CN114075607B - On-site visualization kit for detecting listeria monocytogenes based on SHERLOCK and application - Google Patents
On-site visualization kit for detecting listeria monocytogenes based on SHERLOCK and application Download PDFInfo
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Abstract
The invention discloses a field visualization kit for detecting listeria monocytogenes based on SHERLOCK and application thereof, wherein the kit comprises a T7-RPA primer pair for amplifying a secretory endopeptidase (invasion associated secreted endopeptidase, iap) gene fragment related to listeria monocytogenes invasiveness, and the primer pair consists of an upstream primer and a downstream primer, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; a complex formed by Cas13a protein and crRNA or both; and (3) an RNA fluorescent reporter molecule. In addition, the invention also discloses a method for detecting listeria monocytogenes by using the site visualization kit. By using the kit and the detection method for detecting the listeria monocytogenes, constant-temperature detection at 37-42 ℃ can be realized, and the result is visualized. The method has the advantages of simplicity in operation, rapidness in detection, visualization of results and the like. Thereby providing a technical means for improving the detection level of listeria monocytogenes in the food industry and reducing the infection risk of consumers.
Description
Technical Field
The invention relates to a SHERLOCK field visualization kit for rapidly detecting listeria monocytogenes and application thereof, and belongs to the technical field of molecular biological diagnosis.
Background
Listeria monocytogenes (Listeria monocytogene, abbreviated as listeria monocytogenes) is a gram-positive bacillus-free bacterium that is widely found in nature. Can form biological film on the surface of many objects, is not easy to be cleaned, can grow in the environment of 4 ℃, and is the main pollution bacteria of refrigerated food. Listeria monocytogenes disease is sporadic worldwide, and the death rate can reach 20% -40% in epidemic outbreaks.
At present, the detection of listeria monocytogenes in China comprises bacteriology, immunology and molecular biology. Bacteriology is a gold standard but is time-consuming and laborious, and immunological tests have low sensitivity to non-enriched food pathogen detection. The listeria monocytogenes has extremely low concentration in food, and has high requirement on the sensitivity of detection technology. With the development of molecular biology techniques, nucleic acid amplification methods including PCR, RPA, LAMP have been increasingly used for the identification of listeria monocytogenes. However, the existing molecular biology technology has high requirements on instruments or is easy to pollute in operation, complex in sample treatment and the like, and the amplified product can obtain a detection result only by agarose gel electrophoresis or fluorescent quantitative detection and other equipment, so that the field visualization can not be achieved.
In recent years, gene editing techniques based on CRISPR systems have been hot. CRISPR is essentially an adaptive immune system in bacteria that recognizes and activates the corresponding Cas protein through crRNA to break down the invader's DNA or RNA when re-invaded by a foreign microorganism. Whereas the nonspecific RNA-targeted cleavage activity of CRISPR/Cas13a brings new inspiration for nucleic acid detection. The nucleic acid detection platform SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) established by combining the RPA and the fluorescent reporter molecule has outstanding effect, can improve the sensitivity of the listeria monocytogenes detection technology by 100 times, and does not need any instrument. The detection result can reach the trace detection level of single base, and is efficient, low-cost, simple in operation, time-saving and labor-saving. The advantages enable the SHERLOCK method to be used for rapid detection and diagnosis of listeria monocytogenes in basic-level common laboratories or in the field.
Disclosure of Invention
The invention aims to provide a field visualization kit for detecting listeria monocytogenes based on a SHERLOCK technology and application thereof.
The second object of the present invention is to provide a method for detecting listeria monocytogenes using the above kit.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention discloses a field visualization kit for detecting listeria monocytogenes based on SHERLOCK, which comprises the following components:
1) T7-RPA primer pair for amplifying a listeria monocytogenes invasive related secretory endopeptidase (invasion associated secreted endopeptidase, iap) gene fragment, wherein the primer pair consists of an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;
2) A complex of Cas13a protein and crRNA, or both, the crRNA being used to bind to a ssRNA of interest amplified and transcribed from the T7-RPA primer while binding to and guiding Cas13a protein, the crRNA comprising a repeat sequence for binding to Cas13a and a guide sequence targeting a conserved region of a listeria monocytogenes iap gene; and
3) RNA fluorescent reporter molecules.
Wherein, preferably, the nucleotide sequence of the crDNA is shown as SEQ ID NO. 4.
Preferably, the Cas13a protein is an LwCas13a protein.
