CN101979660A - Brucella detection kit and using method thereof - Google Patents
Brucella detection kit and using method thereof Download PDFInfo
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Abstract
The invention provides a brucella detection kit. The brucella detection kit comprises two pairs of primers, namely internal primers FIP/BIP and external primers F3/B3 which take the OMP25 gene of brucella as a target gene and are designed based on loop-mediated isothermal amplification technology. The brucella detection kit has more comprehensive detection effect and low undetected rate.
Description
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 bacterium that do not move, it can be lived at a variety of families carcass internal memories as intracellular parasitic bacteria. and what the people was caused infection mainly is melitococcus (" Malta fever "), alcaligenes abortus, ox, Brucella ovis, pig Brucella and brucella canis.After the people suffered from, at first the symptom of Chu Xianing was a fever, and body temperature can reach the 38-40 degree, and longer duration is in the prolonged low grade fever state; Somebody's body temperature is wavy, promptly high heat several days, and body temperature lowered several days, began height again, and repeated multiple times is so the cloth disease claims unrestrained shape heat again.Therefore detecting Brucella accurately and rapidly has crucial meaning.
Tradition Brucella detection method, shortcoming can not satisfy the modern requirement that detects far away because its sense cycle is long, program is complicated, required reagent is various etc.With polymerase chain reaction (PCR) technology is that the cause of disease nucleic acid detection technique of representative exists some problems in actual applications, as the special instrument of common polymerase chain reaction (PCR) Technology Need, and there are easy crossed contamination and the loaded down with trivial details shortcoming of operating process.And fluorescence real-time quantitative polymerase chain reaction (real time PCR) though technology has solved the problem of crossed contamination preferably, and has simplified operating process, needs more complicated quantitative assay instrument, therefore is not suitable for field quick detection.And the cost of fluorescent probe is higher in the real-time quantitative polymerase chain reaction PCR technology, has strengthened the difficulty of applying.The immunology detection technology is fast and convenient with low cost, but requires the monoclonal antibody of high quality high stability, otherwise because of accuracy is not enough, can only be the auxiliary detection means at present.So in time use the newest fruits of biotech development significant to satisfying improving constantly of pathogenic micro-organism detection requirement.Wherein constant-temperature amplification (Isothermal Amplification) nucleic acid Fast Detection Technique is the rapid progress on the 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, the low Brucella detection kit of specificity height, loss and using method thereof to be to address the above problem.
Summary of the invention
The objective of the invention is to solve the deficiencies in the prior art part and provide a kind of detect effect more comprehensively, specificity height, Brucella detection kit that loss is low.
Purpose of the present invention can realize by following technical measures: a kind of Brucella detection kit, described Brucella detection kit comprise the OMP25 gene with Brucella be target gene, based on two pairs of primers of loop-mediated isothermal amplification technology design: inner primer FIP/BIP and outer primer F3/B3.
What the present invention detected is the OMP25 gene.This OMP25 gene is the Brucella outer membrane protein gene.
With the BLAST comparison result shows of NCBI website nt database, except that Brucella, do not have other cingula this gene is arranged.
As the preferred implementation of Brucella detection kit of the present invention, described two pairs of 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)
GGTCCTGCTGGAAGTTGCAGCttttAGCATCAAGCCTGACGATTG
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 preferred implementation of Brucella detection kit of the present invention, described Brucella detection kit also comprises Bst archaeal dna polymerase, reaction solution, stable liquid, sample pretreatment liquid, colour developing liquid and positive control solution.
As the more preferably embodiment of Brucella detection kit of the present invention,
Described Bst archaeal dna polymerase: 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 pH 8.0,1～2mmol/LEDTA and 1～1.2 volume %Triton X-100;
Described colour developing liquid is SYBR Green I or Eva Green;
Described stable liquid is paraffin oil;
Described positive control is the Brucella genomic dna;
Described inner primer FIP/BIP respectively is 1.2～2.0 μ mol/L, and the concentration of outer primer F3/B3 respectively is 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/L KCl, 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 pH 8.0,2mmol/L EDTA and 1.2 volume %Triton X-100;
Described colour developing liquid 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 to be formed, and the bottom of tube cavity is provided with the dividing plate of the longitudinal extension that is divided into A, two cavitys of B.
More preferably embodiment as Brucella detection kit of the present invention, working fluid or colour developing liquid are housed respectively in two cavitys of described A, B, seal up for safekeeping by stable liquid on liquid upper strata in two cavitys, and described working fluid is mixing of reaction solution and Bst archaeal dna polymerase.
