CN110541022B - Mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system - Google Patents
Mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system Download PDFInfo
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Abstract
The invention belongs to the technical field of nucleic acid detection, and relates to a detection kit and a detection method for mycobacterium tuberculosis complex based on a CRISPR-Cas12a system. The kit improves the detection sensitivity by adopting a recombinase polymerase amplification technology, activates the bypass cutting activity of the Cas12a after utilizing the CRISPR-Cas12a specific target mycobacterium tuberculosis complex target sequence, and can sensitively and specifically detect the mycobacterium tuberculosis complex from the sputum. The invention has the advantages of no invasion, frequent and multiple detection, high detection speed and the like. Compared with the current PCR detection method, the method does not need a thermal cycler with a temperature rise and drop function, can detect the Mycobacterium tuberculosis complex in the sputum through fluorescence reading, has the advantages of low cost, simple and convenient operation, high sensitivity, good specificity and the like, and is suitable for large-scale clinical application.
Description
(I) technical field
The invention relates to a detection kit and a detection method for mycobacterium tuberculosis complex based on a CRISPR-Cas12a system.
(II) background of the invention
Tuberculosis (TB) is an infectious disease that is a long-term threat to human health, the causative agent of which is mycobacterium Tuberculosis, which is infected in about one third of the world's population. According to the statistics of the world health organization, about 1000 million new tuberculosis cases are shared in 2017 all over the world, and about 160 million people die of tuberculosis. China belongs to one of 22 tuberculosis high-burden countries in the world, and the tuberculosis people live on the second place in the world. Every year, about 100 thousands of new cases exist in China, and about 13 thousands of people die of tuberculosis. Therefore, tuberculosis remains a global health problem that threatens human health to date.
Studies have shown that the main pathogens causing Tuberculosis in humans are Mycobacterium Tuberculosis Complex (MTBC), which mainly includes Mycobacterium Tuberculosis, mycobacterium bovis, mycobacterium africanum, mycobacterium microti, etc. Non-tuberculous Mycobacteria (NTM) refer to Mycobacteria other than Mycobacterium tuberculosis, mycobacterium bovis and Mycobacterium leprae, are widely distributed in the environment (such as wet soil, marsh and river), and belong to opportunistic pathogens. In clinical diagnosis, in the lung disease caused by NTM infection, the symptoms and signs are similar to those of tuberculosis caused by MTBC infection, so that the lung disease is difficult to distinguish. However, in medical practice, since the therapeutic drugs for the two diseases are different, the MTBC and NTM need to be identified rapidly and accurately in clinical diagnosis so as to select the correct drug for treatment.
At present, the diagnosis of tuberculosis mainly depends on the examination of pathogens, and the common methods are smear staining microscopy, separation culture, immunological diagnosis, molecular biological diagnosis and the like. Wherein, the isolation culture method is the current gold standard for diagnosing tuberculosis, but the culture needs 4 to 8 weeks, thereby delaying clinical diagnosis and treatment. The smear staining microscopy method is simple and rapid to operate, but the method has low sensitivity and poor specificity. The immunological diagnosis has poor specificity and high false positive rate due to the cross of the existing antigen or antibody and other microorganisms. The molecular biological diagnosis has the advantages of rapidness and sensitivity, and specific DNA fragments can be used for distinguishing the mycobacterium tuberculosis complex in-vivo strains. The Gene Xpert full-automatic detection system recommended by WHO adopts the PCR technology to detect the mycobacterium tuberculosis complex, has good sensitivity and specificity, does not need to wait for results for a long time, but has expensive instruments and equipment and is difficult to popularize in low-income areas. In recent years, various isothermal amplification techniques such as LAMP and RPA have appeared and are applicable to in-situ detection, but all of them have problems such as lack of effective means for detecting amplified products. Therefore, it is highly desirable to establish a simple, fast and highly sensitive detection technique that can be applied in the field.
The Recombinase Polymerase Amplification (RPA) is a constant temperature Amplification technology that has been developed in recent years, and a mixture of three enzymes, i.e., a Recombinase capable of binding single-stranded nucleic acid (oligonucleotide primer), a single-stranded DNA binding protein (SSB), and a strand displacement DNA Polymerase, is active at room temperature, and the optimal reaction temperature is about 37 ℃. The RPA belongs to an isothermal amplification technology, has low requirements on instruments and equipment, can complete the reaction only by a constant-temperature water bath, and does not need a precise instrument.
CRISPR-Cas is an acquired immune defense system of bacteria against viral and plasmid infection, present in almost all archaea and about 50% of modern bacteria. CRISPR-Cas systems are divided into two major categories, and the CRISPR system in the first major category can only play a role in an effect complex consisting of a plurality of Cas proteins; the second broad category of Cas relies on a single Cas effector protein (e.g., cas9, cpf1, C2, etc.) to function.
