CN105936941A - Mycobacterium tuberculosis PCR-LFB detection kit - Google Patents
Mycobacterium tuberculosis PCR-LFB detection kit Download PDFInfo
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- CN105936941A CN105936941A CN201610521609.2A CN201610521609A CN105936941A CN 105936941 A CN105936941 A CN 105936941A CN 201610521609 A CN201610521609 A CN 201610521609A CN 105936941 A CN105936941 A CN 105936941A
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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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a mycobacterium tuberculosis PCR-LFB detection kit. On the basis of analysis on the gene sequence of a mycobacterium tuberculosis composite group IS6110, conserved gene sequences are selected to design a pair of specific oligonucleotide primers and a pair of specifically label oligonucleotide probes , and a PCR technology and a lateral flow biosensor are used to detect mycobacterium tuberculosis. The technology has the advantages that the clinical sample detection only needs a common PCR instrument and a disposable lateral flow biosensor, and thus the molecular diagnosis technology can be popularized in professional medical detection units, especially basic medical units. Furthermore, the detection time is short, the detection efficiency is high, and the specificity and accuracy are high. The detection sensitivity is prominently higher than that of common PCR detection method in pathogen qualitative analysis; the operation is simple, the technology is easy to popularize, and the repeatability of experiment results is good.
Description
Technical field:
The invention belongs to pathogen detection field, be specifically related to mycobacterium tuberculosis PCR-LFB detection kit.
Background technology:
Tuberculosis (tuberculosis, TB), as a kind of great infectious disease of people, threatens the life security of the mankind.Full generation
Boundary newly sends out tuberculosis case and there are about 10,000,000, and death toll there are about 2,000,000.Patient's number about 2,000,000 is increased every year newly, extremely in China
Number of dying about 250,000.By the end of 2013, the whole world there are about 9,000,000 people and suffers from tuberculosis, and wherein Chinese patients accounts for the 11% of sum；
Dead in addition with 1,500,000 tuberculosis patients, it is tuberculosis and HIV (human immunodeficiency virus) coinfection patient including 360,000 people.
Detection method lungy mainly has clinical diagnosis, etiological diagnosis, serodiagnosis, diagnosis of molecular biology etc.
Method.But clinical diagnosis accuracy is low；Etiological diagnosis smear-positive rate is low；Serodiagnosis cost is high, operator
Asking high, require the highest to laboratory equlpment, meanwhile, the method is also unfavorable for the early diagnosis of infectious disease.Existing frequently-used and detection
The method that accuracy is high is mainly the diagnosis of molecular biology by PCR means.Standard PCR technology, needs in operation
PCR amplified matter is detected by agarose gel electrophoresis, and the method operation is complicated, equipment investment is high, simultaneously need to nucleic acid dye
EB, thus add the pollution to experimental situation.For these reasons, Standard PCR technology still cannot function as conventional sense technology
It is widely used.Along with the progress of technology, quantitative fluorescent PCR (qPCR) technology due to have detection quickly, sensitivity high etc. excellent
Point was detected by the early stage being more and more applied to infectious disease.But what this technology needs were expensive determines Fluorescent Quantitative PCR instrument, and needs
Professional operates.At present, its application is concentrated mainly on developed country or specialty testing agency with good conditionsi, and this is the most greatly
Limit popularization and the application of this technology.
A kind of disposable lateral flow biosensor of this research and utilization realizes the detection to detection nucleic acid amplification thing, detection knot
Fruit gets final product interpretation by naked eyes, and the time is less than 5 minutes.Sensitivity is more than 100 times of conventional electrophoretic method.This technology has broken away from core
Acid detection is to expensive device (such as electrophresis apparatus, fluorescence detector) and the dependence to specialized personnel.This is specialized laboratory, basic unit
Laboratory, the detection carrying out mycobacterium tuberculosis provide new approaches.
