CN102719548B - Kit for detecting brucella and use method thereof - Google Patents
Kit for detecting brucella and use method thereof Download PDFInfo
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- CN102719548B CN102719548B CN201210228880.9A CN201210228880A CN102719548B CN 102719548 B CN102719548 B CN 102719548B CN 201210228880 A CN201210228880 A CN 201210228880A CN 102719548 B CN102719548 B CN 102719548B
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Abstract
The invention provides a kit for detecting brucella. The kit comprises two pairs of primers, i.e., an inner primer FIP/BIP and an outer primer F3/B3, which adopt an OMP25 gene of the brucella as the target gene and are designed on the basis of loop-mediated isothermal amplification technology. The kit provided by the invention has the advantages that the detecting effect is more comprehensive and the loss rate is low.
Description
The application is the applying date: on November 16th, 2010, and application number: 201010545462.3, denomination of invention: the dividing an application of Brucella detection kit and using method thereof.
Technical field
The present invention relates to biological detection reagent, be specifically related to a kind of Brucella detection kit and using method thereof.
Background technology
Brucella is a kind of gram-negative not motion bacterium, it can be lived at a variety of carcass internal memories as intracellular parasitic bacteria. and what people was caused to infection is mainly melitococcus (" Malta fever "), alcaligenes abortus, ox, Brucella ovis, pig Brucella and brucella canis.After people suffers from, the symptom first occurring is fever, and body temperature can reach 38-40 degree, and the time length is long, in prolonged low grade fever state; Somebody's body temperature is wavy, and high heat is several days, and body temperature lowers several days, starts again height, repeated multiple times, so cloth disease claims again unrestrained shape heat.Therefore detecting accurately and rapidly Brucella tool is of great significance.
Tradition Brucella detection method, because the shortcomings such as its sense cycle is long, program is complicated, required reagent is various can not meet modern measure requirement far away.There are in actual applications some problems in the cause of disease nucleic acid detection technique that polymerase chain reaction (PCR) technology of take is representative, as the special instrument of Common Polymerase Chain Reaction (PCR) Technology Need, and there is easy crossed contamination and the loaded down with trivial details shortcoming of operating process.And Fluorescence quantitative real-time polymerase chain reaction (real time PCR) is although technology has solved preferably the problem of crossed contamination and simplified operating process, need more complicated quantitative assay instrument, be not therefore suitable for field quick detection.And in real-time quantitative polymerase chain reaction PCR technology, the cost of fluorescent probe is higher, has strengthened the difficulty of applying.Immunology detection technology is fast and convenient with low cost, but requires the monoclonal antibody of high quality high stability, otherwise because accuracy is inadequate, can only be auxiliary detection means at present.So the newest fruits of using in time biotech development is significant to improving constantly of meeting that the pathogenic microorganism examination requires.Wherein constant-temperature amplification (Isothermal Amplification) nucleic acid Fast Detection Technique is the rapid progress on cause of disease nucleic acid detection technique, and the loop-mediated isothermal amplification technology of now having set up (LAMP) has a lot of superiority.
Therefore, need a kind of detect effect more comprehensively, specificity is high, loss is low Brucella detection kit and using method thereof to be to address the above problem.
Summary of the invention
The object of the invention is to solve the deficiencies in the prior art part and provide a kind of detect effect more comprehensively, specificity is high, loss is low Brucella detection kit.
Object of the present invention can realize by following technical measures: a kind of Brucella detection kit, described Brucella detection kit comprises that take the OMP25 gene of Brucella is target gene, two pairs of primers based on loop-mediated isothermal amplification technology design: inner primer FIP/BIP and outer primer F3/B3.
What the present invention detected is OMP25 gene.This OMP25 gene is Brucella outer membrane protein gene.
With the BLAST comparison result shows of NCBI website nt database, except Brucella, without other cingula, there is this gene.
