阅读说明：本技术 基于CRISPR-Cas12a系统的结核分枝杆菌复合群检测试剂盒 (mycobacterium tuberculosis complex detection kit based on CRISPR-Cas12a system ) 是由 刘景丰 刘小龙 许海坡 蔡志雄 董秀清 于 2019-08-09 设计创作，主要内容包括：本发明属于核酸检测技术领域,涉及一种基于CRISPR-Cas12a系统的结核分枝杆菌复合群的检测试剂盒及方法。该试剂盒通过采用重组酶聚合酶扩增技术提高检测灵敏度,利用CRISPR-Cas12a特异靶向结核分枝杆菌复合群靶序列后激活Cas12a的旁路切割活性,可从痰中灵敏、特异地检出结核杆菌分枝复合群。本发明具有无侵入性、可频繁多次检测、检测速度快等优势。相比于目前的PCR检测方法,本发明不需要有升降温功能的热循环仪,通过荧光读数即可检测痰液中的结核分枝杆菌复合群,具有成本低、操作简便、灵敏度高、特异性好等优点,适用于临床的大规模应用。(the invention belongs to the technical field of nucleic acid detection, and relates to a detection kit and a 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 Cas12a after utilizing a CRISPR-Cas12a specific target mycobacterium tuberculosis complex target sequence, and can sensitively and specifically detect the mycobacterium tuberculosis complex from 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.)

1. A detection kit for Mycobacterium tuberculosis complex based on CRISPR-Cas12a system mainly comprises a primer for specifically amplifying the IS1081 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’。

2. the kit of claim 1, wherein the kit further comprises a Cas12a protein and a signaling reporter probe having the sequence: 5 '-FAM-TTTTT-BHQ 1-3'.

3. A method for detecting mycobacterium tuberculosis complex based on CRISPR-Cas12a system, the 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-BHQ 1-3';

(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 amount of fluorescence was 1-fold greater than that of the negative control.

4. the method of claim 3, characterized in that the CRISPR reaction system consists of: 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100. mu.g/ml BSA, 36nMgRNA, 50 nMSDNA-FQ, 50nM Cas12 a.

(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 causative bacteria of Tuberculosis in humans are the Mycobacterium Tuberculosis Complex (MTBC), which mainly includes Mycobacterium Tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti, and the like. 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. The isolation culture method is the current gold standard for diagnosing tuberculosis, but the culture requires 4-8 weeks, and clinical diagnosis and treatment are delayed. The smear staining microscopy method is simple and rapid to operate, but the method has low sensitivity and poor specificity. The immunological diagnosis is caused by the fact that the existing antigen or antibody is crossed with other microorganisms, so that the specificity is poor, and the false positive rate is high. 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 reaction only by a constant-temperature water bath kettle, 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. The CRISPR-Cas system is divided into two major types, and the CRISPR system of the first major type can only play a role in an effect complex consisting of a plurality of Cas proteins; the second broad class of Cas relies on a single Cas effector protein (e.g., Cas9, Cpf1, C2C1, C2C2, etc.) to function.