In 2015, zhang Feng et al found CRISPR-associated protein endonuclease Cas12a (previously referred to as Cpf 1), which is an RNA-guided specific DNA endonuclease like the commonly used Cas9 protein, but has its own features compared to Cas9, cas12a, such as only need of crRNA to guide specific cleavage of double-stranded DNA, and generation of sticky ends, etc. Cas12a, once it recognizes and cleaves the target DNA designated by the crRNA sequence, is transferred to an enzymatic "activated" state where it can fragment any non-target single-stranded DNA. This effect is referred to as bypass cleavage activity. In 2018, the Doudna team establishes a CRISPR-based nucleic acid detection method DETETR based on the combination of RNA guide and DNA targeting endonuclease bypass cutting effect of Cas12a and DNA isothermal amplification. The principle is that firstly, a fragment containing a target sequence is obtained through a large amount of amplification by an RPA method, then an amplification product is added into a Cas12a detection system (comprising gRNA, crRNA, cas12a protein and ssDNA probe), once a Cas12a and gRNA complex recognizes and cuts the target sequence, the activity of cutting a non-specific single strand can be triggered, a single-strand reporter molecule in the detection system is cut, and fluorescence is emitted, so that the existence of the target sequence is confirmed, and the method can be used for detecting DNA viruses and SNP. The detection method does not need expensive reagents and special instruments, has low cost, simple and convenient operation and high detection sensitivity, can reach the single-molecule detection level, has strong specificity and is very convenient to detect the target sequence DNA.
Disclosure of the invention
The invention aims to provide a mycobacterium tuberculosis complex detection kit and a detection method based on a CRISPR-Cas12a system.
The technical scheme adopted by the invention is as follows:
a detection kit for Mycobacterium tuberculosis complex based on CRISPR-Cas12a system mainly comprises a primer for specifically amplifying an IS1081 gene (target gene) of the Mycobacterium tuberculosis complex, gRNA and a fluorescent reporter molecule ssDNA-FQ,
the primer sequences are as follows:
an upstream primer:
5’-CCAAGCTGCGCCAGGGCAGCTATTTCCCGGAC-3’；
a downstream primer:
5’-TTGGCCATGATCGACACTTGCGACTTGGA-3’；
the gRNA sequence is:
5’-UAAUUUCUACUAAGUGUAGAUGACCAGGCGCUCCAUCCGGC
-3’；
the sequence of the fluorescent reporter molecule ssDNA-FQ is as follows:
5’-FAM-TTTTT-BHQ1-3’。
the detection sensitivity is improved by adopting a recombinase polymerase amplification technology, the CRISPR-Cas12a system is utilized to activate the bypass cutting activity of the Cas12a after the target sequence of the mycobacterium tuberculosis complex is specifically targeted under the guidance of the gRNA, the mycobacterium tuberculosis complex can be detected from sputum and other specimens in a high-sensitivity and specific manner, and an expensive thermal cycler is not needed.
In CRISPR-Cas systems, cas protein initiates "bypass cleavage" activity upon recognition of a target sequence under the guidance of a gRNA (guide RNA). And (3) adding a fluorescent reporter molecule into the system, and realizing the conversion of the sequence information to be detected to a fluorescent signal by using the Cas12a enzyme bypass cleavage activity. By coupling RPA to Cas12a protein, two-stage amplification of "sequence amplification" (RPA completed) plus "enzymatic cascade" (Cas enzyme completed) can be achieved, exceeding the sensitivity of qPCR, a single-stage amplification. In addition, the RPA amplification mode does not need complex temperature change, so that the dependence on precision instruments such as a qPCR instrument is eliminated, and the CRISPR-Cas technology has wide application prospect in the field diagnosis of tuberculosis.