Preferably, the listeria monocytogenes invasive related secretory endopeptidase (invasion associated secreted endopeptidase, iap) gene fragment is located in GenBank ID:985140 from 545 to 678 of the listeria monocytogenes iap gene.
Preferably, the kit further comprises RPA basic lyophilized powder, mgOAc, T7 transcriptase, 10×RPA reaction buffer solution and NTP Mix; wherein the RPA basic lyophilized powder comprises a recombinase, a single-stranded DNA binding protein and a strand displacement polymerase.
Preferably, the kit further comprises a positive control and a negative control, wherein the positive control is the genome DNA of the listeria monocytogenes; the negative control is DEPC water.
Furthermore, the invention also provides application of the field visualization kit in detecting listeria monocytogenes in samples, wherein preferably, the samples comprise dairy products and meat products (raw/cooked meat (such as beef, pork and the like) tissue samples).
Furthermore, the invention also provides a method for detecting listeria monocytogenes by using the field visualization kit, which comprises the following steps:
step (1) extracting genome DNA in a sample to be detected;
step (2) carrying out a SHERLOCK reaction by taking the genome DNA extracted in the step (1) as a template;
among them, the reaction system used for carrying out the SHERLOCK reaction is preferably 50. Mu.L, and comprises: 4mg of RPA Basic lyophilized powder, 10. Mu.M of each of the upstream and downstream primers 2.4. Mu.L, 100nM of Cas13a protein, 10. Mu.L of 10 XRPA reaction buffer, 100nM of crRNA, 2. Mu.L of NTP Mix 6. Mu. L, T7 transcriptase, 5. Mu.M of RNA fluorescent reporter, 100ng of template, 2.5. Mu.L of 280mM MgOAc aqueous solution and DEPC water were made up to 50. Mu.L;
and (3) analyzing the detection result according to the fluorescence intensity after the SHERLOCK reaction in the step (2) is finished.
Preferably, the step (2) further comprises carrying out a SHERLOCK reaction by using the genome DNA of the listeria monocytogenes and DEPC water as templates; the SHERLOCK reaction result with the genomic DNA of the listeria monocytogenes as a template is used as a positive control, and the SHERLOCK reaction result with DEPC water as a template is used as a negative control.
Wherein, preferably, the reaction conditions of the SHERLOCK reaction in the step (2) are as follows: the reaction temperature is 37-42 ℃, the reaction time is 5-40 min, preferably the reaction temperature is 40 ℃, the reaction time is 40min, the operation process of the analysis and detection result can be a conventional method in the field, and the operation process can be specifically room temperature blue light transmission detection, fluorescent quantitative PCR detection and analysis of the detection result of an enzyme-labeled instrument. Preferably, the fluorescence generated by the reaction system is observed under a blue light transmission instrument with the wavelength of 470-520 nm, if the amplification product of the reaction system presents fluorescence, the sample contains the listeria monocytogenes, and if the amplification product of the reaction system does not present fluorescence, the sample does not contain the listeria monocytogenes.
Preferably, the RNA fluorescent reporter may be any suitable commercial product, and in the practice of the present invention, the RNA fluorescent reporter (RNase Alert) is a product of bleiss company, cat# M20801-P001 (a).
Wherein, preferably, the concentration of the ssRNA of the listeria monocytogenes used for detection by the detection kit is not less than 10fM.
Further, the kit may further comprise a sample nucleic acid extraction reagent; the method can be used for extracting sample nucleic acid by adopting the current commercial kit, and the specific experiment of the invention adopts a commercial kit extraction method and a boiling-cracking method, and further, the nucleic acid extraction reagent comprises lysozyme, buffer solution, lysate, elution buffer solution, a collecting pipe and the like.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a field visualization kit for detecting listeria monocytogenes based on SHERLOCK; the invention also provides a method for detecting listeria monocytogenes by using the on-site visualization kit, and by using the method, the polluted sample in food is successfully detected, the operation time is shortened, the detection time is less than 40 minutes, and meanwhile, no cross reaction is observed for any gram positive strain or food background natural flora, so that the method has high specificity and sensitivity. The fluorescent RNA reporter molecule is combined to perform on-site visual detection on the listeria monocytogenes, only a blue light transmission instrument with the wavelength of 470-520 nm is used for irradiation, and whether the amplification product of the reaction system shows fluorescence or not is observed, so that the detection result can be judged. Or quantifying the fluorescence intensity by using an enzyme-labeled instrument and fluorescence quantitative PCR. It does not require expensive equipment and reduces the cost and time of diagnosis for field detection. The primer set has the advantages of strong specificity, good sensitivity, simple operation, quick detection, visual result and the like, so that the SHERLOCK method established by the invention can be used for field visual detection of the listeria monocytogenes, thereby providing a technical means for improving the level of the listeria monocytogenes detected in the food industry and reducing the infection risk of consumers.
