CN110499377A - A kind of primer and probe, kit and application detecting mycobacterium tuberculosis and its drug resistant gene - Google Patents
A kind of primer and probe, kit and application detecting mycobacterium tuberculosis and its drug resistant gene Download PDFInfo
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- CN110499377A CN110499377A CN201910859195.8A CN201910859195A CN110499377A CN 110499377 A CN110499377 A CN 110499377A CN 201910859195 A CN201910859195 A CN 201910859195A CN 110499377 A CN110499377 A CN 110499377A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention discloses a kind of detection mycobacterium tuberculosis and its primer and probe, the kits and application of drug resistant gene, kit includes: amplification reaction solution, enzyme mixation and chip, it include the primer in the amplification reaction solution, the chip includes shell and is fixed on the enclosure interior for detecting the test strips of mycobacterium tuberculosis and its drug resistant gene；The test strips include successively overlapped sample pad, label pad, solid phase carrier and the blotting paper being arranged in backlight；The probe is crosslinked on the solid phase carrier.Testing conditions needed for kit of the present invention are simple, and detection speed is fast, and testing cost is low, are especially suitable for carrying out that tuberculosis and its Drug Resistance Detection are advanced to basic medical unit, promote the pathogen positive rate of tuberculosis with to the detecting field with inspection formula；The present invention can detect the nucleic acid of mycobacterium tuberculosis complex and cross reaction does not occur with non-tuberculous mycobacteria, and the present invention includes two kinds of medicament-resistant mutation detections.
Description
Technical field
The present invention relates to field of biotechnology more particularly to the primers of a kind of detection mycobacterium tuberculosis and its drug resistant gene
And probe, kit and application.
Background technique
Mycobacterium tuberculosis is a kind of ancient pathogen, can cause human tuberculosis, be the important prestige of human health
The side of body, referred to as white pestilence, the World Health Organization is using tulase recall rate as the important indicator of tuberculosis diagnosis and treatment, tuberculosis branch bar
Bacterium detection of nucleic acids has become the important means of detection mycobacterium tuberculosis complex.It is lungy to detect the method mainly used
Including quantitative fluorescent PCR (polymerase chain reaction) and a variety of constant-temperature amplification schemes.
Fluorescent quantitative PCR technique is nucleic acid detection technique most mature at present, and sensitivity and specificity are higher, Er Qieyin
Object design is relatively easy, also multiplex amplification easy to accomplish, therefore, can not only carry out mycobacterium tuberculosis detection but also carry out tuberculosis point
Branch bacillus Drug Resistance Detection, therefore, this method is the mainstream of current tuberculosis detection technique.However, due to being expanded using alternating temperature
Technology, needs place and the reviewer of a large amount of instrument and equipment and profession, and operates also relatively complicated；Furthermore, it is necessary to examine
It surveys the time and detection sample size causes hospital to need to accumulate and just can be carried out detection after a certain amount of sample, to make detection of nucleic acids
The timeliness of mycobacterium tuberculosis complex substantially reduces.
Mycobacterium tuberculosis is detected using constant-temperature amplification method, due to using isothermal amplification technology, no longer needs to hold high
Expensive detecting instrument realizes lifting temperature function, detection overall cost decline, further, since the most suitable amplification where constant-temperature amplification
Temperature also has certain use to the hospital for having had construction molecular diagnostic laboratories in fluorescent quantitation.It is answered in terms of tuberculosis at present
Main isothermal amplification technology includes:
Cross primer isothermal amplification technology is a kind of technology that amplification chain is opened using primer interleaving techniques, however, due to
The technology primer quantity itself is more, and there are technology barriers on multiplex amplification.
RPA technology (recombinase polymerase amplification, recombinase polymeric enzymatic amplification technology) is
The technology detected using recombinase, the technology amplification rate is fast, and this method has the spy of simple and quick suitable on-site test
Point, but due to the ready-made system of system purchase in research process, and probe used is nfo probe, testing cost is higher, difficult
To realize commercialization.
Therefore, prior art Shortcomings need to improve.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of detection mycobacterium tuberculosis and its drug resistant gene
Primer and probe, kit and application.
