阅读说明：本技术 一种基于试纸条的Cas12a酶的微生物快速诊断技术 (Test strip-based microorganism rapid diagnosis technology for Cas12a enzyme ) 是由 万逸 刘红 葛晓林 于 2019-10-23 设计创作，主要内容包括：将Cas12a检测系统与胶体金快速诊断试纸条技术结合,首先通过PCR或恒温扩增技术实现目的片段的扩增,然后进行Cas12a检测反应,最后试纸条判读结果。此结合意义在于不需要特异性标记引物,避免扩增产物稀释和探针杂交等流程,试纸条可以实现多种微生物的检测,扩大了试纸条的适用性。将Cas12a检测系统的广泛性、稳定性、特异性、灵敏性与试纸条检测的直观、快速、方便性结合,为现场检测提供最佳检测法,利于及时诊断,在核酸分子诊断领域具有重要的应用潜力。(The Cas12a detection system is combined with the colloidal gold rapid diagnosis test strip technology, amplification of a target fragment is realized through a PCR (polymerase chain reaction) or constant-temperature amplification technology, then Cas12a detection reaction is carried out, and finally the test strip judges the result. The combination significance lies in that no specific labeled primer is needed, the procedures of dilution of amplification products, probe hybridization and the like are avoided, the test strip can realize the detection of various microorganisms, and the applicability of the test strip is enlarged. The universality, stability, specificity and sensitivity of the Cas12a detection system are combined with the intuition, rapidness and convenience of test strip detection, so that an optimal detection method is provided for field detection, timely diagnosis is facilitated, and the method has important application potential in the field of nucleic acid molecule diagnosis.)

1. A test strip-based microorganism rapid diagnosis technology of Cas12a enzyme is characterized in that: the method comprises the following steps:

the method comprises the following steps: using microbial nucleic acid as a template, and amplifying a target fragment with a proper length by using a specific primer;

step two: adding the amplification product obtained in the step one into a Cas12a detection system, and performing a cleavage reaction under the mediation of specific crRNA;

step three: and D, directly detecting the cutting product obtained in the step two by using a colloidal gold immune test strip.

2. According to the microorganism rapid diagnosis technology of the Cas12a enzyme based on the test strip, the method is characterized in that: the first step is as follows: according to the specific gene of a specific microorganism, a specific conserved region is determined, a specific primer is designed, the nucleic acid of the microorganism is extracted, and the designed specific primer is utilized to amplify a target fragment.

3. According to the microorganism rapid diagnosis technology of the Cas12a enzyme based on the test strip, the method is characterized in that: the third step is as follows: and after the second step is finished, directly incubating the cutting product and the test strip, and judging the result after 2-5 min.

4. The test strip-based rapid microbial diagnosis technology for Cas12a enzyme of claim 1, wherein the specific crRNA comprises the crRNA sequence SEQ ID NO 1-7 for Listeria, Klebsiella pneumoniae, Streptococcus, Staphylococcus aureus, Escherichia coli, Shigella and Salmonella.

5. The test strip-based rapid microbial diagnosis technology of Cas12a enzyme according to claim 2, wherein the specific genes of specific microorganisms include: listeria genusiapKlebsiella pneumoniaekheGenus StreptococcustufStaphylococcus aureusnucEscherichia colichuAShigellaipaHSalmonellainvASpecific gene classes include: pathogenic genes, resistance genes and housekeeping gene variable regions of the microorganism.

6. The test strip-based rapid microbial diagnosis technology of Cas12a enzyme of claim 2, wherein the specific primers comprise primer pairs of SEQ ID NO 8-21 for Listeria, Klebsiella pneumoniae, Streptococcus, Staphylococcus aureus, Escherichia coli, Shigella and Salmonella.

7. The test strip-based rapid microbial diagnosis technology of Cas12a enzyme of claim 2, wherein the amplification technology of the target fragment comprises: polymerase Chain Reaction (PCR), nucleic acid dependent amplification detection technology (NASBA), loop-mediated isothermal amplification technology (LAMP), helicase dependent isothermal amplification of DNA (HDA), Recombinase Polymerase Amplification (RPA).