As the preferred implementation of Brucella detection kit of the present invention, two pairs of described primers are
Outer primer F3(1): (SEQ ID NO 1)
CCTTTGCTGGCTGGAACT
Outer primer B3(1): (SEQ ID NO 2)
GCAATACCAGCCGTGAGG
Inner primer FIP(1): (SEQ ID NO 3)
GTCCTTGGACTTCTTGGCCCAGttttTCCAGCAGGACCAGATCG
Inner primer BIP(1): (SEQ ID NO 4)
GGCCTGGAAGTCAAGCAGGGttttACGGCATAACCGGGTTCA
Or
Outer primer F3(2): (SEQ ID NO 5)
CAAGACCAGCACCGTTGG
Outer primer B3(2): (SEQ ID NO 6)
GGTTCAGGTCGTAGCCGA
Inner primer FIP(2): (SEQ ID NO 7)
GGTCCTGCTGGAAGTTCCAGCttttAGCATCAAGCCTGACGATTG
Inner primer BIP(2): (SEQ ID NO 8)
CGGTGTTGAAGGTGATGCAGGTttttTTCAA?AGCCCTGCTTGACTT
Or
Outer primer F3(3): (SEQ ID NO 9)
CGTCGGCTACGACCTGAA
Outer primer B3(3): (SEQ ID NO 10)
ACCGGCCAGATCATAGTTCT
Inner primer FIP(3): (SEQ ID NO 11)
TCGTCCAAGCCGTTGTTAAGCTttttCCCGGTTATGCCGTACCT
Inner primer BIP(3): (SEQ ID NO 12)
GTGCCGGTCTCGAAGCCAAGttttTGTTGCCGTACTGGGTGTA
Or
Outer primer F3(4): (SEQ ID NO 13)
ATTGCCGGTTCGCAGATC
Outer primer B3(4): (SEQ ID NO 14)
GCGGATATCCTGCGTGTC
Inner primer FIP(4): (SEQ ID NO 15)
TCAGCTTGGCTTCGAGACCGttttCTTAACAACGGCTTGGACGA
Inner primer BIP(4): (SEQ ID NO 16)
GGCCGCGTTGAGTACCGTTAttttAGCTTGTTGCGAACAGTCG。
As the preferred implementation of Brucella detection kit of the present invention, described Brucella detection kit also comprises BstDNA polysaccharase, reaction solution, stable liquid, sample pretreatment liquid, nitrite ion and positive control solution.
As the more preferably embodiment of Brucella detection kit of the present invention,
Described BstDNA polysaccharase: enzyme concn 4-10U/ μ L;
Described reaction solution contains: 1.6～2mmol/L dNTP, 20～25mmol/L Tris-HCl, 10～12.5mmol/L KCl, 10～12.5mmol/L(NH
4)
2sO
4, 8～10mmol/L MgSO
4, 0.1～0.125 volume %TritonX-100,0.8～1mol/L trimethyl-glycine;
Described sample pretreatment liquid contains: the Tris-HCl of 10～20mmol/L pH8.0,1～2mmol/L EDTA and 1～1.2 volume %Triton X-100;
Described nitrite ion is SYBR Green I or Eva Green;
Described stable liquid is paraffin oil;
Described positive control is Brucella genomic dna;
Described inner primer FIP/BIP is respectively 1.2～2.0 μ mol/L, and the concentration of outer primer F3/B3 is respectively 0.2～0.25 μ mol/L.
As the most preferred embodiment of Brucella detection kit of the present invention,
Described BstDNA polysaccharase: enzyme concn 8U/ μ L;
Described reaction solution contains: 2mmol/L dNTP, 25mmol/L Tris-HCl, 12.5mmol/LKCl, 12.5mmol/L(NH
4)
2sO
4, 10mmol/L MgSO
4, 0.125 volume %TritonX-100,1mol/L trimethyl-glycine;
Described sample pretreatment liquid contains: the Tris-HCl of 20mmol/L pH8.0,2mmol/LEDTA and 1.2 volume %Triton X-100;
Described nitrite ion is SYBR Green I;
The concentration of described inner primer FIP/BIP is 0.2 μ mol/L;
The concentration of described outer primer F3/B3 is 1.6 μ mol/L.
Preferred implementation as Brucella detection kit of the present invention, described Brucella detection kit also comprises reaction tubes, described reaction tubes covers two portions by body and pipe and forms, and the bottom of tube cavity is provided with the dividing plate extending longitudinally that is divided into A, two cavitys of B.