The present invention also provides a kind of using method of Brucella detection kit, and this method comprises the steps:
(1) testing sample is centrifugal, remove supernatant, obtain precipitation;
(2) precipitation with step (1) adds sample pretreatment liquid, mix, and cooled on ice after the boiling water bath deactivation, high speed centrifugation, supernatant are the sample template DNA;
(3) in reaction vessel, add Bst archaeal dna polymerase 0.9～1.8 volume parts, reaction solution 38～40 volume parts, stable liquid 52～54.5 volume parts, sample template DNA 4.5～9 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 colour developing liquid respectively, mixing, the sample sets colour developing is identical with control group then positive, otherwise negative;
Inner primer FIP/BIP in the described step (3) and outer primer F3/B3 for be target gene with the OMP25 gene of Brucella, based on two pairs of primers of loop-mediated isothermal amplification technology design.
Use the preferred implementation of the method for Brucella detection kit as the present invention, described two pairs of 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。
Use the preferred implementation of the method for Brucella detection kit as the present invention, in the 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, is not the method for diagnosis or treatment disease.Brucella detection kit of the present invention can detect whether contain Brucella in the food, and food safety is significant.Simultaneously, Brucella detection kit of the present invention can also play the effect of detection to the pollution whether various other materials are subjected to Brucella.
The present invention is said based on loop-mediated isothermal amplification technology (loop-mediated isothermal amplication of DNA, abbreviation LAMP) method of rapid detection Brucella, be utilize the Bst archaeal dna polymerase 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 the specific recognition target sequence, start the endless chain replacement(metathesis)reaction, start complementary strand in target DNA district synthetic, and result's complementary sequence on same chain goes round and begins again and is formed with the very stem of polycyclic Cauliflower structure-circular DNA mixture.In the LAMP reaction process, pyrophosphate ion of separating out from dNTP and the Mg the reaction soln
2+In conjunction with, produce by product---magnesium pyrophosphate milky white precipitate, can be by the visual inspection result of determination.The LAMP reaction is to finish in 45～90 minutes under constant temperature (63～65 ℃) condition.This comparatively gentle temperature condition and do not have temperature cycle that required instrument is oversimplified, overcome conventional P CR inherent detection time long, pollute easily and detect shortcoming such as cost height.In addition, this detection method is lower to testing staff's technical quality requirement, and actually operating is very easy, does not need special reagent and plant and instrument, helps setting up rapid screening system with low cost.The 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 the fluorescence real-time quantitative PCR technology) are compared, can find that this technology is equivalent to or is better than round pcr on 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 detect cost far below fluorescent quantitative PCR technique.At present in the national standard with microorganism separation and Culture and morphology be accredited as main, in conjunction with the passing method that biochemical analysis and serological typing are identified, preliminary evaluation needs 2～3 days, finishes probation report and needs 10～15 days; Adopt detection kit of the present invention only to need 2 hours.And, having added colour developing liquid in the reaction system of the present invention, 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 it is low to detect cost; 2. gene quick diagnosis kit of the present invention is used six sections, four primers, and whether the existence that just can judge target substance according to whether increasing to be, so has high specific; 3. detection kit of the present invention amplification fast and efficient can be finished amplification less than 1 hour, and the productive rate height; 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 of the present invention is identified easy, pyrophosphate ion of separating out from dNTP and the Mg the reaction soln
2+In conjunction with, produce by product---the magnesium pyrophosphate precipitation, can identify by visual inspection, and after adding colour developing liquid, yin and yang attribute colour development difference as a result is remarkable, checking rate height, more obviously reliable; 6. owing to selected the OMP25 gene of high conservative property to design primer, make that the accuracy rate of detection kit detection Brucella of the present invention is higher as target gene; 7. in detection kit of the present invention, adopt special reaction tubes, reduced the aerosol contamination of heavy, 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 (large fragment), Bst archaeal dna polymerase (big 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 outer membrane protein 25 genes
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: the Bst archaeal dna polymerase places 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, place container;
(4) preparation sample pretreatment liquid: sample pretreatment liquid contains 20mmol/L Tris-HCl (pH 8.0), 2mmol/L EDTA and 1.2 volume %Triton X-100, places container;
(5) purchase stable liquid: paraffin oil places container;
(6) purchase colour developing liquid: SYBR Green I places container;
(7) extract positive control: extract the Brucella genomic dna, place container;
(8) above-mentioned 7 containers are dressed up test kit, encapsulation.
