CN113462795A - Combined detection method for rapidly detecting Listeria monocytogenes, system and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of food safety detection and biology, and particularly relates to a combined detection method for rapidly detecting Listeria monocytogenes, a system and application thereof. The method comprises the steps of firstly extracting genome DNA of the listeria monocytogenes, obtaining a DNA fragment carrying a T7 promoter through RAA amplification, obtaining a target RNA through T7RNA polymerase in vitro transcription, exciting the cutting function of a Cas13a-crRNA compound to the single-stranded RNA when the target RNA exists, shearing a quenching group in an RNA fluorescence reporter molecule, and realizing the rapid detection of the listeria monocytogenes through fluorescence reading. The invention has short detection time, strong specificity, high sensitivity and low requirement on equipment, does not need expensive quantitative PCR instruments, only needs constant temperature metal bath and simple fluorescence detectors, and is suitable for being used in a field or a basic laboratory.
Description
Technical Field
The invention belongs to the field of food safety detection and biotechnology, and particularly relates to a combined detection method for rapidly detecting Listeria monocytogenes (hereinafter, Listeria monocytogenes), a system and application thereof, namely a recombinase-mediated isothermal amplification of nucleic acid (RAA) and CRISPR/Cas13a combined detection method.
Background
Listeria Monocytogenes (LM), abbreviated as Listeria monocytogenes, is a facultative intracellular parasitic zoonosis pathogenic bacterium belonging to aerobic or facultative anaerobic gram-positive bacteria. The bacteria can penetrate blood brain barrier, intestinal barrier and placenta barrier to cause meningitis, septicemia, gastroenteritis, abortion, etc.; the bacterium can be slowly propagated in a low-temperature environment, is an important pathogen threatening refrigerated foods, and is classified as one of four important food-borne pathogenic microorganisms by the world health organization. Therefore, people pay attention to the rapid and accurate detection of listeria monocytogenes in food.
Conventional methods for detecting listeria include: conventional separation and culture methods, immunological methods and molecular biological methods. The traditional separation culture method carries out detection and biochemical identification by identifying a culture medium, although the operation is simple, the time consumption is long, and the detection efficiency is low; although the immunological method has high flux and high detection speed, when the content of a target detection object is low, false negative results are easy to occur, and the detection sensitivity and specificity are poor; molecular biological methods such as PCR, fluorescent quantitative PCR and the like have high sensitivity, but the required instruments are expensive and have higher requirements on operators, and the amplification detection time needs at least 2 hours, so that the basic popularization and the field requirements are difficult to meet. In conclusion, there is a need to establish a rapid, specific, sensitive, and low-cost method for detecting Listeria monocytogenes.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a Recombinase mediated isothermal Amplification (RAA) and CRISPR/Cas13a combined detection method for detecting Listeria monocytogenes, which has the advantages of strong specificity, high sensitivity, simple operation and low equipment requirement, and is suitable for the technical fields of food safety detection and the like.
A combined detection method for rapidly detecting Listeria monocytogenes comprises the following steps:
(1) carrying out isothermal amplification on the bacterial sample DNA at 38 ℃ by using a specific primer carrying a T7 promoter and an RAA amplification system (recombinase, polymerase and single-stranded DNA binding protein) to obtain a T7-DNA target fragment;
(2) designing a guide RNA (crRNA) aiming at a target of a target gene;
(3) commercial T7RNA polymerase, NTP, RAA product, Cas13a protein, crRNA, and fluorescent reporter were mixed for isothermal incubation, and signals were collected and recorded using a fluorescence detector.
Wherein the content of the first and second substances,
the amplification primer carrying the T7 promoter in the step (1) is as follows:
the upstream primer F is TAATACGACTCACTATAGGGCCGAAGTTTACATTCAAGCTATTATTTAC;
TAAAACGTGATTCATTAACACTCAGCATTG as downstream primer;
the amplified fragment is the hly gene 258-529-th base and is 272bp in total.
The target gene in the step (2) is crRNA of a specific target listeriolysin coding gene (hly gene) in a detection system, and the sequence is as follows:
5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUAAAUACAUUAGUGGAAAG-3', wherein the second half-sequence GUAAAUACAUUAGUGGAAAG is complementary to the target RNA sequence.