Technical scheme is as follows: providing a kind of primer and spy for detecting mycobacterium tuberculosis and its drug resistant gene
Needle, comprising: the primer with nucleotide sequence shown in SEQ ID NO.1-10, and, there is core shown in SEQ ID NO.11-15
The probe of nucleotide sequence；In SEQ ID NO.1/SEQ ID NO.3/SEQ ID NO.5/SEQ ID NO.7/SEQ ID NO.9
In shown nucleotide sequence, from 5 ' end label biotins；In SEQ ID NO.11/SEQ ID NO.12/SEQ ID NO.13/
In nucleotide sequence shown in SEQ ID NO.14/SEQ ID NO.15, from 5 ' end label amino.
Further, in SEQ ID NO.11/SEQ ID NO.12/SEQ ID NO.13/SEQ ID NO.14/SEQ ID
In nucleotide sequence shown in NO.15, from 5 ' end label quenching groups, from 3 ' end mark fluorescent groups.
The present invention also provides a kind of detection mycobacterium tuberculosis and its kits of drug resistant gene, comprising: amplification reaction solution,
Enzyme mixation and chip, include primer as described above in the amplification reaction solution, and the chip includes shell and is fixed on institute
Enclosure interior is stated for detecting the test strips of mycobacterium tuberculosis and its drug resistant gene.
The test strips include successively overlapped sample pad, label pad, solid phase carrier and the water suction being arranged in backlight
Paper.
Probe as described above is crosslinked on the solid phase carrier.
Further, include on the solid phase carrier nature controlling line location of C, mycobacterium tuberculosis site, the site InhA15,
The site KatG, the site RopB and IC internal standard, mycobacterium tuberculosis site is bright to indicate mycobacterium tuberculosis infection, the site InhA15
Bright 15 site of expression InhA gene promoter mutates, the site the KatG and site RopB is bright indicates that the site is not dashed forward
Become, the site IC is internal standard site, and the kit is able to carry out two kinds of drug-tolerant gene mutation detections.
The amplification reaction solution further include: TrisAc buffer, potassium acetate, PEG20000, dNTPs, phosphocreatine, creatine
Kinases, disodium creatine phosphate, DTT and BSA；The enzyme mixation includes: T4 UVSX albumen, T4 UvSY albumen, T4gp32
Albumen, BSU archaeal dna polymerase or klenow segment, the amplification reaction solution and the enzyme mixation are divided in two pipes, are made
The two is uniformly mixed with preceding.
It is linked in the NC film after the probe and sampling liquid cooperation, the sampling liquid includes probe, glycerol, sulphur
Willow mercury, trypan blue and 10 × PBS.
The label pad includes any one of colloidal gold, color latex microballoon and quantum dot, in the label pad subscript
Remember SA.
The solid phase carrier is any one of NC film, sheet glass, pvdf membrane.
Further, the kit further includes dilution, nucleic acid containging interior traget, magnesium acetate, and the dilution is added
It is sealed in chip reservoir with masking foil, the magnesium acetate is added the sample application zone of reservoir and air-dries.
Further, plasmid or plasmid bacterial containing following segment are designated as in described:
ttgcatcagaagaactaaattttgatacttactggctagagcatcattttactgaatttggactaaca
ggtaacctgtttgttgcttgtgctaacttacttggtcgaaccaccaaactgaatgttggtactatgattgttcttc
caacagctcaccctgcacgtcagatggaagatttattacttttagatca。
The solid phase carrier is NC film, and the label pad is color latex microballoon.
The dilution includes ammonium acetate, potassium acetate, Triton X-100, trisodium citrate and water.
Include on the solid phase carrier nature controlling line location of C, mycobacterium tuberculosis site, the site InhA15, the site KatG,
The site RopB and IC internal standard, mycobacterium tuberculosis site is bright to indicate mycobacterium tuberculosis infection, the bright expression in the site InhA15
15 site of InhA gene promoter mutates, the site the KatG and site RopB is bright indicates that the site does not mutate, and IC
Point is internal standard site, and the kit is able to carry out two kinds of drug-tolerant gene mutation detections.
The amplification reaction solution further include: TrisAc buffer, potassium acetate, PEG20000, dNTPs, phosphocreatine, creatine
Kinases, disodium creatine phosphate, DTT and BSA；The enzyme mixation includes: T4 UVSX albumen, T4 UvSY albumen, T4gp32
Albumen, BSU archaeal dna polymerase or klenow segment, the amplification reaction solution and the enzyme mixation are divided in two pipes, are made
The two is uniformly mixed with preceding；
It is linked in the NC film after the probe and sampling liquid cooperation, the sampling liquid includes probe, glycerol, sulphur
Willow mercury, trypan blue and 10 × PBS；
The label pad includes any one of colloidal gold, color latex microballoon and quantum dot, in the label pad subscript
Remember SA；
The solid phase carrier is any one of NC film, sheet glass, pvdf membrane.