Preparation technology is summarized as follows:
1, with behind inner primer FIP/BIP and the outer primer F3/B3 synthesizing and purifying, quantitatively preparation, concentration detects, the sampling quality inspection;
2, determine the quality inspection of sampling with carrying out concentration with the liquid asepsis packing of above-mentioned (2)～(4) step preparation, and according to experiment;
3, with the stable liquid packing, the sampling quality inspection;
4, with the positive control sample preparations, packing, sampling quality inspection;
5, assembling test kit.
In other embodiment of Brucella detection kit of the present invention, the primer that is adopted 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: the Bst archaeal dna polymerase places 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, place container;
(4) preparation sample pretreatment liquid: sample pretreatment liquid contains 10mmol/L Tris-HCl (pH 8.0), 1mmol/L EDTA and 1.0 volume %Triton X-100, places container;
(5) purchase stable liquid: paraffin oil places container;
(6) purchase colour developing liquid: EVA Green I places container;
(7) extract positive control: extract the Brucella genomic dna, place container;
(8) above-mentioned 7 containers are dressed up test kit, encapsulation.
Other are with embodiment 1.
In other embodiment of Brucella detection kit of the present invention, the primer that is adopted 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 kit
1 materials and methods
1.1 material
1.1.1 bacterial strain
The present invention adopts bacterial strain that 28 strains are arranged, and is mainly derived from disease prevention and control center, heart Guangzhou, clinical isolates strain and environment separation bacterial strain.See table 1 for details.
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 bacterium liquid that the isolation identification of clinical separation strain will be slight muddiness has precipitation not form mycoderm directly is applied to blood agar, on LIA or the brucella agar flat board, under little aerobic (10% carbonic acid gas) and aerobic conditions, putting 36 ± 1 ℃ cultivated 3 days, take out then, 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;
Mix 1.3.2 in above-mentioned bacterial sediment, add 100 μ L sample pretreatment liquid, boil in the boiling water after 20 minutes and placed cooled on ice immediately 10 minutes, centrifugal 2 minutes of 10000rpm, supernatant is the sample template DNA.
1.4, adopt the test kit of embodiment 1 or embodiment 2 to carry out the reaction process of loop-mediated isothermal amplification technique
1.4.1 prepare reaction system at 200 μ L reaction tubess: 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 with the reaction tubes for 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, mixing, also add SYBR Green I mixing in heliotropism control tube (Brucella genomic dna) and the negative control pipe (deionized water) simultaneously, if the same shows green with the positive control pipe of reaction tubes is then positive, as if reaction tubes the same with the negative control pipe manifest orange then negative.
1.6 electrophoresis
Prepare 0.2% agarose gel electrophoresis.Carry out the color reaction observations with SYBR Green I as dyestuff.
1.7 specific degree test
1.7.1 pure bacterial strain LAMP detects and with the LAMP method 25 strain bacteriums increased, and is green positive according to the color reaction observations, orange feminine gender, verification method specificity.
1.7.2 the several bacterial strains hybrid dna detects the DNA equal-volume mixed solution that to Brucella and Salmonellas, singly increases listeria spp, streptococcus aureus with LAMP and gets 2.5ul and make LAMP and detect.
1.8 sensitivity test is cultivated 24 ± 2 hours with Brucella in brucella broth after, get 1mL bacterium liquid and be suspended in the 5mL stroke-physiological saline solution, with 10 times of doubling dilutions to 10 of physiological saline
-10Select 3 suitable concentration levels to get 100ul and be tiled in blood agar, on LIA or the brucella agar flat board, make 3 flat boards respectively, cultivated 3 days for 36 ± 1 ℃, get the flat board of colony number between 30～300 and make plate count, the mean of the colony number of 3 flat boards of this concentration level is calculated bacterial concentration, is bacterium colony mean number * extension rate * 10; Simultaneously, each concentration level is got 1mL, extracts DNA, makes LAMP and detects.
1.9 test of replica test specific degree and sensitivity test repeat respectively 2 times.
2 results
2.1 the foundation of Brucella LAMP detection method
2.2 the specific degree test Brucella detected result positive, the non-Brucella of 7 strains is all negative, 17 routine samples of Brucella and Brucella, Salmonellas, singly increase listeria spp, 4 kinds of DNA of bacteria mixed solutions of streptococcus aureus are positive, as shown in table 2.The visualizingre agent box has high specific as a result.
| Test sample | The result | Test sample | The result | Test sample | The 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 |
| The Listeria monocytogenes Salmonella | Negative | Brucella 7 | Positive | Brucella 16 | Positive |
| Yersinia entero-colitica | Negative | Brucella 8 | Positive | Brucella 17 | Positive |
| The beta hemolysis suis | Negative | Brucella 9 | Positive | The DNA of bacteria mixed solution | Positive |
2.3 sensitivity test
Through the bacterium colony plate count, select the 9th extent of dilution to carry out reading, average colony number is 126cfu, calculates that the bacterium original liquid concentration is 1.26 * 10
11Cfu/mL, the LAMP method can detect the 4th extent of dilution, is 1.26 * 10
7Cfu/mL.