The transcription mode in the step (3) is in vitro transcription, the incubation temperature is 38 ℃, the RAA product (T7-DNA) in the reaction system forms a fragment carrying target RNA under the action of T7RNA polymerase, the fragment is specifically identified by crRNA in a Cas13a-crRNA compound, the Cas13a cleavage function is activated at the same time, the RNA enzyme activity which is not limited by a sequence after the Cas13a is activated is utilized, a fluorescence reporter molecule added in the detection system is sheared, the fluorescence reporter molecule is separated from a quenching group, fluorescence is obtained, and fluorescence signal acquisition is carried out, so that the detection of the target gene is realized.
The in vitro transcription of T7RNA polymerase and the reaction of Cas13a protein are carried out in the same system, namely, the transcription system and the Cas13a shearing system are mixed together and incubated in an environment at 38 ℃ to simplify the experimental operation.
In addition, the application provides a listeria hly gene target sequence, a RAA amplification primer pair, a guide RNA and a detection system.
The RAA primer pair for amplifying the target sequence of the hly gene of the listeria, wherein the upstream primer (F) comprises a T7 promoter, and the amplified fragment comprises the target sequence which is complementary to crRNA, and the sequence is as follows:
target RNA sequence:
5’-CUUUCCACUAAUGUAUUUAC-3’
RAA amplification primer sequences:
F:TAATACGACTCACTATAGGGCCGAAGTTTACATTCAAGCTATTATTTAC
R:TAAAACGTGATTCATTAACACTCAGCATTG
RAA amplified fragment sequence:
GCCGAAGTTTACATTCAAGCTATTATTTACAGCTTTAAATGCTGTACCAAATTTCGCAATTAATTGTGATTCACTGTAAGCCATTTCGTCATCATAATCAATTTTTGCACTTACATTTGGATAAGCTTGAGCATATTTTTCATTCCATCTTTCCACTAATGTATTTACTGCGTTGTTAACGTTTGATTTAGTGGCATTTTTTACAACTATTTTATTGTCTTGATTAGTCATACCTGGCAAATCAATGCTGAGTGTTAATGAATCACGTTTTA。
the sequence of the crRNA for the specific target Listeria monocytogenes hly gene of the CRISPR/Cas13a system is as follows:
5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUAAAUACAUUAGUGGAAAG-3', in sequence format: 5 '-CRISPR/Cas 13a protein specific binding sequence-crRNA target binding sequence-3', wherein the second half sequence GUAAAUACAUUAGUGGAAAG is complementary to the target RNA sequence.
A RAA-CRISPR/Cas13a system for detecting Listeria comprises a Listeria monocytogenes hly gene target sequence, a RAA amplification primer pair and a guide RNA (namely crRNA) designed aiming at the target sequence of a target gene.
In addition, the invention provides application of the CRISPR-Cas13a system for detecting Listeria.
The invention comprises an RAA amplification primer pair and crRNA of a specific target Listeria monocytogenes hemolysin coding gene (hly gene). Firstly, extracting genome DNA of the listeria monocytogenes, obtaining a DNA fragment carrying a T7 promoter through RAA amplification, obtaining target RNA through in vitro transcription of T7RNA polymerase, exciting the cutting function of a Cas13a-crRNA compound to single-stranded RNA when the target RNA exists, shearing a quenching group in an RNA fluorescence reporter molecule, and realizing the rapid detection of the listeria monocytogenes through fluorescence reading. The invention has short detection time, strong specificity, high sensitivity and low requirement on equipment, does not need expensive quantitative PCR instruments, only needs constant temperature metal bath and simple fluorescence detectors, and is suitable for being used in a field or a basic laboratory.
The invention provides a target sequence for specific detection of Listeria monocytogenes and specific crRNA targeting the target sequence based on RAA and CRISPR/Cas13a nucleic acid detection technology through reasonable design, and the crRNA can activate CRISPR/Cas13a by combining the target sequence, thereby realizing rapid, sensitive and specific detection of Listeria monocytogenes. The detection sensitivity of the invention is 10CFU/ml, which is equivalent to fluorescent quantitative PCR, and the detection time is 30 min.