The present invention also provides the preparation methods of a kind of detection mycobacterium tuberculosis and its drug resistant gene kit, specifically include
Following steps:
The first step prepares amplification reaction solution and enzyme mixation.
Second step prepares chip, specifically includes:
1) sampling liquid is prepared；
2) point sample on solid phase carrier；
3) label pad is prepared, label pad is marked into upper SA；
4) bonding pad treatment fluid is prepared, and prepares bonding pad；
5) sample pad treatment fluid is prepared, and prepares sample pad；
6) backlight prelaminar part pad pasting is torn, sticks bonding pad, bonding pad leading portion stretches out solid phase carrier and connects with sample pad
Touching；
7) prepared and diluted liquid；
8) the above-mentioned dilution prepared is added into chip reservoir, then seals buffer area with masking foil；
9) magnesium acetate solution is prepared, magnesium acetate is added to the water and is made into magnesium acetate solution；
10) to reservoir sample application zone be added the 10 prepared magnesium acetate solutions of μ L, natural air drying 2 hours；
11) clip for opening chip, is added gasket, clip is then added on gasket；
12) upwards, adding mouth is closed with sealing strip；
13) clip is closed, two dimensional code is pasted.
Further, amplification reaction solution described in 1mL includes: the vinegar of TrisAc buffer, 70mM that 20mM pH value is 7.4
Sour potassium, the dNTPs of weight percent 7%PEG20000,50mM, 100 μM of phosphocreatines, 20ng/ μ L creatine kinase,
The primer of the disodium creatine phosphate of 100ng/ μ L, the BSA and 20pM of 20 μM of DTT, 10 μ g/mL, above-mentioned component is by anti-
Final concentration is answered to calculate.
Enzyme mixation described in 1mL includes: the T4 UVSX albumen of 120ng/ μ L, the T4UvSY albumen of 30ng/ μ L, 40ng/ μ L
T4gp32 albumen, 0.1U/ μ L BSU archaeal dna polymerase or klenow segment, above-mentioned component by reaction final concentration calculate.
The amplification reaction solution and the enzyme mixation are divided in two pipes, are uniformly mixed the two using preceding.
Sampling liquid described in 1mL includes: the probe of 100 μ L, glycerol, the 1mg of 500 μ L final concentration of 50% final concentration of
0.1% thimerosal, the trypan blue of 0.1mg final concentration of 0.01%, 100 μ L 10 × PBS, wherein the probe includes: 100
IC P of the final concentration of 10nM of μ L, TB P of the 200 final concentration of 20nM of μ L, ROB P of the 350 final concentration of 35nM of μ L, 400 μ L are whole
Concentration is the chemical reaction of KatS P, the 60 final concentration of 0.06mg/ml of μ L of InhA P of 40nM, the 180 final concentration of 18nM of μ L
Quality Control.
The point sample on solid phase carrier specifically: pad pasting in the middle part of backlight is torn, solid phase carrier is sticked, uses point sample instrument
The above-mentioned sampling liquid prepared is successively put according to 3 × 2 arrays and is formed on solid phase carrier, the solid phase carrier put dries 8 in 80 DEG C
Hour；Or 37 DEG C of drying 30min, then UV crosslinking 2min.
Dilution described in 1mL includes: the ammonium acetate of 0.02Mg, the potassium acetate of 0.02Mg, 20 μ L final concentration of 2%
Triton X-100, the trisodium citrate of 0.04Mg, water.
The present invention also provides the applications of a kind of detection mycobacterium tuberculosis and its drug resistant gene kit, using as described above
Kit carry out the detection of mycobacterium tuberculosis and its drug resistant gene, comprising the following specific steps
Step S1 extracts nucleic acid；
Step S2 prepares reaction system, the amplification reaction solution is proportionally mixed with the enzyme mixation, and composition is anti-
Answer system；
Step S3, by after extraction nucleic acid and reaction system mix；
Step S4, by the obtained hybrid reaction system of the step S3 be placed under 30-41 DEG C of constant temperature carry out 20min~
The amplified reaction of 35min；
The obtained hybrid reaction system of the step S4 is diluted and hybridizes colour developing by step S5；
The interpretation of reaction result: step S6 carries out interpretation by nucleic acid detection apparatus.
Further, nucleic acid and 140nM magnesium acetate containging interior traget is added before the step S4.