in 2015, zhanfeng et al discovered CRISPR-associated protein endonuclease Cas12a (formerly Cpf1), which is an RNA-guided specific DNA endonuclease like the commonly used Cas9 protein, but has its own features compared to Cas9 and Cas12a, such as only crRNA is needed to guide specific cleavage of double-stranded DNA, and generate cohesive ends, etc. Cas12a, once it recognizes and cleaves the target DNA designated by the crRNA sequence, is transferred into 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 detect based on the combination of RNA-guided and DNA-targeted alternative cleavage effect of endonuclease 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 (including gRNA, crRNA, Cas12a protein and ssDNA probe), once a Cas12a and gRNA complex recognizes and cuts the target sequence, the cleavage activity of a non-specific single strand can be triggered, a single-strand reporter molecule in the detection system is cut, fluorescence is emitted, and therefore 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 specifically target the target sequence of the mycobacterium tuberculosis complex and activate the bypass cutting activity of Cas12a, the mycobacterium tuberculosis complex can be detected from sputum and other samples 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 TTCATCGTCGTGGCAGGTGTTGGG CTGATTTTGGTCAACCCAGCACC TGCCAGGACGGGCTACGGATGTACACGGCGACGACGGTATGGGAG GATGTC CGGTCTTGCTCCGGTCATGTCCGGTGAATGTGCTGCCAACA TCCTGGGGACCGTCCAGCGAGTTTCACCACACCT TGGGGCACCTTC TGTCACTGCTCGGTGCTGTGGATTGGTGTCAAGTTACGTCCAGGGG TGTGGTGTACGGGCAG GTAAGGCCGGTGGGCGTGTCGTAGCCCAGT AGTGGGCGGTCATCGCGTGATCCTTCGAAACGACCAGCAAAAGTCA ATCGAAGGAAATGACGCAATGACCTCTTCTCATCTTATCGACACCGA GCAGCTTCTGGCTGACCAACTCGCACAG GCGAGCCCGGATCTGCTG CGCGGGCTGCTCTCGACGTTCATCGCCGCCTTGATGGGGGCTGAAG CCGACGCCCT GTGCGGGGCGGGCTACCGCGAACGCAGCGATGAGC GGTCCAATCAGCGCAACGGCTACCGCCACCGTGATTTCGA CACCCG TGCCGCAACCATCGACGTCGCGATCCCCAAGCTGCGCCAGGGCAGC TATTTCCCGGACTGGCTGCTGC AGCGCCGCAAGCGAGCTGAACGCG CACTGACCAGCGTGGTGGCGACCTGCTACCTGCTGGGAGTATCCAC TCGC CGGATGGAGCGCCTGGTCGAAACACTTGGTGTGACAAAGCTT TCCAAGTCGCAAGTGTCGATCATGGCCAAAGAG CTCGACGAAGCCG TAGAGGCGTTTCGGACCCGCCCGCTCGATGCCGGCCCGTATACCTTC CTCGCCGCCGACGC CCTGGTGCTCAAGGTGCGCGAGGCAGGCCGC GTCGTCGGAGTGCACACCTTGATCGCCACCGGCGTCAACGCCGA GG GCTACCGAGAGATCCTGGGCATCCAGGTCACCTCCGCCGAGGACGG GGCCGGCTGGCTGGCGTTCTTCCGCG ACCTGGTCGCCCGCGGCCTG TCCGGGGTCGCGCTGGTCACCAGCGACGCCCACGCCGGCCTGGTG GCCGCGATC GGCGCCACCCTGCCCGCAGCGGCCTGGCAGCGCTGC AGAACCCACTACGCAGCCAATCTGATGGCAGCCACCCCG AAGCCCT CCTGGCCGTGGGTGCGCACCCTGCTGCACTCCATCTACGACCAGCC CGACGCCGAATCAGTTGTTGC CCAATATGATCGGGTACTCGACGCTC TGACCGACAAACTCCCCGCGGTGGCCGAGCACCTCGACACCGCCC GCA CCGACCTGCTGGCGTTCACCGCCTTCCCCAAGCAGATCTGGCG CCAAATCTGGTCCAACAACCCCCAGGAACGCC TCAACCGAGAGGT ACGACGCCGAACCGACGTCGTGGGCATCTTCCCCGACCGCGCCTCG ATCATCCGCCTCGTC GGAGCCGTCCTCGCCGAACAACACGACGAAT GGATCGAAGGACGGCGCTACCTGGGCCTCGAGGTCCTCACCCGA GC CCGAGCAGCACTGACCAGCACCGAAGAACCCGCCAAGCAGCAAAC CACCAACACCCCAGCACTGACCACCTA GACTGCCACCCGAAGGATC 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 are provided.

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-BHQ 1-3'.

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 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 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’；

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 multiplied Reaction Buffer, 1 multiplied basic-Mix, 1.2-2.4 mM dNTP, 0.625 mu L of 20 multiplied 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: and 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-BHQ 1-3';

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 [email protected] ℃), 36nM gRNA, 50nM ssDNA-FQ, 50nM Cas12 a.

(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 MgCl2, 100. mu.g/ml BSA, 36nM gRNA, 50nM ssDNA-FQ, 50nM Cas12 a.

The detection steps are all completed under a constant temperature condition, and complex temperature change is not needed, so that the dependence on precision 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 the mycobacterium tuberculosis complex IS1081 gene, and detecting by using CRISPR technology, in a CRISPR-Cas12a system, Cas12a protein IS guided by guide RNA, and after recognizing a target sequence, the activity of 'bypass cutting' IS started. 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" (Cas12a enzyme completed) can be achieved, exceeding the sensitivity of qPCR for such 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.

in addition, by selecting a target sequence and 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, 0.004483amol of the Mycobacterium tuberculosis complex can be detected at the minimum, no cross reaction exists between the Mycobacterium tuberculosis complex and other common human pathogenic bacteria such as nontuberculous mycobacteria, escherichia coli, staphylococcus aureus and the like, and 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 CRISPR-Cas12 a-based detection of mycobacterium tuberculosis complex.

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 for detecting Mycobacterium tuberculosis complex.

FIG. 4 is a diagram showing the detection result of specificity of Mycobacterium tuberculosis complex by CRISPR-Cas12 a.

(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.