Specifically, the IS1081 gene has the following sequence (SEQ ID No. 1):
GAATTCGATCGCCGAGCCGACAAGACATGCCAGCGCAACCCGC TTCATCGTCGTGGCAGGTGTTGGGCTGATTTTGGTCAACCCAGCACC TGCCAGGACGGGCTACGGATGTACACGGCGACGACGGTATGGGAG GATGTCCGGTCTTGCTCCGGTCATGTCCGGTGAATGTGCTGCCAACA TCCTGGGGACCGTCCAGCGAGTTTCACCACACCTTGGGGCACCTTC TGTCACTGCTCGGTGCTGTGGATTGGTGTCAAGTTACGTCCAGGGG TGTGGTGTACGGGCAGGTAAGGCCGGTGGGCGTGTCGTAGCCCAGT AGTGGGCGGTCATCGCGTGATCCTTCGAAACGACCAGCAAAAGTCA ATCGAAGGAAATGACGCAATGACCTCTTCTCATCTTATCGACACCGA GCAGCTTCTGGCTGACCAACTCGCACAGGCGAGCCCGGATCTGCTG CGCGGGCTGCTCTCGACGTTCATCGCCGCCTTGATGGGGGCTGAAG CCGACGCCCTGTGCGGGGCGGGCTACCGCGAACGCAGCGATGAGC GGTCCAATCAGCGCAACGGCTACCGCCACCGTGATTTCGACACCCG TGCCGCAACCATCGACGTCGCGATCCCCAAGCTGCGCCAGGGCAGC TATTTCCCGGACTGGCTGCTGCAGCGCCGCAAGCGAGCTGAACGCG CACTGACCAGCGTGGTGGCGACCTGCTACCTGCTGGGAGTATCCAC TCGCCGGATGGAGCGCCTGGTCGAAACACTTGGTGTGACAAAGCTT TCCAAGTCGCAAGTGTCGATCATGGCCAAAGAGCTCGACGAAGCCG TAGAGGCGTTTCGGACCCGCCCGCTCGATGCCGGCCCGTATACCTTC CTCGCCGCCGACGCCCTGGTGCTCAAGGTGCGCGAGGCAGGCCGC GTCGTCGGAGTGCACACCTTGATCGCCACCGGCGTCAACGCCGAGG GCTACCGAGAGATCCTGGGCATCCAGGTCACCTCCGCCGAGGACGG GGCCGGCTGGCTGGCGTTCTTCCGCGACCTGGTCGCCCGCGGCCTG TCCGGGGTCGCGCTGGTCACCAGCGACGCCCACGCCGGCCTGGTG GCCGCGATCGGCGCCACCCTGCCCGCAGCGGCCTGGCAGCGCTGC AGAACCCACTACGCAGCCAATCTGATGGCAGCCACCCCGAAGCCCT CCTGGCCGTGGGTGCGCACCCTGCTGCACTCCATCTACGACCAGCC CGACGCCGAATCAGTTGTTGCCCAATATGATCGGGTACTCGACGCTC TGACCGACAAACTCCCCGCGGTGGCCGAGCACCTCGACACCGCCC GCACCGACCTGCTGGCGTTCACCGCCTTCCCCAAGCAGATCTGGCG CCAAATCTGGTCCAACAACCCCCAGGAACGCCTCAACCGAGAGGT ACGACGCCGAACCGACGTCGTGGGCATCTTCCCCGACCGCGCCTCG ATCATCCGCCTCGTCGGAGCCGTCCTCGCCGAACAACACGACGAAT GGATCGAAGGACGGCGCTACCTGGGCCTCGAGGTCCTCACCCGAGC CCGAGCAGCACTGACCAGCACCGAAGAACCCGCCAAGCAGCAAAC CACCAACACCCCAGCACTGACCACCTAGACTGCCACCCGAAGGATC ACGCGAGGAACCTTCACTCGTACACCACGTCCCTGGCCTTGGCCTG GTGTCAGGCCCAGCTG
the sequence can be specifically used for detecting mycobacterium tuberculosis complex, can also be used as a guide sequence (higher CRISPR-DT software score) for high-efficiency detection of CRISPR-Cas technology, and is also a target sequence for detection, and is shown as SEQ ID NO. 2: GACCAGGCGCUCCAUCCGGC.
The gRNA sequence is composed of: 5 '-anchoring sequence-guide sequence-3', the guide sequence is matched with an anchoring sequence 5'-UAAUUUCUACUAAGUGUAGAU-3' of LbCas12a, namely the gRNA sequence (SEQ ID NO. 3) is UAAUUUCUACUAAGUGUAGA UGACCAGGCGCUCCAUCCGGC, and the detection effect is better.
The kit can further include a Cas12a protein and a signaling reporter probe having the sequence: 5'-FAM-TTTTT-BHQ1-3'.
The Cas12a protein can be derived from LbCas12a, asCas12a, fnCas12a and the like, but the anchor sequence and other reagent components need to be adjusted according to different source proteins. The primer group, the gRNA are matched with the LbCas12a and a specific probe sequence, so that a good detection effect is achieved.
