CN109811072A - CRISPR detection primer group and application thereof for mycobacterium tuberculosis complex - Google Patents
CRISPR detection primer group and application thereof for mycobacterium tuberculosis complex Download PDFInfo
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Abstract
The CRISPR detection primer group and application thereof that the present invention relates to a kind of for mycobacterium tuberculosis complex, belongs to technical field of gene detection.The primer sets include amplimer to and crRNA；The amplimer receives the shared conserved sequence of mycobacterium and mycobacterium microti to for expanding mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium africanum, bank；The crRNA includes anchor series and go-ahead sequence, and the anchor series and cas protein binding, the go-ahead sequence match with the targeted rna segment in the shared conserved sequence.Pass through the above-mentioned primer sets based on CRISPR detection technique, the shared conserved sequence of mycobacterium and mycobacterium microti, which is received, for mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium africanum, bank with CRISPR detection carries out genetic test, the on-site test of mycobacterium tuberculosis complex can be quickly carried out, and had the advantages that special high, sensitive high, simple.
Description
Technical field
The present invention relates to the technical field of gene detection based on CRISPR technology, are used for tuberculosis branch more particularly to one kind
CRISPR detection primer group of the compound group of bacillus and application thereof.
Background technique
Tuberculosis (tuberculosis, TB) is one of ten big causes of the death of whole world death population.According to the World Health Organization
(world health organization, WHO) report, new hair tuberculosis 10,400,000 of whole world estimation in 2016, wherein only having
6300000 are found and are reported, 4,100,000 gaps are still had (data are derived from " global tuberculosis report in 2017 ").Cause
This, prevention and control lungy are related to the public health security of country, and the key for controlling transmission of tuberculosis is exactly accurately to examine early stage
It is disconnected.
Pathogenic bacteria lungy are mycobacterium tuberculosis complex (Mycobacterium tuberculosis
Complex, MTBC), including mycobacterium tuberculosis (M.tuberculosis), Mycobacterium bovis (M.bovis), African branch bar
Bacterium (M.africanum), bank receive mycobacterium (M.canettii) and mycobacterium microti (M.microti) etc..
Diagnosis lungy at present depends on the inspection to pathogen, and common method is smear staining microscopy, separation
Culture, immunology diagnosis and diagnosis of molecular biology etc..Wherein, isolated culture is the goldstandard of current diagnosis tuberculosis, but is trained
Support 4~8 weeks time of needs, delay clinical diagnosis and treatment.Smear staining Microscopical Method For Detection is easy to operate, and quickly, but the method is sensitive
Spend low, poor specificity.Immunology diagnosis then intersects since existing antigen or antibody exist with other microorganisms, causes it special
Property is poor, false positive rate is high.Diagnosis of molecular biology has quick, sensitive advantage, and may be used also using the DNA fragmentation of specificity
To distinguish strain in M tuberculosis complex.The GeneXpert automatic detection system that WHO is recommended to use uses PCR skill
Art detects mycobacterium tuberculosis complex, and sensitivity specificity is preferable, and is not required to fall into a long wait as a result, still instrument
Device equipment price is expensive, it is difficult to universal in middle and low income area.In recent years also there are such as LAMP, a variety of permanent isothermal duplications such as RPA
Technology can be used for on-site test, but there is the problems such as lacking more effective amplified production detection means.Therefore, it needs to build
A kind of vertical scene, simple, quickly and highly sensitive detection technique of can be applied to.
Recombinase amplification technique (RPA, Recombinase Polymerase Amplification, recombinase polymerase
Isothermal amplification technique) it is a kind of isothermal amplification technology risen in recent years, by the mixture of three kinds of enzymes, single-chain nucleic acid can be combined
Recombinase, single-stranded DNA binding protein (SSB) and the strand displacement archaeal dna polymerase of (Oligonucleolide primers), also there is work at normal temperature
Property, for optimal reaction temperature at 37 DEG C or so, whole process can generally obtain within ten minutes can detect horizontal amplified production.
But from principle, the essence of single-stage amplified reaction is simultaneously had not been changed, compared to real-time quantitative PCRs most currently on the market
(Q-PCR) detection mode can not have substantive sensitivity enhancement.
