CN109280711A - LAMP detection primer group, detection kit and its detection method of mycobacterium kansasii - Google Patents
LAMP detection primer group, detection kit and its detection method of mycobacterium kansasii Download PDFInfo
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- CN109280711A CN109280711A CN201810473250.5A CN201810473250A CN109280711A CN 109280711 A CN109280711 A CN 109280711A CN 201810473250 A CN201810473250 A CN 201810473250A CN 109280711 A CN109280711 A CN 109280711A
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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
Abstract
The present invention provides a kind of LAMP primer group for detecting mycobacterium kansasii, including outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer LF and ring primer LB.The present invention also provides detection kits and its detection method comprising above-mentioned primer sets.Detection kit includes primer liquid, reaction solution, archaeal dna polymerase, yin and yang attribute control and fluorescent dye (or color developing agent).Detection method is the DNA by extracting sample to be tested, then in the presence of archaeal dna polymerase and primer sets, to sample DNA templates in 65 DEG C or so progress isothermal duplications, determine in measuring samples whether contain mycobacterium kansasii finally by color change in real-time fluorescence quantitative PCR instrument detection fluorescence or addition color developing agent observation tube.Kit provided by the invention and detection method have specificity and sensitivity well, and amplification is quickly, efficiently, easily operated, can be used for the quick detection of mycobacterium kansasii in clinical samples.
Description
Technical field
The present invention relates to the quick detections of technical field of molecular biology more particularly to mycobacterium kansasii, specially
A kind of LAMP detection primer group, detection kit and its detection method of mycobacterium kansasii.
Background technique
Mycobacterium kansasii is a kind of slow growth non-tuberculous mycobacteria, belongs to I group in Runyon classification, i.e. light
Chromogenic group.The micro- sem observation of direct smear is shown in that the bacterium is coarseer, also visible particulate bacillus form and the arrangement of thallus fence sample.
After mycobacterium kansasii grows about 6 weeks on suitable culture medium (such as Michaelis culture medium, Russell medium), it can be formed thick
Rough or smooth medium sized yellow color colonies, as incubation time extends, colony colour is slowly changed into reddish orange.Kansas
Mycobacteria may be present in water and soil in environment, and the most common source is tap water, will not generally infect the mankind and cause
Disease and the human world is caused to propagate.But when under special circumstances, if any basic tuberculosis or patients with low immunity, which can cause
The outer disease of pulmonary disease, lung even whole body disseminated infections.
Mycobacterium kansasii infects common clinical symptoms and includes cough, pectoralgia, hemoptysis and expiratory dyspnea etc., common
Imageology is unilateral upper lung section shade, these performances and phthisical clinical symptoms and Imageology all compared with class
Seemingly, therefore such diagnosis basis all lacks specificity in terms of mycobacterium kansasii Infect And Diagnose.In terms of laboratory diagnosis, lead to
Mycobacterium kansasii and other mycobacterias (including mycobacterium tuberculosis compound) can be identified by crossing culture and biochemical identification, but
This tradition discrimination method is cumbersome and takes a long time, and the repeatability and stability of result are not satisfactory；Interferon
Release test be considered as identify tuberculosis and Nontuberculous mycobacterial infections advantageous weapon, but due to mycobacterium kansasii with
Mycobacterium tuberculosis is the same to generate CFP-10 and EAST-6, therefore interferon release test can not identify Kansas branch bar
Bacterium disease and tuberculosis；Molecular biology method can rapidly identify mycobacterium kansasii, common method include regular-PCR,
Real-time PCR and DNA probe hybridization etc., but these methods are required using specific apparatus such as PCR instruments at present, and some methods make
Amplification target position also lacks good specificity, these deficiencies cause clinical new to the detection of research and development application mycobacterium kansasii
The demand of method is more urgent.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) is this generation
The new nucleic acid amplification technologies of one kind just to come out of recording, the technology carry out being not necessarily to transformation temperature when nucleic acid amplification, under isothermal conditions
Entire amplified reaction can be completed, and final result judgement can observe by the naked eye color change and can be realized, therefore
LAMP is not needed being capable of the special installations such as accurate temperature controlling and the PCR instrument of progress fluorescence monitoring.Because LAMP can make up for it PCR method
The deficiency of a little aspects, so the technology has been widely applied in terms of mycobacterium tuberculosis clinical detection, but is bearing at present
Application is not yet received in Sa Si mycobacteria context of detection LAMP.
