CN116837125A - Kit for rapidly detecting vibrio parahaemolyticus based on LAMP-CRISPR/Cas12b integrated system and method thereof - Google Patents
Kit for rapidly detecting vibrio parahaemolyticus based on LAMP-CRISPR/Cas12b integrated system and method thereof Download PDFInfo
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Abstract
The invention relates to a kit for rapidly detecting vibrio parahaemolyticus based on an LAMP-CRISPR/Cas12b integrated system and a method thereof, wherein the kit comprises an LAMP primer, an sgRNA guide sequence, cas12b enzyme and ssDNA fluorescent probes. The invention has the following advantages: the kit for visually detecting the vibrio parahaemolyticus comprises the steps of carrying out LAMP amplification on a sample to be detected, guiding a CRISPR-Cas12b system to carry out recognition and combination on LAMP amplification products and cut target double-stranded DNA under the mediation of a sgRNA guide sequence, activating a nonspecific nuclease function, then randomly cutting ssDNA fluorescent probes in the system to obtain a cut product, and finally judging whether cut FAM groups exist in a reaction system by detecting fluorescent signals of the cut product or using a colloidal gold test strip in a visual way. The LAMP-CRISPR/cas12b technology for detecting the vibrio parahaemolyticus provided by the invention detects the vibrio parahaemolyticus through double specificity of LAMP amplification and sgRNA recognition, has strong specificity, can be as low as 37CFU/mL in sensitivity, and can rapidly detect the vibrio parahaemolyticus polluted on food on site.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a technology for rapidly detecting vibrio parahaemolyticus by isothermal amplification and CRISPR/Cas12b specific cleavage of a detection target gene and a corresponding detection kit.
Background
Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a gram-negative halophilic bacterium which is widely distributed in marine products such as marine environments, fishes, shrimps and shellfish and is a common pathogenic bacterium in aquaculture industry. Food for human food poisoning caused by Vibrio parahaemolyticus is mainly marine products (fish, shrimp, crab, shellfish, etc. and products thereof), and can be detected in a large amount in fresh water products. Parahaemolytic vibrio food poisoning usually occurs in summer and autumn, and has acute onset and short latency, and symptoms mainly include diarrhea, enterospasm, nausea, emesis, fever and other typical gastroenteritis reactions, and severe cases can cause septicemia. Vibrio parahaemolyticus is distributed worldwide, especially in coastal areas, and the proportion of food poisoning caused by Vibrio parahaemolyticus in coastal cities in China in bacterial food poisoning events is higher than that of salmonella, diarrheal escherichia coli, staphylococcus aureus and the like, and the position is first. Therefore, the rapid and accurate detection of vibrio parasoluble in foods such as seafood is of great significance in controlling and preventing the bacteria from being popular in aquatic products or aquaculture environments and guaranteeing the food safety and health of people. Vibrio parahaemolyticus is one of important items for food safety monitoring, food poisoning source investigation and in-out aquatic product detection in China.
At present, conventional PCR, nested PCR, semi-nested PCR, fluorescent quantitative PCR and other technologies are established and developed at home and abroad to detect the vibrio parahaemolyticus, and the sensitivity and the accuracy of detection are continuously improved. At present, food safety national standard food microbiology test for detecting food-borne vibrio parahaemolyticus, namely vibrio parahaemolyticus test (GB 4789.7-2013), is a gold standard for detecting vibrio parahaemolyticus in China, and the method is based on bacterial culture, and comprises the following detection steps: sample preparation, enrichment, separation, pure culture, preliminary identification, definitive identification, serological test (selected), kanagawa test (selected). The operation is complicated, and the period is long (the total detection time is 48-120 h). In the detection of import and export foods in China, 5 vibrio parahaemolyticus detection are adopted according to the standard, and the molecular biological detection methods different from the traditional culture method of GB4789.7-2013 are respectively as follows: SN/T1869-2007 PCR method for rapid detection of various pathogenic bacteria in food, SN/T2424-2010 Rapid Vibrio parahaemolyticus identification detection method for Inlet and outlet food, real-time fluorescence PCR method for identification detection method, SN/T2754.5-2011 part 5 of detection method for Loop-mediated isothermal amplification (LAMP) of pathogenic bacteria in outlet food: vibrio parahaemolyticus, SN/T4603-2016 (export food and water body) detection method of common pathogenic genes of toxic Vibrio parahaemolyticus multiple PCR and multiple real-time fluorescence PCR method, SN/T5364.1-2021 (export food and water body) detection method of pathogenic bacteria microdroplet digital PCR method part 1): vibrio parahaemolyticus. The PCR and fluorescent quantitative PCR methods referred to herein require relatively expensive instrumentation and are performed in the laboratory and are not suitable for rapid detection in the field; in addition, in most cases, the number of contaminated vibrio parahaemolyticus bacteria in food may be small, and bacteria increase is required; the PCR method has low detection sensitivity, and if the number of polluted bacteria in food is low, the detection limit can not be reached after the bacteria increase, so that the detection can be missed.