Summary of the invention
Based on this, it is necessary in view of the above-mentioned problems, providing a kind of CRISPR detection for mycobacterium tuberculosis complex
This primer sets is used in the genetic test based on CRISPR technology by primer sets, can rapid field detection mycobacterium tuberculosis it is multiple
Gregarious (mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium africanum, bank receive mycobacterium, mycobacterium microti) has special
High, sensitive high, simple advantage.
A kind of CRISPR detection primer group for mycobacterium tuberculosis complex, including amplimer to and crRNA；Institute
It states amplimer and receives mycobacterium and vole point to for expanding mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium africanum, bank
The shared conserved sequence of branch bacillus；The crRNA includes anchor series and go-ahead sequence, the anchor series and cas albumen knot
It closes, the go-ahead sequence matches with the targeting DNA fragmentation in the shared conserved sequence.
Above-mentioned primer sets receive mycobacterium and field for mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium africanum, bank
The shared conserved sequence of mycobacterium muris is designed, and utilizes CRISPR (Cluste redregularly inter
Spacedshort palindromic repeats, the short palindrome repetitive sequence in Regularity interval) technology detected,
In CRISPR Cas system, Cas albumen starts after identifying target sequence under the guidance of crRNA (CRISPR-derivedRNA)
" subsidiary cutting " activity.Fluorescent reporter molecule is added in system, borrows Cas enzyme and is attached to cleavage activity, realizes sequence letter to be checked
Cease the conversion to fluorescence signal.By the coupling of RPA and Cas albumen, " sequence amplification " (RPA completion) can be realized plus " enzyme
The two-stage amplification of rush cascade " (completion of Cas enzyme), to surmount the sensitivity of this single-stage amplification of Q-PCR.In addition, because RPA expands
Increasing mode changes without complicated temperature, so that the dependence to precision instruments such as Q-PCR instrument is got rid of, so that CRISPR-Cas skill
Art has broad application prospects in terms of field diagnostic lungy
The shared conserved sequence is as shown in SEQ ID NO:1 in one of the embodiments,.Inventor is in early-stage study
On the basis of, it through excessive kind of screening and compares, it is found that shared conserved sequence shown in SEQ ID NO:1 both can be specific is used for tuberculosis
The detection of the compound group of mycobacteria, and can be detected with CRISPR-Cas technique to high-efficiency.
The go-ahead sequence is as shown in SEQ ID NO:2 in one of the embodiments,.It chooses shown in SEQ ID NO:2
Sequence is go-ahead sequence, namely the targeting sequence of detection, and SEQ ID NO:2 sequence is AUCAGCUCGGUCUUGUAUAG, is had
Preferable detection effect.
The crRNA sequence is as shown in SEQ ID NO:3 in one of the embodiments,.Sequence shown in SEQ ID NO:3
Composition are as follows: 5 '-anchor series-go-ahead sequence -3 ', with above-mentioned go-ahead sequence cooperation LbCas12a anchor series 5 ' -
UAAUUUCUACUAAGUGUAGAU-3 ', i.e. SEQ ID NO:3 sequence are UAAUUUCUAC UAAGUGUAG A UAUCAGCUCG
GUCUUGUAUA G has preferable detection effect.
The amplimer is to as follows in one of the embodiments:
Positive amplimer: 5 '-GGTCGGAAGCTCCTATGACAATGCACTAGCC-3 ' (SEQ ID NO:4)
Reversed amplimer: 5 '-TTGAGCGTAGTAGGCAGCCTCGAGTTCGAC-3 ' (SEQ ID NO:5).
The present inventor has found after passing through lot of experiments, is cooperated again with above-mentioned amplimer progress RPA amplification above-mentioned
CrRNA has preferable amplification efficiency and sensitivity.
The invention also discloses the above-mentioned CRISPR detection primer group for mycobacterium tuberculosis complex exploitation and/or
Prepare the application in the product with Diagnosis of Tuberculosis and/or prognosis evaluation purposes.
It should be understood that the said goods can be kit, it is also possible to integrated detection equipment etc..
The invention also discloses a kind of for detecting the kit of mycobacterium tuberculosis complex, including above-mentioned for tying
The CRISPR detection primer group of the compound group of core mycobacteria.
The kit further includes cas12a albumen, signal reports probe in one of the embodiments,.The cas12a egg
It is white from LbCas12a, AsCas12a and FnCas12a etc., but need to be adjusted according to separate sources albumen its anchor series and
Other agent formulations.
