CN117867140A - System for detecting brucella based on RPA-CRISPR-Cas12a and application thereof - Google Patents

System for detecting brucella based on RPA-CRISPR-Cas12a and application thereof Download PDF

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CN117867140A
CN117867140A CN202310752976.3A CN202310752976A CN117867140A CN 117867140 A CN117867140 A CN 117867140A CN 202310752976 A CN202310752976 A CN 202310752976A CN 117867140 A CN117867140 A CN 117867140A
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brucella
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陈泽良
韩小虎
范首东
崔扬书翰
张燚
焦洋
祁安东
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Dongwo Tongtai Fengcheng Bioengineering Co ltd
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Abstract

The invention relates to the technical field of gene detection, in particular to a system for detecting brucella based on RPA-CRISPR-Cas12a and application thereof. The RPA primer designed by the invention uses the RNA of brucella as a template to carry out specific amplification, the obtained amplification product contains a target sequence which can be identified by CrRNA, the target sequence is cut after the CrRNA identifies the target sequence, the target sequence has the cutting activity on a nonspecific single-chain reporter molecule, a fluorescent tag peptide is connected to a T base at the 5' end of the single-chain reporter molecule, and an LF test strip is used for detecting whether the single-chain reporter molecule is cut, so that the visual detection of the brucella is realized. The system provided by the invention can carry out field detection under the constant temperature condition (37 ℃) without a large instrument, and has the characteristics of good specificity, high sensitivity, simple operation, lower requirement and short time consumption.

Description

System for detecting brucella based on RPA-CRISPR-Cas12a and application thereof
Technical Field
The invention relates to the technical field of gene detection, in particular to a system for detecting brucella based on RPA-CRISPR-Cas12a and application thereof.
Background
Brucellosis is a zoonosis caused by brucellosis, and is also a zoonosis with the greatest popularity and harm in the world. Brucella is an intracellular parasitic gram-negative bacterium, and can infect human beings and more than 60 other mammals, so that the host has abortion, fever, joint injury and other disease characteristics, and huge economic loss is brought to animal husbandry.
The existing laboratory diagnosis technical standards of brucellosis mainly comprise tiger red plate agglutination test (RBT), full milk circle test (MRT, only cows), test tube agglutination test (SAT), complement Fixation Test (CFT), anti-human globulin test (AGT, only human), indirect enzyme-linked immunosorbent assay (IELISA), competitive enzyme-linked immunosorbent assay (CELISA), complement fixation enzyme-linked immunosorbent assay (CF-ELISA), colloidal gold technique (GICT), fluorescence polarization test (FPA) and various nucleic acid diagnosis techniques. Wherein RBT and MRT are limited to the primary screening of Brucella. Whereas conventional serological tests, typified by SAT, have the phenomena of false positives and false negatives.
Although most of the detection methods of brucella have higher sensitivity and good specificity, the methods often require complex operation steps and complicated sample processing procedures, and also depend on specific experimental instruments and professional operators.
Disclosure of Invention
In order to solve the problems, the invention provides a system for detecting Brucella based on RPA-CRISPR-Cas12a and application thereof. The system provided by the invention can carry out field detection under the constant temperature condition (37 ℃) without a large instrument, and has the characteristics of good specificity, high sensitivity, simple operation, lower requirement and short time consumption.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a system for detecting brucella based on RPA-CRISPR-Cas12a, which comprises: RPA primers, crRNA, single-stranded reporter molecules, and LF test strips; the RPA primer comprises F and R;
the nucleotide sequence of F is shown as SEQ ID NO. 1;
the nucleotide sequence of R is shown as SEQ ID NO. 2;
the nucleotide sequence of the CrRNA is shown as SEQ ID NO. 3;
the nucleotide sequence of the single-chain reporter molecule is 5'-TTATTATT-3'; the 5 'end of the single-chain reporter molecule is connected with a fluorescent labeling peptide, and the 3' end of the single-chain reporter molecule is connected with biotin.
Preferably, the fluorescent-labeled peptide comprises FITC or FAM.
Preferably, the system further comprises an RPA reactant and a CRISPR-Cas12a reactant.
The invention also provides application of the system in detecting Brucella for non-diagnosis purpose.