The invention also relates to a method for detecting the mycobacterium tuberculosis complex based on the CRISPR-Cas12a system, which comprises the following steps:
(1) Extracting sample nucleic acid: taking a sample to be detected, and extracting sample DNA;
(2) Amplification of RPA: amplifying the DNA of the sample to be detected extracted in the step (1) by using a target gene amplification primer through an RPA method to obtain an amplification product;
the primer sequences are as follows:
an upstream primer:
5’-CCAAGCTGCGCCAGGGCAGCTATTTCCCGGAC-3’；
a downstream primer:
5’-TTGGCCATGATCGACACTTGCGACTTGGA-3’；
RPA reaction system: the total volume was 12.5 μ L: the kit comprises 0.125-0.625 mu L (with the concentration of 10 mu M) of RPA upstream amplification primer, 0.125-0.625 mu L (with the concentration of 10 mu M) of RPA downstream amplification primer, 1 × Reaction Buffer,1 × basic-Mix, 1.2-2.4 mM dNTP,0.625 mu L of 20 × Core Reaction Mix, 14-28 mM MgOAc, 1 mu L of genomic DNA of a sample to be detected, and the balance of DEPC water for supplementing 12.5 mu L. And (3) amplification procedure: reacting for 30-60 min at constant temperature of 37 ℃.
(3) And (3) CRISPR reaction detection: adding a fluorescent reporter molecule, cas12a protein, gRNA and a detection reagent into the reaction tube in the step (2), carrying out CRISPR reaction detection, and reading a detection signal;
the gRNA sequence is:
5’- UAAUUUCUACUAAGUGUAGAUGACCAGGCGCUCCAUCCGGC -3’；
the sequence of the fluorescent reporter molecule ssDNA-FQ is as follows: 5'-FAM-TTTTT-BHQ1-3';
CRISPR-Cas12a system reaction: each 20uL of the final line contained 1 XBuffer (50mM NaCl,10mM Tris-HCl,10mM MgCl2, 100. Mu.g/ml BSA, pH 7.9@25 ℃), 36nM gRNA,50nM ssDNA-FQ,50nM Cas12a.
(4) And (4) judging a result: the cumulative fluorescence value obtained by the fluorescence detector was used as the signal intensity, and the analysis was performed according to the following criteria:
negative judgment standard: the fluorescence amount is less than or equal to 1 time of the fluorescence amount of the negative control;
positive judgment standard: the amount of fluorescence was 1-fold greater than that of the negative control.
The CRISPR reaction system comprises the following components: 50mM NaCl,10mM Tris-HCl,10mM MgCl 2 ，100μg/ml BSA，36nM gRNA，50nM ssDNA-FQ，50nM Cas12a。
The detection steps are all completed under a constant temperature condition without complex temperature change, so that the dependence on precise instruments such as a qPCR instrument is eliminated, and the method has a wide application prospect.
Compared with the prior art, the invention has the following beneficial effects:
the invention relates to a fluorescence detection method for target sequence amplification of a mycobacterium tuberculosis complex by using alternative cleavage activity of Cas12a under the guide of gRNA. Aiming at mycobacterium tuberculosis complex IS1081 gene, CRISPR technology IS used for detection, and in a CRISPR-Cas12a system, cas12a protein IS guided by guide RNA to recognize a target sequence and then start the activity of bypass cutting. And (3) adding a fluorescent reporter molecule into the system, and realizing the conversion of the information of the sequence to be detected to a fluorescent signal by using the cleavage activity attached by the Cas12a enzyme. By coupling RPA to Cas12a protein, two-stage amplification of "sequence amplification" (RPA completed) plus "enzymatic cascade" (Cas 12a enzyme completed) can be achieved, exceeding the sensitivity of qPCR, a single-stage amplification. In addition, the RPA amplification mode does not need complex temperature change, so that the dependence on precise instruments such as a qPCR instrument and the like is eliminated, and the CRISPR-Cas technology has wide application prospect in the field diagnosis of tuberculosis.
In addition, by selecting a target sequence, designing and screening an amplification primer pair and a gRNA, a clinically practical primer group with high amplification efficiency, good sensitivity and strong specificity is finally obtained, the detection time of the mycobacterium tuberculosis complex is shortened, the detection can be completed within 120-150 min, the mycobacterium tuberculosis complex of 0.004483amol can be detected at the lowest, and the primer group has no cross reaction with other common human pathogenic bacteria such as nontuberculous mycobacteria, escherichia coli, staphylococcus aureus and the like, so that the practical operability of the CRISPR-Cas technology in tuberculosis field diagnosis is greatly improved.
(IV) description of the drawings
FIG. 1 is a schematic diagram of detection of Mycobacterium tuberculosis complex based on CRISPR-Cas12 a.
Fig. 2 is a schematic diagram of the results of validation of the activity of the purified Cas12a protein.
FIG. 3 is a diagram showing the sensitivity investigation result of CRISPR-Cas12a detection of Mycobacterium tuberculosis complex.