Drawings
FIG. 1 is a screen for T7-RPA primers for the listeria monocytogenes iap gene;
FIG. 2 shows the results of three amplifications of Listeria monocytogenes T7-RPA and SHERLOCK;
wherein: nucleic acid electrophoresis identification of positive samples in lanes 1-3;
FIG. 3 shows the results of designed listeria monocytogenes T7-RPA primer specific amplification;
wherein: lanes 1 are nucleic acid electrophoretic identifications of positive samples of Listeria monocytogenes, lanes 2-7 are genomic samples of Salmonella (Salmonella typhimurium), vibrio parahaemolyticus (Vibrio Parahemolyticus), escherichia coli (Escherichia coli), yersinia enterocolitica (Yersinia enterocolitica), shigella flexneri (Shigella flexneri), staphylococcus aureus (Staphylococcus aureus), and lane 8 is a negative control.
Fig. 4 is a sequence map of LwCas13a protein (2751 … 6209=3459 bp);
FIG. 5 is a coomassie brilliant blue staining identification of LwCas13a protein induced expression, purification and concentration;
wherein: left: eluting proteins with eluents of different imidazole concentrations; 1. eluting with a buffer containing 220mM imidazole; 2. eluting with a buffer containing 150mM imidazole 3. Eluting with a buffer containing 80mM imidazole; m. marker; right: purification and concentration of LwCas13a protein; 1. protein eluted with 80mM imidazole; 2. dialyzing the digested proteins; 3. concentrating the protein; m. marker;
FIG. 6 is a time-fluorescence curve of SHERLOCK detection of Listeria monocytogenes for the candidate 3 gene primer-crRNA complex;
FIG. 7 is a fluorescent histogram of SHERLOCK detection of Listeria monocytogenes using the optimal iap gene primer-crRNA complex;
FIG. 8 is a visual and histogram of SHERLOCK reaction specificity fluorescence for different species;
wherein: column 1 is a listeria monocytogenes positive standard; columns 2 to 8 are salmonella, vibrio parahaemolyticus, escherichia coli, yersinia small intestine, shigella flexneri, staphylococcus aureus and negative control, respectively;
FIG. 9 is a visual and histogram of fluorescence for detecting sensitivity of different concentrations of a template of Listeria monocytogenes by SHERLOCK;
wherein: the template concentrations for columns 1 to 8 were 150ng,15ng,1.5ng,150pg,15pg,1.5fg,150ag, respectively, for the negative control.
FIG. 10 is a graph showing the results of sensitivity detection of an artificial labeled milk sample;
wherein: 1-7 Listeria monocytogenes bacteria concentration is 5.34×10 respectively 6 CFU/mL、5.34×10 5 CFU/mL、5.34×10 4 CFU/mL、5.34×10 3 CFU/mL、5.34×10 2 CFU/mL、5.34×10 1 CFU/mL、5.34×10 0 CFU/mL；
FIG. 11 is a graph showing the results of sensitivity detection of an artificial labeled beef sample;
wherein: 1-7 Listeria monocytogenes bacteria concentration is 5.34×10 respectively 6 CFU/g、5.34×10 5 CFU/g、5.34×10 4 CFU/g、5.34×10 3 CFU/g、5.34×10 2 CFU/g、5.34×10 1 CFU/g、5.34×10 0 CFU/g。
Detailed Description
The following describes the embodiments of the present invention further with reference to the drawings and examples. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
The experimental procedures, without specific conditions noted in the examples below, were selected according to methods and conditions conventional in the art, or according to the commercial specifications. Reagents and starting materials were commercially available, for which specific components were not identified in the examples below. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
The basic principle of the method for detecting the listeria monocytogenes DNA based on the SHERLOCK provided by the invention is as follows: the method comprises the steps of firstly amplifying a specific DNA fragment through a T7-RPA amplification technology, then carrying out transcription to obtain ssRNA, then specifically combining the target fragment through crRNA, finally utilizing the RNA nonspecific enzyme cleavage activity of Cas13a to cut an RNA fluorescent reporter molecule, and detecting the listeria monocytogenes through a fluorescent signal.