Summary of the invention:
The invention aims to solve mycobacterium tuberculosis detection apparatus expensive, the problem being unfavorable for promoting, and put forward one
Mycobacterium tuberculosis PCR-LFB detection kit.
Mycobacterium tuberculosis specific oligonucleotide primer and through the oligonucleotide probe of specific markers,
Its primer sequence:
Forward primer: 5-CGATCGTGGTCCTGC-3 ',
Downstream primer: 5 '-AACTACGGTGTTTACGGTGC-3 '.
Modified oligonucleotide probe is:
P1:5’-GCGGCAAAGC-FAM-3’；
P2: 5-Biotin-GAGGGCATCGA-3’。
Mycobacterium tuberculosis PCR-LFB detection kit, it includes: primer, probe and lateral flow biosensor, described
Primer, probe be above-mentioned mycobacterium tuberculosis specific oligonucleotide primer and the oligonucleotide probe through specific markers；
Described lateral flow biosensor is prepared by following method:
1) prepared by 30nm gold colloidal: have maximum absorption band (ultraviolet scanning spectrum) at 525nm, and OD value is 0.882, gold chloride: lemon
Lemon acid trisodium 1:1.25；
2) gold colloidal and marked by streptavidin: as a example by labelling 1mL gold colloidal, adds 14 μ L 0.2M K2CO3Mixing, makes glue
Body gold pH is near SA isoelectric point, IP；Add SA, hand mixing 10min that 1 μ L concentration is 10 μ g/ μ l；Add 20 μ L10% BSA envelopes
Close 10min, add 20 μ L10% PEG20000 and close 10min, 4 ° of C, 7000rpm, centrifugal 15min, remove supernatant, with (10mM
PBS+0.5% Triton X-100+0.3% BSA, the redissolution liquid of pH 7.4 press original volume redissolve, gold-marking binding pad is cut into 0.9cm
× 30cm size, the label after redissolving with pipettor is layered on gold-marking binding pad, and 37 ° of C are dried 3-4 hour, standby.Every
Film bar treating capacity is 1.809mL；
3) sample pad processes: sample pad is cut into 1.7cm × 30cm size, is layered on by the sample pad treatment fluid prepared with pipettor
In sample pad, 37 DEG C of drying, every film bar treating capacity is 3.417mL；
4) Biotin Yu the BSA coupling activated: Sulfo-NHS-Biotin Yu BSA is that 20:1 is in 4 DEG C of couplings in molar ratio
Night.0.2 μm filtering with microporous membrane, draws in nitrocellulose filter nature controlling line subsequently--on C line.Concrete operation step is: weigh
0.1g BSA is dissolved in 10 mL PBS, regulates pH7.4；Add 5mg Sulfo-NHS-Biotin in 3.7mL BSA
In, mixing, and under the conditions of 4 DEG C, stirring coupling is overnight；0.22 μm filters ,-20 DEG C of preservations.
5) prepared by control line: nitrocellulose filter--the Biotin that on NC film, the coupling of C wire tag is good, it is diluted in proportion
1.0mg/mL, draws on NC film by 1 μ L/cm, and 37 DEG C dry 3-4 hour, standby.
6) prepared by detection line: detects line--T line, labelling Rabbit anti-FAM on NC film, draws at NC film by 1 μ L/cm
On, 37 DEG C of drying.
7) test strips assembles: take the support as test strips of the PVC base plate, and viscous face is supreme, by above-mentioned be ready to draw have C line, T
The NC film of line is bonded at middle position, then pastes gold conjugation pad and makes it be pressed in below NC film end at 2mm, sample pad is glued
It is attached to test strips bottom, finally pastes absorbent paper in the top of PVC base plate so that it is afterbody is pressed on NC film.