The current isothermal amplification technology (such as LAMP, RPA, RAA and the like) has the advantages of short detection time, high sensitivity (for example, research shows that the LAMP can be 100-10000 times higher than the sensitivity of conventional PCR), good specificity, no need of expensive instruments and equipment (a constant temperature water bath pot or constant temperature metal bath) and the like, and the application of the isothermal amplification technology in the aspect of rapid detection of pathogenic microorganisms is concerned by scientific researchers, and has been used for rapid detection of food-borne pathogenic bacteria (such as salmonella, vibrio parahaemolyticus and the like) and viruses (such as novel coronavirus SARS-CoV-2). Since the number of the contaminated vibrio parahaemolyticus bacteria in food is probably low, the temperature amplification technology such as LAMP and RPA which has high sensitivity and is suitable for on-site rapid detection is a preferred choice for developing a related detection method. In addition, because of the non-specific amplification (false positive) that is easily generated in the LAMP and RPA detection processes, the lack of means for detecting positive samples further proves that the problem of non-specific amplification becomes an important factor for preventing related achievements from going to commercial application.
Chinese patent CN105219845a discloses a dual LAMP detection method and primer set for simultaneously detecting vibrio parahaemolyticus and vibrio vulnificus. Although the LAMP detection method based on ompA target gene in this patent can rapidly detect Vibrio parahaemolyticus, the problem of non-specific amplification caused by the high-sensitivity isothermal amplification technology is not solved. The LAMP method adopted in the industry standard SN/T2754.5-2011 for detecting the vibrio parahaemolyticus also has the possibility of non-specific amplification. The CRISPR-Cas system is derived from adaptive immunity of microorganisms and has been widely used in the field of genome editing due to its ease of use and stability. The CRISPR/Cas12a system belongs to the V-type CRISPR-Cas system. Cas12a nuclease recognizes PAM-side DNA target sequences complementary to CRISPR RNA (crRNA) spacers, so some researchers have combined LAMP and Cas12a to detect vibrio parahaemolyticus, but since the enzymes in the LAMP and CRISPR reactions work at different temperatures (Bst DNA polymerase: 60-65 ℃; cas12a:37 ℃), reagents for the CRISPR reaction must be added after the LAMP reaction. Some researchers have taken measures such as adding a soluble polyvinyl alcohol film as an automatic valve to avoid this problem (Yang T, et al biosensors (Basel) 2023,13 (1): 111), but the reliability of the results obtained from such treatments requires further verification and also increases the complexity of the experimental operation. Therefore, there is a need to provide a method for performing isothermal amplification such as LAMP or RPA, which has high sensitivity, short (rapid) detection time, simple operation, and no need for expensive equipment (suitable for on-site detection); but also can realize the detection method of accurate detection (high specificity) and the corresponding detection kit. Whereas the cas12b family is a class of endonucleases mediated by crrnas and tracrRNA (or engineered fusion sgrnas). In addition to having specific "cis-cleaving activity" against dsDNA or ssDNA targets, cas12b, after forming a ternary complex with sgRNA and target DNA, is also activated "trans-cleaving activity" against ssDNA, possibly to non-specifically cleave ssDNA in the system. Cas12b has good heat resistance, so the rapid detection of one-step nucleic acid can be realized by combining with LAMP.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provide a kit for simply, conveniently, rapidly and accurately detecting vibrio parahaemolyticus with high sensitivity.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a kit for rapidly detecting vibrio parahaemolyticus based on an LAMP-CRISPR/Cas12b integrated system comprises an LAMP primer, an sgRNA guide sequence, a Cas12b enzyme and a ssDNA fluorescent probe,
the nucleotide sequence of the LAMP primer is as follows:
F3:5’-AACTACTTCCCAACTCGC-3’，B3:5’-CTCCACTCGAACCAAACT-3’；
FIP：5’-CGATATTGTCCGAAAGGAAATTCGAAGATTTGGTTGATGAAGTTCTCA-3’；
BIP：5’-CTAGACATACACGCTCGTGAAAACCAGTGACAATCTTGGCTTA-3’；
the sgRNA guide sequence is as follows:
5’-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGC CCGUUGAACUUCAAGCGAAGUGGCACACGAGCGUGUAUGUCUAGGUCGA-3’。
preferably, the ssDNA fluorescent probe is a single-stranded nucleotide sequence marked by FAM or FITC at the 5 'end and BHQ1 at the 3' end, or a single-stranded nucleotide sequence marked by FAM or FITC at the 5 'end and Biotin at the 3' end.
Preferably, the nucleotide sequence of the ssDNA fluorescent probe is 5'-FAM-TTTTTT-BHQ1-3', 5'-FITC-TTTTTT-BHQ1-3', or 5'-FAM-TTTTTT-Biotin-3'.
Preferably, the visual detection kit comprises a colloidal gold detection test strip; the colloidal gold binding pad contains an anti-FAM antibody marked by colloidal gold, a detection line T and a quality control line C are arranged on the chromatographic membrane, the detection line T is coated with an anti-mouse antibody, and the quality control line C is coated with ligand streptavidin of Biotin.
Preferably, the kit further comprises positive control vibrio parahaemolyticus ATCC17802 strain genomic DNA and negative control escherichia coli DH5a genomic DNA.
The invention also provides application of the kit in visual detection of vibrio parahaemolyticus.
A method for visually detecting vibrio parahaemolyticus, comprising the steps of:
s1, extracting genome DNA of a sample to be detected;
s2, taking the DNA in the step S1 as a template, adding an LAMP primer into a reaction system to perform LAMP isothermal amplification reaction, and simultaneously adding AapCas12b enzyme, sgRNA and ssDNA fluorescent probes into the reaction system to perform Cas12 b-based CRISPR cleavage reaction, so that the LAMP isothermal amplification and CRISPR cleavage reaction are performed in the same reaction system simultaneously, and the LAMP amplification and CRISPR cleavage are performed simultaneously.