The cas12a albumen is LbCas12a in one of the embodiments,；The signal reports probe sequence are as follows:
5'-6-FAM-TTTTTTTTTTTT-BHQ1-3'.Cooperate LbCas12a and specific probe sequence with above-mentioned primer sets, has preferable
Detection effect.
The invention also discloses a kind of non-diagnostic therapeutic purposes mycobacterium tuberculosis complex detection methods, comprising the following steps:
Sample extraction: taking sample to be tested, extracts wherein DNA；
RPA amplification: with above-mentioned amplimer, the sample to be tested DNA that said extracted obtains is expanded by RPA method, must be expanded
Increase production object；
CRISPR reaction detection: taking above-mentioned amplified production, and signal reports probe, cas albumen and above-mentioned crRNA is added, into
Row CRISPR reaction detection, read detection signal to get.
Above-mentioned detecting step is completed in constant temperature, is changed without complicated temperature, to get rid of to Q-PCR instrument etc.
The dependence of precision instrument has more wide application prospect.
Compared with prior art, the invention has the following advantages:
A kind of CRISPR detection primer group for mycobacterium tuberculosis complex of the invention, for tuberculosis branch bar
Bacterium, Mycobacterium bovis, mycobacterium africanum, bank are received the shared conserved sequence of mycobacterium and mycobacterium microti and are designed,
And detected using CRISPR technology, in CRISPR Cas system, Cas albumen identifies target under the guidance of guide RNA
Starting " subsidiary cutting " activity after sequence.Fluorescent reporter molecule is added in system, borrows Cas enzyme and is attached to cleavage activity, realize
Conversion of the sequence information to be checked to fluorescence signal.By the coupling of RPA and Cas albumen, can be realized " sequence amplification ", (RPA is complete
At) plus the two-stage amplification of " enzymatic cascade " (completion of Cas enzyme), to surmount the sensitivity of this single-stage amplification of Q-PCR.This
Outside, because RPA amplification mode changes without complicated temperature, so that the dependence to precision instruments such as Q-PCR instrument is got rid of, so that
CRISPR-Cas technology has broad application prospects in terms of field diagnostic lungy.
Also, the present inventor also by the selected, amplimer to target sequence to and crRNA design and screening, finally
Obtained that, sensitivity high with amplification efficiency is good and high specificity can clinical practice primer sets, shorten tuberculosis branch bar
The detection time of the compound group of bacterium can complete detection, and the minimum mycobacterium tuberculosis that can detecte 5cfu/mL in 90min
Meet group, with other equal no cross reactions of human body common pathogen such as non-tuberculous mycobacteria, streptococcus pneumonia, greatly improves
CRISPR-Cas technology is in the on-site diagnosis practical operation of tuberculosis.
Detailed description of the invention
Fig. 1 is primer and crRNA sequence amplification efficiency the selection result schematic diagram in embodiment 1；
Fig. 2 is primer and crRNA sequence sensitivity the selection result schematic diagram in embodiment 1；
Fig. 3 is specific detection result schematic diagram in embodiment 1.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing
Give presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to this paper institute
The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more thorough
Comprehensively.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
Material and instrument involved by following embodiment are as follows:
1, experimental material:
Mycobacterium tuberculosis (H37Ra plants of M.tuberculosis), Mycobacterium bovis (M.bovis ATCC 19210),
Mycobacterium africanum (M.africanum), bank receive mycobacterium (M.canettii), mycobacterium microti (M.microti) DNA
Extract is provided by Shanghai Huashan Hospital.15 kinds of non-specific bacterial strain (mycobacterium avium, soil mycobacteria, Amur branch bars
Bacterium, Asia mycobacteria, Mycobacterium scrofulaceum, mycobacterium gordonae, tortoise purulence mycobacteria, is accidentally divided mycobacterium kansasii
Branch bacillus, Mycobacterium graminis, Nocardia brasiliensis, Beijing corynebacterium, legionella pneumophilia, streptococcus pneumonia and pertussis Boulder are special
Salmonella) it is purchased from National Institute for Food and Drugs Control.
Primer is synthesized with signal reports probe by the raw work biology Co., Ltd in Shanghai, and crRNA is had by Jin Sirui biotechnology
The synthesis of limit company, Lbcas12a albumen is by Jin Sirui Biotechnology Co., Ltd expression and purification.Other biochemical reagents are import
Packing or domestic analysis are pure.