Summary of the invention
In view of this, technical problem solved by the invention is that the LAMP detection for providing a kind of mycobacterium kansasii is drawn
Object group.
Technical problem solved by the invention, which also resides in, provides a kind of LAMP detection kit of mycobacterium kansasii.
Technical problem solved by the invention, which also resides in, provides a kind of utilization above-mentioned mycobacterium kansasii LAMP detection examination
The method of agent box detection mycobacterium kansasii.
In order to solve the above-mentioned technical problem, the technical solution adopted in the present invention is as follows:
On the one hand, the invention discloses a kind of LAMP detection primer groups of mycobacterium kansasii, for Kansas branch
The distinctive ECHT gene coded sequence of bacillus designs and optimizes, and the detection primer group includes outer primer F3, outer primer B3, interior draws
Object FIP, inner primer BIP, ring primer LF and ring primer LB, nucleic acid sequence difference are as follows:
Outer primer F3:5 '-GACTGTTGGCGAGTTGGT-3 '
Outer primer B3:5 '-GTCCTGGAACGTCTGCTC-3 '
Inner primer FIP:5 '-GGTGTGGCACATTGCGACAGATATAACTCGAACAGCAC-3 '
Inner primer BIP:5 '-CGGATCAGCGGTATGACGGCCGGCTGCCCGATTAGAT-3 '
Ring primer LF:5 '-GTTCTGCGACGGCGATTC-3 '
Ring primer LB:5 '-GGGATGCGTTGTCCACCA-3 '.
Preferably, the outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer LF and ring primer LB exist
Molar ratio in reaction system is 1:1:4:4:2:2.
On the other hand, the invention also discloses a kind of LAMP detection kit of mycobacterium kansasii, the detection reagents
Contain the LAMP detection primer group of mycobacterium kansasii as claimed in claim 1 or 2 in box.
Preferably, which is characterized in that further include some or all of of following component: (1) archaeal dna polymerase；(2) LAMP is anti-
Answer liquid；(2) fluorescent dye or color developing agent；(3) positive control and negative control.
Preferably, the archaeal dna polymerase is Bst archaeal dna polymerase；LAMP reaction solution include dNTP, reaction buffer,
MgSO4 and glycine betaine.
Preferably, positive control is the carrier cloning containing mycobacterium kansasii ECHT genetic fragment, and negative control is
Water or other solvents without mycobacterium kansasii ECHT genetic fragment.
Preferably, the fluorescent dye is SYBR green, and the color developing agent is hydroxynaphthol blue.
In a preferred embodiment of the invention, the LAMP detection kit of mycobacterium kansasii of the invention, including it is following
Ingredient:
(1) primer sets: including LAMP detection primer group described in claim 1, outer primer, inner primer and ring primer it is dense
Degree is 10 μM；
(2) reaction solution: including dNTP, reaction buffer, MgSO4 and glycine betaine, when use, needs equimultiple to dilute；
(3) Bst archaeal dna polymerase: concentration is 0.16U/ μ L；
(4) fluorescent dye or color developing agent: fluorescent dye is SYBR green, and color developing agent is hydroxynaphthol blue (HNB)；
(5) sealing fluid: for closing paraffin oil；
(6) it is enucleated sour water: for supplementing reaction volume.
Further more, detecting Kansas using above-mentioned mycobacterium kansasii LAMP detection kit the invention also discloses a kind of
The method of mycobacteria, which comprises the following steps:
(1) sample DNA the extraction of measuring samples DNA: is extracted using boiling lysis.(a) according to sputum specimen sliminess
Difference, be suitably added the digestive juice of 1~2 times of volume, then vortex oscillation 30s, place 15min under room temperature.Bronchus
The processing of flushing liquor sample is the same with sputum specimen.1~2 times of volume is suitably added after pleural effusion sample 5000rpm centrifugation 15min
The digestive juice of volume, remainder handle same sputum specimen.Swab specimen is first put into 1ml physiological saline, takes out swab after rotating repeatedly,
Then 5000rpm is centrifuged 15min, and 1ml digestive juice is added, and remainder handles same sputum specimen.(b) the postdigestive liquid of 1ml is drawn extremely
In 1.5ml microcentrifugal tube, 13000rpm is centrifuged 10min.(c) 1ml physiological saline, 13000g centrifugation is added after discarding supernatant liquid
10min.(d) step (c) is repeated after discarding supernatant.Supernatant is removed after centrifugation, 40 μ l DNA extracting solutions are added, and oscillation mixes
2000rpm is centrifuged 5s afterwards, then 37 DEG C of warm bath 30min, then 100 DEG C of heating 10min, and 13000rpm is centrifuged 10min after cooling.