Drawings
FIG. 1 shows the sensitivity contrast before and after the optimization of the CRISPR/Cas13a system
FIG. 2 shows the result of the specificity test of the present detection system
FIG. 3 shows the detection effect of the detection system for different detection durations
FIG. 4 shows the anti-interference capability of the detection system
FIG. 5 shows the detection capability of the detection system for simulating the detection of milk samples
Detailed Description
The invention will be further illustrated and described with reference to specific examples, which are not intended to limit the scope of the invention.
The experimental procedures referred to in the following examples are conventional unless otherwise specified.
Materials, reagents, instruments and the like used in the following examples are conventional commercially available products unless otherwise specified.
The following experiments are quantitative and are all set up as two replicates and the results averaged.
The invention relates to a RAA and CRISPR/Cas13a combined method for detecting Listeria monocytogenes, which is used for extracting a sample genome DNA to be detected and detecting through two-step reaction (namely RAA amplification and T7 transcription/Cas 13a reaction). The RAA product is T7-DNA; the T7 transcription and CRISPR/Cas13a reaction are carried out simultaneously, in the co-incubation process of the obtained target RNA, crRNA, fluorescent reporter molecule and CRISPR/Cas13a, the target RNA is specifically identified by crRNA in a Cas13a-crRNA compound, the cutting function of Cas13a is activated simultaneously, the RNA enzyme activity which is not limited by sequence after being activated by CRISPR/Cas13a is utilized to cut a quenching group of the fluorescent reporter molecule, fluorescence is obtained, fluorescence data collection is carried out, and the detection of the gene of Listeria monocytogenes is realized.
Example 1 establishment of a Listeria monocytogenes detection method based on RAA and CRISPR/Cas13a
First, design and synthesize crRNA
1. Design of crRNA specifically targeting Listeria monocytogenes hly gene
The CRISPR/Cas13a belongs to a CRISPR Class2 type VI system, no clear CRISPR/Cas13acrRNA design principle exists at present, and according to previous work experience, the design principle of the crRNA of the hly gene of the targeted Listeria monocytogenes is as follows:
(1) crRNA includes: a direct repeat sequence (hereinafter referred to as a direct repeat sequence) combined with the CRISPR/Cas13a protein and a sequence (hereinafter referred to as a binding sequence) ensuring that the direct repeat sequence is combined with target RNA of a target gene, wherein the sequence format is 5 '-direct repeat sequence-binding sequence-3';
(2) the direct repetitive sequence of crRNA must contain a stem-loop structure, and the specific sequence information is determined by the characteristics of CRISPR/Cas13a protein;
(3) the binding sequence of crRNA is about 20bp in length, and the next base at the 3' end of the corresponding target sequence cannot be G.
The specific screening steps of the crRNA of the hly gene of the target Listeria monocytogenes are as follows:
(1) homology analysis is carried out in the NCBI existing genome database to ensure the target sequence specificity of crRNA;
(2) the crRNA target too close to the initiation codon (ATG) is eliminated.
Finally, selecting a crRNA targeting the hly gene of the listeria monocytogenes, wherein the sequence and the target sequence are as follows:
crRNA nucleotide sequence
5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUAAAUACAUUAGUGGAAAG-3’。
Target RNA sequence
5’-CUUUCCACUAAUGUAUUUAC-3’
Synthesis of crRNA
The crRNA complementary to the target bacterial RNA is committed to the relevant bio-companies for synthesis.
Second, RAA isothermal amplification
RAA primer design. The RAA amplification fragment comprises the crRNA targeting fragment, and a T7 promoter sequence is added at the 5' end of the upstream primer, so that the double-stranded DNA product obtained by RAA amplification can be conveniently transcribed. The primer sequence is as follows:
the upstream primer F is TAATACGACTCACTATAGGGCCGAAGTTTACATTCAAGCTATTATTTAC;
TAAAACGTGATTCATTAACACTCAGCATTG as downstream primer;
the amplified fragment is the hly gene 258-529-th base and is 272bp in total.