Hybrid reaction system is placed in the amplified reaction that 20min is carried out under 40 DEG C of constant temperature in the step S4.
Crossing system described in the step S5 includes any one of micro-fluidic, lateral chromatography, diafiltration, when hybridization develops the color
Between be 10min.
Using the above scheme, it is only needed using the process of kit detection mycobacterium tuberculosis and its drug resistant gene of the invention
It is carried out under the conditions of simply, has detection speed fast, the low feature of testing cost is especially suitable for carrying out the inspection with arriving with inspection formula
Field is surveyed, tuberculosis and its Drug Resistance Detection are advanced to basic medical unit, the pathogen positive rate of tuberculosis can be greatly promoted；This hair
The bright nucleic acid that can detect mycobacterium tuberculosis complex and cross reaction, and packet of the present invention does not occur with non-tuberculous mycobacteria
It is detected containing two kinds of medicament-resistant mutations.
Detailed description of the invention
Fig. 1 is the judging result ideograph of the chip provided in one embodiment of the invention；
Fig. 2 is the judging result ideograph of the chip provided in another embodiment of the present invention；
Fig. 3 is the flow chart for the detection that the present invention carries out mycobacterium tuberculosis and its drug resistant gene.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.Particular technique is not specified in embodiment
Or condition person, described technology or conditions (such as write, Huang Peitang with reference to J. Pehanorm Brooker etc. according to the literature in the art
Etc. " Molecular Cloning:A Laboratory guide " translated, the third edition, Science Press) or carry out according to product description.Agents useful for same or
Production firm person is not specified in instrument, and being can be with conventional products that are commercially available.
The present invention provides a kind of primer and probe for detecting mycobacterium tuberculosis and its drug resistant gene, comprising: has SEQ
The primer of nucleotide sequence shown in ID NO.1-10, as shown in table 1；And there is nucleotides sequence shown in SEQ ID NO.11-15
The probe of column, as shown in table 2.Colour developing scheme generally preferably is molecule hybridization, i.e., by the primer mark biotin come real
Show, i.e. the nucleotide shown in SEQ ID NO.1/SEQ ID NO.3/SEQ ID NO.5/SEQ ID NO.7/SEQ ID NO.9
In sequence, from 5 ' end label biotins；Probe marks amino, i.e., in SEQ ID NO.11/SEQ ID NO.12/SEQ ID
In nucleotide sequence shown in NO.13/SEQ ID NO.14/SEQ ID NO.15, from 5 ' end label amino.It is of course also possible to logical
Cross and the probe marked into quenching group and fluorophor, then by selection have 3 ' hold the archaeal dna polymerase of 5 prime excision enzyme activities come
Detection is realized, specifically, in SEQ ID NO.11/SEQ ID NO.12/SEQ ID NO.13/SEQ ID NO.14/SEQ ID
In nucleotide sequence shown in NO.15, from 5 ' end label quenching groups, from 3 ' end mark fluorescent groups.
Table 1, primer sequence table:
Table 2, probe sequence table:
| Title | Sequence | Number |
| TB P: | agatctggcgccaaatctggtccaac | SEQ11 |
| InhA P: | aacggattctggttagcgga | SEQ12 |
| ROB P: | CCAGCGCCGACAGTCGGCGCTTGTGGGTCA | SEQ13 |
| KatS P: | GAACTCGTCGGCCAATTCCTCGGGGT | SEQ14 |
| IC P: | ttcttccaacagctcaccct | SEQ15 |
The present invention also provides a kind of detection mycobacterium tuberculosis and its kits of drug resistant gene, comprising: amplified reaction
Liquid, enzyme mixation and chip include primer as described above in the amplification reaction solution, and the chip includes shell and fixation
It is used to detect the test strips of mycobacterium tuberculosis and its drug resistant gene in the enclosure interior.Specifically, the test strips include
Successively overlapped sample pad, label pad, solid phase carrier and blotting paper in backlight is set.It is crosslinked on the solid phase carrier
Probe as described above, the solid phase carrier include but is not limited to any one of NC film, sheet glass, pvdf membrane, preferably NC film.
The amplification reaction solution further include: TrisAc buffer, potassium acetate, PEG20000, dNTPs, phosphocreatine, creatine
Kinases, disodium creatine phosphate, DTT and BSA；The enzyme mixation includes: T4 UVSX albumen, T4 UvSY albumen, T4gp32
Albumen, BSU archaeal dna polymerase or klenow segment, the amplification reaction solution and the enzyme mixation are divided in two pipes, are made
The two is uniformly mixed with preceding.