The invention provides mycobacterium tuberculosis PCR-LFB detection kit, be to mycobacterium tuberculosis complex
On the basis of IS6110 gene sequencing, choose conserved genetic sequences and design a pair specific oligonucleotide primer and a pair
Through the oligonucleotide probe of specific markers, utilize round pcr to combine lateral flow biosensor and mycobacterium tuberculosis is examined
Survey.This technology only needs the PCR instrument of routine and disposable lateral flow biosensor just can complete the inspection to clinical sample, will
Molecular diagnostic techniques is in professional medical testing agency, and especially basic medical unit is promoted, and has potential using value.
This test kit mainly includes Mycobacterium tuberculosis DNA extracting method, a pair specificity for mycobacterium tuberculosis
Primer, the nucleic probe of two specific markers, positive control, negative control, PCR Buffer, lateral flow biosensor and
Supporting buffer.
After detection ultimate principle (as shown in Figure 1) involved in the present invention: PCR amplification terminates, (amplification complex two ends are respectively
By Biotion and FAM labelling), take 10 μ L PCR amplified matters and 90 μ L developping solutions fully mix in ELISA Plate, formed to be measured
Liquid.The lateral flow biosensor lower specimen pad prepared is immersed in ELISA Plate liquid to be measured.In amplification complex
Biotin site Streptavidin-nano-Au composite in gold mark pad is combined, and forms new complex.With liquid to be measured from lower and
Upper chromatography, is captured by anti-FAM monoclonal antibody when arriving T line, forms sandwich structure.Finally, due in course of reaction
Nanogold particle enrichment at T line, produces macroscopic red stripes；Superfluous nanometer gold-Streptavidin continues layer
Analysis is captured to C line by Biotin, forms macroscopic red stripes, and now testing result is positive.If only nature controlling line in
Redness, testing result is negative.If T line and C line are colourless band, show that this biological sensing material lost efficacy.
Advantages of the present invention:
Time cycle is short, detection efficiency is high in detection；Detection high specificity, accuracy rate is high；It is it carrying out pathogen qualitative analysis
Detection sensitivity name is aobvious higher than regular-PCR detection method；Simple to operate, easy to spread；Experimental result is reproducible.
Accompanying drawing illustrates:
Fig. 1. detection basic principle schematic；
Fig. 2. the abosrption spectrogram of 30nm gold colloidal；
Fig. 3. the assembling assumption diagram of lateral flow biosensor；
Fig. 4. the specific detection result of mycobacterium tuberculosis PCR-LFB method；
Fig. 5. the susceptiveness testing result of mycobacterium tuberculosis PCR-LFB method.
Detailed description of the invention:
Embodiment 1 mycobacterium tuberculosis identifies design and the synthesis of specific nucleic acid sequence
According to the insertion sequence IS6110 gene design specific oligonucleotide primer that mycobacterium tuberculosis complex is total.Primer
Sequence is as follows:
Forward primer: 5-CGATCGTGGTCCTGC-3 ',
Downstream primer: 5 '-AACTACGGTGTTTACGGTGC-3 '.
Modified oligonucleotide probe for hybridizing with IS6110 amplified fragments is:
P1:5’-GCGGCAAAGC-FAM-3’；
P2: 5-Biotin-GAGGGCATCGA-3’。
The specificity of above primer and probe is analyzed by NCBI website Blast tool software, it was demonstrated that this sequence with
In nucleic acid database there is not cross reaction in other sequence, preliminary proof its for Mycobacterium tuberculosis, there is specificity.Few core
Thuja acid primer, probe are synthesized by Shanghai Sheng Gong biological engineering company limited.Amplification gene sequence is as shown in SEP ID NO.1.
The preparation of embodiment 2 lateral flow biosensor
(1) prepared by 30nm gold colloidal: have maximum absorption band (ultraviolet scanning spectrum) at 525nm, as in figure 2 it is shown, OD value is
0.882, gold chloride: trisodium citrate 1:1.25.