The invention also provides a method for detecting Brucella by using the system for non-diagnosis, which comprises the following steps:
taking RNA of a sample to be detected as a template, and carrying out RPA reaction by utilizing the RPA primer to obtain an RPA amplification product;
performing CRISPR-Cas12a reaction on the RPA amplification product, the CrRNA and the single-stranded reporter molecule to obtain a reaction product;
detecting a reaction product by using an LF test strip, and judging whether the sample to be detected contains Brucella or not according to a detection result;
if the T line and the C line of the LF test strip are developed or only the T line is developed, the sample to be tested contains Brucella;
if the C line of the LF test strip is colored and the T line is not colored, the sample to be tested does not contain Brucella.
Preferably, the reaction system of the RPA reaction comprises: f2.4mu. L, R2.4.2.4. Mu.L, primer-free rehydration buffer 29.5. Mu.L, template 2. Mu. L, ddH 2 O11.2. Mu.L and magnesium acetate 2.5. Mu.L.
Preferably, the temperature of the RPA reaction is 37℃and the time is 30min.
Preferably, the CRISPR-Cas12a reaction system is 50 μl, comprising: lb Cas12a1 μ L, crRNA μL, 1 XNE Buffer 5 μL, single-stranded reporter 5 μ L, RPA amplification product 5 μL and the remainder ddH 2 O。
Preferably, the concentration of the Lb Cas12a is 1 μmol/L; the concentration of the crRNA is 1 mu mol/L; the concentration of the single-stranded reporter molecule is 1.5 mu mol/L.
Preferably, the CRISPR-Cas12a reaction is at 37 ℃ for 30min.
The beneficial effects are that:
the invention provides a system for detecting brucella based on RPA-CRISPR-Cas12a, which comprises: RPA primers, crRNA, single-stranded reporter molecules, and LF test strips; the RPA primer comprises F and R; the nucleotide sequence of F is shown as SEQ ID NO. 1; the nucleotide sequence of R is shown as SEQ ID NO. 2; the nucleotide sequence of the CrRNA is shown as SEQ ID NO. 3; the nucleotide sequence of the single-chain reporter molecule is 5'-TTATTATT-3'; the T base at the 5 'end of the single-chain reporter molecule is connected with a fluorescent labeling peptide, and the T base at the 3' end of the single-chain reporter molecule is connected with biotin. The RPA primer designed by the invention uses the RNA of brucella as a template to carry out specific amplification, the obtained amplification product contains a target sequence which can be identified by CrRNA, the CrRNA cuts the target sequence after identifying the target sequence and has the cutting activity on a nonspecific single-chain reporter molecule, a fluorescent tag peptide is connected to a T base at the 5' end of the single-chain reporter molecule, and an LF test strip is used for detecting whether the single-chain reporter molecule is cut or not, so that the visual detection of the brucella is realized. The system provided by the invention can perform field detection under the constant temperature condition (37 ℃) without a large instrument, and has the characteristics of good specificity, high sensitivity (the minimum detection copy number is 10 copies/. Mu.L), simple operation, lower requirement and short time consumption.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a sensitivity detection result in example 3;
FIG. 2 shows the results of the specific assay in example 4.
Detailed Description
The invention provides a system for detecting brucella based on RPA-CRISPR-Cas12a, which comprises: RPA primers, crRNA, single-stranded reporter molecules, and LF test strips; the RPA primer comprises F and R;
the nucleotide sequence of F is shown as SEQ ID NO.1, and is specifically 5'-TTTCTGAGCCTTCCCAATCCAGTCCATGACG-3';
the nucleotide sequence of R is shown as SEQ ID NO.2, and is specifically 5'-CTTCTGAACGACAGCAGCCCCTACGGTACTGAC-3';
the nucleotide sequence of the CrRNA is shown as SEQ ID NO.3, and is specifically 5'-UAAUUUCUACUAAGUGUAGAUCACGAUGGCAACCGCAAUCGC-3';
the nucleotide sequence of the single-chain reporter molecule is 5'-TTATTATT-3'; the T base at the 5 'end of the single-chain reporter molecule is connected with a fluorescent labeling peptide, and the T base at the 3' end of the single-chain reporter molecule is connected with Biotin (Biotin).
The RPA primer of the invention uses the RNA of brucella as a template to carry out specific amplification, and the obtained amplification product contains a target sequence which can be identified by CrRNA; the nucleotide sequence of the amplified product is preferably shown as SEQ ID NO.4, in particular Wherein the bolded sequence is the spacer DNA of the Cas12a system and the underlined sequence is the Cas12a target PAM.
In the present invention, the fluorescent-labeled peptide preferably includes FITC or FAM, more preferably FAM. The method of the present invention for linking the fluorescent-labeled peptide to biotin is not particularly limited, and may be any linking method known to those skilled in the art.