8. The test strip-based rapid diagnostic technique for microorganisms of Cas12a enzyme according to claim 3, wherein the Cas12a enzyme comprises: amino acid coccus BV3L6Cas12a (Acidaminococcus sp. BV3L6Cas12a, AsCas12 a), streptomyces ND2006 Cas12a (lachrispiraceae bacterium ND2006 Cas12a, LbCas12 a), francisella novida U112 Cas12a (francisella subsp. novicida U112 Cas12a, FnCas12 a), klebsiella Cas12a (prevotelladisiensis Cas12a, PdCas12 a), Moraxella bovis Cas12a (Moraxella novoculi 237Cas12a, MbCas12 a), macadimia porphyria Cas12a (porphyromonacaca Cas12a, PmCas12 a), and the like.

9. The test strip-based rapid microbial diagnosis technology for Cas12a enzyme according to claim 3, wherein the Reporter sequence is SEQ ID NO. 22, and is characterized by a compound modified short nucleotide sequence with a nucleotide length of 6nt-20nt, wherein the 5 'end of the short nucleotide sequence is labeled with Fluorescein Isothiocyanate (FITC) or carboxyfluorescein (FAM), and the 3' end of the short nucleotide sequence is labeled with Biotin (Biotin).

10. According to the microorganism rapid diagnosis technology of the Cas12a enzyme based on the test strip, the method is characterized in that: the diagnosis technology provides an optimal detection method for field detection, can realize amplification of a target fragment through PCR or isothermal amplification, combines the universality, stability and specificity of a Cas12a detection system with the simplicity, convenience and rapidness of test strip detection, has the characteristics of simplicity, rapidness, specificity, sensitivity, stability and the like, and is favorable for timely diagnosis.

Technical Field

The invention designs a simple, convenient, rapid and specific microbial diagnosis technology, and belongs to the technical field of microbial detection

Background

Research and development of microbial detection technology are always one of the key and hot directions in the detection field. The traditional microorganism detection method has complicated detection steps and long detection period, can not completely meet the detection requirements of agricultural products and import and export inspection and quarantine, but is still widely applied to the detection of microorganisms and is used as an official detection standard for being applied to enterprises and related detection departments for a long time. However, with the rapid development of industrialization and the advancement of modern logistics technology, the demand for rapid detection of microorganisms is increasing.

In recent years, with the rapid development of molecular biology, new technologies and new methods for rapidly, accurately and specifically detecting microorganisms are emerging, the microbial detection technology advances from a culture level to a molecular level and develops towards instrumentation, automation and standardization, and the high efficiency, accuracy and reliability of microbial detection work are improved.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins (Cas) thereof form a CRISPR-Cas system, and a CRISPR/Cas technology formed by modifying the CRISPR/Cas system becomes a powerful gene editing tool and is widely applied to gene function research and gene modification and treatment. In addition to being used as a gene editing tool, class II Cas proteins have the characteristic of 'dependent cleavage', have been developed into a rapid, low-cost and highly sensitive nucleic acid detection tool, and have important application potential in the field of nucleic acid molecular diagnosis.

In 2015, Zhang Feng et al found a class II type V CRISPR effector protein Cas12 a. Cas12a can bind to target double-stranded DNA and complete cleavage under crRNA guidance, simplifying experimental design compared to Cas9 systems. Cas12a has the activity of non-specifically cleaving single stranded DNA after it specifically binds to and cleaves the target dsDNA. The nucleic acid amplification technology is combined with Cas12a, a non-specific cleavage substrate is labeled, an amplification product activates the auxiliary cleavage activity of Casl2a, and finally fluorescence is detected.

Both the target and the substrate in Cas12a detection are DNA, and the stability is strong, so the system has low requirements on experimental operating environment and can be applied to on-site rapid detection. The Cas12a detection system does not need to rely on expensive equipment and medical staff experience, brings great convenience to developing countries lacking advanced equipment and trained personnel, and has wide application potential in the aspects of single nucleotide polymorphism analysis, cancer screening, bacterial and viral infection detection, drug resistance screening and the like.

The test paper strip technology for fast diagnosis of colloidal gold is a new in vitro diagnosis technology developed in the 90 s of 20 th century, and is a technology for marking known antigen or antibody with colloidal gold and making the colloidal gold gather to form a macroscopic form through antigen-antibody specific binding reaction, so as to achieve the purpose of fast, simple, fast and accurate detection of target object to be detected. The technology is established on the basis of a monoclonal antibody preparation technology and a colloidal gold labeling technology, and integrates several technologies such as an immunoaffinity technology, a imprinting technology, a chromatography technology and the like. In recent years, the method is developed rapidly, and the technology has the advantages of convenience, rapidness, specificity, sensitivity, strong stability, no need of special equipment and reagents, intuitive result judgment and the like, and is widely applied to the field of biomedicine, particularly medical inspection.