(2) gold colloidal and Streptavidin (SA) labelling: as a example by labelling 1mL gold colloidal, add 14 μ L 0.2M K2CO3
Mixing, makes gold colloidal pH near SA isoelectric point, IP；Add 1 μ LSA(10 μ g/ μ l), hand mixing 10min；Add 20 μ L10%
BSA closes 10min, adds 20 μ L10% PEG20000 and closes 10min, 4 ° of C, 7000rpm, centrifugal 15min, removes supernatant, use
The liquid (10mM PBS+0.5% Triton X-100+0.3% BSA, pH 7.4) that redissolves is pressed original volume and is redissolved, and gold-marking binding pad is cut into
0.9cm × 30cm size, the label after redissolving with pipettor is layered on gold-marking binding pad, and 37 ° of C are dried 3-4 hour, standby
With.Every film bar treating capacity is 1.809mL.
(3) sample pad processes: sample pad is cut into 1.7cm × 30cm size, the sample pad prepared is processed with pipettor
Liquid is layered in sample pad, 37 DEG C of drying, standby.Every film bar treating capacity is 3.417mL.
(4) Biotin Yu the BSA coupling activated: Sulfo-NHS-Biotin Yu BSA is that 20:1 is in 4 DEG C of idols in molar ratio
Connection is overnight.0.2 μm filtering with microporous membrane, draws subsequently on nitrocellulose filter nature controlling line (C line).Concrete operation step is: claim
Take 0.1g BSA to be dissolved in 10 mL PBS, regulate pH7.4；Add 5mg Sulfo-NHS-Biotin in 3.7mL BSA
In, mixing, and under the conditions of 4 DEG C, stirring coupling is overnight；0.22 μm filters ,-20 DEG C of preservations.
(5) prepared by control line: the Biotin that the upper C wire tag coupling of nitrocellulose filter (NC film) is good, is diluted in proportion
1.0mg/mL, draws on NC film by 1 μ L/cm, and 37 DEG C dry 3-4 hour, standby.
(6) prepared by detection line: detect line (T line) labelling Rabbit anti-FAM(1mg/mL on NC film) draw by 1 μ L/cm
On NC film, 37 DEG C of drying, standby.
(7) test strips assembles: take the support as test strips of the PVC base plate, and viscous face is supreme, by above-mentioned be ready to draw have C line,
The NC film of T line is bonded at middle position, then pastes gold conjugation pad and makes it be pressed in below NC film end at 2mm, by sample pad
It is pasted onto test strips bottom, finally pastes absorbent paper in the top of PVC base plate so that it is afterbody is pressed on NC film, has assembled
Become, as shown in Figure 3.
The detection method of embodiment 3 mycobacterium tuberculosis
(1) process and the Mycobacterium tuberculosis DNA of patient's sputum specimen extracts: add 4% sodium hydroxide solution of 2 times of sputum volumes,
Vibration mixing, room temperature places 30min, period vibration mixing so that it is fully liquefy；6000 × g is centrifuged 10 min, abandons supernatant；Add
1.5 mL 0.01mol/L PBS (pH 7.8), mixing of fully vibrating.6000 × g is centrifuged 10 min, abandons supernatant.Collect precipitation
Thing, add 100 L DNA extraction liquid (10 mmol Tris, 150mmol NaCL, 0.01% Triton X-100,0.05%
Tween 20,200 g/ L E.C. 3.4.21.64) mixing of fully vibrating, 60 DEG C of water-bath 15min；95 DEG C of heating 5 min, brief centrifugation；Add
Equal-volume chloroform, vibration mixing, 12 000 × g is centrifuged 5 min；Take supernatant 5 μ L, be directly used in PCR amplification.
(2) PCR amplified reaction program:
PCR reaction system forms: 10 × PCR buffer, 0.2 mM dNTP, the 0.5 each primer of μm ol and probe, 1 μ L template
DNA, 0.5 UTaqDNA polymerase, DDW benefit is 25 μ L to cumulative volume.PCR reaction condition: 95 DEG C of denaturations 30s；95 DEG C of changes
Property 5s, 59 DEG C annealing extend 15s, totally 40 circulations；Subsequently, 95 DEG C of degeneration 5s, 50 DEG C of annealing extension 15s, totally 5 circulations, 4 DEG C
Preserve.