The probe and sampling liquid cooperation after is linked in the NC film, the sampling liquid further include glycerol, thimerosal,
Trypan blue and 10 × PBS.
The label pad includes but is not limited to any one of colloidal gold, color latex microballoon and quantum dot, preferably colour
Latex beads mark SA in the label pad.
The kit further includes dilution, nucleic acid containging interior traget, magnesium acetate, and chip reservoir is added in the dilution
Interior to be sealed with masking foil, the dilution includes ammonium acetate, potassium acetate, Triton X-100, trisodium citrate and water.The vinegar
Sour magnesium is added the sample application zone of reservoir and air-dries.Plasmid or plasmid bacterial containing segment in table 3 are designated as in described:
Table 3, interior label sequence table:
Please refer to Fig. 1 and Fig. 2, include on the solid phase carrier nature controlling line location of C, mycobacterium tuberculosis site,
The site InhA15, the site KatG, the site RopB and IC internal standard, mycobacterium tuberculosis site is bright to indicate mycobacterium tuberculosis sense
Dye, bright 15 site of expression InhA gene promoter in the site InhA15 mutate, and the bright expression in the site KatG and the site RopB should
Site does not mutate, and the site IC is internal standard site, and the kit is able to carry out two kinds of drug-tolerant gene mutation detections.
Referring to Fig. 1, when the kit has the function of to detect mycobacterium tuberculosis and drug resistant gene simultaneously: chemistry is anti-
Quality Control is answered to remove identification related locus as mark, mycobacterium tuberculosis site is bright to indicate mycobacterium tuberculosis infection, InhA15
(C → T) occurs for bright 15 site of expression InhA gene promoter in site, the site the KatG and site RopB is bright indicates that the site is not sent out
Raw mutation.The site IC is internal standard site.
Referring to Fig. 2, there is detection mycobacterium tuberculosis or a certain drug resistant gene locus when part is using the kit
Function, chemical reaction Quality Control as mark goes identification the site IC be internal standard site.The one kind mentioned in any table 1 can be added
Or several sequences.
It is specific to wrap the present invention also provides the preparation method of a kind of detection mycobacterium tuberculosis and its drug resistant gene kit
Include following steps:
The first step prepares amplification reaction solution and enzyme mixation,
The amplification reaction solution includes: the TrisAc buffer that 20mM pH value is 7.4, the potassium acetate of 70mM, weight percent
Than for 7%PEG20000,50mM dNTPs, 100 μM of phosphocreatines, the creatine kinase of 20ng/ μ L, 100ng/ μ L phosphoric acid flesh
Acid disodium salt, 20 μM of DTT, 10 μ g/mL BSA and 20pM the primer.
The enzyme mixation includes: the T4 UVSX albumen of 120ng/ μ L, the T4 UvSY albumen of 30ng/ μ L, 40ng/ μ L
T4gp32 albumen, the BSU archaeal dna polymerase of 0.1U/ μ L or klenow segment, the amplification reaction solution and the enzyme mixation point
In two pipes, the two is uniformly mixed using preceding.
Second step prepares chip, specifically includes:
1) preparation of sampling liquid: preparing the sampling liquid of 5 kinds of probes according to table 3,
Table 3 contains the following substances in 1ml sampling liquid:
Table 4, the additional amount of 5 kinds of probes:
2) point sample on solid phase carrier；
Pad pasting in the middle part of backlight is torn, NC film is sticked, with point sample instrument by the above-mentioned sampling liquid prepared according to 3 × 2 arrays
Successively point is formed on NC film, and the NC film put is dried 8 hours in 80 DEG C；Or 37 DEG C of drying 30min, then UV crosslinking 2min.Make
Probe is crosslinked in solid phase carrier NC film.
3) label pad is prepared, color latex microballoon is marked into upper SA.
4) bonding pad treatment fluid is prepared, and prepares bonding pad.
5) sample pad treatment fluid is prepared, and prepares sample pad.
6) backlight prelaminar part pad pasting is torn, sticks bonding pad, bonding pad leading portion stretches out NC film 0.5cm, connects with sample pad
Touching.
7) prepared and diluted liquid, it is as shown in table 5 that 1mL dilutes formula of liquid:
Table 5, the formula of dilution:
8) the above-mentioned dilution prepared of 105 μ L is added into chip reservoir, then seals buffer area with masking foil.
9) magnesium acetate solution is prepared, 25 μM of g magnesium acetates are added to the water and are made into the magnesium acetate solution of 100mL.