Electrophoresis result also meets The above results.
2.4 the test of replica test specific degree repeats twice, as a result unanimity.Sensitivity test repeats twice, order-of-magnitude agreement.
Embodiment 4 adopts the Brucella detection kit of reaction tubes
The Brucella detection kit of present embodiment, the reagent that adopts is identical with embodiment 1 with primer, test kit also comprises reaction tubes, reaction tubes covers two portions by body and pipe to be formed, the bottom of tube cavity is provided with the dividing plate of the longitudinal extension that is divided into A, two cavitys of B, wherein: the LAMP reaction solution of 22.0 μ L and the Bst archaeal dna polymerase of 0.5 μ L are housed in the cavity A, and the liquid upper strata is sealed up for safekeeping by paraffin; The LAMP reaction solution liquid of 2.0 μ L is housed in the cavity B, and the liquid upper strata is also sealed up for safekeeping by paraffin, with this reaction tubes-20 ℃ preservation.
Present embodiment adopts reaction tubes as container, Brucella is carried out LAMP detect, and 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 each 2.5 μ L of negative control sample, detected sample and positive control sample respectively, rifle head pierce through the protection liquid layer during application of sample, sample adds in the reaction tubes A chamber; the tight pipe lid of lid is also carried out mark, moves to reaction zone.
With above-mentioned reaction tubes all in 65 ℃ of isothermal reaction 1h.
Reaction finishes, and reaction tubes is inverted whipping 1 time, is just putting whipping again 1 time, and working fluid and colour developing liquid thorough mixing are evenly observed the back.
If the same shows green with the positive control pipe of reaction tubes is then positive, as if reaction tubes the same with the negative control pipe manifest orange then negative.
In the LAMP reaction process, colour developing liquid and working fluid sealed state is good, not have to take place situation about revealing mutually, and the later stage is during color reaction, is inverted the whipping operation and makes colour developing liquid and working fluid mixing, and the result that develops the color 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 has been done detailed description with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from the essence and the scope of technical solution of the present invention.
Claims (10)
1. Brucella detection kit, it is characterized in that, described Brucella detection kit comprise the OMP25 gene with Brucella be target gene, based on two pairs of primers of loop-mediated isothermal amplification technology design: inner primer FIP/BIP and outer primer F3/B3.
2. Brucella detection kit according to claim 1 is characterized in that, described two pairs of primers are
Outer primer F3 (1):
CCTTTGCTGGCTGGAACT
Outer primer B3 (1):
GCAATACCAGCCGTGAGG
Inner primer FIP (1):
GTCCTTGGACTTCTTGGCCCAGttttTCCAGCAGGACCAGATCG
Inner primer BIP (1):
GGCCTGGAAGTCAAGCAGGGttttACGGCATAACCGGGTTCA
Or
Outer primer F3 (2):
CAAGACCAGCACCGTTGG
Outer primer B3 (2):
GGTTCAGGTCGTAGCCGA
Inner primer FIP (2):
GGTCCTGCTGGAAGTTCCAGCttttAGCATCAAGCCTGACGATTG
Inner primer BIP (2):
CGGTGTTGAAGGTGATGCAGGTttttTTCAAAGCCCTGCTTGACTT
Or
Outer primer F3 (3):
CGTCGGCTACGACCTGAA
Outer primer B3 (3):
ACCGGCCAGATCATAGTTCT
Inner primer FIP (3):
TCGTCCAAGCCGTTGTTAAGCTttttCCCGGTTATGCCGTACCT
Inner primer BIP (3):
GTGCCGGTCTCGAAGCCAAGttttTGTTGCCGTACTGGGTGTA
Or
Outer primer F3 (4):
ATTGCCGGTTCGCAGATC
Outer primer B3 (4):
GCGGATATCCTGCGTGTC
Inner primer FIP (4):
TCAGCTTGGCTTCGAGACCGttttCTTAACAACGGCTTGGACGA
Inner primer BIP (4):
GGCCGCGTTGAGTACCGTTAttttAGCTTGTTGCGAACAGTCG。
3. Brucella detection kit according to claim 1 is characterized in that, described Brucella detection kit also comprises Bst archaeal dna polymerase, reaction solution, stable liquid, sample pretreatment liquid, colour developing liquid and positive control solution.