In the invention, the C line of the LF test strip is preferably coated with a substance capable of binding with biotin, more preferably with streptavidin; the T line of the LF test strip is preferably coated with an antibody which can be combined with the fluorescent labeling peptide. The LF test strip of the present invention is preferably Milenia HybriDetect kit from TwitDx under the designation MILENIA01.
In the present invention, the system preferably further comprises an RPA reactant and a CRISPR-Cas12a reactant; the RPA reaction reagent preferably comprises an RPA kit; the RPA KIT is preferably a TwitAmp DNA amplification KIT purchased from Miere (Shanghai) Biotechnology Co., ltd, and has the product number of TABASRT01KIT; the CRISPR-Cas12a reagent preferably comprises a Cas12a kit; the Cas12a kit is preferably purchased from a CRISPR Cas12a DNA detection kit of Shenzhen Yizhen Biotechnology Co., ltd, with the product number of D-F-CAS12-1S.
The system provided by the invention has high sensitivity and specificity, and the reaction temperature is controlled at 37 ℃ and can be easily achieved without depending on a precise machine; the RPA reagent and the CRISPR-Cas12a reagent can be all commercial kits, detection can be completed by referring to the instruction book, the professional of personnel is not required, and real-time, on-site and visual detection can be achieved.
The invention also provides application of the system in detecting Brucella for non-diagnosis purpose. The system provided by the invention can be used for detecting the Brucella for the purpose of non-diagnosis, and is only used for obtaining an intermediate value of whether the Brucella is contained in the sample to be detected.
The invention also provides a method for detecting Brucella by using the system for non-diagnosis, which comprises the following steps:
taking RNA of a sample to be detected as a template, and carrying out RPA reaction by utilizing the RPA primer to obtain an RPA amplification product;
performing CRISPR-Cas12a reaction on the RPA amplification product, the CrRNA and the single-stranded reporter molecule to obtain a reaction product;
detecting a reaction product by using an LF test strip, and judging whether the sample to be detected contains Brucella or not according to a detection result;
if the T line and the C line of the LF test strip are developed or only the T line is developed, the sample to be tested contains Brucella;
if the C line of the LF test strip is colored and the T line is not colored, the sample to be tested does not contain Brucella.
The invention uses the RNA of the sample to be detected as a template, and uses the RPA primer to carry out RPA reaction to obtain the RPA amplification product. In the present invention, the sample to be measured preferably includes: one or more of blood, tissue, stool, and body fluids. The method for extracting the RNA of the sample to be detected is not particularly required, and the method or the commercially available kit which are well known to the person skilled in the art can be used for extracting the RNA.
In the present invention, the reaction system of the RPA reaction preferably includes: f2.4mu. L, R2.4.2.4. Mu.L, primer-free rehydration buffer 29.5. Mu.L, template 2. Mu. L, ddH 2 O11.2. Mu.L and magnesium acetate 2.5. Mu.L; the concentrations of F and R in the reaction system are each preferably 10. Mu.M; the concentration of magnesium acetate is preferably 40mmol/L. The components of the reaction system of the RPA reaction of the invention, except F, R and templates, are preferably derived from the RPA kit described in the above technical scheme.
In the present invention, the temperature of the RPA reaction is preferably 37 ℃ and the time is preferably 30min.
After the RPA amplification product is obtained, CRSPR-Cas 12a reaction is carried out on the RPA amplification product, the CrRNA and the single-chain reporter molecule to obtain a reaction product.
In the present invention, the CRISPR-Cas12a reaction system is preferably 50 μl, preferably comprising: lb Cas12a1 μ L, crRNA μL, 1 XNE Buffer 5 μL, single-stranded reporter 5 μ L, RPA amplification product 5 μL and the remainder ddH 2 O; the concentration of Lb Cas12a is preferably 1 μmol/L; the concentration of crRNA is preferably 1. Mu. Mol/L; the concentration of the single-stranded reporter molecule is preferably 1.5. Mu. Mol/L. The components of the reaction system for the CRISPR-Cas12a reaction of the present invention are preferably derived from the Cas12a kit described in the above technical scheme, except for crRNA, RPA amplification products and single-stranded reporter molecules.
In the present invention, the CRISPR-Cas12a reaction is preferably at 37 ℃ for 30min.