However, because the colloidal gold test strip is a novel immunolabeling technology based on the application of colloidal gold as a tracer marker to antigen and antibody, a certain colloidal gold test strip can only detect a certain corresponding antigen or a certain class of antigen due to a specific antibody carried by the colloidal gold test strip, a detection object is single and fixed, and one test strip can only detect 1 or 1 class of substances. In the aspect of microorganism detection, specific antigens or antibodies of certain microorganisms have the problems of poor representativeness, unstable expression, difficulty in preparation and the like, so that the detection purpose cannot be achieved by using a colloidal gold immune test strip, and the applicability of test strip detection is limited to a great extent.

The Cas12a detection system is combined with a colloidal gold rapid diagnosis test strip technology, a target fragment with a proper length is amplified by a specific primer, then Cas12a detection reaction is carried out, and finally the test strip visually judges the result. The combination significance lies in that no specific marker primer is needed, the processes of dilution of amplification products, probe hybridization and the like are avoided, no restriction of strict correspondence of antigen and antibody exists, and the detection of various microorganisms can be realized by 1 test strip, so that the applicability and the application range of the test strip are enlarged, the advantages of intuition, rapidness, convenience, maturity, low price and the like of the immune colloidal gold test strip are maintained, and the test strip has the characteristics of high sensitivity and high specificity of the Cas12a detection method. The combination of the two technologies provides an optimal detection method for on-site detection, has the characteristics of simplicity, convenience, rapidness, specificity, sensitivity, stability and the like, and is favorable for timely diagnosis. Therefore, the detection method has wide application prospect.

Disclosure of Invention

A test strip-based microorganism rapid diagnosis technology of Cas12a enzyme is characterized in that: the method comprises the following steps:

the method comprises the following steps: using microbial nucleic acid as a template, and amplifying a target fragment with a proper length by using a specific primer;

step two: adding the amplification product obtained in the step one into a Cas12a detection system, and performing a cleavage reaction under the mediation of specific crRNA;

step three: and D, directly detecting the cutting product obtained in the step two by using a colloidal gold immune test strip.

According to the microorganism rapid diagnosis technology of the Cas12a enzyme based on the test strip, the method is characterized in that: the first step is as follows: according to the specific gene of a specific microorganism, a specific conserved region is determined, a specific primer is designed, the nucleic acid of the microorganism is extracted, and the designed specific primer is utilized to amplify a target fragment.

According to the microorganism rapid diagnosis technology of the Cas12a enzyme based on the test strip, the method is characterized in that: the second step is as follows: and (3) adding the Target fragment amplified in the step one into a Cas12a cleavage reaction, and performing cleavage for 10min-20min, wherein a Cas12a cleavage system comprises a Cas12a protein, specific crRNA, a Target and a Reporter.

According to the microorganism rapid diagnosis technology of the Cas12a enzyme based on the test strip, the method is characterized in that: the third step is as follows: and after the second step is finished, directly incubating the cutting product and the test strip, and judging the result after 2-5 min.

The specific crRNA comprises a specific crRNA sequence SEQ ID NO of 1-7 aiming at Listeria, Klebsiella pneumoniae, Streptococcus, Staphylococcus aureus, Escherichia coli, Shigella and Salmonella.

The nucleotide sequence shown in SEQ ID NO. 1 is:

5’- TAATTTCTACTGTTGTAGATTGTTCAAGACATTATGTCATGGA -3’；

the nucleotide sequence shown in SEQ ID NO. 2 is:

5’- TAATTTCTACTGTTGTAGATTCAATAACACCGAGCAGGAAGTT -3’；

the nucleotide sequence shown in SEQ ID NO. 3 is:

5’- TAATTTCTACTGTTGTAGATAGAATACGATTTCCCAGGTGATG -3’；

the nucleotide sequence shown in SEQ ID NO. 4 is:

5’- TAATTTCTACTGTTGTAGATAAGTCTAAGTAGCTCAGCAAATG -3’；

the nucleotide sequence shown in SEQ ID NO. 5 is:

5’- TAATTTCTACTGTTGTAGATGTGTCTTTGGTACTGGCGGCACG -3’；

the nucleotide sequence shown in SEQ ID NO. 6 is:

5’- TAATTTCTACTGTTGTAGATCGGGAACCATGCTGTCACGGCAT -3’；

the nucleotide sequence shown in SEQ ID NO. 7 is:

5’- TAATTTCTACTGTTGTAGATGTCTGGCATTATCGATCAGTACC -3’；

a microbial rapid diagnosis technology of Cas12a enzyme based on test paper strips detects a listeria crRNA sequence shown as SEQID NO. 1.