More preferably embodiment as Brucella detection kit of the present invention, in described A, two cavitys of B, working fluid or nitrite ion are housed respectively, seal up for safekeeping by stable liquid on liquid upper strata in two cavitys, and described working fluid is that reaction solution and BstDNA polysaccharase mix.
The present invention also provides a kind of using method of Brucella detection kit, and the method comprises the steps:
(1) testing sample is centrifugal, remove supernatant, be precipitated;
(2) precipitation of step (1) is added to sample pretreatment liquid, mix, cooled on ice after boiling water bath deactivation, high speed centrifugation, supernatant is sample template DNA;
(3) in reaction vessel, add BstDNA polysaccharase 0.9～1.8 volume parts, reaction solution 38 ~ 40 volume parts, stable liquid 52～54.5 volume parts, sample template DNA4.5 ~ 9 volume parts, each 2 volume parts of inner primer FIP/BIP, each 4 volume parts of outer primer F3/B3, isothermal reaction;
(4) in above-mentioned reaction vessel and positive control, add respectively nitrite ion, mix, sample sets colour developing is identical with control group positive, otherwise negative;
Inner primer FIP/BIP in described step (3) and outer primer F3/B3 are target gene, two pairs of primers based on loop-mediated isothermal amplification technology design for take the OMP25 gene of Brucella.
As the present invention, use the preferred implementation of the method for Brucella detection kit, two pairs of described primers are
Outer primer F3(1): (SEQ ID NO 1)
CCTTTGCTGGCTGGAACT
Outer primer B3(1): (SEQ ID NO 2)
GCAATACCAGCCGTGAGG
Inner primer FIP(1): (SEQ ID NO 3)
GTCCTTGGACTTCTTGGCCCAGttttTCCAGCAGGACCAGATCG
Inner primer BIP(1): (SEQ ID NO 4)
GGCCTGGAAGTCAAGCAGGGttttACGGCATAACCGGGTTCA
Or
Outer primer F3(2): (SEQ ID NO 5)
CAAGACCAGCACCGTTGG
Outer primer B3(2): (SEQ ID NO 6)
GGTTCAGGTCGTAGCCGA
Inner primer FIP(2): (SEQ ID NO 7)
GGTCCTGCTGGAAGTTCCAGCttttAGCATCAAGCCTGACGATTG
Inner primer BIP(2): (SEQ ID NO 8)
CGGTGTTGAAGGTGATGCAGGTttttTTCAAAGCCCTGCTTGACTT
Or
Outer primer F3(3): (SEQ ID NO 9)
CGTCGGCTACGACCTGAA
Outer primer B3(3): (SEQ ID NO 10)
ACCGGCCAGATCATAGTTCT
Inner primer FIP(3): (SEQ ID NO 11)
TCGTCCAAGCCGTTGTTAAGCTttttCCCGGTTATGCCGTACCT
Inner primer BIP(3): (SEQ ID NO 12)
GTGCCGGTCTCGAAGCCAAGttttTGTTGCCGTACTGGGTGTA
Or
Outer primer F3(4): (SEQ ID NO 13)
ATTGCCGGTTCGCAGATC
Outer primer B3(4): (SEQ ID NO 14)
GCGGATATCCTGCGTGTC
Inner primer FIP(4): (SEQ ID NO 15)
TCAGCTTGGCTTCGAGACCGttttCTTAACAACGGCTTGGACGA
Inner primer BIP(4): (SEQ ID NO 16)
GGCCGCGTTGAGTACCGTTAttttAGCTTGTTGCGAACAGTCG。
As the present invention, use the preferred implementation of the method for Brucella detection kit, in step (3), the reaction conditions of isothermal reaction is 63～65 ℃ of temperature, reaction times 45～90min.
The present invention uses the method for Brucella detection kit, not the method for diagnosis or treatment disease.Brucella detection kit of the present invention can detect in food whether contain Brucella, and food safety is significant.Meanwhile, the effect of detection is played in the pollution that whether Brucella detection kit of the present invention can also be subject to Brucella to various other materials.
