical
Amplification fluorescent signal, it is reproducible.
Embodiment 3
Primed probe and positive quality control product sequence are same as Example 1.
Table 4 is the kit of embodiment 3
Samples sources and DNA extractions：
Brucella suis sample DNA is provided by Liaoning international travel health care center, and blood fluke sample is by Jiangsu Province
Prevention and cure of snail fever research institute provides, for the DNA of japonice ovum extraction；Salmonella and Escherichia coli sample are by Zhejiang state
Border traveling health care center provides, and salmonella and staphylococcus aureus extract DNA by heating 90 DEG C of 10min, have extracted
DNA is saved backup at -80 DEG C,
Buffer is reacted to prepare：5 reaction tubes are taken, respectively by following operation, 27 μ L reaction buffers is drawn, adds in 2 μ L
The mixture of probe and primer, is fully mixed；It is general anti-that 49 μ L reagents of buffer solution after being mixed are added to RAA basic fluorescences
It answers in Reagent Tube, freeze-dried powder is made fully to dissolve and be mixed；
1 μ L feminine genders quality-control product is separately added into 5 prepared pipes, 1 μ L have extracted brucella sample DNA, 1 μ L
Japonice ovum DNA, 1 μ L salmonellas sample DNA, 1 μ L Escherichia coli sample DNA are template, and each reaction tube carries out abundant
It is mixed, each reaction tube total volume is 50 μ L；
Detecting instrument uses the RAA-F1620 fluorescence detectors that Jiangsu Qi Tian genes bio tech ltd provides；
Instrument is arranged to：39 DEG C of reaction temperature, 20 minutes reaction time.
The reaction tube being mixed is put into RAA-F1620 fluorescence detectors, is reacted 20 minutes at 39 DEG C.
Testing result is as shown in Figure 3.The results show only has brucella sample DNA substantially to have amplification, other samples
And negative quality-control product does not expand, is feminine gender.
Above example is repeated, identical amplification fluorescent signal can be obtained, it is reproducible.Embodiment three shows the present invention's
Kit specificity is good.
Above example illustrates quickly detect brucella using RAA technologies using the present invention, easy to operate,
Time used reduces significantly, and large-scale instrument and equipment is not required, suitable for screening on a large scale.Detection side provided by the present invention
Method high sensitivity, specificity are good, easy to operate, rapid, and low to detection material quality requirement.
Preferable the foregoing is merely the present invention implements example, is not intended to limit the invention, all spirit in the present invention
Within principle, any modifications, equivalent replacements and improvements are made should all be included in the protection scope of the present invention.
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Claims (6)
- The brucellar probe of 1.RAA Fluorometric assays, which is characterized in thatThe probe is the terminal sequence of 5 ' terminal sequences/fluorescent reporter group //idSp//quenching group/3 '；Wherein：The nucleotide sequence of the 5 ' terminal sequence is as shown in SEQ ID NO.1；The nucleotide sequence of the 3 ' terminal sequence is as shown in SEQ ID NO.2；The idSp is 1 base room；The fluorescent reporter group is selected from any one of FAM, HEX, TET, JOE, VIC, ROX, Cy3 or Cy5；The quenching group is selected from any one of TAMRA, Eclipse, BHQ1, BHQ2, BHQ3 or DABCYL.
- 2. a kind of brucella RAA Fluorometric assay kits, which is characterized in that the kit includes claim 1 institute The probe and following primer stated：The nucleotide sequence of sense primer is as shown in SEQ ID NO.3；The nucleotides sequence of anti-sense primer Row are as shown in SEQ ID NO.4.
- 3. brucella RAA Fluorometric assay kits according to claim 2, which is characterized in that the probe Final concentration of 0.02-0.05mM, the sense primer and the final concentration of 0.05-0.1mM of anti-sense primer.
- 4. brucella RAA Fluorometric assay kits according to claim 2, which is characterized in that the kit Further include the general reaction reagent of reagent RAA basic fluorescences and reaction buffer.
- 5. brucella RAA Fluorometric assay kits according to claim 4, which is characterized in that the reaction is slow Fliud flushing includes：Concentration is the Tris-HCl Buffer of 450mM；The MgAc of 260mM；The PEG 10000 of 20%W/V.
- 6. brucella RAA Fluorometric assay kits according to claim 2, which is characterized in that the kit Further include following reagent：Negative quality-control product, positive quality control product and/or critical positive quality control product；It is brucellar genomic DNA fragment that the positive quality control product, which contains, and concentration is 1.0 × 104IU/mL；The critical positive quality control product is containing brucellar genomic DNA fragment, and concentration is 1.0 × 103IU/mL；The feminine gender quality-control product is the reagent for not containing brucellar genomic fragment.
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| CN108913563A (en) * | 2018-07-05 | 2018-11-30 | 宁夏大学 | A kind of micro-fluidic genetic chip of dish-style and related kit and purposes |
| CN109439727A (en) * | 2018-04-25 | 2019-03-08 | 内蒙古华星康为生物科技有限公司 | A kind of method and kit detecting microorganism |
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| CN112941212A (en) * | 2021-03-23 | 2021-06-11 | 大连海关技术中心 | Universal primer group, kit and method for on-site detection of brucella |
| CN118460752A (en) * | 2024-07-11 | 2024-08-09 | 中国疾病预防控制中心传染病预防控制所 | Primer group, kit and method for detecting brucella based on RAA-CRISPR/Cas13a technology |
| CN118460752B (en) * | 2024-07-11 | 2024-10-29 | 中国疾病预防控制中心传染病预防控制所 | Primer group, kit and method for detecting brucella based on RAA-CRISPR/Cas13a technology |
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| CN112941212A (en) * | 2021-03-23 | 2021-06-11 | 大连海关技术中心 | Universal primer group, kit and method for on-site detection of brucella |
| CN118460752A (en) * | 2024-07-11 | 2024-08-09 | 中国疾病预防控制中心传染病预防控制所 | Primer group, kit and method for detecting brucella based on RAA-CRISPR/Cas13a technology |
| CN118460752B (en) * | 2024-07-11 | 2024-10-29 | 中国疾病预防控制中心传染病预防控制所 | Primer group, kit and method for detecting brucella based on RAA-CRISPR/Cas13a technology |
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Application publication date: 20180518 |