(e) supernatant comprising DNA profiling is transferred to another after being centrifuged to go in the 1.5ml microcentrifugal tube of nucleic acid, sets -20 DEG C of guarantors
It deposits spare；
(2) isothermal amplification: the measuring samples DNA profiling obtained in 2 μ L steps (1) or positive and negative control is taken to add
Enter and mixed in LAMP reaction system, 65 DEG C of isothermal reaction 50min are expanded, and then 80 DEG C of isothermal reaction 10min terminate reaction；
LAMP detection primer group comprising mycobacterium kansasii described in claims 1 or 2 in the LAMP reaction system；
(3) result judges: being judged by the real-time fluorescence detection data of analysis of fluorescence quantitative PCR apparatus, or passed through
The variation that liquid color in color developing agent observation PCR pipe is added carrys out judging result.
Preferably, the LAMP reaction system is to contain in 20 μ L reaction systems:
Outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer LF and ring primer LB concentration are 10 μM,
Additional amount is respectively 0.5 μ L, 0.5 μ L, 2 μ L, 2 μ L, 1 μ L, 1 μ L；
The additional amount of 0.16U/ μ L Bst archaeal dna polymerase is 0.4 μ L；
0.4 μ L of fluorescent dye or color developing agent；
10 μ L of reaction solution includes 4mM dNTP, 12mM MgSO in reaction solution4, 1.6mM glycine betaine, 40mM Tris-HCl,
20mM KCl、20mM(NH4)SO4, 0.1%TritonX-100；
The measuring samples DNA extracted in 2 μ L steps (1)；
With deionized water polishing to 20 μ L.
As the important indicator for influencing detection effect, the selection of testing goal segment is most important.In calculating detection, examine
The length for considering mycobacterium kansasii, using complete series detection and unrealistic, time and effort consuming and may be to testing result, this hair
It is bright by consulting literatures and search Genebank database find the distinctive nucleic acid sequence of mycobacterium kansasii, and by with its
The detection of its target gene is compared to verify the specificity of the nucleic acid sequence, finally the determining peculiar nucleic acid of mycobacterium kansasii
Sequence is target fragment of the ECHT gene order as amplification, can in conjunction with the technical effect in above-mentioned analysis and the embodiment of the present invention
Know, the present invention include at least it is following the utility model has the advantages that
(1) easy to operate: DNA extraction step is simple and effective, and template can be directly used for following amplification reaction, and isothermal duplication can
Selection is not necessarily to the special installations such as PCR instrument；
(2) amplification is quick: isothermal duplication only needs 60min, after the completion of amplification result can direct interpretation, entire detection process institute
It takes time within 2h.
(3) specificity is high: detection primer group includes six specific primers, can only effectively expand mycobacterium kansasii,
Mycobacterium tuberculosis compound, Mycobacterium chelonei, mycobacterium abscessus, mycobacterium avium and branch bar intracellular cannot effectively be expanded
Other pathogen such as bacterium.
(4) sensitivity is high: the minimum that can be detected is 100 copies.
(5) avoid pollution: fluorescent dye or color developing agent are added before the reaction, without addition of uncapping after reaction, can effectively prevent
The only diffusion and pollution of amplified production.
Detailed description of the invention
Fig. 1 is specific analysis of fluorescence curve graph.S type curve is the amplification curve of mycobacterium kansasii.
Fig. 2 is that specificity analysis visually observes figure.Upper row's PCR pipe respectively corresponds mycobacterium kansasii, knot from left to right
Core mycobacteria, Mycobacterium chelonei, mycobacterium abscessus, mycobacterium avium, lower row's PCR pipe respectively correspond intracellular point from left to right
Branch bacillus, mycobacterium smegmatis, escherichia coli, staphylococcus aureus, Candida albicans.