10) to reservoir sample application zone be added the 10 prepared magnesium acetate solutions of μ L, natural air drying 2 hours.
11) clip for opening chip, is added gasket, clip is then added on gasket.
12) upwards, adding mouth is closed with sealing strip
13) clip is closed, two dimensional code is pasted.
Referring to Fig. 3, making the present invention also provides the application of a kind of detection mycobacterium tuberculosis and its drug resistant gene kit
The detection of mycobacterium tuberculosis and its drug resistant gene is carried out with kit as described above, comprising the following specific steps
Step S1 extracts nucleic acid；
Step S2 prepares reaction system, the amplification reaction solution is proportionally mixed with the enzyme mixation, and composition is anti-
Answer system；
Step S3, by after extraction nucleic acid and reaction system mix；
Step S4, by the obtained hybrid reaction system of the step S3 be placed under 30-41 DEG C of constant temperature carry out 20min~
The amplified reaction of 35min；
The obtained hybrid reaction system of the step S4 is diluted and hybridizes colour developing by step S5；
The interpretation of reaction result: step S6 carries out interpretation by nucleic acid detection apparatus.
Further, nucleic acid and 140nM magnesium acetate containging interior traget is added before the step S4.
Hybrid reaction system is placed in the amplified reaction that 20min is carried out under 40 DEG C of constant temperature in the step S4.
Crossing system described in the step S5 includes any one of micro-fluidic, lateral chromatography, diafiltration, when hybridization develops the color
Between be 10min.
In conclusion only need to be in letter using the process of kit detection mycobacterium tuberculosis and its drug resistant gene of the invention
It is carried out under conditions of list, has detection speed fast, the low feature of testing cost is especially suitable for carrying out the detection with arriving with inspection formula
, tuberculosis and its Drug Resistance Detection are advanced to basic medical unit, the pathogen positive rate of tuberculosis can be greatly promoted；The present invention
The nucleic acid of mycobacterium tuberculosis complex can be detected and cross reaction does not occur with non-tuberculous mycobacteria, and the present invention includes
Two kinds of medicament-resistant mutation detections.
The above is merely preferred embodiments of the present invention, be not intended to restrict the invention, it is all in spirit of the invention and
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within principle.
Sequence table
<110>Shenzhen Xin Si microorganism Science and Technology Ltd.
<120>primer and probe, the kit and application of a kind of detection mycobacterium tuberculosis and its drug resistant gene
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agatctggcg ccaaatctgg tccaac 26
<210> 2
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gagcagcact gaccagcacc gaagaac 27
<210> 3
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
caattcatgg accagaacaa cccgctgtcg 30
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ctcaggggtt tcgatcgggc ac 22
<210> 5
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tgacacaaca caaggacgca catgacagga 30
<210> 6
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tgatgattcc gctaaccaga a 21
<210> 7
<211> 26
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 7
Gly Thr Ala Ala Gly Gly Ala Cys Gly Cys Gly Ala Thr Cys Ala Cys
1 5 10 15
Cys Ala Gly Cys Gly Gly Cys Ala Thr Cys
20 25
<210> 8
<211> 26
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
Gly Ala Ala Cys Thr Cys Gly Thr Cys Gly Gly Cys Cys Ala Ala Thr
1 5 10 15
Thr Cys Cys Thr Cys Gly Gly Gly Gly Thr
20 25
<210> 9
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
catcagaaga actaaatttt gatacttact gg 32
<210> 10
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gatctaaaag taataaatct tccatctgac 30
<210> 11
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
agatctggcg ccaaatctgg tccaac 26
<210> 12
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
aacggattct ggttagcgga 20
<210> 13
<211> 30
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 13
Cys Cys Ala Gly Cys Gly Cys Cys Gly Ala Cys Ala Gly Thr Cys Gly
1 5 10 15
Gly Cys Gly Cys Thr Thr Gly Thr Gly Gly Gly Thr Cys Ala
20 25 30
<210> 14
<211> 26
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 14
Gly Ala Ala Cys Thr Cys Gly Thr Cys Gly Gly Cys Cys Ala Ala Thr
1 5 10 15
Thr Cys Cys Thr Cys Gly Gly Gly Gly Thr
20 25
<210> 15
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ttcttccaac agctcaccct 20
<210> 16
<211> 193
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
ttgcatcaga agaactaaat tttgatactt actggctaga gcatcatttt actgaatttg 60
gactaacagg taacctgttt gttgcttgtg ctaacttact tggtcgaacc accaaactga 120
atgttggtac tatgattgtt cttccaacag ctcaccctgc acgtcagatg gaagatttat 180
tacttttaga tca 193
Claims (10)
1. the primer and probe of a kind of detection mycobacterium tuberculosis and its drug resistant gene characterized by comprising have SEQ ID
The primer of nucleotide sequence shown in NO.1-10, and, the probe with nucleotide sequence shown in SEQ ID NO.11-15；In
In nucleotide sequence shown in SEQ ID NO.1/SEQ ID NO.3/SEQ ID NO.5/SEQ ID NO.7/SEQ ID NO.9,
From 5 ' end label biotins；In SEQ ID NO.11/SEQ ID NO.12/SEQ ID NO.13/SEQ ID NO.14/SEQ ID
In nucleotide sequence shown in NO.15, from 5 ' end label amino.