FIG. 4 is a diagram showing the result of CRISPR-Cas12a detection of Mycobacterium tuberculosis complex specificity.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the cas12a protein can be derived from LbCas12a, asCas12a, fnCas12a and the like, but the anchoring sequence and other reagent components need to be adjusted according to different source proteins.
Example 1: CRISPR-Cas12a protein expression, purification and activity verification
1) The plasmid for expressing the LbCas12a protein is purchased from the addge, the BL21 strain is transformed, and the LB plate is cultured overnight at 37 ℃;
2) Selecting a single bacterium on an LB flat plate, inoculating the single bacterium into 5mL of LB culture medium (100 ug/mL Amp), and culturing at 37 ℃ for 3-5 h;
3) Inoculating 5mL of bacterial liquid into 1L of LB liquid culture medium (100 ug/mL Amp), culturing at 37 ℃ until OD60 is approximately equal to 0.6, adding 0.3mmol/L of IPTG, and inducing at 16 ℃ for 16h;
4) Placing the cultured bacterial liquid into a centrifuge, centrifuging for 10-15 min at 5000r/min, discarding supernatant, collecting thalli, suspending the centrifuged thalli precipitate in 25mL lysis buffer (50 mM Tris-HCl, pH 7.5, 500mM NaCl,5% (v/v) glycerol,1mM TCEP, 0.5mM PMSF and 0.25mg/mL lysozyme);
5) Placing in an ice bath, carrying out ultrasonic crushing for 26 times in each cycle, carrying out ultrasonic treatment for 6s at an interval of 5s, carrying out 2-4 cycles according to the crushing effect, centrifuging the crushed thallus at 4 ℃ and 18000r/min for 30min, collecting supernate, and placing at 4 ℃;
6) The Ni column is balanced by a lysis buffer solution, then the sample is loaded, after the combination of 30min, the mixture is stirred once every 5 to 10min, and the mixture is kept stand and drained;
7) The non-specific binding proteins were washed with 2-3 column volumes of lysis buffer, and eluted with 15mL of elution buffer (20 mM Tris-HCl, pH 7.5, 200mM NaCl,5% (v/v) glycerol and 1mM TCEP), which was added in two portions to increase the elution efficiency.
8) Carrying out TEV enzyme digestion on the expressed protein, then carrying out ion exchange and gel filtration chromatography, and finally storing the protein by using an elution buffer solution;
9) Measuring concentration, adding glycerol of the same volume, mixing, and standing at-20 deg.C;
10 Agarose gel electrophoresis verified the "side-cleavage" activity of CRISPR-Cas12 a: in addition to other components of the CRISPR-Cas system, whether the target and non-specific sequences can be cleaved in the presence or absence of Cas12a, as shown in figure 2;
11 CRISPR-Cas12a real-time fluorescence detection further validated: in addition to the other components of the CRISPR-Cas system, the presence or absence of a fluorescent signal in the presence of Cas12a. There is a fluorescent signal indicating that the target sequence and the signal reporter molecule are cleaved.
Example 2: CRISPR-Cas12a detection mycobacterium tuberculosis complex target sequence selection, gRNA and amplification primer screening
1) Target selection: the IS1081 insert was present in all Mycobacterium tuberculosis complex, although it has a lower copy number (5-7 repeats) than IS 6110. In order to avoid missing detection to the maximum extent, the insertion sequence IS1081 IS selected as a target for recombinase polymerase amplification reaction primer design and target of CRISPR-Cas12a targeted detection.
2) gRNA screening: obtaining the sequence information of an insertion sequence IS1081 from NCBI, designing gRNA by using CRISPR-DT software, scoring, selecting gRNA with score not lower than 0.5, and screening suitable gRNA by using IS1081 plasmid. Specifically, the 20uL system contained 1 XBuffer (50 mM NaCl,10mM Tris-HCl,10mM MgCl) 2 100. Mu.g/ml BSA, pH 7.9@25 ℃ C.), 36nM gRNA,50nM ssDNA-FQ,50nM Cas12a,20ng/ul IS1081 plasmid. The ssDNA-FQ sequence is 5'-FAM-TTTTT-BHQ1-3'. FAM fluorescence is collected by a fluorescence collector (such as ABI 7500) at the temperature of 37 ℃ for 20 min-2 h and at intervals of 1-1.25 min. The gRNA sequence for detecting the mycobacterium tuberculosis complex is shown in SEQ ID NO.3, 5'-UAAUUUCUACUAAGUGUAGAUGACCAGGCGCUCCAUCCGGC-3'. The IS1081 plasmid IS formed by inserting an insertion sequence IS1081 into a PUC57 vector, and the insertion sequence IS1081 IS shown as a sequence SEQ ID NO. 1.