2, laboratory apparatus
Metal bath, centrifuge, vortex instrument, fluorescence detection equipment etc..
Embodiment 1
The design and screening of mycobacterium tuberculosis complex CRISPR detection primer sequence.
One, the design of sequence.
1, target sequence is selected.
Inventor through excessive kind of screening and compares on the basis of early-stage study, selectes the sequence as shown in SEQ ID NO:1
As mycobacterium tuberculosis (M.tuberculosis), Mycobacterium bovis (M.bovis), mycobacterium africanum
(M.africanum), bank receives the target sequence of mycobacterium (M.canettii) and mycobacterium microti (M.microti), should
Conserved sequence of the sequence as above-mentioned strain can detect above-mentioned tuberculosis pathogenic bacteria.SEQ ID NO:1 sequence is as follows:
AAAGACCGCGTCGGCTTTCTTCGCGGCCGAGCTCGACCGGCCAGCACGCTAATTACCCGGTTCATCGCC
GATCATCAGGGCCACCGCGAGGGCCCCGATGGTTTGCGGTGGGGTGTCGAGTCGATCTGCACACAGCTGACCGAGCT
GGGTGTGCCGATCGCCCCATCGACCTACTACGACCACATCAACCGGGAGCCCAGCCGCCGCGAGCTGCGCGATGGCG
AACTCAAGGAGCACATCAGCCGCGTCCACGCCGCCAACTACGGTGTTTACGGTGCCCGCAAAGTGTGGCTAACCCTG
AACCGTGAGGGCATCGAGGTGGCCAGATGCACCGTCGAACGGCTGATGACCAAACTCGGCCTGTCCGGGACCACCCG
CGGCAAAGCCCGCAGGACCACGATCGCTGATCCGGCCACAGCCCGTCCCGCCGATCTCGTCCAGCGCCGCTTCGGAC
CACCAGCACCTAACCGGCTGTGGGTAGCAGACCTCACCTATGTGTCGACCTGGGCAGGGTTCGCCTACGTGGCCTTT
GTCACCGACGCCTACGCTCGCAGGATCCTGGGCTGGCGGGTCGCTTCCACGATGGCCACCTCCATGGTCCTCGACGC
GATCGAGCAAGCCATCTGGACCCGCCAACAAGAAGGCGTACTCGACCTGAAAGACGTTATCCACCATACGGATAGGG
GATCTCAGTACACATCGATCCGGTTCAGCGAGCGGCTCGCCGAGGCAGGCATCCAACCGTCGGTCGGAGCGGTCGGA
AGCTCCTATGACAATGCACTAGCCGAGACGATCAACGGCCTATACAAGACCGAGCTGATCAAACCCGGCAAGCCCTG
GCGGTCCATCGAGGATGTCGAGTTGGCCACCGCGCGCTGGGTCGACTGGTTCAACCATCGCCGCCTCTACCAGTACT
GCGGCGACGTCCCGCCGGTCGAACTCGAGGCTGCCTACTACGCTCAACGCCAGAGACCAGCCGCCGGCTGA
2, amplimer to and crRNA design.
For above-mentioned shared conserved sequence, a plurality of crRNA and corresponding amplimer pair are designed, following table lists few examples
Property primer sequence.
Table 1.crRNA and primer sequence
Two, the screening of sequence.
1, amplification efficiency screens
1.1 method
Mycobacterium tuberculosis (M.tuberculosis) bacterial concentration is calculated using colony counting method, respectively with 10 times of multiple proportions
It is diluted to 5.0 × 104-5.0×10-1Cfu/mL bacterium solution, takes 5.0 × 103Cfu/mL bacterium solution 1mL, is mentioned using bacterial genomes DNA
It takes kit (TIANGEN Biotech (Beijing) Co., Ltd., article No. DP320) to extract bacterium solution genomic DNA, is buffered with 20 μ L TE
Liquid dissolution, taking 2 μ L genomic DNAs respectively is template progress RPA amplification, while using mycobacterium avium nucleic acid DNA as negative control,
It is carried out according to following operation:
A.RPA amplification
Amplification system:
RPA reaction system is 50 μ L: including RPA upstream primer 0.5-2.5 μ L (10 μM of concentration), RPA downstream primer
0.5-2.5 μ L (10 μM of concentration), 41.5 μ L of RPA enzyme premixed liquid (wherein contain: phosphocreatine (concentration 20-80mM), creatine kinase
(concentration 50-150mM), dNTPs (100-300 μM of concentration), ATP (concentration 20-80mM), DTT (concentration 1-10mM), potassium acetate
(concentration 50-200mM), recombinase uxsX (50-300ng/ μ L), uxsY (10-100ng/ μ L), single strand binding protein (200-
1000ng/ μ L), Bsu polymerase (10-100ng/ μ L)), magnesium acetate 0.5-2 μ L (concentration 280mM), sample gene to be tested group DNA
2μL。
Amplification program: 37 DEG C of reaction 15-60min of constant temperature.