RAA isothermal amplification. Carrying out pre-enrichment treatment on a sample to be detected, and extracting the genomic DNA of the sample by using a genomic extraction kit; the RAA primers provided by the present invention were used to amplify samples using genomic DNA as a template, and the amplification system is shown in Table 2.
TABLE 2 RAA amplification System
Adding the mixed solution of 47.5 μ L into a basic reaction unit filled with lyophilized powder (including recombinase, DNA polymerase and single-stranded DNA binding protein), respectively adding 2.5 μ L Buffer B into a reaction tube cover, closing the tube cover to fully dissolve the lyophilized powder and the solution, turning upside down and stirring to mix uniformly, collecting instantly, and reacting at 38 deg.C for 10-20 min; meanwhile, the DNA of the sample is replaced by water as a template to be used as a negative control.
Third, based on CRISPR/Cas13a system detection Listeria monocytogenes
The RAA product obtained in the experiment is used as a template for in vitro transcription, a CRISPR/Cas13a detection system is prepared and mixed with a transcription system, and the T7 transcription-CRISPR/Cas 13a detection system is shown in Table 3.
When formulating the system, crRNA can be premixed with CRISPR/Cas13a protein separately to ensure that both are fully bound. And incubating the mixed systems together in a constant-temperature metal bath at 38 ℃, reacting for 10-20min, and collecting fluorescence by using a fluorescence reader with excitation wavelength of 484 nm. The RAA product in table 3 was replaced with water and the other reagent components were kept unchanged as a negative control.
TABLE 3T 7 transcription and CRISPR/Cas13a reaction System
The reaction can also be incubated at constant temperature and collected with fluorescence by means of a quantitative PCR instrument, and the parameters are set as follows: the excitation wavelength is 484nm, the emission wavelength is 529nm, the fluorescence intensity is measured every 30-60s, and the total time is 30 min.
Example 2 sensitivity testing of the present detection System
The detection was carried out according to the method in example 1 with the bacterial genomes at different concentrations as templates to examine the sensitivity of the method of the present invention. The method comprises the following specific steps: diluting the fresh bacterial liquid to 100-108CFU/mL, 1mL each was taken to extract a genome, RAA amplification and T7 transcription and CRISPR/Cas13a detection were performed according to the detection procedure in example 1 using the genome as a template, and water as a negative control.
And (3) displaying a detection result: the lower limit of detection is 101CFU/mL, the system sensitivity is higher. The results are shown in FIG. 1.
Example 3 specificity test of the present detection System
The genomes of listeria monocytogenes, salmonella, escherichia coli, staphylococcus aureus, enterobacter faecium, yersinia were used as RAA amplification templates, respectively, and the detection was performed according to the method in example 1, while water was used as a negative control.
And (3) displaying a detection result: the system has strong specificity and no cross reaction exists in the detection process. The results are shown in FIG. 2.
Example 4 detection Effect of the detection System for different incubation durations
At 101And (3) using the CFU/mL bacterial liquid genome as an RAA amplification template, detecting according to the method in the embodiment 1, wherein the RAA amplification time is 10, 20 and 30min, the CRISPR/Cas13a reaction time is 10, 20 and 30min, and collecting and detecting fluorescence.
And (3) displaying a detection result: when the recombinase-mediated amplification time is 10min, the T7-CRISPR/Cas13a system can detect enough fluorescence intensity after reaction for 20min so that the negative control of the system presents difference; when the recombinase mediated amplification time is prolonged to 20min, the T7-CRISPR/Cas13a system can obviously distinguish negative and positive samples by fluorescence value difference only within 10 min. The total detection time of the two time distribution methods is 30min, and the shortest time consumption result of the detection system is shown in fig. 3.
Example 5 detection of interference rejection capability of the inventive System
Will 101-104After CFU/mL Listeria monocytogenes and the interfering bacterium liquid are fully mixed, genome DNA is extracted, and the genome DNA is used as an RAA amplification template to carry out detection according to the method in the embodiment 1. The interfering bacterium solution is 107CFU/mL Salmonella and 107CFU/mL Escherichia coli mixed bacterial liquid.
And (3) displaying a detection result: the system is able to withstand strong interference, i.e. at 107Under the condition that the CFU/mL salmonella and the escherichia coli coexist, the lower detection limit is still stable at 101CFU/mL, not affected by interfering bacteria. The results are shown in FIG. 4.