The present invention is said based on loop-mediated isothermal amplification technology (loop-mediated isothermal amplicat ion of DNA, abbreviation LAMP) method of rapid detection Brucella, be utilize BstDNA polysaccharase and according to two couple of target-gene sequence design special in, outer primer (being inner primer FIP/BIP and outer primer F3/B3), six isolated areas on specific recognition target sequence, start endless chain replacement(metathesis)reaction, in target DNA district, start complementary strand synthetic, go round and begin again stem-circular DNA mixture of the Cauliflower structure that is formed with a lot of rings of result complementary sequence on same chain.In LAMP reaction process, the pyrophosphate ion of separating out from dNTP and the Mg reaction soln
2+in conjunction with, produce by product---magnesium pyrophosphate milky white precipitate, can be by visual inspection result of determination.LAMP reaction is in 45～90 minutes, to complete under constant temperature (63～65 ℃) condition.This comparatively gentle temperature condition and do not have temperature cycle that required instrument is simplified, overcome normal PCR intrinsic detection time long, easily pollute and the shortcoming such as testing cost height.In addition, this detection method requires lower to testing staff's technical quality, and actually operating is very easy, does not need special reagent and plant and instrument, is conducive to set up rapid screening system with low cost.LAMP method is a kind of gene amplification method of easy, quick, high degree of specificity.Constant temperature gene amplification technology and round pcr (comprising fluorescence real-time quantitative PCR technology) are compared, can find that this technology is equivalent to or is better than round pcr in the methodology indexs such as sensitivity, specificity and sensing range, and do not rely on any special plant and instrument and can realize on-the-spot high-throughput rapid detection, and testing cost is far below fluorescent quantitative PCR technique.In national standard, take microorganism separation and Culture and Morphological Identification at present as master, in conjunction with the passing method of biochemical analysis and serological typing evaluation, and preliminary evaluation needs 2～3 days, completes probation report and needs 10～15 days; Adopt detection kit of the present invention only to need 2 hours.And, in reaction system of the present invention, having added nitrite ion, qualification result is more visual and clear.
Compared with prior art, the present invention has following beneficial effect: 1. detection kit of the present invention only needs just energy amplified reaction of a steady temperature, does not need special reagent and equipment, and testing cost is low; 2. six sections of gene quick diagnosis kit of the present invention application, whether four primers, just can judge the existence of target substance, so have high specific according to whether increasing; 3. detection kit of the present invention amplification fast and efficient, can complete amplification less than 1 hour, and productive rate is high; 4. detection kit of the present invention is highly sensitive, and amplification template only needs 10 copies or still less; 5. gene quick diagnosis kit evaluation of the present invention is easy, the pyrophosphate ion of separating out from dNTP and the Mg reaction soln
2+in conjunction with, produce by product---magnesium pyrophosphate precipitation, can identify by visual inspection, and add after nitrite ion, yin and yang attribute result colour development difference is remarkable, and checking rate is high, more obviously reliable; 6. owing to having selected the OMP25 gene of high conservative property to design primer as target gene, make the accuracy rate of detection kit detection Brucella of the present invention higher; 7. in detection kit of the present invention, adopt special reaction tubes, reduced the possibility of Aerosol Pollution, simultaneously handled easily.
Embodiment
For making the present invention easier to understand, will further set forth specific embodiments of the invention below.