B. the CRISPR reaction based on cas12a
CRISPR reaction system:
Above-mentioned RPA amplified production (22 μ L), 1 μ L of Lbcas12a albumen (1-5 μM of concentration), crRNA1 μ L (1-5 μM of concentration),
1 μ L of signal reports probe (5 ' 6-FAM-TTTTTTTTTTTT-BHQ1-3 ', 1-10 μM of concentration).
Reaction condition and time: 37 DEG C of reactions 30-60min, every 1min read FAM fluorescent value.
Interpretation of result: the accumulation fluorescent value for using fluorescence detector to obtain is carried out as signal strength according to following standard
Analysis determines:
Negative judgment criteria: fluorescence volume is less than or equal to 2 times of negative control fluorescence volume.
Positive judgment criteria: fluorescence volume is greater than 2 times of negative control fluorescence volume.
Negative control group be each experimental group be correspondingly arranged non-tuberculous mycobacteria (mycobacterium avium) nucleic acid is added
Negative signal group of the DNA as template.
1.2 result
Detection screening has been carried out to specificity crRNA in upper table and its corresponding each 5 pairs of primer pairs combination, has finally selected one
The good specific crRNA-3 sequence of group amplification efficiency, the selection result are as shown in Figure 1.
As a result indicate that there is optimal amplification efficiency using crRNA-3 cooperation primer pair 12.
2, sensitivity is screened
1.1 method
Mycobacterium tuberculosis bacterial concentration is calculated using colony counting method, respectively with 10 times of doubling dilutions at 5.0 × 104-
5.0×10-1Cfu/mL bacterium solution takes each 1mL of the bacterium solution of corresponding extension rate, using bacterial genomes DNA extraction kit (Tiangeng
Biochemical technology (Beijing) Co., Ltd) the bacterium solution genomic DNA that extracts each extension rate, is dissolved with 20 μ L TE, takes 2 μ L respectively
Genomic DNA is that template carries out RPA amplification, while using mycobacterium avium nucleic acid DNA as negative control, imitating to the amplification screened
The preferable crRNA-3 sequence of rate and corresponding primer pair 11,12 and 15 carry out sensitivity screening, are examined according to the method described above
It surveys.
1.2 result
As a result as shown in Fig. 2, with 12 (SEQ ID NO:4- of crRNA-3 sequence (SEQ ID NO:3) and corresponding primer pair
5), minimum to can detecte 5 × 100The mycobacterium tuberculosis complex of cfu/mL has optimal sensitivity.
3, specificity is investigated
1.1 method
CrRNA-3 sequence (SEQ ID NO:3) and corresponding primer pair 12 (SEQ ID NO:4-5) are chosen, according to above-mentioned
Method is to mycobacterium tuberculosis (M.tuberculosis), Mycobacterium bovis (M.bovis), mycobacterium africanum
(M.africanum), bank receives mycobacterium (M.canettii), mycobacterium microti (M.microti) genomic DNA and 15 kinds
Non-specific bacterial strain (mycobacterium avium, soil mycobacteria, Amur mycobacteria, mycobacterium kansasii, Asia branch bar
Bacterium, Mycobacterium scrofulaceum, mycobacterium gordonae, tortoise purulence mycobacteria, mycobacterium fortuitum, Mycobacterium graminis, Brazilian promise Cattell
Bacterium, Beijing corynebacterium, legionella pneumophilia, streptococcus pneumonia and Bordetella pertussis) it is expanded, while with bird branch bar
Bacterium nucleic acid DNA is negative control, examines the specificity of this method.