Example 1 screening of primers and T7-RPA amplification detection
The bacterial genome extraction kit and the DNA purification recovery kit used in the invention are products of Tiangen company.
The invention designs three pairs of primers aiming at secretory endopeptidase (invasion associated secreted endopeptidase, iap) genes related to listeria monocytogenes invasion together, and the genes are highly conserved and consistent and exist in genomes of all pathogenic serotypes of listeria monocytogenes; the iap sequence is referred to GenBank accession number 985140. The nucleotide sequences of the primers are shown in Table 1:
T7-RPA primers used in Table 1
The lower case base is the T7 promoter. The primer is synthesized by vinca biosciences of vinca biosciences. The optimal primer pair is screened out through T7-RPA reaction.
In the invention, the nucleotide sequence of the amplified iap gene conserved region is shown as SEQ ID NO.3, and specifically comprises the following steps:
5’-GCCTAAAGTAGCAGAAACGAAAGAAACTCCAGTAGTAGATCAAAATGCTACTACACACGCTGTTAAAAGCGGTGACACTATTTGGGCTTTATCCGTAAAATACGGTGTTTCTGTTCAAGACATTATGTCATGG-3’
the genome of Listeria monocytogenes (Listeria monotygenes) 1/2C, salmonella (Salmonella typhimurium), vibrio parahaemolyticus (Vibrio Parahemolyticus), escherichia coli (Escherichia coli), yersinia enterocolitica (Yersinia enterocolitica), shigella flexneri (Shigella flexneri) and Staphylococcus aureus (Staphylococcus aureus) was extracted using a bacterial genome extraction kit (Tiangen Biotechnology Co., ltd.) and referring to the kit instructions, and finally the DNA was dissolved with 50. Mu.L of water to obtain an amplification template. All strains were maintained by the institute of zoonotic study at Jilin university. T7-RPA reactions were performed using primers iap-1F and iap-1R from different bacterial genomes as templates, respectively. The reaction procedure is: 40 ℃ for 20min. FIG. 1 shows that iap-1F/R amplification was optimal in three pairs of primers; FIG. 2 shows good reproducibility of the iap-1F/R amplified Listeria monocytogenes genome template, and FIG. 3 shows good specificity of the reaction of iap-1F/R amplification of each bacterial genome T7-RPA. The T7-RPA reaction system is shown in Table 2.
TABLE 2T 7-RPA reaction System of example 1
| Reaction components | Dosage (mu L) |
| Upstream primer (10. Mu.M) | 2.4μL |
| Downstream primer (10. Mu.M) | 2.4μL |
| RPA Basic freeze-dried powder | 4mg |
| 10 XRPA reaction buffer | 29.5μL |
| DNA template | 1.5μL |
| Sterile water | 11.7μL |
| 280mM MgOAc | 2.5μL |
| Totals to | 50μL |
Example 2 preparation of crRNA
In the invention, the preparation and purification method of crRNA is as follows:
the pre-crRNA comprises a 37nt repeat region at the 5 'end, a target sequence and a PFS at the 3' end (the sequence can be combined with LwCas13a protein, the sequence is 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3'), an annealing primer is designed by taking a single-stranded DNA sequence of the pre-crRNA as an amplification template, and an upstream primer iap-crRNA-F is designed to comprise a T7 sequence (5'-TAATACGACTCACTATAGGG-3') and a 20nt repeat sequence (5'-GATTTAGACTACCCCAAAAA-3'); the downstream primer iap-crRNA-R was designed to contain the reverse complement of the target sequence of about 20nt and the reverse complement of the upstream crRNA repeat, and the primer sequences are shown in Table 3. Subsequently, the double-stranded DNA after PCR annealing was recovered, and 1. Mu.g was used as a transcription template after detecting the concentration by using a Nanodrop spectrophotometer, and the transcription system was shown in Table 4. Transcription to crRNA using T7 transcriptase takes place, and the RNase-free DNase I is used to degrade the template DNA after overnight transcription at 37 ℃. The crRNA obtained was purified using a column RNA rapid purification kit (manufacturer). And measuring the concentration by using a spectrophotometer, and subpackaging and storing in a refrigerator at-80 ℃ for standby. The nucleotide sequence of the crRNA is shown as SEQ ID NO.4, and specifically comprises the following steps:
gauuuagacuaccccaaaaacgaaggggacuaaaacacggauaaagcccaaauaguguc。
TABLE 3 primers for crRNA sequences
| Name of the name | Sequence 5'-3' |
| iap-crRNA-F | TAATACGACTCACTATAGGGGATTTAGACTACCCCAAAAA |
| iap-crRNA-R | GGATAAAGCCCAAATAGTGTTTTTTGGGGTAGTCTAAATC |
TABLE 4 crRNA transcription System
| Reaction components | Dosage (mu L) |
| T7 transcriptase | 2μL |
| T7 reaction buffer | 3μL |
| NTP Mix | 7μL |
| Pre-crRNA double-stranded DNA template | 1μg |
| RNase-free water | Is added to 20 mu L |
EXAMPLE 3 expression purification of LwCas13a protein
1. Induction expression of LwCas13a proteins
1. Transformation
LwCas13a protein expression plasmid is transformed into Rosetta competent cells according to the reference specification, the transformed bacterial liquid is added to LB plates with corresponding resistance, and the liquid is smeared by a coating rod. Single colonies were picked into LB liquid medium of the corresponding resistance and shake-cultured overnight at a constant temperature of 37 ℃. Extracting plasmid, sequencing and identifying, and storing the accurate monoclonal glycerol bacteria.