S3, judging a detection result: the reaction tube is arranged in an instrument capable of simultaneously carrying out constant-temperature reaction and fluorescence signal detection, and the detection result can be judged by collecting fluorescence signals in a reaction system and according to fluorescence signal curves at different time points; or the reaction tube is placed in a constant-temperature water bath kettle and a constant-temperature metal bath, and a fluorescent signal is detected by using a handheld ultraviolet lamp after the reaction is finished. Or the reaction product obtained in the step S2 is subjected to color development detection by using a colloidal gold test strip, and if a red strip appears in the detection line of the sample to be detected or red strips appear in the positions of the detection line of the sample to be detected and the quality control line of the negative sample, the detection of vibrio parahaemolyticus in the sample to be detected is indicated; if the detection line does not have a red band, and the quality control line has a red band, the fact that the vibrio parahaemolyticus is not detected in the sample to be detected is indicated.
Preferably, the reaction system of the step S2 is as follows: F3/B3 primers were each 0.2. Mu.M, FIP/BIP primers were each 1.6. Mu.M, 6mM MgSO4, 5. Mu.l 10X Isothermal Amplification Buffer II, 320units/mL Bst 3.0DNA Polymerase,50nM AapCas12b enzyme, 50nM sgRNA,250nM ssDNA,DNA template 5.0. Mu.L, ddH20 was added to 50. Mu.L, and the mixture was reacted at 60℃for 40-60 minutes after mixing.
Preferably, the reaction tube in the step S2 can perform constant temperature reaction and fluorescence signal detection at the same time; or reacting in water bath or metal bath at 60 ℃ for 40-60 minutes, and observing the result under a handheld ultraviolet lamp or performing visual detection by using a colloidal gold test strip for color development.
Compared with the prior art, the invention has the following advantages:
the kit for visually detecting the vibrio parahaemolyticus comprises the steps of carrying out LAMP amplification on a sample to be detected, guiding a CRISPR/Cas12b system to carry out recognition and combination on LAMP amplification products and cut target double-stranded DNA under the mediation of a sgRNA guiding sequence, activating a nonspecific nuclease function, then obtaining a cut product by a ssDNA fluorescent probe in any cut system, and finally judging through fluorescent signal detection of the cut product. The LAMP-CRISPR/cas12b technology for detecting the vibrio parahaemolyticus provided by the invention detects the vibrio parahaemolyticus through double specificity of LAMP amplification and sgRNA recognition, has strong specificity, can be as low as 37CFU/mL in sensitivity, and can be used for detecting and preventing the infection of the vibrio parahaemolyticus earlier. The kit has the characteristics of low cost, convenient operation, less time consumption, high sensitivity, strong specificity and the like, and the whole detection reaction process is carried out in an isothermal environment at 60 ℃, so that the dependence of a large laboratory instrument can be effectively eliminated.
The foregoing summary is provided merely for the purpose of the specification and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the present invention will become readily apparent by reference to the following detailed description and the entire text.
Drawings
FIG. 1 is a flow chart of the visual detection of Vibrio parahaemolyticus based on the LAMP-CRISPR/Cas12b system of the present invention;
FIG. 2 is a diagram showing the results of LAMP detection specificity experiments of Vibrio parahaemolyticus according to the present invention;
FIG. 3 is a screening chart of the sgRNA of the opaR gene of Vibrio parahaemolyticus according to the present invention;
FIG. 4 is a graph showing the experimental results of sensitivity of the LAMP-CRISPR/Cas12b one-step method for detecting Vibrio parahaemolyticus according to the present invention;
FIG. 5 is a graph showing the results of a specific assay for detecting Vibrio parahaemolyticus by the LAMP-CRISPR/Cas12b one-step method of the present invention.
FIG. 6 is a diagram of the detection result of the lateral flow detection test strip for detecting Vibrio parahaemolyticus by the LAMP-CRISPR/Cas12b one-step method of the invention.
Detailed Description
Reference will now be made in detail to specific embodiments of the invention. While the invention will be described in conjunction with these specific embodiments, it will be understood that they are not intended to limit the invention to these specific embodiments. On the contrary, these embodiments are intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the invention as defined by the appended claims. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the present invention.
When used in conjunction with the description herein and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The present invention will be described in further detail in connection with the following.
In combination with the above, the first object of the present invention is to provide a simple, rapid, highly sensitive and accurate method for detecting Vibrio parahaemolyticus.
The second object of the invention is to provide the application of the kit in visual detection of vibrio parahaemolyticus.
A third object of the present invention is to provide a kit for visual detection of Vibrio parahaemolyticus.
The aim of the invention is achieved by combining LAMP isothermal amplification and CRISPR specific cleavage based on heat-resistant Cas12b, and the technical scheme can be implemented by completing detection under the same reaction temperature of the same reaction system, and the specific scheme is as follows:
a kit for rapidly detecting vibrio parahaemolyticus based on an LAMP-CRISPR/Cas12b integrated system comprises an LAMP primer, an sgRNA guide sequence, a Cas12b enzyme and a ssDNA fluorescent probe,
the nucleotide sequence of the LAMP primer is as follows:
F3:5’-AACTACTTCCCAACTCGC-3’，B3:5’-CTCCACTCGAACCAAACT-3’；
FIP：5’-CGATATTGTCCGAAAGGAAATTCGAAGATTTGGTTGATGAAGTTCTCA-3’；
BIP：5’-CTAGACATACACGCTCGTGAAAACCAGTGACAATCTTGGCTTA-3’；
the sgRNA guide sequence is as follows:
5’-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGC CCGUUGAACUUCAAGCGAAGUGGCACACGAGCGUGUAUGUCUAGGUCGA-3’。
the invention designs a specific LAMP amplification primer aiming at the conserved region of the opaR gene DNA sequence in the vibrio parahaemolyticus genome, and prepares an sgRNA guide sequence through reverse transcription and purification; the target DNA in the sample to be detected is subjected to LAMP amplification to enrich the nucleic acid detection target, and the Cas12b enzyme guided by sgRNA can specifically recognize and cut the dsDNA target with PAM; meanwhile, after the Cas12b forms a ternary complex with the sgRNA and the target DNA, the trans-shearing activity of the ssDNA can be activated, the ssDNA in a nonspecific shearing system can be activated, and after FAM and BHQ1 on the ssDNA probe molecule are cut off, fluorescent signals emitted by the FAM are detected, so that specific detection (such as fluorescence) is generated. The above reaction is completed in one step under the same reaction system and the same temperature (60 ℃) for 40-60 minutes.