1.2 result
As a result as shown in figure 3, NC indicates the negative control using distilled water as template in figure, the method for the present invention is only to tuberculosis
(M. tuberculosis mycobacteria, mycobacterium bovis, mycobacterium africanum, bank receive mycobacterium, field to the compound group of mycobacteria
Mycobacterium muris) sample has amplification, and other non-tuberculous mycobacterias show that this method specificity is good then without amplification.
Embodiment 2
It is a kind of for detecting the kit of mycobacterium tuberculosis complex, comprising:
(1) RPA amplification system:
RPA amplimer pair:
Positive: 5 '-GGTCGGAAGCTCCTATGACAATGCACTAGCC-3 ' (SEQ ID NO:4), concentration are 0.1~1 μ
M
Reversed: 5 '-TTGAGCGTAGTAGGCAGCCTCGAGTTCGAC-3 ' (SEQ ID NO:5), concentration are 0.1~1 μ
M。
RPA enzyme premixed liquid: phosphocreatine (concentration 20-80mM), creatine kinase (concentration 50-150mM), dNTPs (concentration
100-300 μM), ATP (concentration 20-80mM), DTT (concentration 1-10mM), potassium acetate (concentration 50-200mM), recombinase uxsX
(50-300ng/ μ L), uxsY (10-100ng/ μ L), single strand binding protein (200-1000ng/ μ L), Bsu polymerase (10-
100ng/ μ L) etc..
Magnesium acetate solution: concentration 10-20mM
(2) CRISPR reaction system:
CrRNA:5 '-UAAUUUCUACUAAGUGUAGAUAUCAGCUCGGUCUUGUAUAG-3 ' (SEQ ID NO:3) is dense
Degree is 20-100nM
Lb cas12a albumen: concentration 20-100nM.
Signal reports probe: 5 ' 6-FAM-TTTTTTTTTTTT-BHQ1-3 ', concentration 50-500nM.
The method for carrying out mycobacterium tuberculosis complex detection using mentioned reagent box is as follows:
(1) DNA for detecting sample is extracted
Detection sample can be mycobacterium tuberculosis complex bacterial strain, and being also possible to clinical sample (mainly includes tissue sample
Sheet, sputum, cerebrospinal fluid, Pleural effusions, urine, fester, blood sample etc.) or other scientific experiment samples.
(2) pathogen nucleic acid is expanded by RPA technology
Amplification system:
RPA reaction system is 50 μ L: including RPA upstream primer 0.5-2.5 μ L (10 μM of concentration), RPA downstream primer
0.5-2.5 μ L (10 μM of concentration), RPA enzyme premixed liquid 41.5 μ L, magnesium acetate 14mM, sample gene to be tested group DNA2 μ L.
Amplification program: 37 DEG C of reaction 15-60min of constant temperature.
(3) the CRISPR reaction based on cas12a
Above-mentioned RPA amplified production (22 μ L), 1 μ L of Lbcas12a albumen (1-5 μM of concentration), crRNA1 μ L (1-5 μM of concentration),
1 μ L of signal reports probe (5 ' 6-FAM-TTTTTTTTTTTT-BHQ1-3 ', 1-10 μM of concentration).
Reaction condition and time: 37 DEG C of reactions 30-60min, every 1min read FAM fluorescent value.
(4) interpretation of result:
In above-mentioned detection process, the signal that is separately connected fluorescent material and quencher since both ends being added in the reaction system
Reporter probe after identifying the targeting DNA with targeting sequence, is activated when cas12a albumen is with the help of crRNA
Cas12a enzyme can degrade, and this has the signal reports probe of signal, to discharge fluorescence signal, realizes detection.
The accumulation fluorescent value for using fluorescence detector to obtain carries out analysis judgement as signal strength, according to following standard:
Negative judgment criteria: fluorescence volume is less than or equal to 2 times of negative control fluorescence volume.
Positive judgment criteria: fluorescence volume is greater than 2 times of negative control fluorescence volume.
Wherein, negative control group be each experimental group be correspondingly arranged non-tuberculous mycobacteria (mycobacterium avium) is added
Negative signal group of the nucleic acid DNA as template.
Embodiment 3
The mycobacterium tuberculosis complex detection of clinical sample is carried out with the kit of above-described embodiment 2.
1. method
(1) samples sources: Infectious Disease, Shanghai Huashan Hospital.
It clinical sample totally 120, is carried out with the GeneXpert detection system and the method for the present invention being widely recognized as in the market blind
Detection.