Fig. 3 is sensitivity analysis fluorescence curve figure.From left to right three S type curves be respectively 106copies/ μ L,
The amplification curve of 104copies/ μ L, 102copies/ μ L concentration template.
Fig. 4 is that sensitivity analysis visually observes figure.The template concentrations of PCR reaction tube amplification from left to right are respectively as follows:
106copies/μL、104copies/μL、102Copies/ μ L and 1copies/ μ L.
Fig. 5 is clinical samples detection fluorescence curve figure.9 parts of clinical samples are detected altogether, wherein 1 part of sample Kansas branch bar
Bacterial examination is surveyed positive.The S type curve on the left side is positive quality control amplification curve.
Fig. 6 is that clinical samples detection visually observes figure.9 parts of clinical samples are detected altogether, wherein 1 part of sample Kansas branch bar
Bacterial examination is surveyed positive.It is the positive pipe of clinical samples amplification that lower row's PCR pipe the 1st, which manages (from left to right), and the lower pipe of row's PCR pipe the 4th, 5 is (from a left side
To the right side) it is respectively positive quality control and negative Quality Control amplification pipe.
Specific embodiment
Following embodiment further illustrates the contents of the present invention, but described example is that part of the invention is implemented
Example, rather than whole embodiments, therefore should not be construed limitation of the present invention.Based on the embodiments of the present invention, noninvasive
To the modifications or substitutions of the method for the present invention, step or condition under the premise of the property made labour, shall fall within the protection scope of the present invention.
Embodiment 1: design of primers
The distinctive gene order of mycobacterium kansasii is found by consulting literatures first, then in the Genebank of NCBI
The gene order is searched in database and verifies its specificity, is then with the sequence using special LAMP primer design software
Target gene carries out design of primers, and finally designed primer is analyzed and optimized on primer-blast.It is final to determine
Primer sequence see SEQ ID No:1 to SEQ ID No:6 respectively.
The comparative test for verifying the peculiar gene order of mycobacterium kansasii includes following amplimer:
Hsp65F:5 '-CAGGAAGCAGTGCTGGAAGA-3 ' SEQ ID No:7；
Hsp65R:5 '-GTGCTCAAAGCCTCACCCTC-3 ' SEQ ID No:8；
ECHTF:5 '-AAACGGTGTGTCGCAATGTG-3 ' SEQ ID No:9；
ECHTR:5 '-GGATATGGCGGTCCCTTCTG-3 ' SEQ ID No:10；
The branches such as mycobacterium kansasii, mycobacterium tuberculosis and Mycobacterium chelonei are detected respectively using above-mentioned two pairs of primers
Two kinds of target genes (i.e. hsp65 and ECHT gene) of bacillus clinical strain compare two kinds of target gene amplification positive findings and analyze it
Accuracy, accuracy is higher, and the specificity for indicating the target gene augmentation detection mycobacterium kansasii is higher, also indicates that the target base
Because being more suitable for being used for the detection of mycobacterium kansasii in clinical samples.
Embodiment 2: mycobacterium kansasii LAMP detection reaction system is established
Different isothermal amplification temperature (60 DEG C, 63 DEG C, 65 DEG C), different primers concentration (5 μM, 10 μM, 15 μM) are set
With different additional amount Bst archaeal dna polymerases (0.2 μ L, 0.4 μ L, 0.6 μ L), more same template expands under each factor different condition
The Ct value of increasing, primer concentration, Bst archaeal dna polymerase additional amount and the amplified reaction temperature of minimum Ct value can be obtained as originally
The end reaction parameter of invention.
It has been determined that highly preferred mycobacterium kansasii LAMP detects reaction system and reaction condition by repetition test,
I.e. isothermal duplication temperature is 65 DEG C；In 20 μ L reaction systems, outer primer F3, outer primer B3, inner primer FIP, inner primer BIP,
Ring primer LF and ring primer LB concentration are 10 μM, and additional amount is respectively 0.5 μ L, 0.5 μ L, 2 μ L, 2 μ L, 1 μ L, 1 μ L, are being reacted
Molar ratio in system is 1:1:4:4:2:2；The additional amount of 0.16U/ μ L Bst archaeal dna polymerase is 0.4 μ L.Specific additional amount
It is as follows:
| Ingredient names | Additional amount (μ L) | Ingredient names | Additional amount (μ L) |
| Reaction solution | 10 | Bst archaeal dna polymerase | 0.4 |
| Outer primer | 1 | Fluorescent dye/color developing agent | 0.4 |
| Inner primer | 4 | Sample DNA | 2 |
| Ring primer | 2 | Add water polishing extremely | 20 |
It include 4mM dNTP, 12mM MgSO in reaction solution4, 1.6mM glycine betaine, 40mM Tris-HCl, 20mM KCl,
20mM(NH4)SO4, 0.1%TritonX-100.