The test paper strip-based microorganism rapid diagnosis technology of Cas12a enzyme is used for detecting the crRNA sequence of Klebsiella pneumoniae as shown in SEQ ID NO. 2.

A microbial rapid diagnosis technology of Cas12a enzyme based on test paper strips detects a crRNA sequence of streptococcus as shown in SEQ ID NO. 3.

The microbial rapid diagnosis technology for detecting the Cas12a enzyme based on the test strip is shown as SEQ ID NO. 4.

The microbial rapid diagnosis technology for detecting the Escherichia coli based on the Cas12a enzyme of the test strip is shown as SEQ ID NO. 5.

The microbial rapid diagnosis technology for detecting the Shigella crRNA based on the Cas12a enzyme of the test strip is shown as SEQ ID NO. 6.

The microbial rapid diagnosis technology of Cas12a enzyme based on test paper strip detects the sequence of the salmonella crRNA as shown in SEQ ID NO. 7.

Genes specific for a particular microorganism include: listeria genusiapKlebsiella pneumoniaekheGenus StreptococcustufStaphylococcus aureusnucEscherichia colichuAShigellaipaHSalmonellainvA。

Specific gene classes include: pathogenic genes, resistance genes and housekeeping gene variable regions of the microorganism.

The specific primers comprise a primer pair SEQ ID NO of 8-21 aiming at Listeria, Klebsiella pneumoniae, Streptococcus, Staphylococcus aureus, Escherichia coli, Shigella and Salmonella.

The nucleotide sequence shown in SEQ ID NO. 8 is:

5’-GCTTTATCCGTTAAATACGGCGTTTCTGT-3’；

the nucleotide sequence shown in SEQ ID NO. 9 is:

5’- GCAGCTTGAGCTGGTGCTTTAGTTG -3’；

the nucleotide sequence shown in SEQ ID NO. 10 is:

5’- GCGAGGTTTACGTCTCAACCGGCTGGGGATC -3’；

the nucleotide sequence shown in SEQ ID NO. 11 is:

5’- GGGATTGAGCGGGTAATAAATGCGGTTGT -3’；

the nucleotide sequence shown as SEQ ID NO. 12 is

5’- TTGCTTGAATTGGTTGAAATGGAAATCCGTGAC -3’；

The nucleotide sequence shown in SEQ ID NO. 13 is:

5’- TACGGAACATTTCAACACCAGTAACAACAGC -3’；

the nucleotide sequence shown in SEQ ID NO. 14 is:

5’- ATTAAGTGCTGGCATATGTATGGCAATTGTTT -3’；

the nucleotide sequence shown in SEQ ID NO. 15 is:

5’- ATTAATTTAACCGTATCACCATCAATCGCTTT -3’；

the nucleotide sequence shown as SEQ ID NO. 16 is:

5’- GCGGCGTGCTGGTTCTTGTCGATGGTGTT -3’；

the nucleotide sequence shown in SEQ ID NO. 17 is:

5’- TGGGCTGAATCAATTTGCCAGGTCCCTT -3’；

the nucleotide sequence shown as SEQ ID NO. 18 is:

5’- TATCGGAAAGGCGGTCAAGGAACGCGGAAAAG -3’；

the nucleotide sequence shown in SEQ ID NO. 19 is:

5’- CAGATTTACTTCTCCATGAGTGACGGACAACAG -3’；

the nucleotide sequence shown in SEQ ID NO. 20 is:

5’- GATTTCCTGATCGCACTGAATATCGTACTGGC -3’；

the nucleotide sequence shown in SEQ ID NO. 21 is:

5’- AATACTCATCTGTTTACCGGGCATACCATCC -3’；

the microbial rapid diagnosis technology of the Cas12a enzyme based on the test strip is characterized in that an upstream primer for detecting the genus Listeria is shown as SEQ ID NO. 8, and a downstream primer is shown as SEQ ID NO. 9.

The test strip-based microorganism rapid diagnosis technology of Cas12a enzyme is characterized in that an upstream primer for detecting Klebsiella pneumoniae is shown as SEQ ID NO. 10, and a downstream primer is shown as SEQ ID NO. 11.