(2) detection method:
Coherent detection step of the invention in reference implementation example 2 detects 120 samples.
120 samples are detected with reference to GeneXpert detection system relevant operation specification detecting step.
2. result
It is as shown in the table using the testing result of two methods:
2 distinct methods of table are to clinical sample testing result
Known to calculating:
Positive coincidence rate=97.06%
Negative match-rate=100%
Total coincidence rate=99.17%
95% credibility interval of macro-forecast value is [0.9389~1.0197]
Value=0.9793 Kappa
The above results illustrate that detection method is consistent with GeneXpert detection method detection effect with higher
Property.
And an example CRISPR testing result of the present invention is feminine gender in the above results, and GeneXpert testing result is the positive
Case, through analyzing, it may be possible to since the sample size difference used causes, because GeneXpert is more than using original sample amount
0.5mL, the nucleic acid of extraction are all used to augmentation detection, but CRISPR method of the invention, are extracting sample genomic dna
Afterwards, 2 μ l has only been taken to be detected；Meanwhile the difference of a sample, it is also possible to there are some other unknown causes to cause.
And consider from sensitivity, method of the invention, the minimum mycobacterium tuberculosis that can detecte 5 × 100cfu/mL is multiple
Gregarious (1 result of embodiment), and according to GeneXpert specification (MTB/RIF 301-3300C, revised edition A2014
03 month), detecting limit estimated value for MTB is 131cfu/mL, is much larger than the method for the present invention.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
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<213>artificial sequence (Artificial Sequence)
<400> 6
uaauuucuac uaaguguaga uucaccgacg ccuacgcucg ca 42
<210> 7
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ctgatccggc cacagcccgt cccgccgatc tcgtccagc 39
<210> 8
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gaggaccatg gaggtggcca tcgtggaagc gaccc 35
<210> 9
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ccacagcccg tcccgccgat ctcgtccagc 30
<210> 10
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gccatcgtgg aagcgacccg ccagcccagg 30
<210> 11
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
gctgatgacc aaactcggcc tgtccgggac ca 32
<210> 12
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
tggtggataa cgtctttcag gtcgagtacg ccttcttgt 39
<210> 13
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
tcgaacggct gatgaccaaa ctcggcctgt cc 32
<210> 14
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ccgtatggtg gataacgtct ttcaggtcga gtacgccttc 40
<210> 15
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ttcggaccac cagcacctaa ccggctgtgg gta 33
<210> 16
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
ggataacgtc tttcaggtcg agtacgcctt cttgttgg 38
<210> 17
<211> 42
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 17
uaauuucuac uaaguguaga uuagcagacc ucaccuaugu gu 42
<210> 18
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
tgaaccgtga gggcatcgag gtggccagat gc 32
<210> 19
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
gatcctgcga gcgtaggcgt cggtgacaaa ggccacgtag 40
<210> 20
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
gccacagccc gtcccgccga tctcgtccag 30
<210> 21
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
agatggcttg ctcgatcgcg tcgaggacca tg 32
<210> 22
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
gaacggctga tgaccaaact cggcctgtcc gggac 35
<210> 23
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
tcctgcgagc gtaggcgtcg gtgacaaagg 30
<210> 24
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
tcgaacggct gatgaccaaa ctcggcctgt cc 32
<210> 25
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
catcgtggaa gcgacccgcc agcccaggat 30
<210> 26
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
cggctgatga ccaaactcgg cctgtccggg ac 32
<210> 27
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
atcgcgtcga ggaccatgga ggtggccatc gt 32
<210> 28
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
cctatgacaa tgcactagcc gagacgatca ac 32
<210> 29
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
ctggtctctg gcgttgagcg tagtaggcag 30
<210> 30
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
gctcctatga caatgcacta gccgagacga tcaacg 36
<210> 31
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
gccaactcga catcctcgat ggaccgccag 30
<210> 32
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
ggaagctcct atgacaatgc actagccgag acgat 35
<210> 33
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
tggtagaggc ggcgatggtt gaaccagtcg ac 32
<210> 34
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
tcctatgaca atgcactagc cgagacgatc aacg 34
<210> 35
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
acgtcgccgc agtactggta gaggcggcga tggtt 35
Claims (10)
1. a kind of CRISPR detection primer group for mycobacterium tuberculosis complex, which is characterized in that including amplimer pair
And crRNA；The amplimer receives branch bar to for expanding mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium africanum, bank
The shared conserved sequence of bacterium and mycobacterium microti；The crRNA includes anchor series and go-ahead sequence, the anchor series with
Cas protein binding, the go-ahead sequence match with the targeting DNA fragmentation in the shared conserved sequence.