(3), after the lateral flow biosensor detection to PCR amplified matter: PCR amplification terminates, 10 μ L amplified productions are taken in life
On the lower specimen pad of thing sensor, subsequently biosensor bottom is put into developping solution (0.01 M PBS, the pH of 90 μ L
7.4) in, reading result, nature controlling line and detection line and take on a red color after 5min, result is positive；If only nature controlling line takes on a red color, result is
Feminine gender, if two lines is the most colourless, interpretation is invalid.
Embodiment 4 test kit specific detection
Respectively to Mycobacterium tuberculosis var.bovis, staphylococcus aureus, escherichia coli, Salmonella, yeast, single increasing Liszt
The genomic DNA of bacterium, detects by above-mentioned PCR-LFB method.Result shows, only Mycobacterium tuberculosis genomic DNA
Testing result is positive, and the testing result of other non-purpose bacterium is all negative, and the method inspection to mycobacterium tuberculosis is described
Measuring tool has specificity, as shown in Figure 4.
Embodiment 5 test kit susceptiveness detects
Extract Mycobacterium tuberculosis DNA, measure its initial concentration, it is carried out gradient dilution.Each reaction amplifying nucleic acid concentration depends on
Secondary for 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg.PCR expansion is carried out respectively using above-mentioned concentration DNA as template
Increase, and use agarose gel electrophoresis and PCR-LFB to carry out comparison and detection.Result shows, the lowest detection of electrophoresis method is limited to
1pg, and PCR-LFB lowest detection is limited to 10fg, as shown in Figure 5.
The clinical sample detection of embodiment 6 mycobacterium tuberculosis specific PCR-LFB method
54 parts of clinical sputum samples are detected by the method and the test kit that use the present invention, compare with culture method and smear method
Relatively.In 54 parts of specimen, PCR-LFB Positive rate is 75.9%, is significantly higher than 16.7% and culture method of smear method
24.1%.Cultivating in positive specimen at 9 parts, PCR method detection total positives, both are positive, and recombination rate is 100%, as shown in table 1.
Embodiment 7
Use method and the test kit of the present invention, select the primer of present invention design and the gene for IS6110 reported
The special primer of (gene order is as shown in SEP ID NO.2) (forward primer: 5 '-CGATCGTGGTCCTGC-3 ', draw by downstream
Thing: 5 '-AACTACGGTGTTTACGGTGC-3 ') carry out detection and compare.The primer sequence of present invention screening, to tuberculosis branch
Bacillus detection has higher sensitivity.Detecting 54 parts of clinical sputum samples, its Positive rate is 75.9%,
Report primer sequence is only 61.3% to the positive rate of these 54 parts of Samples detections.
<110>Jilin Agriculture University
<120>mycobacterium tuberculosis PCR-LFB detection kit
<160> 2
<210> 1
<211> 94
<212> DNA
<213>artificial
<400> 1
ggtcctgcgg gctttgccgc gggtggtccc ggacaggccg agtttggtca tcagccgttc 60
gacggtgcat ctggccacct cgatgccctc acgg 94
<210> 2
<211> 110
<212> DNA
<213>artificial
<400> 2
gggctttgcc gcgggtggtc ccggacaggc cgagtttggt catcagccgt tcgacggtgc 60
atctggccac ctcgatgccc tcacggttca gggttagcca cactttgcgg 110
Claims (3)
1. mycobacterium tuberculosis specific oligonucleotide primer and the oligonucleotide probe through specific markers,
Its primer sequence:
Forward primer: 5-CGATCGTGGTCCTGC-3 ',
Downstream primer: 5 '-AACTACGGTGTTTACGGTGC-3 '；
Modified oligonucleotide probe is:
P1:5’-GCGGCAAAGC-FAM-3’；
P2: 5-Biotin-GAGGGCATCGA-3’。
2. mycobacterium tuberculosis PCR-LFB detection kit, it includes: primer, probe and lateral flow biosensor, its feature
It is: described primer, probe are the mycobacterium tuberculosis specific oligonucleotide primer described in claim 1 and through specific mark
The oligonucleotide probe of note.