Embodiment 3: analysis Evaluation on specificity
Common mycobacteria and common common bacteria are chosen as test object and carrys out evaluation analysis specificity, specifically includes and bears
Sa Si mycobacteria, mycobacterium tuberculosis, Mycobacterium chelonei, mycobacterium abscessus, mycobacterium avium, Mycobacterium intracellulare, shame
10 kinds of microorganisms such as dirty mycobacteria, escherichia coli, staphylococcus aureus, Candida albicans.As the result is shown (attached drawing 1,
2), the present invention fails to detect to other microorganisms other than above-mentioned mycobacterium kansasii, therefore the present invention has well
Analysis specificity.
Embodiment 4: analytical sensitivity evaluation
10 are prepared using the plasmid for having connected target gene6copies/μL、104copies/μL、102Copies/ μ L and
The template of 1copies/ μ L concentration, every kind of concentration take 1 μ L to carry out LAMP detection, observe by the naked eye color change and glimmering respectively
Light PCR instrument monitors analysis of fluorescence and carries out result interpretation, using the detection limit of fluorescence analysis interpretation method as Monitoring lower-cut.Knot
Fruit shows (such as Fig. 3,4) that the lower limit that the present invention detects ureaplasma urealyticum is 100 copies, therefore the present invention has good point
Analyse sensitivity.
Embodiment 5: clinical samples detection
1, reagent prepares
(1) primer, reaction solution, Bst archaeal dna polymerase, fluorescent dye, color developing agent, stoning sour water, paraffin oil, feminine gender are right
According to, positive control, the various composition of corresponding amount is taken to mix (specific additional amount is shown in embodiment 2), is saved at 4 DEG C after brief centrifugation standby
With.
2, sample preparation
(1) according to the difference of sputum specimen sliminess, it is suitably added the digestive juice of 1~2 times of volume, then vortex oscillation
30s places 15min under room temperature.The processing of Bronchial washing sample is the same with sputum specimen.Pleural effusion sample
The digestive juice of 1~2 times of volume volume is suitably added after 5000rpm centrifugation 15min, remainder handles same sputum specimen.Swab specimen is first
It is put into 1mL physiological saline, takes out swab after rotating repeatedly, then 5000rpm is centrifuged 15min, and 1mL digestive juice is added, remaining
Handle same sputum specimen.
(2) the postdigestive liquid of 1mL is drawn into 1.5mL microcentrifugal tube, and 13000rpm is centrifuged 10min.
(3) 1mL physiological saline is added after discarding supernatant liquid, 13000g is centrifuged 10min.It is primary to repeat this step.
(4) supernatant is removed after being centrifuged, and 40 μ L DNA extracting solutions are added, 2000rpm is centrifuged 5s after oscillation mixes, and then 37
DEG C warm bath 30min, then 100 DEG C of heating 10min, it is cooling after 13000rpm be centrifuged 10min.
(5) supernatant comprising DNA profiling another is transferred to after being centrifuged to go in the 1.5mL microcentrifugal tube of nucleic acid,
- 20 DEG C are set if not expanding immediately to save backup.