The ssDNA fluorescent probe is a single-stranded nucleotide sequence marked by FAM at the 5 'end and BHQ1 at the 3' end, or a single-stranded nucleotide sequence marked by FITC at the 5 'end and BHQ1 at the 3' end; after the ssDNA fluorescent probe is cut, FAM and BHQ1 are not on the same ssDNA, so that by detecting FAM fluorescent signals under the excitation of blue-green light, a small instrument, a hand-held ultraviolet lamp or naked eyes are used for detecting whether the vibrio parahaemolyticus exists in a target system.
The nucleotide sequence of the ssDNA fluorescent probe is 5'-FAM-TTTTTT-BHQ1-3', or 5'-FAM-TTTTTT-Biotin-3'.
Positive control vibrio parahaemolyticus ATCC17802 genomic DNA and negative control escherichia coli DH5a genomic DNA are also included.
The kit is applied to visual detection of vibrio parahaemolyticus.
A method for visually detecting vibrio parahaemolyticus, comprising the steps of:
s1, extracting genome DNA of a sample to be detected;
s2, taking the DNA in the step S1 as a template, adding the LAMP primer in the claim 1 into a reaction system to perform LAMP isothermal amplification reaction, and simultaneously adding AapCas12b enzyme, sgRNA (helping AapCas12b to recognize and cut target DNA) and ssDNA fluorescent probe into the reaction system to perform Cas12 b-based CRISPR cutting reaction, so that LAMP isothermal amplification and CRISPR cutting reaction are performed simultaneously in a reaction system, and LAMP amplification and CRISPR cutting are performed simultaneously;
s3, judging a detection result: in a laboratory, placing a reaction tube in an instrument (such as a fluorescent quantitative PCR instrument) capable of performing constant-temperature reaction and fluorescent signal detection, and judging detection results by collecting fluorescent signals in a reaction system and according to fluorescent signal curves at different time points; in the field detection, placing the reaction tube in a water bath or a metal bath at 60 ℃ for reaction for 40-60 minutes, and observing the result under a handheld ultraviolet lamp, wherein if the reaction product has no fluorescent brightness under ultraviolet irradiation or has no brightness under naked eye observation, the vibrio parahaemolyticus is not detected in the sample to be detected; if the reaction product has fluorescent brightness under the irradiation of an ultraviolet lamp or has brightness observed by naked eyes, the detection of the vibrio parahaemolyticus by the sample to be detected is indicated. Or the reaction product obtained in the step S2 is subjected to color development detection by using a colloidal gold test strip, if a red strip appears in the detection line of the sample to be detected or red strips appear in the positions of the detection line of the sample to be detected and the quality control line of the negative sample, the detection of vibrio parahaemolyticus in the sample to be detected is indicated, and if the red strip does not appear in the detection line and the red strip appears in the quality control line, the detection of vibrio parahaemolyticus in the sample to be detected is indicated.
LAMP detection specificity experiment result of vibrio parahaemolyticus: as shown in FIG. 2, A is 2% agarose gel; b, observing SYBR Green I under natural light; c, observing under SYBR Green I ultraviolet light; a2% agarsose gel; SYBR Green I under daylight; SYBR Green I under UV light; m: a DNA molecular mass standard; 1-11: vibrio parahaemolyticus genome, staphylococcus aureus, listeria monocytogenes, clostridium perfringens, shigella flexneri, shigella dysenteriae, escherichia coli O157: h7, salmonella typhimurium, yersinia enterocolitica, campylobacter jejuni genomic DNA, and ddH2O.
Screening of Vibrio parahaemolyticus opaR gene sgRNA: as shown in fig. 3, a is a real-time fluorescent amplification plot; b is 365nm ultraviolet light observation.
The sensitivity experiment result of LAMP-CRISPR/Cas12b one-step method for detecting vibrio parahaemolyticus: as shown in fig. 4, a is a fluorescence signal detected on a real-time fluorescence PCR instrument; b is the observation result under an ultraviolet lamp. The bacterial strain of Vibrio parahaemolyticus ATCC17802 after 10-fold ratio dilution is firstly subjected to plate count, and the colony forming unit of Vibrio parahaemolyticus OD600 nm=1.0 is determined to be 3.7X10 8 CFU/mL. Respectively at 3.7X10 6 -3.7×10 0 Genomic DNA extracted from CFU/mL 7 vibrio parahaemolyticus with different concentrations is used as a detection template, and is detected by a LAMP-CRISPR/Cas12b one-step method. Experimental results show that the minimum detection limit of LAMP-CRISPR one-step method for detecting vibrio parahaemolyticus based on opaR target gene is 3.7X10 1 CFU/mL。
The reaction system of the step S2 is as follows: F3/B3 primers were each 0.2. Mu.M, FIP/BIP primers were each 1.6. Mu.M, 6mM MgSO4, 5. Mu.l 10X Isothermal Amplification Buffer II, 320units/mL Bst 3.0DNA Polymerase,50nM AapCas12b enzyme, 50nM sgRNA,250nM ssDNA,DNA template 5.0. Mu.L, ddH2O were added to 50. Mu.L, and the mixture was reacted at 60℃for 40-60 minutes after mixing.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Vibrio parahaemolyticus ATCC17802 genomic DNA was used as positive control, and E.coli DH5a genomic DNA was used as negative control.