Following shortenings is applicable to the present invention:
LAMP:loop-mediated isothermal amplification, loop-mediated isothermal amplification
DNTP:deoxyribonucleoside triphosphate, deoxynucleoside triphosphate
Bst enzyme: Bst DNA polymerase (largefragment), Bst archaeal dna polymerase (large fragment)
EDTA:ethylenediamine tetraacetic acid, ethylenediamine tetraacetic acid (EDTA)
DNA:deoxyribonucleic acid, thymus nucleic acid
Betaine: trimethyl-glycine
Triton X-100: Triton X-100
OMP25: Brucella outside membrance protein 25 gene
The preparation of embodiment 1 test kit
(1) in the following sequence through the synthetic oligomerization picodna primer of DNA synthesizer:
Outer primer F3(1):
CCTTTGCTGGCTGGAACT
Outer primer B3(1):
GCAATACCAGCCGTGAGG
Inner primer FIP(1):
GTCCTTGGACTTCTTGGCCCAGttttTCCAGCAGGACCAGATCG
Inner primer BIP(1):
GGCCTGGAAGTCAAGCAGGGttttACGGCATAACCGGGTTCA。
(2) purchase archaeal dna polymerase: BstDNA polysaccharase is placed in container;
(3) preparation reaction solution and primer: reaction solution contains 2mmol/LdNTP, 25mmol/L Tris-Cl, 12.5mmol/L KCl, 12.5mmol/L(NH
4)
2sO
4, 10mmol/L MgSO
4, each 1.2 μ mol/L of 0.125 volume %TritonX-100,1mol/L trimethyl-glycine, inner primer FIP/BIP and each 0.25 μ mol/L of outer primer F3/B3, be placed in container;
(4) preparation sample pretreatment liquid: sample pretreatment liquid contains 20mmol/L Tris-HCl(pH8.0), 2mmol/L EDTA and 1.2 volume %Triton X-100, be placed in container;
(5) purchase stable liquid: paraffin oil, is placed in container;
(6) purchase nitrite ion: SYBR Green I, is placed in container;
(7) extract positive control: extract Brucella genomic dna, be placed in container;
(8) above-mentioned 7 containers are dressed up to test kit, encapsulation.
Preparation technology is summarized as follows:
1, by after inner primer FIP/BIP and outer primer F3/B3 synthesizing and purifying, quantitatively preparation, concentration detects, sampling quality inspection;
2, by the liquid asepsis packing of above-mentioned (2) ~ (4) step preparation, and according to experiment, with carrying out concentration, determine sampling quality inspection;
3, by stable liquid packing, sampling quality inspection;
4, by the preparation of positive control sample, packing, sampling quality inspection;
5, assembling test kit.
In other embodiment of Brucella detection kit of the present invention, the primer adopting can also be
Outer primer F3(3):
CGTCGGCTACGACCTGAA
Outer primer B3(3):
ACCGGCCAGATCATAGTTCT
Inner primer FIP(3):
TCGTCCAAGCCGTTGTTAAGCTttttCCCGGTTATGCCGTACCT
Inner primer BIP(3):
GTGCCGGTCTCGAAGCCAAGttttTGTTGCCGTACTGGGTGTA
The preparation of embodiment 2 test kits
(1) in the following sequence through the synthetic oligomerization picodna primer of DNA synthesizer:
Outer primer F3(2):
CAAGACCAGCACCGTTGG
Outer primer B3(2):
GGTTCAGGTCGTAGCCGA
Inner primer FIP(2):
GGTCCTGCTGGAAGTTCCAGCttttAGCATCAAGCCTGACGATTG
Inner primer BIP(2):
CGGTGTTGAAGGTGATGCAGGTttttTTCAAAGCCCTGCTTGACTT
(2) purchase archaeal dna polymerase: BstDNA polysaccharase is placed in container;
(3) preparation reaction solution and primer: reaction solution contains 1.6mmol/LdNTP, 20mmol/L Tris-Cl, 10mmol/L KCl, 10mmol/L(NH
4)
2sO
4, 8mmol/L MgSO
4, each 2.0 μ mol/L of 0.1 volume %TritonX-100,0.8mol/L trimethyl-glycine, inner primer FIP/BIP and each 0.2 μ mol/L of outer primer F3/B3, be placed in container;
(4) preparation sample pretreatment liquid: sample pretreatment liquid contains 10mmol/L Tris-HCl(pH8.0), 1mmol/L EDTA and 1.0 volume %Triton X-100, be placed in container;
(5) purchase stable liquid: paraffin oil, is placed in container;
(6) purchase nitrite ion: EVA Green I, is placed in container;
(7) extract positive control: extract Brucella genomic dna, be placed in container;
(8) above-mentioned 7 containers are dressed up to test kit, encapsulation.
Other are with embodiment 1.