2. Induction
Taking 10mL of fungus liquid cultured overnight, inoculating 1L of corresponding resistant LB liquid culture medium, culturing at 37 ℃ for 1.5-2 h, adding IPTG with the final concentration of 500 mu M, and culturing at 16 ℃ for 24h to induce protein expression. And (5) pre-cooling the bacterial cells in a centrifugal machine at 4 ℃ and centrifuging the bacterial cells for 10min at 8000g, and carrying out the subsequent purification process.
2. Purification of LwCas13a protein
The LwCas13a protein contains 1,152 amino acids with the size of about 150kD, and the plasmid map of the protein is expressed, so that the recombinant LwCas13a protein is provided with 2 tags (His-tag and Strep-tag) after the recombinant LwCas13a protein is used for preliminary purification of the protein, and SUMO enzyme cleavage sites are arranged between the tags and the protein, so that the protein can be separated from a solid-phase medium during purification, and the influence of the tags on the activity of the protein can be prevented.
1. Purification of LwCas13a protein by affinity chromatography
The cells were weighed, 3g of cells were washed twice with 30mL of PBS, the cells were resuspended using Binding Buffer, and lysozyme and protease inhibitor were added at a ratio of 1:100. Ultrasonic crushing, 30% of power, ultrasonic treatment for 2s at intervals of 4s and ultrasonic treatment for 20min, wherein bacterial liquid can be found to be clear after ultrasonic crushing. If the bacterial liquid is still turbid or has no obvious change after the ultrasonic treatment, the buffer liquid dosage can be properly increased, and the diluted bacterial liquid continues to be subjected to the ultrasonic treatment. The well-broken bacterial liquid was placed in a centrifuge tube and centrifuged at 8000rpm,4℃for 40min. The centrifuged supernatant was collected and filtered using a 0.22 μm filter to prepare the subsequent nickel column protein purification. After the nickel column (HisTrap HP column) was equilibrated using Binding Buffer, protein loading was performed. The target protein was bound to the column via His tag, non-specifically bound hetero-protein on the column was eluted using Binding Buffer, 800mM (16% B) was eluted, and samples were collected for electrophoretic identification (FIG. 5).
LwCas13a protein dialysis and SUMO cleavage
In order to prevent the high concentration imidazole in LwCas13a protein solution and the label on the protein from affecting the activity of the protein, the corresponding label structure is removed by dialysis replacement buffer solution and SUMO protease, the target protein obtained by the previous elution is added into a dialysis bag, the dialysis bag is sealed, the dialysis bag is placed into 500mL SUMO digestion buffer solution, the dialysis is carried out under stirring at 4 ℃ for about 1 hour, the protein concentration in the dialysis bag is measured after 2 hours, the SUMO protease is added according to the total protein amount by referring to the specification, and the mixture is spun and digested overnight at 4 ℃, and the sample is collected for electrophoresis identification (FIG. 5).