The invention provides a visual detection method of vibrio parahaemolyticus based on a LAMP-CRISPR/Cas12b system, the flow is shown in figure 1, by carrying out LAMP amplification on a sample to be detected, then under the mediation of an sgRNA guide sequence, the CRISPR/Cas12b system is guided to carry out recognition and combination on LAMP amplification products and cut target double-stranded DNA, the function of nonspecific nuclease is activated, then ssDNA fluorescent probes in a reaction system are cut at will to obtain a cleavage product, finally, the cleavage product is judged through chromogenic detection, and various friendly using terminals can be adopted to display detection results.
Example one, LAMP primer design screening and amplification system determination:
the opaR gene DNA sequence (5'-ATGGACTCAATTGCAAAGAGACCTAGAACTAGGCTTTCTCCTCTTAAACGTAAGCAACAACTCATGGAAATCGCGTTAGAAGTATTTGCACGCCGTGGTATTGGCCGTGGTGGTCACGCAGATATTGCTGAAATTGCGCAAGTGTCTGTTGCAACCGTTTTTAACTACTTCCCAACTCGCGAAGATTTGGTTGATGAAGTTCTCAACCATGTTGTCCGTCAGTTCTCGAATTTCCTTTCGGACAATATCGACCTAGACATACACGCTCGTGAAAACATCGCAAACATCACCAATGCGATGATCGAGCTTGTAAGCCAAGATTGTCACTGGCTGAAAGTTTGGTTCGAGTGGAGCGCATCAACTCGTGATGAAGTTTGGCCTCTATTTGTGTCTACCAACCGCACTAACCAATTACTGGTTCAAAACATGTTCATTAAAGCGATTGAACGCGGTGAAGTGTGTGATCAACACGATTCAGAACACTTGGCAAATCTATTCCACGGTATTTGTTACTCGCTGTTCGTACAAGCAAACCGCTTCAAAGGTGAAGCCGAGTTGAAGGAACTCGTGAGCGCTTACCTAGATATGCTTTGCATCTACAATCGCGAACACTAA-3') in the vibrio parahaemolyticus Colony83 genome (NZ_CP 078705) is designed with the assistance of PrimerExplorer V5 software to obtain a series of primer pairs meeting the LAMP primer design principle, and the primer pairs are synthesized by Beijing qing department biology (PAGE purification); after a series of early detection and screening. Primer screening results are shown below:
F3:5’-AACTACTTCCCAACTCGC-3’，B3:5’-CTCCACTCGAACCAAACT-3’；FIP：5’-CGATATTGTCCGAAAGGAAATTCGAAGATTTGGTTGATGAAGTTCTCA-3’；BIP：5’-CTAGACATACACGCTCGTGAAAACCAGTGACAATCTTGGCTTA-3’；
the size of the amplified fragment is 191bp;
after the optimal primer group is determined, the reaction systems such as primer concentration, mgSO4 concentration, amplification temperature, amplification time and the like are optimized.
The LAMP amplification system after optimization is as follows: 10X Isothermal Amplification Buffer II 2.5. Mu.L, dNTPs mix (10 mM) 3.5. Mu.L, F3 (10. Mu.M) 0.5. Mu.L, B3 (10. Mu.M) 0.5. Mu.L, FIP (10. Mu.M) 4.0. Mu.L, BIP (10. Mu.M) 4.0. Mu.L, bst 3.0DNA polymerase (8000U/mL) 1.0. Mu.L, sample DNA 2.0. Mu.L, ddH2O to 25.0. Mu.L.
Example two, sgRNA screening:
according to the 191bp DNA sequence amplified by LAMP, 6 possible Cas12b shear PAM sites (5 ' -TTN-3 ') and 20-23 bp fragments at the 3' end thereof are selected as targets of potential sgRNA guide sequences. Using plasmid (constructed in the applicant laboratory) carrying T7 promoter and Cas12b DR DNA fragment (synthesized by Beijing qing department Biotechnology Co., ltd.) as template, amplifying with Q5 high-fidelity DNA polymerase to obtain target (20-23 bp) DNA fragment containing T7 promoter, cas12b DR and potential sgRNA guide sequence, purifying and recovering, then using HiScribe TM Reverse transcription of T7 rapid and efficient RNA synthesis kit (NEB) to obtain RNA, and re-usingThe RNA purification kit (NEB) was purified to give each sgRNA. The LAMP-CRISPSR/Cas 12b one-step method is adopted to verify and screen each designed sgRNA. Finally, the sgRNA sequence which can be used for the LAMP-CRISPSR/Cas 12b one-step detection system is obtained as follows:
5’-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAACUUCAAGCGAAGUGGCACACGAGCGUGUAUGUCUAGGUCGA-3’。
specificity and sensitivity determination of the LAMP-CRISPR/cas12b method:
1. method of
(1) Based on the pre-optimized LAMP reaction system based on the opaR target gene, the reaction temperature of LAMP-CRISPR/Cas12b for detecting vibrio parahaemolyticus, the concentration of Cas12b and sgRNA and the like are further optimized, and the stability of the detection reaction is analyzed through collected fluorescent signals (curve and fluorescent intensity value).