In other embodiment of Brucella detection kit of the present invention, the primer adopting can also be
Outer primer F3(4):
ATTGCCGGTTCGCAGATC
Outer primer B3(4):
GCGGATATCCTGCGTGTC
Inner primer FIP(4):
TCAGCTTGGCTTCGAGACCGttttCTTAACAACGGCTTGGACGA
Inner primer BIP(4):
GGCCGCGTTGAGTACCGTTAttttAGCTTGTTGCGAACAGTCG。
The application of embodiment 3 Brucella detection kits
1 materials and methods
1.1 material
1.1.1 bacterial strain
The present invention adopts bacterial strain to have 28 strains, is mainly derived from disease prevention and control center, heart Guangzhou, clinical isolates strain and environment separation bacterial strain.Refer to table 1.
Table 1 strain name and source
1.1.2 key instrument and reagent
1.2 the evaluation of isolated strains
1.2.1 the bacterial classification of the cultivation Brucella stab culture of Brucella is recovered with brucella broth, 36 ± 1 ℃, cultivates 24 ± 2 hours.
1.2.2 the isolation identification of clinical separation strain is directly applied to blood agar by the bacterium liquid that is slight muddiness and has precipitation not form mycoderm, on LIA or brucella agar flat board, under micro-aerobic (10% carbonic acid gas) and aerobic conditions, putting 36 ± 1 ℃ cultivates 3 days, then take out, observe form, and do gramstaining, agglutination test and biochemical test and confirm.
1.3 sample preparation (template DNA extraction)
1.3.1 get the bacterium liquid sample of 1mL liquid culture, centrifugal 2 minutes of 10000rpm, obtains bacterial sediment;
1.3.2 in above-mentioned bacterial sediment, add 100 μ L sample pretreatment liquid to mix, in boiling water, boil after 20 minutes and be placed in immediately cooled on ice 10 minutes, centrifugal 2 minutes of 10000rpm, supernatant is sample template DNA.
1.4, the test kit of employing embodiment 1 or embodiment 2 carries out the reaction process of loop-mediated isothermal amplification technique
1.4.1 in 200 μ L reaction tubes preparation reaction systems: reaction solution and primer be totally 22 μ L, BstDNA polysaccharase 0.5 μ L(4U), stable liquid 30 μ L, template DNA 2.5 μ L.
1.4.2 by the reaction tubes preparing in 65 ℃ of isothermal reactions 1 hour.
1.5 post-reaction treatment
In above-mentioned reaction product, add 2 μ L SYBR Green I, mix, also in heliotropism control tube (Brucella genomic dna) and negative control pipe (deionized water), add SYBR Green I to mix simultaneously, if reaction tubes shows green the same as positive control pipe is positive, if reaction tubes the same with negative control pipe manifest orange negative.
1.6 electrophoresis
Prepare 0.2% agarose gel electrophoresis.The SYBR Green I of usining carries out color reaction observations as dyestuff.
1.7 specific degree tests
1.7.1 pure bacterial strain LAMP detects and by LAMP method, 25 strain bacteriums is increased, according to color reaction observations, green positive, orange feminine gender, verification method specificity.
1.7.2 several bacterial strains hybrid dna detects and with LAMP, Brucella and Salmonellas, single DNA equal-volume mixed solution that increases listeria spp, streptococcus aureus to be got to 2.5ul and make LAMP and detect.
1.8 sensitivity tests are cultivated Brucella after 24 ± 2 hours in brucella broth, get 1mL bacterium liquid and are suspended in 5mL stroke-physiological saline solution, with 10 times of doubling dilutions to 10 of physiological saline
-10.Select 3 suitable concentration levels to get 100ul and be laid in blood agar, on LIA or brucella agar flat board, make respectively 3 flat boards, cultivate 3 days for 36 ± 1 ℃, get the flat board of colony number between 30～300 and make plate count, the mean of 3 dull and stereotyped colony numbers of this concentration level is calculated bacterial concentration, is bacterium colony mean number * extension rate * 10; Meanwhile, each concentration level is got 1mL, extracts DNA, makes LAMP and detects.
1.9 replica test specific degree tests and sensitivity test repeat respectively 2 times.