3) Amplification of RPA:
RPA primer design: primers were designed using Primer Premier 5.0, according to the TwistAmp assay design Manual. Optimization was performed using twist amp assay (liquid, base).
RPA reaction system: the total volume was 12.5 μ L: the kit comprises 0.24 mu M of RPA upstream amplification primer, 0.24 mu M of RPA downstream amplification primer, 1 × Reaction Buffer,1 × basic E-Mix,1.8mM dNTP,0.625 mu L of 20 × Core Reaction Mix,28mM MgOAc, 1 mu L of genome DNA of a sample to be detected, and the balance of 12.5 mu L of DEPC water. And (3) amplification procedure: reacting for 30-60 min at constant temperature of 37 ℃.
The RPA amplification primers selected were:
an upstream amplification primer: 5'-CCAAGCTGCGCCAGGGCAGCTATTTCCCGGAC-3' downstream amplification primer: 5'-TTGGCCATGATCGACACTTGCGACTTGGA-3'.
4) Detection of CRISPR-Cas12a in combination with RPA amplification: RPA amplification total volume 12.5 μ Ι _: the kit comprises 0.24 mu M of RPA upstream amplification primer, 0.24 mu M of RPA downstream amplification primer, 1 × Reaction Buffer,1 × Basic E-Mix,1.8mM dNTP,0.625 mu L of 20 × Core Reaction Mix,28mM MgOAc, 1 mu L of genomic DNA of a sample to be detected, and the balance of 12.5 mu L of DEPC water. And (3) amplification procedure: the reaction was carried out at a constant temperature of 37 ℃ for 60min. After the reaction is finished, the CRISPR-Cas12a system reaction components are added so that the 20uL final system contains 1 XBuffer (50mM NaCl,10mM Tris-HCl,10mM MgCl) 2 100. Mu.g/ml BSA, pH 7.9@25 ℃ C.), 36nM gRNA,50nM ssDNA-FQ,50nM Cas12a. Reacting at the constant temperature of 37 ℃ for 120min, and collecting FAM fluorescence once every 1.25-1.5 min by using a fluorescence collection system of a real-time fluorescence PCR instrument. The principle is shown in fig. 1.
5) Analysis of results
In the detection process, because the signal reporting probe with two ends respectively connected with the fluorescent group and the quenching group is added into the reaction system, after the Cas12a protein recognizes the target DNA with the target sequence with the help of the gRNA, the activated Cas12a enzyme can degrade the signal reporting probe with the signal, thereby releasing the fluorescent signal and realizing the detection.
The cumulative fluorescence value obtained by the fluorescence detector was used as the signal intensity, and the analysis was performed according to the following criteria:
negative judgment standard: the amount of fluorescence is less than or equal to 1 time the amount of fluorescence of the negative control.
Positive judgment standard: the amount of fluorescence was 1-fold greater than that of the negative control.
Wherein the negative control group is a negative signal group which is correspondingly arranged for each experimental group and takes the non-tubercle mycobacterium nucleic acid DNA as a template.
Example 3: examination of sensitivity and specificity of reaction system
1) Reaction system sensitivity investigation: IS1081 plasmid was used as template and serially diluted in gradient (448.3amol, 44.83amol,4.483amol,0.4483amol,0.08966amol, 0.04483 amol), non-tuberculous mycobacterial DNA was used as negative template, H was used as negative template 2 O was used as a blank control, each was repeated 3 times, and the system sensitivity was examined. The results are shown in FIG. 3. The lowest detection limit is 0.04483amol, the linear regression equation is Y =136613+84616x, R 2 ＝0.973。
2) Reaction system specificity investigation: selecting pseudomonas aeruginosa, escherichia coli, mycoplasma pneumoniae, streptococcus pneumoniae, klebsiella pneumoniae, haemophilus influenzae, staphylococcus aureus, mycobacterium intracellulare, mycobacterium avium, mycobacterium kansasii, mycobacterium scrofulae, mycobacterium gordonae, huang Fenzhi, mycobacterium chelonii, mycobacterium thuringiensis, mycobacterium abscessus, mycobacterium fortuitum, mycobacterium smegmatis and other strain genome DNAs (deoxyribonucleic acids) for system specificity inspection, simultaneously taking a mycobacterium tuberculosis H37Rv genome as a positive control, and taking a mycobacterium tuberculosis H37Rv genome as a negative control 2 O as a negative control. The results are shown in FIG. 4. M. tuberculosis H37Rv is distinguishable from other non-M.tuberculosis complex groups.