(2) Respectively, the extracted vibrio parahaemolyticus, shigella dysenteriae and escherichia coli O157: h7, staphylococcus aureus, salmonella typhimurium, yersinia enterocolitica and listeria monocytogenes genome are used as templates, ultrapure sterile water is used as a blank control group, and the specificity of the detection system is analyzed by adopting a LAMP-CRISPR/Cas12b one-step method. Fluorescence signal was collected using a fluorescent quantitative PCR instrument to select FAM fluorescence channels. The reaction temperature was set to 60℃and the fluorescence signal was measured every 1min for 60 times for 1 hour, and then the detection result was determined by the fluorescence signal curve. After the reaction is finished, the measured PCR tube is placed under an ultraviolet lamp for observation, the positive reaction tube has green fluorescence reaction, and the negative reaction tube and the blank control group have no obvious effect.
(3) Firstly, the number of viable bacteria of the vibrio parahaemolyticus in each ml of bacterial liquid when the vibrio parahaemolyticus is cultured to the bacterial liquid concentration OD600 nm=1.0 is measured by adopting a plate counting method. Then, 10-fold gradient dilution was performed on the vibrio parahaemolyticus bacterial solution with a concentration of OD600 nm=1.0 with sterilized PBS, and 1ml of each concentration bacterial solution genome was extracted. And then the LAMP-CRISPR/Cas12b one-step detection method is used for measuring the system sensitivity.
2. Results
1. Specificity analysis of the detection system:
extracting vibrio parahaemolyticus, listeria monocytogenes and escherichia coli O157 respectively: h7, staphylococcus aureus, salmonella typhimurium, shigella dysenteriae, campylobacter jejuni genome and ultrapure sterile water are used as detection templates, and the specificity of the established LAMP-CRISPR/Cas12b one-step vibrio parahaemolyticus detection method is analyzed. The result shows that when only the genome DNA of vibrio parahaemolyticus is used as a detection template, an amplification curve appears in real-time fluorescence detection, and when other genome DNA of 6 food-borne pathogens or ultrapure sterile water is used as a template, no obvious fluorescence signal appears. When the reaction tube is placed under an ultraviolet lamp for observation, only the reaction tube taking the vibrio parahaemolyticus genome DNA as a template shows obvious green fluorescence, and other groups have no fluorescence reaction. Therefore, the established LAMP-CRISPR/Cas12b one-step method has good specificity for detecting the vibrio parahaemolyticus system, and as shown in figure 5, A is a fluorescent signal curve graph on a real-time fluorescent PCR instrument; b is the result observed under 365nm ultraviolet and the like.
Fourth embodiment is a kit for rapid detection of vibrio parahaemolyticus:
a kit for visually detecting vibrio parahaemolyticus based on an LAMP-CRISPR/Cas12b integrated detection system comprises LAMP primers, sgRNA guide sequences, ssDNA fluorescent probes, an LAMP reaction system and reagents required by a CRISPR/Cas12b cleavage detection system, the genomic DNA of vibrio parahaemolyticus ATCC17802 strain as a positive control, and the genomic DNA of escherichia coli DH5a as a negative control.
The required instrument: an instrument (such as a fluorescent quantitative PCR instrument and a small special instrument) capable of simultaneously carrying out isothermal amplification and fluorescent signal detection, or a constant-temperature water bath (constant-temperature metal bath) and a handheld ultraviolet lamp.
The LAMP primer:
F3:5’-AACTACTTCCCAACTCGC-3’，B3:5’-CTCCACTCGAACCAAACT-3’；FIP：5’-CGATATTGTCCGAAAGGAAATTCGAAGATTTGGTTGATGAAGTTCTCA-3’；BIP：5’-CTAGACATACACGCTCGTGAAAACCAGTGACAATCTTGGCTTA-3’；
the sgRNA guide sequence:
5’-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAG GUGGCAAAGCCCGUUGAACUUCAAGCGAAGUGGCACACGAGCGUGUAUGUCUAG GUCGA-3’；
the ssDNA fluorescent probe:
the fluorescent probe is used for detecting whether vibrio parahaemolyticus exists in a target system under the excitation of blue-green light or naked eyes under the condition of 5'-FAM-TTTTTT-BHQ 1-3'.
The method for visually detecting the vibrio parahaemolyticus by using the kit comprises the following steps of:
(1) Extracting genome DNA of a sample to be detected;
(2) The LAMP isothermal amplification and CRISPR cleavage reactions were performed simultaneously in one reaction system using the DNA of step S1 as a template, and in one reaction system, LAMP primer, bst 3.0DNA polymerase, aapCas12b enzyme, sgRNA, ssDNA fluorescent probe (5 '-FAM,3' -BHQ 1), and the like.
(2) And (3) carrying out color development or naked eye observation on the reaction product under blue-green light, wherein if the cleavage product has no fluorescent brightness or no brightness under the blue-green light irradiation, the detection of vibrio parahaemolyticus in the sample to be detected is indicated, and if the cleavage product has fluorescent brightness or brightness under the blue-green light irradiation, the detection of vibrio parahaemolyticus in the sample to be detected is indicated.