2 results
The foundation of 2.1 Brucella LAMP detection methods
2.2 specific degree test Brucella detected results are positive, and the non-Brucella of 7 strain is all negative, and the 17 routine samples of Brucella and Brucella, Salmonellas, list increase listeria spp, 4 kinds of DNA of bacteria mixed solutions of streptococcus aureus are positive, as shown in table 2.Result visualizingre agent box has high specific.
| Detect sample | Result | Detect sample | Result | Detect sample | Result |
| Negative control | Negative | Brucella 1 | Positive | Brucella 10 | Positive |
| Positive control | Positive | Brucella 2 | Positive | Brucella 11 | Positive |
| Streptococcus aureus | Negative | Brucella 3 | Positive | Brucella 12 | Positive |
| Shigellae | Negative | Brucella 4 | Positive | Brucella 13 | Positive |
| Vibrio parahaemolyticus | Negative | Brucella 5 | Positive | Brucella 14 | Positive |
| Salmonellas | Negative | Brucella 6 | Positive | Brucella 15 | Positive |
| Listeria monocytogenes Salmonella | Negative | Brucella 7 | Positive | Brucella 16 | Positive |
| Yersinia entero-colitica | Negative | Brucella 8 | Positive | Brucella 17 | Positive |
| Beta hemolysis suis | Negative | Brucella 9 | Positive | DNA of bacteria mixed solution | Positive |
2.3 sensitivity test
Through bacterium colony plate count, select the 9th extent of dilution to carry out reading, average colony number is 126cfu, calculates that bacterium original liquid concentration is 1.26 * 10
11cfu/mL, LAMP method can detect the 4th extent of dilution, is 1.26 * 10
7cfu/mL.
Electrophoresis result also meets the above results.
2.4 replica test specific degree tests repeat twice, and result is consistent.Sensitivity test repeats twice, order-of-magnitude agreement.
Embodiment 4 adopts the Brucella detection kit of reaction tubes
The Brucella detection kit of the present embodiment, the reagent and the primer that adopt are identical with embodiment 1, test kit also comprises reaction tubes, reaction tubes covers two portions by body and pipe and forms, the bottom of tube cavity is provided with the dividing plate extending longitudinally that is divided into A, two cavitys of B, wherein: the LAMP reaction solution of 22.0 μ L and the BstDNA polysaccharase of 0.5 μ L are housed in cavity A, and liquid upper strata is sealed up for safekeeping by paraffin; The LAMP reaction solution liquid that 2.0 μ L are housed in cavity B, liquid upper strata is also sealed up for safekeeping by paraffin, by ℃ preservation of these reaction tubes-20.
The present embodiment adopts reaction tubes as container, and Brucella is carried out to LAMP detection, is provided with positive controls and negative control group simultaneously, specifically comprises the steps:
Get three of the reaction tubess of above-mentioned preparation; adopt liquid-transfering gun to add respectively each 2.5 μ L of negative control sample, detected sample and positive control sample, rifle head pierce through the protection liquid layer during application of sample, sample adds in reaction tubes A chamber; cover tightly pipe and cover and carry out mark, move to reaction zone.
By above-mentioned reaction tubes all in 65 ℃ of isothermal reaction 1h.
Reaction finishes, and reaction tubes is inverted to whipping 1 time, is more just putting whipping 1 time, makes working fluid fully mix rear observation with nitrite ion.
If reaction tubes shows green the same as positive control pipe is positive, if reaction tubes the same with negative control pipe manifest orange negative.
In LAMP reaction process, nitrite ion and working fluid sealed state are good, there is no to occur situation about revealing mutually, and the later stage during color reaction, is inverted whipping operation nitrite ion and working fluid are mixed, and colour developing result is clear.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention is explained in detail with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.