2. the CRISPR detection primer group according to claim 1 for mycobacterium tuberculosis complex, which is characterized in that
The shared conserved sequence is as shown in SEQ ID NO:1.
3. the CRISPR detection primer group according to claim 1 for mycobacterium tuberculosis complex, which is characterized in that
The go-ahead sequence is as shown in SEQ ID NO:2.
4. the CRISPR detection primer group according to claim 3 for mycobacterium tuberculosis complex, which is characterized in that
The crRNA sequence is as shown in SEQ ID NO:3.
5. according to the described in any item CRISPR detection primer groups for mycobacterium tuberculosis complex of claim 3-4,
It is characterized in that, the amplimer is to as follows:
Positive amplimer: 5 '-GGTCGGAAGCTCCTATGACAATGCACTAGCC-3 ' (SEQ ID NO:4)
Reversed amplimer: 5 '-TTGAGCGTAGTAGGCAGCCTCGAGTTCGAC-3 ' (SEQ ID NO:5).
6. the described in any item CRISPR detection primer groups for mycobacterium tuberculosis complex of claim 1-5 are being developed
And/or preparation has the application in Diagnosis of Tuberculosis and/or the product of prognosis evaluation purposes.
7. a kind of for detecting the kit of mycobacterium tuberculosis complex, which is characterized in that including any one of claim 1-5
The CRISPR detection primer group for mycobacterium tuberculosis complex.
8. according to claim 7 for detecting the kit of mycobacterium tuberculosis complex, which is characterized in that further include
Cas12a albumen, signal reports probe.
9. according to claim 7 for detecting the kit of mycobacterium tuberculosis complex, which is characterized in that described
Cas12a albumen is LbCas12a；The signal reports probe sequence are as follows: 5 ' -6-FAM-TTTTTTTTTTTT-BHQ1-3 '.
10. a kind of non-diagnostic therapeutic purposes mycobacterium tuberculosis complex detection method, which comprises the following steps:
Sample extraction: taking sample to be tested, extracts wherein DNA；
RPA amplification: it with the amplimer in claim any one of 1-5, is obtained by RPA method amplification said extracted to be measured
Sample DNA obtains amplified production；
CRISPR reaction detection: taking above-mentioned amplified production, and any one of signal reports probe, cas albumen and claim 1-5 is added
In crRNA, carry out CRISPR reaction detection, read detection signal to get.
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| CN110106290A (en) * | 2019-05-31 | 2019-08-09 | 华南理工大学 | A kind of field fast detection method and kit being used to detect ASFV based on CRISPR/Cas system |
| CN110184329A (en) * | 2019-05-31 | 2019-08-30 | 华南理工大学 | A kind of one-step method nucleic acid detection method and kit based on CRISPR/Cas and constant-temperature amplification |
| CN110241237A (en) * | 2019-06-24 | 2019-09-17 | 浙江大学 | A kind of kit for clostridium perfringen detection |
| CN110541022A (en) * | 2019-08-09 | 2019-12-06 | 福建医科大学孟超肝胆医院(福州市传染病医院) | mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system |
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| CN114457073A (en) * | 2021-06-08 | 2022-05-10 | 山东舜丰生物科技有限公司 | Method for detecting mycobacterium paratuberculosis based on CRISPR technology |
| CN114457073B (en) * | 2021-06-08 | 2023-07-18 | 山东舜丰生物科技有限公司 | Method for detecting mycobacterium paratuberculosis based on CRISPR technology |
| CN114480682A (en) * | 2022-01-13 | 2022-05-13 | 潍坊医学院 | Composition and kit for detecting mycobacterium tuberculosis and application of composition and kit |
| CN114921576A (en) * | 2022-06-29 | 2022-08-19 | 湖南工程学院 | Reagent, kit and detection method for detecting mycobacterium bovis |
| CN114921576B (en) * | 2022-06-29 | 2023-11-10 | 湖南工程学院 | Reagent, kit and detection method for detecting mycobacterium bovis |
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