(3) And (3) judging a detection result: in a laboratory, placing a reaction tube in an instrument (such as a fluorescent quantitative PCR instrument) capable of simultaneously carrying out constant-temperature reaction and fluorescent signal detection, and judging detection results by collecting fluorescent signals in a reaction system and according to fluorescent signal curves at different time points; in the field detection, placing the reaction tube in a water bath at 60 ℃ for 40-60 minutes, and observing the result under a handheld ultraviolet lamp, wherein if the reaction product has no fluorescent brightness under ultraviolet irradiation or is observed with naked eyes to have no brightness, the vibrio parahaemolyticus is not detected in the sample to be detected; if the reaction product has fluorescent brightness under the irradiation of an ultraviolet lamp or has brightness observed by naked eyes, the detection of the vibrio parahaemolyticus by the sample to be detected is indicated.
The LAMP-CRISPR/Cas12b integrated detection reaction system for vibrio parahaemolyticus in the step (2) comprises the following steps: F3/B3 primers each 0.2. Mu.M, FIP/BIP primers each 1.6. Mu.M, 6mM MgSO4, 5. Mu.l 10X Isothermal Amplification Buffer II, 320units/mL Bst 3.0DNA Polymerase,50nM AapCas12b enzyme, 50nM sgRNA,250nM ssDNA,DNA template 5.0. Mu.L, ddH2O to 50. Mu.L. Mixing evenly and then placing at 60 ℃ to react for 40-60 minutes.
Fifth embodiment of the colloidal gold detection kit for visually detecting Vibrio parahaemolyticus
A kit for visually detecting vibrio parahaemolyticus based on an LAMP-CRISPR/Cas12b integrated detection system comprises LAMP primers, sgRNA guide sequences, ssDNA fluorescent probes, an LAMP reaction system and reagents required by a CRISPR/Cas12b cleavage detection system, the genomic DNA of vibrio parahaemolyticus ATCC17802 strain as a positive control, and the genomic DNA of escherichia coli DH5a as a negative control.
The FAM (FITC) -biotin immunoassay system required for visual detection is a commercial lateral flow assay test strip (CRISPR).
The LAMP primer:
F3:5’-AACTACTTCCCAACTCGC-3’，B3:5’-CTCCACTCGAACCAAACT-3’；FIP：5’-CGATATTGTCCGAAAGGAAATTCGAAGATTTGGTTGATGAAGTTCTCA-3’；BIP：5’-CTAGACATACACGCTCGTGAAAACCAGTGACAATCTTGGCTTA-3’；
the sgRNA guide sequence:
5’-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCA GGUGGCAAAGCCCGUUGAACUUCAAGCGAAGUGGCACACGAGCGUGUAUGUCUA GGUCGA-3’；
the ssDNA fluorescent probe:
after the fluorescent probe is cut, the FAM is separated from the Biotin, and then a lateral flow detection test strip (CRISPR) detects the cut FAM group, and whether the vibrio parahaemolyticus exists in a target system is judged according to whether a red strip appears at the detection line.
The method for visually detecting the vibrio parahaemolyticus by using the kit comprises the following steps of:
(1) Extracting genome DNA of a sample to be detected;
(2) LAMP isothermal amplification and CRISPR cleavage reactions were performed simultaneously in one reaction system using the DNA of step S1 as a template, in one reaction system, LAMP primer, bst 3.0DNA polymerase, aapCas12b enzyme, sgRNA, ssDNA fluorescent probe (5 '-FAM,3' -Bio), and the like.
The LAMP-CRISPR/Cas12b integrated detection reaction system for vibrio parahaemolyticus in the step (2) comprises the following steps: F3/B3 primers each 0.2. Mu.M, FIP/BIP primers each 1.6. Mu.M, 6mM MgSO4, 5. Mu.l 10X Isothermal Amplification Buffer II, 320units/mL Bst 3.0DNA Polymerase,50nM AapCas12b enzyme, 50nM sgRNA,100nM ssDNA,DNA template 5.0. Mu.L, ddH2O to 50. Mu.L. Mixing evenly and then placing at 60 ℃ to react for 40-60 minutes.
(3) A lateral flow test strip (CRISPR) was placed in the reaction tube above, and the liquid level was not allowed to exceed the MAX line. The results were prevented from interpretation for 7-10 minutes.
(3) And (3) judging a detection result: as shown in fig. 6, the detection line and the quality control line both appear or the test strip appears obvious red stripes, and the result is positive; only the detection line showed a red band, and the result was negative. In the figure, the test strip 1 is used for detecting the genome DNA of vibrio parahaemolyticus, and the test strips 2-6 are used for sequentially detecting salmonella and enterohemorrhagic escherichia coli O157: h7, listeria monocytogenes, staphylococcus aureus, and campylobacter jejuni genomic DNA.
In the sixth embodiment, the kit for rapidly detecting vibrio parahaemolyticus based on LAMP-CRISPR/Cas12b integrated detection system is used for detecting clinical samples:
from 9 months 2022 to 3 months 2023, 50 parts of sea fish samples are collected from sea market such as Shanghai Ming region, fengxian region, pingjiang region, jinshan region and Qing Pu region, dai vegetable market, shengxian super market and Dai supermarket respectively, and the vibrio parahaemolyticus carried in the food is detected by a kit (adopting a fluorescent quantitative PCR instrument) for detecting vibrio parahaemolyticus by adopting an LAMP-CRISPR/Cas12b integrated detection system and a national standard method (GB 4789.7-2013). The experimental result shows that in 50 clinical samples detected, the LAMP-CRISPR/Cas12b one-step detection method is adopted to detect the vibrio parahaemolyticus in 6 sea fish samples, and the detection result is consistent with that of the national standard method, and the coincidence rate is 100%. This suggests that the LAMP-CRISPR/Cas12b integrated detection system utilizing Vibrio fulgidus can be used for rapid detection of clinical samples.