LwCas13a protein concentration
To increase protein activity, proteins were ultrafiltration concentrated and replaced with Storage Buffer using a millipore ultrafiltration centrifuge tube. Pre-cooling the protein solution at 4 ℃ and centrifuging at 3500rpm for 5min, concentrating the protein solution after enzyme digestion, adding 1-2 mL Storage Buffer when 500-1000 mu L of protein solution is remained in the tube, centrifuging again, collecting the protein solution in a clean ep tube, and freezing and storing the protein solution at-80 ℃ (figure 5).
Example 4 establishment and optimization of the reaction System
1. Nucleic acid extraction and template ssRNA preparation
For DNA samples, 100. Mu.L of tissue supernatant or sample is taken, bacterial DNA extraction is performed according to the specification of a DNA extraction kit (commercially available, for example, a kit purchased from TIANGEN), and finally, 50. Mu.L of water is used for dissolving the DNA to obtain an amplification template; T7-RPA amplification, after the concentration of the RPA product is purified and measured, DNA is transcribed into ssRNA by T7 transcriptase, and the ssRNA is frozen at-80 ℃ after being purified by using a column type RNA rapid purification kit (biotechnology).
After extracting the genome of each bacterium according to the method described in example 1, the concentration was measured by using a Nanodrop spectrophotometer, and after the amplification of T7-RPA (conditions and system refer to example 1), the transcription was carried out in vitro at 37℃overnight, and after purification and split charging, the transcription product was stored at-80℃and ready for subsequent detection.
2. Screening of optimal primers and crRNA
In the experimental process of the invention, three pairs of primers aiming at three different targets and three corresponding crRNAs are designed together, and are shown in tables 5 and 6:
TABLE 5 T7-RPA primer design
| Primer name | Sequence 5'-3' |
| prfA-F | taatacgactcactatagggTATGCGGAATCACGCGGCTGGAAA |
| prfA-R | ACGCTCATCTTGCATCGAAACGACA |
| hly-F | taatacgactcactatagggATTGATTATGATGACGAAATGGCTTA |
| hly-R | TGACTTCTTCTTGCATTTTCCCTTCA |
| iap-F | taatacgactcactatagggACTATTTGGGCTTTATCCG |
| iap-R | CTGTTTGTTGTTGCGTTGC |
TABLE 6 crRNA design
The reaction system and conditions for the experiments described in the present invention are shown in Table 7. Under the condition that the concentration of other components in the reaction system is unchanged, the same nucleic acid sample is subjected to SHERLOCK-LM detection by using the designed three pairs of primers and 3 crRNAs respectively, wherein the reaction condition is 37 ℃ for 40min. As a result, it was found that the fluorescence-time curve and fluorescence intensity of the reaction system containing iap-crRNA and its primer pair were significantly better than those of the reaction system containing prfA-crRNA and its primer pair and that of the reaction system containing hly-crRNA and its primer pair (FIGS. 6 and 7), so that iap-crRNA and its primer pair were selected for the subsequent reaction system optimization study.
TABLE 7 SHERLOCK detection System
| Reaction components | Dosage of |
| LwaCas13a protein | 100nM |
| Protein reaction buffer | 4μL |
| crRNA | 100nM |
| RNase Alert | 5μM |
| RNase inhibitor | 1μL |
| ssRNA templates | 100ng |
| RNase free water | Is added to 20 mu L |
The PCR tube containing the SHERLOCK reaction system is put into a fluorescent quantitative PCR instrument, the excitation light wavelength of a channel is set to 490nm, the emission light wavelength is set to 520nm, the fluorescent value is read once per minute at 37 ℃, and the fluorescent intensity change in the system is detected in a circulating way for 40 times.
3. Establishment of one-step SHERLOCK detection system
The invention optimizes the concentration of each component and the reaction time of the SHERLOCK detection system, and carries out one-step SHERLOCK detection by using the system and the conditions shown in the table 8:
table 8 one-step SHERLOCK detection system
| Reaction components | Dosage of |
| Upstream and downstream primers | 2.4 mu L each |
| RPA basic freeze-dried powder | 4mg |
| LwaCas13a protein | 100nM |
| 10 XRPA reaction buffer | 10μL |
| crRNA | 100nM |
| NTP Mix | 6μL(1mM each) |
| T7 transcriptase | 2μL |
| RNase Alert | 5μM |
| MgOAc | 2.5μL |
| DNA template | 100ng |
| RNase free water | Is added to 50 mu L |
Reaction conditions: the detection reaction condition is 40 ℃, the fluorescence value is read every 1min, and the cycle is 40 times.