4. Brucella detection kit according to claim 3 is characterized in that,
Described Bst archaeal dna polymerase: 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 pH 8.0,1～2mmol/LEDTA and 1～1.2 volume %Triton X-100;
Described colour developing liquid is SYBR Green I or Eva Green;
Described stable liquid is paraffin oil;
Described positive control is the Brucella genomic dna;
Described inner primer FIP/BIP respectively is 1.2～2.0 μ mol/L, and the concentration of outer primer F3/B3 respectively is 0.2～0.25 μ mol/L.
5. Brucella detection kit according to claim 4 is characterized in that,
Described BstDNA polysaccharase: enzyme concn 8U/ μ L;
Described reaction solution contains: 2mmol/L dNTP, 25mmol/L Tris-HCl, 12.5mmol/L KCl, 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 pH 8.0,2mmol/L EDTA and 1.2 volume %Triton X-100;
Described colour developing liquid is SYBR Green I;
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.
6. according to claim 3,4 or 5 described Brucella detection kit, it is characterized in that, described Brucella detection kit also comprises reaction tubes, described reaction tubes covers two portions by body and pipe to be formed, and the bottom of tube cavity is provided with the dividing plate of the longitudinal extension that is divided into A, two cavitys of B.
7. Brucella detection kit according to claim 6, it is characterized in that, working fluid or colour developing liquid are housed respectively in two cavitys of described A, B, and seal up for safekeeping by stable liquid on the liquid upper strata in two cavitys, and described working fluid is mixing of reaction solution and Bst archaeal dna polymerase.
8. a method of using Brucella detection kit as claimed in claim 3 is characterized in that this method comprises the steps:
(1) testing sample is centrifugal, remove supernatant, obtain precipitation;
(2) precipitation with step (1) adds sample pretreatment liquid, mix, and cooled on ice after the boiling water bath deactivation, high speed centrifugation, supernatant are the sample template DNA;
(3) in reaction vessel, add Bst archaeal dna polymerase 0.9～1.8 volume parts, reaction solution 38～40 volume parts, stable liquid 52～54.5 volume parts, sample template DNA 4.5～9 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 colour developing liquid respectively, mixing, the sample sets colour developing is identical with control group then positive, otherwise negative;
Inner primer FIP/BIP in the described step (3) and outer primer F3/B3 for be target gene with the OMP25 gene of Brucella, based on two pairs of primers of loop-mediated isothermal amplification technology design.
9. the method for use Brucella detection kit according to claim 8 is characterized in that described two pairs of primers are
Outer primer F3 (1):
CCTTTGCTGGCTGGAACT
Outer primer B3 (1):
GCAATACCAGCCGTGAGG
Inner primer FIP (1):
GTCCTTGGACTTCTTGGCCCAGttttTCCAGCAGGACCAGATCG
Inner primer BIP (1):
GGCCTGGAAGTCAAGCAGGGttttACGGCATAACCGGGTTCA
Or
Outer primer F3 (2):
CAAGACCAGCACCGTTGG
Outer primer B3 (2):
GGTTCAGGTCGTAGCCGA
Inner primer FIP (2):
GGTCCTGCTGGAAGTTCCAGCttttAGCATCAAGCCTGACGATTG
Inner primer BIP (2):
CGGTGTTGAAGGTGATGCAGGTttttTTCAAAGCCCTGCTTGACTT
Or
Outer primer F3 (3):
CGTCGGCTACGACCTGAA
Outer primer B3 (3):
ACCGGCCAGATCATAGTTCT
Inner primer FIP (3):
TCGTCCAAGCCGTTGTTAAGCTttttCCCGGTTATGCCGTACCT
Inner primer BIP (3):
GTGCCGGTCTCGAAGCCAAGttttTGTTGCCGTACTGGGTGTA
Or
Outer primer F3 (4):
ATTGCCGGTTCGCAGATC
Outer primer B3 (4):
GCGGATATCCTGCGTGTC
Inner primer FIP (4):
TCAGCTTGGCTTCGAGACCGttttCTTAACAACGGCTTGGACGA
Inner primer BIP (4):
GGCCGCGTTGAGTACCGTTAttttAGCTTGTTGCGAACAGTCG。
10. according to Claim 8 or the methods of 9 described use Brucella detection kit, it is characterized in that,
In the step (3), the reaction conditions of isothermal reaction is 63～65 ℃ of temperature, reaction times 45～90min.
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