The invention and its embodiments have been described above with no limitation, and the actual construction is not limited to the embodiments of the invention as shown throughout. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.
Claims (9)
1. A kit for rapidly detecting vibrio parahaemolyticus based on an LAMP-CRISPR/Cas12b integrated system is characterized by comprising an LAMP primer, an sgRNA guide sequence, cas12b enzyme and ssDNA fluorescent probes,
the nucleotide sequence of the LAMP primer is as follows:
F3:5’-AACTACTTCCCAACTCGC-3’，B3:5’-CTCCACTCGAACCAAACT-3’；
FIP：5’-CGATATTGTCCGAAAGGAAATTCGAAGATTTGGTTGATGAAGTTCTCA-3’；
BIP：5’-CTAGACATACACGCTCGTGAAAACCAGTGACAATCTTGGCTTA-3’；
the sgRNA guide sequence is as follows:
5’-GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGA ACUUCAAGCGAAGUGGCACACGAGCGUGUAUGUCUAGGUCGA-3’。
2. the kit for rapidly detecting vibrio parahaemolyticus based on the LAMP-CRISPR/Cas12b integrated system according to claim 1, which is characterized in that: the ssDNA fluorescent probe is a single-stranded nucleotide sequence marked by FAM or FITC at the 5 'end and BHQ1 at the 3' end, or a single-stranded nucleotide sequence marked by FAM or FITC at the 5 'end and Biotin at the 3' end.
3. The kit for rapidly detecting vibrio parahaemolyticus based on the LAMP-CRISPR/Cas12b integrated system according to claim 1, which is characterized in that: the nucleotide sequence of the ssDNA fluorescent probe is 5 '-FAM-TTTTTTTT-BHQ 1-3', 5'-FITC-TTTTTT-BHQ1-3' or 5'-FAM-TTTTTT-Biotin-3'.
4. The kit for rapidly detecting vibrio parahaemolyticus based on the LAMP-CRISPR/Cas12b integrated system according to claim 1, which is characterized in that: positive control vibrio parahaemolyticus ATCC17802 genomic DNA and negative control escherichia coli DH5a genomic DNA are also included.
5. Use of the kit according to any one of claims 1 to 4 for the visual detection of vibrio parahaemolyticus.
6. A method for visually detecting vibrio parahaemolyticus, comprising the steps of:
s1, extracting genome DNA of a sample to be detected;
s2, taking the DNA in the step S1 as a template, adding an LAMP primer into a reaction system to perform LAMP isothermal amplification reaction, and simultaneously adding AapCas12b enzyme, sgRNA and ssDNA fluorescent probes into the reaction system to perform Cas12 b-based CRISPR cleavage reaction, so that the LAMP isothermal amplification and the CRISPR cleavage reaction are performed in the same reaction system simultaneously, and the LAMP amplification and CRISPR cleavage are performed simultaneously;
s3, judging a detection result: placing an instrument capable of performing constant temperature reaction and fluorescence signal detection in a reaction tube, and judging a detection result by collecting fluorescence signals in a reaction system according to fluorescence signal curves at different time points; or the reaction tube is placed in a constant-temperature water bath kettle or a constant-temperature metal bath, and a fluorescent signal is detected by using a handheld ultraviolet lamp after the reaction is finished;
or the reaction product obtained in the step S2 is subjected to color development detection by using a colloidal gold test strip, if a red strip appears in the detection line of the sample to be detected or red strips appear in the positions of the detection line of the sample to be detected and the quality control line of the negative sample, the detection of vibrio parahaemolyticus in the sample to be detected is indicated, and if the red strip does not appear in the detection line and the red strip appears in the quality control line, the detection of vibrio parahaemolyticus in the sample to be detected is indicated.
7. A method for visually detecting Vibrio parahaemolyticus according to claim 6, wherein: the visual detection comprises an immunochromatography detection-lateral flow colloidal gold detection test strip by a FAM or FITC-biotin report system, a detection line T and a quality control line C are arranged on a chromatographic membrane of the test strip, a gold-labeled anti-FAM or FITC antibody is coated on a sample loading area of the chromatographic membrane, streptavidin is coated on the quality control line C, and an anti-mouse antibody is coated on the detection line T.
8. The method for visually inspecting vibrio parahaemolyticus according to claim 6, wherein the reaction system of step S2 is: F3/B3 primers were each 0.2. Mu.M, FIP/BIP primers were each 1.6. Mu.M, 6mM MgSO4, 5. Mu.l 10X Isothermal Amplification Buffer II, 320units/mL Bst 3.0DNA Polymerase,50nM AapCas12b enzyme, 50nM sgRNA,250nM ssDNA,DNA template 5.0. Mu.L, ddH2O were added to 50. Mu.L, and the mixture was reacted at 60℃for 40 to 60 minutes after mixing.
9. The method for visual detection of Vibrio parahaemolyticus according to claim 6, wherein the reaction tube of step S2 is arranged to perform a constant temperature reaction and a fluorescent signal detection simultaneously; or reacting in water bath or metal bath at 60 ℃ for 40-60 minutes, and observing the result under a handheld ultraviolet lamp or performing visual detection by using a colloidal gold test strip for color development.
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