2. primed probe group according to claim 1, which is characterized in that in SEQ ID NO.11/SEQ ID NO.12/
In nucleotide sequence shown in SEQ ID NO.13/SEQ ID NO.14/SEQ ID NO.15, from 5 ' end label quenching groups, certainly
3 ' end mark fluorescent groups.
3. the kit of a kind of detection mycobacterium tuberculosis and its drug resistant gene characterized by comprising amplification reaction solution, enzyme
Mixed liquor and chip include primer described in claim 1 in the amplification reaction solution, and the chip includes shell and fixation
It is used to detect the test strips of mycobacterium tuberculosis and its drug resistant gene in the enclosure interior；
The test strips include successively overlapped sample pad, label pad, solid phase carrier and the blotting paper being arranged in backlight；
The described in any item probes of claim 1-2 are crosslinked on the solid phase carrier.
4. kit according to claim 3, which is characterized in that include nature controlling line location of C, knot on the solid phase carrier
Core mycobacterium site, the site InhA15, the site KatG, the site RopB and IC internal standard, mycobacterium tuberculosis site is bright to be indicated
Mycobacterium tuberculosis infection, bright 15 site of expression InhA gene promoter in the site InhA15 mutate, the site KatG and RopB
Site is bright to indicate that the site does not mutate, and the site IC is internal standard site, and the kit is able to carry out two kinds of drug resistant genes
Abrupt climatic change.
The amplification reaction solution further include: TrisAc buffer, potassium acetate, PEG20000, dNTPs, phosphocreatine, creatine swash
Enzyme, disodium creatine phosphate, DTT and BSA；The enzyme mixation includes: T4 UVSX albumen, T4 UvSY albumen, T4gp32 egg
White, BSU archaeal dna polymerase or klenow segment, the amplification reaction solution and the enzyme mixation are divided in two pipes, are used
It is preceding to be uniformly mixed the two；
It is linked in the NC film after the probe and sampling liquid cooperation, the sampling liquid includes probe, glycerol, sulphur willow
Mercury, trypan blue and 10 × PBS；
The label pad includes any one of colloidal gold, color latex microballoon and quantum dot, marks SA in the label pad；
The solid phase carrier is any one of NC film, sheet glass, pvdf membrane.
5. kit according to claim 4, which is characterized in that further include dilution, nucleic acid containging interior traget, acetic acid
Magnesium, the dilution are added in chip reservoir and are sealed with masking foil, and the magnesium acetate is added the sample application zone of reservoir and air-dries.
6. kit according to claim 5, which is characterized in that be designated as plasmid or plasmid containing following segment in described
Bacterium:
ttgcatcagaagaactaaattttgatacttactggctagagcatcattttactgaatttggactaacaggta
acctgtttgttgcttgtgctaacttacttggtcgaaccaccaaactgaatgttggtactatgattgttcttccaac
agctcaccctgcacgtcagatggaagatttattacttttagatca；
The solid phase carrier is NC film, and the label pad is color latex microballoon,
The dilution includes ammonium acetate, potassium acetate, Triton X-100, trisodium citrate and water.