A test strip-based microorganism rapid diagnosis technology of Cas12a enzyme is used for detecting an upstream primer of streptococcus as shown in SEQ ID NO. 12, and a downstream primer as shown in SEQ ID NO. 13.

An upstream primer for detecting staphylococcus aureus is shown as SEQ ID NO. 14, and a downstream primer is shown as SEQ ID NO. 15 by using a test strip-based microorganism rapid diagnosis technology of Cas12a enzyme.

The test strip-based microorganism rapid diagnosis technology of Cas12a enzyme is characterized in that an upstream primer for detecting Escherichia coli is shown as SEQ ID NO. 16, and a downstream primer is shown as SEQ ID NO. 17.

The microorganism rapid diagnosis technology based on the Cas12a enzyme of the test strip detects the upstream primer of Shigella as shown in SEQ ID NO. 18, and the downstream primer as shown in SEQ ID NO. 19.

The test strip-based microorganism rapid diagnosis technology of Cas12a enzyme is characterized in that an upstream primer for detecting salmonella is shown as SEQ ID NO. 20, and a downstream primer is shown as SEQ ID NO. 21.

The method for extracting the microbial nucleic acid comprises the following steps: extracting the kit and quickly extracting, wherein the quick extraction method comprises a boiling method, an enzymolysis method, an alkali cracking method, a magnetic bead method and a microwave method.

The amplification technology of the target fragment comprises the following steps: polymerase Chain Reaction (PCR), nucleic acid dependent amplification detection technology (NASBA), loop-mediated isothermal amplification technology (LAMP), helicase dependent isothermal amplification of DNA (HDA), Recombinase Polymerase Amplification (RPA).

Cas12a enzymes include: aminococcus acidilactici BV3L6Cas12a (amidechococcus sp. BV3L6Cas12a, AsCas12 a), streptomyces ND2006 Cas12a (Lachnospiraceae bacteria ND2006 Cas12a, LbCas12 a), francisella novida U112 Cas12a (francisella subtsp. novicida U112 Cas12a, FnCas12 a), klebsiella Cas12a (prevolella disiensis Cas12a, PdCas12 a), Moraxella bovis Cas12a (Moraxella novoculi 237Cas12 Cas 2, MbCas12 Cas a), macadimia porphyrins 12a (porphyromonas cass 12 Cas a, pm68612 Cas 8), and the like.

The Reporter sequence SEQ ID NO:22 is characterized in that the short nucleotide sequence modified by the compound has the nucleotide length of 6nt-20nt, the 5 'end of the short nucleotide sequence is marked with Fluorescein Isothiocyanate (FITC) or carboxyl Fluorescein (FAM), and the 3' end of the short nucleotide sequence is marked with Biotin (Biotin).

The nucleotide sequence shown in SEQ ID NO. 22 is: 5 '-FAM/ACACACACACACACAC/Biotin-3'.

The gold-labeled antibody contained in the colloidal gold immune test strip comprises: anti-FAM antibody, anti-FITC antibody.

According to the microorganism rapid diagnosis technology of the Cas12a enzyme based on the test strip, the method is characterized in that: the diagnosis technology provides an optimal detection method for field detection, can realize amplification of a target fragment through PCR or isothermal amplification, combines the universality, stability and specificity of a Cas12a detection system with the simplicity, convenience and rapidness of test strip detection, has the characteristics of simplicity, rapidness, specificity, sensitivity, stability and the like, and is favorable for timely diagnosis.

Description of the drawings:

FIG. 1 is a schematic diagram of the detection of the present invention;

FIG. 2 is a graph showing the results of an example of the specificity of detecting Shigella dysenteriae crRNA according to the present invention;

FIG. 3 is a graph showing results of examples of 7 microorganisms detected according to the present invention;

FIG. 4 is a graph showing the results of an example of the sensitivity of the present invention for detecting Shigella dysenteriae;

FIG. 5 is a graph showing the results of an example of the present invention in detecting Shigella dysenteriae after rapid treatment.

The specific implementation mode is as follows:

the invention is further illustrated by the following examples.

A test strip-based microbial rapid diagnosis technology for a Cas12a enzyme, the method comprising the following steps:

the method comprises the following steps: using microbial nucleic acid as a template, and amplifying a target fragment with a proper length by using a specific primer;

step two: adding the amplification product obtained in the step one into a Cas12a detection system for carrying out a cleavage reaction;

step three: and D, directly detecting the cutting product obtained in the step two by using a colloidal gold immune test strip.