Example 6 testing of the ability of the system of the invention to detect Listeria in milk
Take 1mL of 100-102CFU/mL Listeria monocytogenes artificially pollutes 9mL milk, then 90mL BHI liquid culture medium is added for pre-enrichment treatment, samples are taken when the milk is cultured for 0, 2, 4 and 6 hours, genome DNA is extracted from all samples, and the samples are used as RAA amplification templates and are detected according to the method in the example 1.
And (3) displaying a detection result: the detection lower limit of the CRISPR/Cas13a-RAA system in a simulated sample (milk) is still kept at 101CFU/mL level, high sensitivity and stable detection capability. The results are shown in FIG. 5.
The above description is only a preferred embodiment of the present invention, and it will be apparent to those skilled in the art that various modifications and optimization can be made without departing from the technical principles of the present invention, and these modifications and optimization should also be considered as the protection scope of the present invention. Therefore, modifications made without departing from the technical spirit of the present invention are within the scope of the claims of the present invention.
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Claims (8)
1. A joint detection method for rapidly detecting Listeria monocytogenes is characterized by comprising the following steps:
(1) carrying out isothermal amplification on the bacterial sample DNA by using an amplification primer carrying a T7 promoter and an RAA amplification system to obtain a T7-DNA target fragment;
(2) a target design guide RNA, crRNA, against the listeria hly gene;
(3) commercial T7RNA polymerase, NTP, RAA product, Cas13a protein, crRNA, fluorescent reporter molecule and Listeria in vitro transcribed target-containing RNA fragment are mixed for isothermal incubation, and signals are collected and recorded by a fluorescence detector.
Wherein, the amplification primer carrying the T7 promoter is:
the upstream primer F is TAATACGACTCACTATAGGGCCGAAGTTTACATTCAAGCTATTATTTAC;
TAAAACGTGATTCATTAACACTCAGCATTG as downstream primer;
the amplified fragment is the 258-529 th base of the hly gene and is 272bp in total;
the target gene is crRNA of a specific target listeriolysin coding gene in a detection system, and the sequence is as follows:
5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUAAAUACAUUAGUGGAAAG-3’。
2. the joint detection method according to claim 1, characterized in that: the RAA amplification system in the step (1) comprises recombinase, DNA polymerase and single-stranded DNA binding protein.
3. The joint detection method according to claim 1, characterized in that: the steps (1) and (3) are both incubated at 38 ℃.
4. The joint detection method according to claim 1, characterized in that: and (3) forming a fragment carrying the target RNA by the RAA product T7-DNA in the reaction system in the step (3) under the action of T7RNA polymerase, specifically recognizing the fragment by the crRNA in the Cas13a-crRNA complex, simultaneously activating the cutting function of Cas13a, and shearing the fluorescent reporter molecule added in the detection system by utilizing the RNA enzyme activity which is not limited by the sequence after the activation of Cas13a so as to separate the fluorescent reporter molecule from the quenching group, obtain fluorescence and collect a fluorescent signal, thereby realizing the detection of the target gene.
5. The RAA primer pair for amplifying the target sequence of the hly gene of the listeria, wherein the upstream primer (F) comprises a T7 promoter, and the amplified fragment comprises the target sequence which is complementary to crRNA, and the sequence is as follows:
target RNA sequence:
5’-CUUUCCACUAAUGUAUUUAC-3’
RAA amplification primer sequences:
F:TAATACGACTCACTATAGGGCCGAAGTTTACATTCAAGCTATTATTTAC
R:TAAAACGTGATTCATTAACACTCAGCATTG。
6. a crRNA for a CRISPR/Cas13a system, specifically targeting a listeria monocytogenes hly gene, has the sequence as follows:
5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGUAAAUACAUUAGUGGAAAG-3’。
7. a CRISPR-Cas13a system for detecting listeria comprising the listeria monocytogenes hly gene target sequence of claim 5, a RAA amplification primer pair, and the crRNA of claim 6.
8. Use of the CRISPR-Cas13a system of claim 7 for the detection of listeria.
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