After a reaction product is obtained, the invention utilizes an LF test strip to detect the reaction product, and judges whether the sample to be detected contains Brucella or not according to the detection result; if the T line and the C line of the LF test strip are developed or only the T line is developed, the sample to be tested contains Brucella; if the C line of the LF test strip is colored and the T line is not colored, the sample to be tested does not contain Brucella; if the C line and the T line of the LF test strip are not developed, the LF test strip is polluted, and the test strip needs to be replaced for re-detection.
When the sample to be detected does not contain Brucella, the RPA primer cannot be amplified to obtain an amplification product containing a target sequence, so that the CRISPR-Cas12a reaction cannot cut a single-chain reporter molecule, biotin on the single-chain reporter molecule is entrapped in a C line by the streptavidin coated by colloidal gold, the C line is colored, and the T line is not colored;
when the sample to be detected contains Brucella, the RPA primer is amplified to obtain an amplified product containing a target sequence, CRISPR-Cas12a reacts to cut a single-chain reporter molecule, a Biotin (Biotin) marked part of the cut reporter molecule is firstly trapped on a C line, a FITC or FAM marked part continues to flow upwards, and the T line is trapped by a corresponding colloidal gold-coated antibody, so that the C line does not develop color, the T line develops color, but when the single-chain reporter molecule in a reaction system is not completely cut, the single-chain reporter molecule which is trapped on the C line and is not cut is developed, namely, both the T line and the C line develop color.
The method for detecting the reaction product by using the LF test strip has no special requirement, and the method can be operated according to the specification of the LF test strip.
The method of the invention can be used for detecting the brucella for the purpose of non-diagnosis, and is only used for obtaining an intermediate value of whether the brucella is contained in the sample to be detected.
For further explanation of the present invention, a system for detecting Brucella based on RPA-CRISPR-Cas12a and its application are provided in the following detailed description with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the invention.
Example 1
A system for detecting brucella based on RPA-CRISPR-Cas12a, which consists of RPA primer, crRNA, single-chain reporter, LF test strip (Milenia HybriDetect KIT from twist dx, cat No. milania 01), RPA KIT (twist amp DNA amplification KIT from merry (Shanghai) biotechnology limited, cat No. TABASRT01 KIT), cas12a KIT (CRISPR Cas12a DNA detection KIT from Shenzhen facile biotechnology limited, cat No. D-F-Cas 12-1S); the RPA primer consists of F and R;
the nucleotide sequence of F is shown as SEQ ID NO. 1;
the nucleotide sequence of R is shown as SEQ ID NO. 2;
the nucleotide sequence of the CrRNA is shown as SEQ ID NO. 3;
the nucleotide sequence of the single-chain reporter molecule is 5'-FAM-TTATTATT-Biotin-3'.
Example 2
A method for detecting brucella for non-diagnostic purposes, consisting of the steps of:
taking RNA of a sample to be detected as a template, and carrying out RPA reaction by using an RPA primer and an RPA kit in the system described in the embodiment 1 to obtain an RPA amplification product;
the RPA reaction system comprises: f2.4mu. L, R2.4.2.4. Mu.L, primer-free rehydration buffer 29.5. Mu.L, template 2. Mu. L, ddH 2 O11.2. Mu.L and magnesium acetate2.5. Mu.L; the concentration of F and R in the reaction system is 10 mu M; the concentration of the magnesium acetate is 40mmol/L; the RPA reaction condition is that the reaction is carried out for 30min at 37 ℃;
performing a CRISPR-Cas12a reaction on the RPA amplification product, crRNA, single-stranded reporter molecule, and Cas12a kit in the system of example 1 to obtain a reaction product;
the CRISPR-Cas12a reaction system comprises: lb Cas12a1 μ L, crRNA μl, 1 XNE Buffer 5 μl, single Strand reporter 5 μ L, RPA amplification product 5 μl and ddH 2 O is added to 50 mu L; the concentration of the Lb Cas12a is 1 mu mol/L; the concentration of the crRNA is 1 mu mol/L; the concentration of the single-chain reporter molecule is 1.5 mu mol/L; the CRISPR-Cas12a reaction condition is that the reaction is carried out for 30min at 37 ℃;
detecting a reaction product by using the LF test strip (detection according to the specification) in the system described in the embodiment 1, and judging whether the sample to be detected contains Brucella or not according to the detection result;
if the T line and the C line of the LF test strip are developed or only the T line is developed, the sample to be tested contains Brucella;
if the C line of the LF test strip is colored and the T line is not colored, the sample to be tested does not contain Brucella;
if the C line and the T line of the LF test strip are not developed, the LF test strip is polluted, and the test strip needs to be replaced for re-detection.