4. Specificity and sensitivity based on SHERLOCK detection method
Templates for specific assays were tested using the genomes extracted in example 1, including listeria monocytogenes (Listeria monotygenes) 1/2C, salmonella (Salmonella typhimurium), vibrio parahaemolyticus (Vibrio Parahemolyticus), escherichia coli (Escherichia coli), yarrowia enterocolitica (Yersinia enterocolitica), shigella flexneri (Shigella flexneri), staphylococcus aureus (Staphylococcus aureus), with the addition of DEPC water as a negative control. The one-step SHERLOCK assay system was followed to verify the specificity of the method of the invention. The detection result of FIG. 8 shows that SHERLOCK has high specificity to the fluorescence intensity of the listeria monocytogenes gene, other bacteria and negative control have no fluorescence reaction, the method has no cross reaction in the detection process, the specificity is good, and the visualization can be realized.
The listeria monocytogenes genome template used for sensitivity detection is quantified and subjected to gradient dilution, and the prepared template concentration is 150ng,15ng,1.5ng,150pg,15pg,1.5fg and 150ag respectively per mu L, and DEPC water is used as a negative control. Compared with the negative control, the fluorescence intensities of the fluorescence histograms of fig. 9 all have statistical differences (one way ANOVA test, p=0.0023, P <0.0001;bars represent mean ±s.e.m.), and the result shows that the sensitivity of the method of the invention can reach 1.5fg, and simultaneously, the visualization can be realized.
Example 5 manually labeling a milk sample, a beef sample.
The invention adopts a boiling-lysate method aiming at the extraction of listeria monocytogenes in food samples, and the specific method is as follows:
manually adding a standard milk sample: and counting the listeria monocytogenes bacterial liquid through a flat plate to obtain the bacterial liquid concentration. Milk was boiled for 10min and centrifuged at 12000rpm for 2min to remove impurities such as fatty proteins, leaving a supernatant. Mixing the bacterial liquid with the supernatant of milk to obtain final concentration of Listeria monocytogenes in milk of 5.34×10 6 And (3) centrifuging at 12000rpm for 5min at CFU/mL, collecting thalli, washing twice with sterile physiological saline, adding 100 mu L of physiological saline to resuspend thalli, boiling for 10min with boiling water, centrifuging, adding lysate, standing at 37 ℃ for 10min, centrifuging again to obtain a supernatant genome, and diluting the supernatant with a 10-fold ratio as a template for analyzing the detection sensitivity of Listeria monocytogenes in the milk sample. The prepared sample was detected using the one-step SHERLOCK detection system and reaction conditions established in example 5, the detection result was visually observed using a blue light transmittance meter, as a control, T7-RPA amplification was performed, and the amplification result was detected using 2% agarose gel electrophoresis.
Manually adding a labeled beef sample: counting Listeria monocytogenes by plate to obtain bacterial liquid concentration, mincing beef into meat paste with small meat mincer, mixing thallus with meat paste to obtain Listeria monocytogenes in meat pasteThe final concentration of the bacteria is 5.34 multiplied by 10 6 CFU/g, re-suspending meat paste with sterile physiological saline of the same volume, filtering the meat paste, centrifuging the filtrate at 12000 rpm/min for 2min to collect thalli, washing the thalli twice with the sterile physiological saline, adding 100 mu L of physiological saline to re-suspend thalli, boiling with boiling water for 10min, centrifuging to add lysate, standing at 37 ℃ for 10min, centrifuging again to obtain a supernatant genome, and diluting the supernatant genome with a 10-fold ratio as a template for analyzing the detection sensitivity of Listeria monocytogenes in beef samples. The prepared sample was detected using the one-step SHERLOCK detection system and reaction conditions established in example 5, the detection result was visually observed using a blue light transmittance meter, as a control, T7-RPA amplification was performed, and the amplification result was detected using 2% agarose gel electrophoresis.