7. it is a kind of detection mycobacterium tuberculosis and its drug resistant gene kit preparation method, which is characterized in that specifically include with
Lower step:
The first step prepares amplification reaction solution and enzyme mixation；
Second step prepares chip, specifically includes:
1) sampling liquid is prepared；
2) point sample on solid phase carrier；
3) label pad is prepared, label pad is marked into upper SA；
4) bonding pad treatment fluid is prepared, and prepares bonding pad；
5) sample pad treatment fluid is prepared, and prepares sample pad；
6) backlight prelaminar part pad pasting is torn, sticks bonding pad, bonding pad leading portion stretches out solid phase carrier and contacts with sample pad；
7) prepared and diluted liquid；
8) the above-mentioned dilution prepared is added into chip reservoir, then seals buffer area with masking foil；
9) magnesium acetate solution is prepared, magnesium acetate is added to the water and is made into magnesium acetate solution；
10) to reservoir sample application zone be added the 10 prepared magnesium acetate solutions of μ L, natural air drying 2 hours；
11) clip for opening chip, is added gasket, clip is then added on gasket；
12) upwards, adding mouth is closed with sealing strip；
13) clip is closed, two dimensional code is pasted.
8. the preparation method of kit according to claim 7, which is characterized in that amplification reaction solution described in 1mL includes:
Potassium acetate, weight percent 7%PEG20000,50mM of TrisAc buffer, 70mM that 20mM pH value is 7.4
DNTPs, 100 μM of phosphocreatines, the creatine kinase of 20ng/ μ L, the disodium creatine phosphate of 100ng/ μ L, 20 μM of DTT, 10 μ
The primer of the BSA and 20pM of g/mL, above-mentioned component are calculated by reaction final concentration；
Enzyme mixation described in 1mL includes: the T4 UVSX albumen of 120ng/ μ L, the T4 UvSY albumen of 30ng/ μ L, 40ng/ μ L
T4gp32 albumen, the BSU archaeal dna polymerase of 0.1U/ μ L or klenow segment, above-mentioned component are calculated by reaction final concentration；
The amplification reaction solution and the enzyme mixation are divided in two pipes, are uniformly mixed the two using preceding；
Sampling liquid described in 1mL includes: the probe of 100 μ L, glycerol, the 1mg of 500 μ L final concentration of 50% final concentration of
0.1% thimerosal, the trypan blue of 0.1mg final concentration of 0.01%, 100 μ L 10 × PBS, wherein the probe includes: 100
IC P of the final concentration of 10nM of μ L, TB P of the 200 final concentration of 20nM of μ L, ROB P of the 350 final concentration of 35nM of μ L, 400 μ L are whole
Concentration is the chemical reaction of KatS P, the 60 final concentration of 0.06mg/ml of μ L of InhA P of 40nM, the 180 final concentration of 18nM of μ L
Quality Control；
The point sample on solid phase carrier specifically: pad pasting in the middle part of backlight is torn, solid phase carrier is sticked, it will be upper with point sample instrument
It states the sampling liquid prepared and is successively put according to 3 × 2 arrays and is formed on solid phase carrier, the solid phase carrier put is dried 8 hours in 80 DEG C；
Or 37 DEG C of drying 30min, then UV crosslinking 2min；
Dilution described in 1mL includes: the Triton of the ammonium acetate of 0.02Mg, the potassium acetate of 0.02Mg, 20 μ L final concentration of 2%
Trisodium citrate, the water of X-100,0.04Mg.
9. the application of a kind of detection mycobacterium tuberculosis and its drug resistant gene kit, which is characterized in that using such as claim 3
~6 described in any item kits carry out the detection of mycobacterium tuberculosis and its drug resistant gene, comprising the following specific steps
Step S1 extracts nucleic acid；
Step S2 prepares reaction system, the amplification reaction solution is proportionally mixed with the enzyme mixation, anabolic reaction body
System；
Step S3, by after extraction nucleic acid and reaction system mix；
The obtained hybrid reaction system of the step S3 is placed in progress 20min~35min under 30-41 DEG C of constant temperature by step S4
Amplified reaction；
The obtained hybrid reaction system of the step S4 is diluted and hybridizes colour developing by step S5；
The interpretation of reaction result: step S6 carries out interpretation by nucleic acid detection apparatus.
10. the application of kit according to claim 9, which is characterized in that be added before the step S4 and contain internal standard
Nucleic acid and 140nM magnesium acetate；
Hybrid reaction system is placed in the amplified reaction that 20min is carried out under 40 DEG C of constant temperature in the step S4；
Crossing system described in the step S5 includes any one of micro-fluidic, lateral chromatography, diafiltration, and hybridization developing time is
10min。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111187857A (en) * | 2020-02-14 | 2020-05-22 | 深圳市芯思微生物科技有限公司 | Primer pair, kit and preparation method and application of kit for detecting novel coronavirus through isothermal amplification |
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Application publication date: 20191126 |