Example 3
Sensitivity detection
The PCR product containing the detection target sequence is used for the detection object, and the preparation method of the PCR product containing the detection target sequence is as follows:
using the method described in example 2, using RNA of a sample to be tested containing brucella as a template, and using the RPA primer and the RPA kit in the system described in example 1 to perform an RPA reaction, an RPA amplification product is obtained, that is, a PCR product containing a detection target sequence.
The copy number is measured and then diluted by a multiple ratio to obtain 10 10 ～10 0 The probes/. Mu.L of the test substance. The test sample diluted with different ratios was used as a test sample, and the method described in example 2 was usedThe method is used for detection, and the detection result is shown in figure 1.
As can be seen from FIG. 1, the detection copy number was positive at 10 copies/. Mu.L and negative at 1 copy/. Mu.L, showing the lowest detection copy number was 10 copies/. Mu.L.
Example 4
Specificity test
Brucella, proteus pennii, vibrio alginolyticus, vibrio river, pseudomonas aeruginosa and Klebsiella pneumoniae were detected by the method described in example 2, and the detection results are shown in FIG. 2.
As can be seen from FIG. 2, only the strips for detecting Brucella showed positive in the presence of red bands near the detection line, and the strips for detecting other viruses showed negative in the absence of bands near the detection line. The final result shows that the specificity of the detection method is good.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (10)

1. A system for detecting brucella based on RPA-CRISPR-Cas12a, comprising: RPA primers, crRNA, single-stranded reporter molecules, and LF test strips; the RPA primer comprises F and R;
the nucleotide sequence of F is shown as SEQ ID NO. 1;
the nucleotide sequence of R is shown as SEQ ID NO. 2;
the nucleotide sequence of the CrRNA is shown as SEQ ID NO. 3;
the nucleotide sequence of the single-chain reporter molecule is 5'-TTATTATT-3'; the 5 'end of the single-chain reporter molecule is connected with a fluorescent labeling peptide, and the 3' end of the single-chain reporter molecule is connected with biotin.
2. The system of claim 1, wherein the fluorescent-labeled peptide comprises FITC or FAM.
3. The system of claim 1 or 2, further comprising an RPA reactant and a CRISPR-Cas12a reactant.
4. Use of a system according to any one of claims 1 to 3 for the detection of brucella for non-diagnostic purposes.
5. A method for detecting brucella for non-diagnostic purposes using the system of any one of claims 1 to 3, comprising the steps of:
taking RNA of a sample to be detected as a template, and carrying out RPA reaction by utilizing the RPA primer to obtain an RPA amplification product;
performing CRISPR-Cas12a reaction on the RPA amplification product, the CrRNA and the single-stranded reporter molecule to obtain a reaction product;
detecting a reaction product by using an LF test strip, and judging whether the sample to be detected contains Brucella or not according to a detection result;
if the T line and the C line of the LF test strip are developed or only the T line is developed, the sample to be tested contains Brucella;
if the C line of the LF test strip is colored and the T line is not colored, the sample to be tested does not contain Brucella.
6. The method of claim 5, wherein the reaction system of the RPA reaction comprises: f2.4mu. L, R2.4.2.4. Mu.L, primer-free rehydration buffer 29.5. Mu.L, template 2. Mu. L, ddH 2 O11.2. Mu.L and magnesium acetate 2.5. Mu.L.
7. The method of claim 5 or 6, wherein the RPA reaction is carried out at a temperature of 37 ℃ for a period of 30 minutes.
8. The method of claim 5, wherein the CRISPR-Cas12a reaction system is 50 μl, comprising: lb Cas12a1 μ L, crRNA μL, 1 XNE Buffer 5 μL, single-stranded reporter 5 μL5. Mu.L of RPA amplification product and the remaining ddH 2 O。
9. The method of claim 8, wherein the concentration of Lb Cas12a is 1 μmol/L; the concentration of the crRNA is 1 mu mol/L; the concentration of the single-stranded reporter molecule is 1.5 mu mol/L.
10. The method of claim 5 or 8, wherein the CRISPR-Cas12a reaction is at a temperature of 37 ℃ for a time of 30min.
CN202310752976.3A 2023-06-25 2023-06-25 System for detecting brucella based on RPA-CRISPR-Cas12a and application thereof Pending CN117867140A (en)

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