The detection sensitivity results of the invention on the Listeria monocytogenes in the artificial marked milk sample are shown in figure 10 (1-7: the concentration of the Listeria monocytogenes is 5.34 multiplied by 10 respectively) 6 CFU/mL、5.34×10 5 CFU/mL、5.34×10 4 CFU/mL、5.34×10 3 CFU/mL、5.34×10 2 CFU/mL、5.34×10 1 CFU/mL、5.34×10 0 CFU/mL), it can be seen that the minimum detection concentration of Listeria monocytogenes in milk samples in the T7-RPA reaction is 5.34×10 4 CFU/mL, while in SHERLOCK, the minimum detection concentration for Listeria monocytogenes in milk samples is 5.34×10 1 CFU/mL. The detection result of the invention on the Listeria monocytogenes in the artificial labeled beef sample is shown in figure 11 (1-7: the concentration of the Listeria monocytogenes is 5.34 multiplied by 10 respectively) 6 CFU/g、5.34×10 5 CFU/g、5.34×10 4 CFU/g、5.34×10 3 CFU/g、5.34×10 2 CFU/g、5.34×10 1 CFU/g、5.34×10 0 CFU/g), it can be seen that in the T7-RPA reaction, the minimum detection concentration of Listeria monocytogenes in beef samples is 5.34×10 5 CFU/g, but in SHERLOCK, the minimum detection concentration of Listeria monocytogenes on the artificial added-mark beef sample is 5.34×10 2 CFU/g。
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
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Claims (9)
1. The field visualization kit for detecting listeria monocytogenes based on SHERLOCK is characterized by comprising the following components:
1) T7-RPA primer pair for amplifying a listeria monocytogenes invasive related secretory endopeptidase (invasion associated secreted endopeptidase, iap) gene fragment located at GenBank ID:985140, the primer pair consists of an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2;
2) A complex formed by a Cas13a protein and a crRNA, or both, wherein the crRNA is used for combining a target ssRNA obtained after amplification and transcription of the T7-RPA primer, and simultaneously combining and guiding the Cas13a protein, the crRNA comprises a repetitive sequence for combining with the Cas13a and a guide sequence for targeting a listeria monocytogenes iap gene conservation region, the nucleotide sequence of the crDNA is shown as SEQ ID NO.4, and the Cas13a protein is LwCas13a protein; and
3) RNA fluorescent reporter molecules.
2. The site visualization kit according to claim 1, wherein the kit further comprises RPA basic lyophilized powder, mgOAc, T7 transcriptase, 10 x RPA reaction buffer, and NTP Mix; wherein the RPA basic lyophilized powder comprises a recombinase, a single-stranded DNA binding protein and a strand displacement polymerase.
3. The field visualization kit of claim 2, further comprising a positive control and a negative control, the positive control being a genomic DNA of listeria monocytogenes; the negative control is DEPC water.
4. Use of the field visualization kit of any one of claims 1-3 for detecting listeria monocytogenes in dairy or meat products.
5. A method for detecting listeria monocytogenes in milk or meat products using the site visualization kit of any of claims 1-3, comprising the steps of:
step (1): extracting genome DNA in a sample to be detected;
step (2): taking the genomic DNA extracted in the step (1) as a template to carry out a SHERLOCK reaction;
step (3): and (3) after the SHERLOCK reaction in the step (2) is finished, analyzing a detection result according to the fluorescence intensity.
6. The method of claim 5, further comprising performing a SHERLOCK reaction using the genomic DNA of Listeria monocytogenes and DEPC water, respectively, as templates in step (2); the SHERLOCK reaction result with the genomic DNA of the listeria monocytogenes as a template is used as a positive control, and the SHERLOCK reaction result with DEPC water as a template is used as a negative control.
7. The method according to claim 5, wherein the reaction system used for carrying out the SHERLOCK reaction in the step (2) is 50. Mu.L, comprising: 4mg RPA Basic lyophilized powder, 10. Mu.M upstream and downstream primer each 2.4. Mu.L, 100nM Cas13a protein, 10 XRPA reaction buffer 10. Mu. L, crRNA 100nM, NTP Mix 6. Mu. L, T7 transcriptase 2. Mu.L, 5. Mu.M RNA fluorescent reporter, 100ng template, 280mM MgOAc2.5. Mu.L of aqueous solution and DEPC water were made up to 50. Mu.L.
8. The method of claim 5, wherein the reaction conditions of the shorlock reaction in step (2) are: the reaction temperature is 37-42 ℃, the reaction time is 5-40 min, the fluorescence generated by the reaction system is observed under a blue light transmission instrument with the wavelength of 470-520 nm, if the amplification product of the reaction system presents fluorescence, the sample contains the listeria monocytogenes, and if the amplification product of the reaction system does not present fluorescence, the sample does not contain the listeria monocytogenes.
9. The method of claim 8, wherein the reaction conditions of the shorlock reaction in step (2) are: the reaction temperature was 40℃and the reaction time was 40min.
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