3, sample is added
It takes the addition of 2 μ L templates to have in the PCR pipe of various reacted constituents and mix, marks good rear brief centrifugation.Using glimmering
Fluorescent dye is added when optical analysis in the reaction system, color developing agent is then added in the reaction system when using observation method of naked eye
HNB。
4, isothermal duplication
(1) fluorescence analysis: PCR reaction tube is put on the sample rack of fluorescence quantitative PCR instrument, the basis in analysis software
Placement order works out sample names and yin and yang attribute control；Be arranged amplification condition are as follows: 65 DEG C of reaction 50min, 90 DEG C of reaction 10min,
25 DEG C of cooling 1min, fluorescence detection channel select the channel FAM；Using fluorescence quantitative PCR instrument monitoring and analysis of fluorescence signal, sample
Amplification curve is S-type, and signal value is higher than threshold value and is just determined as the positive, no amplification curve or amplification curve is straight is determined as yin
Property, it only just can be carried out sample results interpretation (such as Fig. 5) under the premise of yin and yang attribute results of comparison meets；
(2) observation method of naked eye: PCR reaction tube is put into 65 DEG C of reaction 50min in dry bath pot, then rises to 90 DEG C of reactions
10min, brief centrifugation after cooling；Visual results, navy blue are that amplification is negative, light blue positive to expand, only yin-yang
Property results of comparison meet could interpretation sample amplification (such as Fig. 6).
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Sequence table
<110>The Third People's Hospital of Shenzhen (hepatopathy research institute of Shenzhen)
<120>LAMP detection primer group, detection kit and its detection method of mycobacterium kansasii
<130> 2017
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
GACTGTTGGCGAGTTGGT 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 2
GTCCTGGAACGTCTGCTC 18
<210> 3
<211> 38
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
GGTGTGGCACATTGCGACAGATATAACTCGAACAG CAC 38
<210> 4
<211> 37
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
CGGATCAGCGGTATGACGGCCGGCTGCCCGATTAGAT 37
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 5
GTTCTGCGACGGCGATTC 18
<210> 6
<211> 18
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 6
GGGATGCGTTGTCCACCA 18
Claims (10)
1. a kind of LAMP detection primer group of mycobacterium kansasii, which is characterized in that distinctive for mycobacterium kansasii
ECHT gene coded sequence designs and optimizes, and the detection primer group includes outer primer F3, outer primer B3, inner primer FIP, interior draws
Object BIP, ring primer LF and ring primer LB, nucleic acid sequence difference are as follows:
Outer primer F3:5 '-GACTGTTGGCGAGTTGGT-3 '
Outer primer B3:5 '-GTCCTGGAACGTCTGCTC-3 '
Inner primer FIP:5 '-GGTGTGGCACATTGCGACAGATATAACTCGAACAGCAC-3 '
Inner primer BIP:5 '-CGGATCAGCGGTATGACGGCCGGCTGCCCGATTAGAT-3 '
Ring primer LF:5 '-GTTCTGCGACGGCGATTC-3 '
Ring primer LB:5 '-GGGATGCGTTGTCCACCA-3 '.
2. LAMP detection primer group according to claim 1, which is characterized in that it is characterized in that, outer primer F3, outer primer
B3, inner primer FIP, inner primer BIP, the molar ratio of ring primer LF and ring primer LB in the reaction system are 1:1:4:4:2:2.
3. a kind of LAMP detection kit of mycobacterium kansasii, which is characterized in that containing such as right in the detection kit
It is required that the LAMP detection primer group of mycobacterium kansasii described in 1 or 2.
4. according to the LAMP detection kit of claim 3 mycobacterium kansasii, which is characterized in that it is characterized in that, also wrapping
Include some or all of of following component: (1) archaeal dna polymerase；(2) LAMP reaction solution；(2) fluorescent dye or color developing agent；(3) positive
Property control and negative control.
5. the kit according to claim 4 based on intelligent constant-temperature amplification technique detection staphylococcus aureus, special
Sign is: the archaeal dna polymerase is Bst archaeal dna polymerase；LAMP reaction solution includes dNTP, reaction buffer, MgSO4 and beet
Alkali.
6. the kit according to claim 4 based on intelligent constant-temperature amplification technique detection staphylococcus aureus, special
Sign is: positive control is the carrier cloning containing mycobacterium kansasii ECHT genetic fragment, and negative control is without bearing Sa
The water or other solvents of this mycobacteria ECHT genetic fragment.
7. according to the LAMP detection kit of claim 3 mycobacterium kansasii, which is characterized in that the fluorescent dye is
SYBR green, the color developing agent are hydroxynaphthol blue.