Claims (4)
1. a Brucella detection kit, it is characterized in that, described Brucella detection kit comprises that take the OMP25 gene of Brucella is target gene, two pairs of primers based on loop-mediated isothermal amplification technology design: inner primer FIP/BIP and outer primer F3/B3, two pairs of described primers are
Outer primer F3:
CGTCGGCTACGACCTGAA
Outer primer B3:
ACCGGCCAGATCATAGTTCT
Inner primer FIP:
TCGTCCAAGCCGTTGTTAAGCTttttCCCGGTTATGCCGTACCT
Inner primer BIP:
GTGCCGGTCTCGAAGCCAAGttttTGTTGCCGTACTGGGTGTA
Or
Outer primer F3:
ATTGCCGGTTCGCAGATC
Outer primer B3:
GCGGATATCCTGCGTGTC
Inner primer FIP:
TCAGCTTGGCTTCGAGACCGttttCTTAACAACGGCTTGGACGA
Inner primer BIP:
GGCCGCGTTGAGTACCGTTAttttAGCTTGTTGCGAACAGTCG；
Described Brucella detection kit also comprises BstDNA polysaccharase, reaction solution, stable liquid, sample pretreatment liquid, nitrite ion and positive control solution;
Described BstDNA polysaccharase: enzyme concn 4-10U/ μ L;
Described reaction solution contains: 1.6～2mmol/LdNTP, 20～25mmol/LTris-HCl, 10～12.5mmol/LKCl, 10～12.5mmol/L(NH
4)
2sO
4, 8～10mmol/LMgSO
4, 0.1～0.125 volume %TritonX-100,0.8～1mol/L trimethyl-glycine;
Described sample pretreatment liquid contains: the Tris-HCl of 10～20mmol/LpH8.0,1～2mmol/LEDTA and 1～1.2 volume %TritonX-100;
Described nitrite ion is SYBRGreenI or EvaGreen;
Described stable liquid is paraffin oil;
Described positive control solution is Brucella genomic dna;
Described inner primer FIP/BIP is respectively 1.2～2.0 μ mol/L, and the concentration of outer primer F3/B3 is respectively 0.2～0.25 μ mol/L.
2. Brucella detection kit according to claim 1, is characterized in that,
Described BstDNA polysaccharase: enzyme concn 8U/ μ L;
Described reaction solution contains: 2mmol/LdNTP, 25mmol/LTris-HCl, 12.5mmol/LKCl, 12.5mmol/L(NH
4)
2sO
4, 10mmol/LMgSO
4, 0.125 volume %TritonX-100,1mol/L trimethyl-glycine;
Described sample pretreatment liquid contains: the Tris-HCl of 20mmol/LpH8.0,2mmol/LEDTA and 1.2 volume %TritonX-100;
Described nitrite ion is SYBRGreenI;
The concentration of described inner primer FIP/BIP is 1.6 μ mol/L;
The concentration of described outer primer F3/B3 is 0.2 μ mol/L.
3. Brucella detection kit according to claim 1 and 2, it is characterized in that, described Brucella detection kit also comprises reaction tubes, described reaction tubes covers two portions by body and pipe and forms, and the bottom of tube cavity is provided with the dividing plate extending longitudinally that is divided into A, two cavitys of B.
4. Brucella detection kit according to claim 3, it is characterized in that, in described A, two cavitys of B, working fluid or nitrite ion are housed respectively, seal up for safekeeping by stable liquid on the liquid upper strata in two cavitys, and described working fluid is that reaction solution and BstDNA polysaccharase mix.
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| CN103602721B (en) * | 2013-07-16 | 2015-07-01 | 黄耀江 | LAMP primer for detecting Brucella and kit containing same |
| CN104263839A (en) * | 2014-10-13 | 2015-01-07 | 天津市动物疫病预防控制中心 | LAMP-LFD (loop-mediated isothermal amplification and lateral flow dipstick) detection kit and detection method for brucella |
| CN106319054B (en) * | 2016-08-23 | 2019-07-19 | 中央民族大学 | Detect the primer and detection method of brucella |
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| CN101712924A (en) * | 2009-12-18 | 2010-05-26 | 广州华峰生物科技有限公司 | Reaction tube used in loop-mediated isothermal amplification technique and use method thereof |
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| CN101565753A (en) * | 2008-04-29 | 2009-10-28 | 广州华峰生物科技有限公司 | Rapid diagnostic kit for staphylococcus aureus gene based on loop-mediated isothermal amplification technology and detecting method thereof |
| CN101654706A (en) * | 2008-05-30 | 2010-02-24 | 广州华峰生物科技有限公司 | Rapid diagnostic reagent kit of legionella pneumophilia genes based on loop-mediated isothermal amplification technique and detecting method thereof |
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