Mycobacterium tuberculosis PCR-LFB detection kit the most according to claim 2, it is characterised in that: described is lateral
Stream biosensor, is prepared by following method:
1) prepared by 30nm gold colloidal: have maximum absorption band (ultraviolet scanning spectrum) at 525nm, and OD value is 0.882, gold chloride: lemon
Lemon acid trisodium 1:1.25；
2) gold colloidal and marked by streptavidin: as a example by labelling 1mL gold colloidal, adds 14 μ L 0.2M K2CO3Mixing, makes glue
Body gold pH is near SA isoelectric point, IP；Add SA, hand mixing 10min that 1 μ L concentration is 10 μ g/ μ l；Add 20 μ L10% BSA envelopes
Close 10min, add 20 μ L10% PEG20000 and close 10min, 4 ° of C, 7000rpm, centrifugal 15min, remove supernatant, with (10mM
PBS+0.5% Triton X-100+0.3% BSA, the redissolution liquid of pH 7.4 press original volume redissolve, gold-marking binding pad is cut into 0.9cm
× 30cm size, the label after redissolving with pipettor is layered on gold-marking binding pad, and 37 ° of C are dried 3-4 hour, standby；Every
Film bar treating capacity is 1.809mL；
3) sample pad processes: sample pad is cut into 1.7cm × 30cm size, is layered on by the sample pad treatment fluid prepared with pipettor
In sample pad, 37 DEG C of drying, every film bar treating capacity is 3.417mL；
4) Biotin Yu the BSA coupling activated: Sulfo-NHS-Biotin Yu BSA is that 20:1 is in 4 DEG C of couplings in molar ratio
Night；
0.2 μm filtering with microporous membrane, draws in nitrocellulose filter nature controlling line subsequently--on C line；Concrete operation step is: weigh
0.1g BSA is dissolved in 10 mL PBS, regulates pH7.4；Add 5mg Sulfo-NHS-Biotin in 3.7mL BSA
In, mixing, and under the conditions of 4 DEG C, stirring coupling is overnight；0.22 μm filters ,-20 DEG C of preservations；
5) prepared by control line: nitrocellulose filter--the Biotin that on NC film, the coupling of C wire tag is good, it is diluted to 1.0mg/ in proportion
ML, draws on NC film by 1 μ L/cm, and 37 DEG C dry 3-4 hour, standby；
6) prepared by detection line: detects line--T line, labelling Rabbit anti-FAM on NC film, draws on NC film by 1 μ L/cm, and 37
DEG C dry；
7) test strips assembles: take the support as test strips of the PVC base plate, and viscous face is supreme, by above-mentioned be ready to draw have C line, T line
NC film is bonded at middle position, then pastes gold conjugation pad and makes it be pressed in below NC film end at 2mm, sample pad is pasted onto
Test strips bottom, finally pastes absorbent paper in the top of PVC base plate so that it is afterbody is pressed on NC film.
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| CN107043807A (en) * | 2017-01-19 | 2017-08-15 | 昆明医科大学 | The assay method of Survival probability of bacteria after a kind of mycobacterium tuberculosis infection human body |
| CN107523624A (en) * | 2017-09-19 | 2017-12-29 | 中国疾病预防控制中心传染病预防控制所 | A kind of combination AUDG and the more intersection constant-temperature amplification methods for keeping away molecular recognition system certainly |
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| CN107523624A (en) * | 2017-09-19 | 2017-12-29 | 中国疾病预防控制中心传染病预防控制所 | A kind of combination AUDG and the more intersection constant-temperature amplification methods for keeping away molecular recognition system certainly |
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