8. according to the LAMP detection kit of claim 3 mycobacterium kansasii, which is characterized in that including following component:
(1) primer sets: including LAMP detection primer group described in claim 1, and the concentration of outer primer, inner primer and ring primer is equal
It is 10 μM；
(2) reaction solution: including dNTP, reaction buffer, MgSO4 and glycine betaine, when use, needs equimultiple to dilute；
(3) Bst archaeal dna polymerase: concentration is 0.16U/ μ L；
(4) fluorescent dye or color developing agent: fluorescent dye is SYBR green, and color developing agent is hydroxynaphthol blue (HNB)；
(5) sealing fluid: for closing paraffin oil；
(6) it is enucleated sour water: for supplementing reaction volume.
9. a kind of method of mycobacterium kansasii LAMP detection kit detection mycobacterium kansasii, which is characterized in that packet
Include following steps:
(1) sample DNA the extraction of measuring samples DNA: is extracted using boiling lysis.(a) according to the difference of sputum specimen sliminess
It is different, it is suitably added the digestive juice of 1~2 times of volume, then vortex oscillation 30s, places 15min under room temperature.Irrigation of bronchus
The processing of liquid sample is the same with sputum specimen.1~2 times of volume volume is suitably added after pleural effusion sample 5000rpm centrifugation 15min
Digestive juice, remainder handles same sputum specimen.Swab specimen is first put into 1ml physiological saline, takes out swab after rotating repeatedly, then
5000rpm is centrifuged 15min, and 1ml digestive juice is added, and remainder handles same sputum specimen.(b) the postdigestive liquid of 1ml is drawn to 1.5ml
In microcentrifugal tube, 13000rpm is centrifuged 10min.(c) 1ml physiological saline, 13000g centrifugation is added after discarding supernatant liquid
10min.(d) step (c) is repeated after discarding supernatant.Supernatant is removed after centrifugation, 40 μ l DNA extracting solutions are added, and oscillation mixes
2000rpm is centrifuged 5s afterwards, then 37 DEG C of warm bath 30min, then 100 DEG C of heating 10min, and 13000rpm is centrifuged 10min after cooling.
(e) supernatant comprising DNA profiling is transferred to another after being centrifuged to go in the 1.5ml microcentrifugal tube of nucleic acid, sets -20 DEG C of guarantors
It deposits spare；
(2) isothermal amplification: the measuring samples DNA profiling obtained in 2 μ L steps (1) or positive and negative control is taken to be added
It is mixed in LAMP reaction system, 65 DEG C of isothermal reaction 50min are expanded, and then 80 DEG C of isothermal reaction 10min terminate reaction；Institute
State the LAMP detection primer group comprising mycobacterium kansasii described in claims 1 or 2 in LAMP reaction system；
(3) result judges: being judged by the real-time fluorescence detection data of analysis of fluorescence quantitative PCR apparatus, or passes through addition
The variation of liquid color carrys out judging result in color developing agent observation PCR pipe.
10. the side of mycobacterium kansasii LAMP detection kit detection mycobacterium kansasii according to claim 9
Method, which is characterized in that LAMP reaction system is to contain in 20 μ L reaction systems:
Outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer LF and ring primer LB concentration are 10 μM, are added
Amount is respectively 0.5 μ L, 0.5 μ L, 2 μ L, 2 μ L, 1 μ L, 1 μ L；
The additional amount of 0.16U/ μ L Bst archaeal dna polymerase is 0.4 μ L；
0.4 μ L of fluorescent dye or color developing agent；
10 μ L of reaction solution includes 4mM dNTP, 12mM MgSO4,1.6mM glycine betaine, 40mM Tris-HCl, 20mM in reaction solution
KCl、20mM(NH4)SO4, 0.1%TritonX-100；
The measuring samples DNA extracted in 2 μ L steps (1)；
With deionized water polishing to 20 μ L.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897889A (en) * | 2019-04-17 | 2019-06-18 | 北京天恩泽基因科技有限公司 | A kind of LAMP(ring mediated isothermal amplification) product visible detection method |