Example 4: clinical sputum specimen genome DNA extraction and detection
193 parts of clinical sputum specimen is selected, wherein sputum culture is positive 140 parts of mycobacterium tuberculosis complex and negative 53 parts of mycobacterium tuberculosis complex. The samples are extracted by QIAamp DNA Mini Kit (Qiagen) genome, amplified by RPA, detected by CRISPR-Cas12a fluorescence detection system and finally analyzed by results. The final test results were as follows: the detection result shows that the mycobacterium tuberculosis complex has 139 positive parts and 54 negative parts, and the positive coincidence rate is 99.3 percent compared with the sputum culture result. The results are shown in Table 1.
Table 1: summary table of results of CRISPR-Cas12a detection of sputum culture samples
Sequence listing
<110> Fujian medical university Meng Chao Hospital for liver and gallbladder (infectious disease Hospital, fuzhou city)
<120> Mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213> Unknown (Unknown)
<400> 1
ccaagctgcg ccagggcagc tatttcccgg ac 32
<210> 2
<211> 29
<212> DNA
<213> Unknown (Unknown)
<400> 2
ttggccatga tcgacacttg cgacttgga 29
<210> 3
<211> 41
<212> RNA
<213> Unknown (Unknown)
<400> 3
uaauuucuac uaaguguaga ugaccaggcg cuccauccgg c 41
<210> 4
<211> 1430
<212> RNA
<213> Unknown (Unknown)
<400> 4
gaacgacgcc gagccgacaa gacagccagc gcaacccgcc acgcgggcag gggggcgagg 60
caacccagca ccgccaggac gggcacggag acacggcgac gacggaggga ggagccggcg 120
cccggcagcc gggaaggcgc caacaccggg gaccgccagc gagcaccaca ccggggcacc 180
cgcacgccgg gcgggagggc aagacgccag gggggggacg ggcaggaagg ccgggggcgg 240
cgagcccaga ggggcggcac gcggacccga aacgaccagc aaaagcaacg aaggaaagac 300
gcaagacccc cacacgacac cgagcagccg gcgaccaacc gcacaggcga gcccggacgc 360
gcgcgggcgc ccgacgcacg ccgccgaggg ggcgaagccg acgcccggcg gggcgggcac 420
cgcgaacgca gcgagagcgg ccaacagcgc aacggcaccg ccaccggacg acacccggcc 480
gcaaccacga cgcgcgaccc caagcgcgcc agggcagcac ccggacggcg cgcagcgccg 540
caagcgagcg aacgcgcacg accagcgggg gcgaccgcac cgcgggagac caccgccgga 600
ggagcgccgg cgaaacacgg ggacaaagcc caagcgcaag gcgacaggcc aaagagccga 660
cgaagccgag aggcgcggac ccgcccgccg agccggcccg aacccccgcc gccgacgccc 720
gggccaaggg cgcgaggcag gccgcgcgcg gaggcacacc gacgccaccg gcgcaacgcc 780
gagggcaccg agagaccggg caccaggcac cccgccgagg acggggccgg cggcggcgcc 840
cgcgaccggc gcccgcggcc gccggggcgc gcggcaccag cgacgcccac gccggccggg 900
gccgcgacgg cgccacccgc ccgcagcggc cggcagcgcg cagaacccac acgcagccaa 960
cgaggcagcc accccgaagc ccccggccgg gggcgcaccc gcgcacccac acgaccagcc 1020
cgacgccgaa cagggcccaa agacgggacc gacgccgacc gacaaacccc cgcggggccg 1080
agcacccgac accgcccgca ccgaccgcgg cgcaccgccc cccaagcaga cggcgccaaa 1140
cggccaacaa cccccaggaa cgcccaaccg agaggacgac gccgaaccga cgcggggcac 1200
ccccgaccgc gcccgacacc gcccgcggag ccgcccgccg aacaacacga cgaaggacga 1260
aggacggcgc accgggcccg aggcccaccc gagcccgagc agcacgacca gcaccgaaga 1320
acccgccaag cagcaaacca ccaacacccc agcacgacca ccagacgcca cccgaaggac 1380
acgcgaggaa cccaccgaca ccacgcccgg ccggccgggc aggcccagcg 1430
Claims (3)
1. A detection kit for Mycobacterium tuberculosis complex based on CRISPR-Cas12a system mainly comprises a primer for specifically amplifying an IS1081 gene of the Mycobacterium tuberculosis complex, gRNA, a fluorescent reporter molecule ssDNA-FQ and a Cas12a protein;
the primer sequences are as follows:
an upstream primer:
5’- CCAAGCTGCGCCAGGGCAGCTATTTCCCGGAC-3’；
a downstream primer:
5’- TTGGCCATGATCGACACTTGCGACTTGGA-3’；
the gRNA sequence is:
5’- UAAUUUCUACUAAGUGUAGAUGACCAGGCGCUCCAUCCGGC -3’；
the sequence of the fluorescent reporter molecule ssDNA-FQ is as follows:
5’-FAM-TTTTT-BHQ1-3’。
2. a non-diagnostic purpose CRISPR-Cas12a system based mycobacterium tuberculosis complex detection method, comprising:
(1) Extracting nucleic acid of a sample: taking a sample to be detected, and extracting sample DNA;
(2) Amplification of RPA: amplifying the DNA of the sample to be detected extracted in the step (1) by using a target gene amplification primer through an RPA method to obtain an amplification product;
the primer sequences are as follows:
an upstream primer:
5’- CCAAGCTGCGCCAGGGCAGCTATTTCCCGGAC-3’；
a downstream primer:
5’- TTGGCCATGATCGACACTTGCGACTTGGA-3’；
(3) And (3) CRISPR reaction detection: adding a fluorescent reporter molecule, cas12a protein, gRNA and a detection reagent into the reaction tube in the step (2), carrying out CRISPR reaction detection, and reading a detection signal;
the gRNA sequence is:
5’- UAAUUUCUACUAAGUGUAGAUGACCAGGCGCUCCAUCCGGC -3’；
the sequence of the fluorescent reporter molecule ssDNA-FQ is as follows: 5'-FAM-TTTTT-BHQ1-3';
(4) And (4) judging a result: the cumulative fluorescence value obtained by the fluorescence detector was used as the signal intensity, and the analysis was performed according to the following criteria:
negative judgment standard: the fluorescence amount is less than or equal to 1 time of the fluorescence amount of the negative control;
positive judgment standard: the fluorescence amount is1 times larger than that of the negative control.
3. The method of claim 2, characterized in that the CRISPR reaction system consists of: 50mM NaCl,10mM Tris-HCl,10mM MgCl 2 100 μ g/ml BSA,36nM gRNA,50nM ssDNA-FQ and 50nM Cas12a.
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| CN112941153B (en) * | 2019-12-11 | 2022-09-09 | 清华大学 | DNA detection method |
| WO2021155775A1 (en) * | 2020-02-03 | 2021-08-12 | 苏州克睿基因生物科技有限公司 | Method and kit for dectecting target nucleic acid |
| BR112022015365A2 (en) * | 2020-02-06 | 2022-09-20 | The Administrators Of The Tulane Educational Fund | CRISPR-BASED ASSAY FOR TB DETECTION IN BODY FLUIDS |
| CN111394490B (en) * | 2020-05-15 | 2020-12-29 | 中国人民解放军军事科学院军事医学研究院 | CRISPR-Cas12a detection primer group for eupolyphaga and application thereof |
| CN111808931A (en) * | 2020-07-15 | 2020-10-23 | 南方科技大学 | One-step RPA-CRISPR nucleic acid detection method, kit and application |
| CN112522429B (en) * | 2020-12-16 | 2021-07-16 | 中国人民解放军军事科学院军事医学研究院 | Method and reagent set for detecting bacillus anthracis by RPA (reverse transcriptase polymerase chain reaction) combined CRISPR (clustered regularly interspaced short palindromic repeats) technology |
| CN112941051A (en) * | 2021-04-14 | 2021-06-11 | 浙江优诺生物科技有限公司 | FENM protein mutant and application thereof and kit containing mutant |
| CN113373223A (en) * | 2021-06-01 | 2021-09-10 | 武汉大学 | CRISPR-Cas system for FLT3-D835Y mutation detection and application thereof |
| 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 |
| CN114107300B (en) * | 2021-11-30 | 2023-10-27 | 宁夏医科大学 | CRRNA for detecting rifampicin resistance point mutation of mycobacterium tuberculosis based on CRISPR-Cas technology |
| CN114085918B (en) * | 2021-12-16 | 2023-10-24 | 重庆医科大学 | RPA-CRISPR/Cas12a-FQ system and application thereof in environmental water body biological detection |
| CN114231530B (en) * | 2021-12-20 | 2024-03-15 | 大连理工大学 | Cas12a- C CrRNA system and application thereof |
| 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 |
| CN114752656B (en) * | 2022-04-18 | 2022-12-23 | 暨南大学 | Method for rapidly detecting vibrio parahaemolyticus based on chemically enhanced CE-RAA-CRISPR |
| CN116064868B (en) * | 2022-09-30 | 2023-12-29 | 青岛农业大学 | Method for detecting aquatic pathogenic vibrio splendidus |
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