| CN110273015A (en) * | 2019-07-31 | 2019-09-24 | 四川省疾病预防控制中心 | A kind of mycobacterium kansasii visible detection method based on LAMP technology |
| CN110872614A (en) * | 2019-12-05 | 2020-03-10 | 深圳市第三人民医院 | Cas12 a/gRNA-based method for rapidly identifying mycobacteria |
| CN112538540A (en) * | 2020-11-26 | 2021-03-23 | 中国医学科学院北京协和医院 | Kit and system for detecting mycobacterium kansasii |
| CN113249516A (en) * | 2020-02-10 | 2021-08-13 | 华南理工大学 | Primer group of 2019-nCoV and 2019-nCoV nucleic acid detection kit |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005204582A (en) * | 2004-01-23 | 2005-08-04 | Asahi Kasei Corp | Oligonucleotide and method for detecting atypical acid-fast bacterium group by using the same |
| CN1995380A (en) * | 2006-01-05 | 2007-07-11 | 中国人民解放军总医院第二附属医院 | Method for detecting and identifying mycobacterium tuberculosis strain and its dedicated reagent kit |
| CN101175852A (en) * | 2005-05-13 | 2008-05-07 | 和光纯药工业株式会社 | Primer and probe for use in detection of mycobacterium kansasii and method for detection of mycobacterium kansasii using the same |
| CN101285062A (en) * | 2008-04-29 | 2008-10-15 | 博奥生物有限公司 | Process for abstracting bacterial DNA from phlegm, kit and uses thereof |
| CN107099601A (en) * | 2017-05-27 | 2017-08-29 | 中国人民解放军白求恩国际和平医院 | RT LAMP detect Primer composition, corresponding kit and the detection method of viable bacteria of Mycobacterium tuberculosis through |
-
2018
- 2018-05-16 CN CN201810473250.5A patent/CN109280711A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005204582A (en) * | 2004-01-23 | 2005-08-04 | Asahi Kasei Corp | Oligonucleotide and method for detecting atypical acid-fast bacterium group by using the same |
| CN101175852A (en) * | 2005-05-13 | 2008-05-07 | 和光纯药工业株式会社 | Primer and probe for use in detection of mycobacterium kansasii and method for detection of mycobacterium kansasii using the same |
| CN1995380A (en) * | 2006-01-05 | 2007-07-11 | 中国人民解放军总医院第二附属医院 | Method for detecting and identifying mycobacterium tuberculosis strain and its dedicated reagent kit |
| CN101285062A (en) * | 2008-04-29 | 2008-10-15 | 博奥生物有限公司 | Process for abstracting bacterial DNA from phlegm, kit and uses thereof |
| CN107099601A (en) * | 2017-05-27 | 2017-08-29 | 中国人民解放军白求恩国际和平医院 | RT LAMP detect Primer composition, corresponding kit and the detection method of viable bacteria of Mycobacterium tuberculosis through |
Non-Patent Citations (2)
| Title |
|---|
| ELVIRA RICHTER等: "Identification of Mycobacterium kansasii by Using a DNA Probe (AccuProbe) and Molecular Techniques", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| MIAO YANG等: "Isolation of a DNA Probe for Identification of Mycobacterium kansasii, Including the Genetic Subgroup", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109897889A (en) * | 2019-04-17 | 2019-06-18 | 北京天恩泽基因科技有限公司 | A kind of LAMP(ring mediated isothermal amplification) product visible detection method |
| CN110273015A (en) * | 2019-07-31 | 2019-09-24 | 四川省疾病预防控制中心 | A kind of mycobacterium kansasii visible detection method based on LAMP technology |
| CN110273015B (en) * | 2019-07-31 | 2023-10-13 | 四川省疾病预防控制中心 | LAMP technology-based visual detection method for mycobacterium kansasii |
| CN110872614A (en) * | 2019-12-05 | 2020-03-10 | 深圳市第三人民医院 | Cas12 a/gRNA-based method for rapidly identifying mycobacteria |
| CN113249516A (en) * | 2020-02-10 | 2021-08-13 | 华南理工大学 | Primer group of 2019-nCoV and 2019-nCoV nucleic acid detection kit |
| CN113249516B (en) * | 2020-02-10 | 2023-04-25 | 华南理工大学 | Primer group of 2019-nCoV and 2019-nCoV nucleic acid detection kit |
| CN112538540A (en) * | 2020-11-26 | 2021-03-23 | 中国医学科学院北京协和医院 | Kit and system for detecting mycobacterium kansasii |
| CN112538540B (en) * | 2020-11-26 | 2022-11-22 | 中国医学科学院北京协和医院 | Kit and system for detecting mycobacterium kansasii |
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