CN117025807B - RPA-CRISPR/Cas12a primer group, gRNA and probe, kit and detection method - Google Patents
RPA-CRISPR/Cas12a primer group, gRNA and probe, kit and detection method Download PDFInfo
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Abstract
The invention belongs to the field of biology, and discloses an RPA-CRISPR/Cas12a primer group, gRNA and a probe for detecting haemophilus parasuis, wherein the RPA primer group, the gRNA and the probe are included; the RPA primer group comprises an upstream primer and a downstream primer as shown in SEQ ID NO.1 and SEQ ID NO. 2; the sequence of the gRNA is shown as SEQ ID NO. 3; the sequence of the probe is shown as SEQ ID NO. 4; the 5-end and the 3-end of the probe are respectively a fluorescent group and a quenching group. The method has the advantages of low detection limit and high detection accuracy. Meanwhile, the invention also discloses a kit and a method for detecting haemophilus parasuis.
Description
Technical Field
The invention relates to the field of biology, in particular to an RPA-CRISPR/Cas12a primer group, gRNA and a probe for detecting haemophilus parasuis, a kit and a detection method.
Background
Haemophilus parasuis is one of the most common bacterial diseases in modern livestock breeding, can be infected and onset in all age groups, is a disease caused by haemophilus parasuis, and has clinical symptoms mainly including multiple serositis, arthritis, meningitis, dyspnea, body temperature rise and the like, and can be mixed with other pathogens for infection or secondary infection. Haemophilus parasuis belongs to gram-negative bacteria, and the bacteria grow well in a culture medium containing serum and NAD, and can form obvious satellite colonies when co-cultured with staphylococcus aureus. The strain has 15 serotypes, and the main serotypes in China are types 4, 5 and 12, which cause serious economic loss to the breeding industry.
The recombinase polymerase isothermal amplification technique (Recombinase polymerase amplification, RPA) is a highly sensitive isothermal amplification technique that can be performed at 37-42 ℃ by amplifying a target with a recombinase, a polymerase, a single-stranded binding protein, and a primer. The RPA has the advantages of mild reaction condition, high amplification efficiency and simple equipment, and is widely applied to rapid detection of bacteria, fungi, viruses, parasites and the like in recent years, thereby being applicable to the requirements of basic level and field detection.
CRISPR-Cas is an acquired immune system widely found in archaea and bacteria, records the entry of exogenous nucleic acids, and when the same exogenous nucleic acid enters again, it is sheared by Cas proteins. Researchers find that Cas12a can cleave dsDNA targets in cis-systems with gRNA as a guide, while having trans-cleavage activity triggered by hybridization of gRNA with complementary target sequences, which can cleave single-stranded DNA of non-target sequences. Based on this finding, researchers applied Cas12a to pathogen detection, designed a reporter molecule with single-stranded DNA bound to a quencher and a fluorophore, and when a target sequence is present in a test sample, gRNA bound to the target sequence to form a complex, activating its nuclease activity to cleave the target sequence, while trans-cleavage activity cleaves the reporter molecule at will, and determining the presence or absence of a result based on the fluorescent signal.
In recent years, due to the need of a rapid and convenient pathogen detection method, doudna combines RPA with CRISPR detection aiming at human papilloma virus detection, and establishes a DNA endonuclease targeting CRISPR trans-reporting system, and from this, the CRISPR detection method is increasingly applied to pathogen detection of various pathogenic bacteria such as SARS-CoV-2, methicillin-resistant staphylococcus aureus, parasites and the like.
CN116463462a discloses an RPA-CRISPR/Cas12a detection method of IBRV virus and application thereof, which discloses detecting DNA of IBRV with a minimum initial template concentration of 20pg/μl within 30 min.
Since copies/. Mu.L is the expression unit for plasmid detection, the expression unit for virus and bacteria is generally pg/. Mu.L; in view of the prior art, there are few cases where the lower limit of detection for bacteria based on RPA-CRISPR/Cas12a is below 1 pg/. Mu.L.
The technical problem that the present case solves is: how to establish accurate detection of haemophilus parasuis based on the RPA-CRISPR/Cas12a method.
Disclosure of Invention
The first aim of the invention is to provide an RPA-CRISPR/Cas12a primer group, gRNA and a probe for detecting haemophilus parasuis, which have the advantages of low detection limit and high detection accuracy.
Meanwhile, the invention also discloses a kit and a method for detecting haemophilus parasuis.
In order to achieve the first object, the present invention provides the following technical solutions:
an RPA-CRISPR/Cas12a primer group, gRNA and a probe for detecting haemophilus parasuis, comprising the RPA primer group, the gRNA and the probe;
the RPA primer group comprises an upstream primer and a downstream primer as shown in SEQ ID NO.1 and SEQ ID NO. 2;
the sequence of the gRNA is shown as SEQ ID NO. 3;
the sequence of the probe is 5'-FAM-TTATT-BHQ1-3';
the 5-end and the 3-end of the probe are respectively a fluorescent group and a quenching group.
As shown in table 1 below:
TABLE 1
| Nucleic acid sequence (5 '-3') | |
| RPA F1 | 5’-AATCTGTAACTGTGGCAGCAGGTTATACTCA-3’（SEQ ID NO.1） |
| RPA R1 | 5’-AGTACCATAAGAGTAGTTTCCATACACGCCA-3’（SEQ ID NO.2） |
| gRNA1 | 5’-UAAUUUCUACUAAGUGUAGAUUACAACACCAUGACCACCAUCAA-3’（SEQ ID NO.3） |
| Probe with a probe tip | 5'-FAM-TTATT-BHQ1-3' |
Meanwhile, the invention also discloses a kit which contains the RPA-CRISPR/Cas12a primer group, the gRNA and the probe for detecting haemophilus parasuis.
Meanwhile, the invention also discloses a method for detecting haemophilus parasuis by using the primer group, the gRNA and the probe, which is characterized by comprising the following steps:
step 1: extracting DNA of a sample to be detected;
step 2: performing a nucleic acid amplification reaction on the DNA obtained in the step 1 by using an RPA primer group and a nucleic acid amplification reagent to obtain a nucleic acid amplification product;
step 3: mixing the gRNA, the probe and the Cas12a enzyme with the nucleic acid amplification product obtained in the step 2, and performing CRISPR/Cas12a reaction on the nucleic acid amplification product;
step 4: the detection result is determined by fluorescence detection.
In the above method, the system of step 2 is:
2.4. Mu.L of the upstream primer at a concentration of 10. Mu. Mol/L;
2.4. Mu.L of the downstream primer at a concentration of 10. Mu. Mol/L;
Primer Free Rehydration Buffer 29.5μL；
11.2 mu L of deionized water;
mixing the above materials, adding into a freeze-drying tube, blowing, mixing, dividing into two parts, adding 1 μl of DNA template into each tube, mixing, and adding 1.25 μl of MgoAc with concentration of 280 mmol/L into each tube.
In the above method, the system of step 3 is:
1.5. Mu.L of Cas12a enzyme at a concentration of 1. Mu. Mol/L;
gRNA at a concentration of 1. Mu. Mol/L, 1.5. Mu.L;
NEB buffer 2.1 mu L of 10 times unit;
1.35. Mu.L of ssDNA reporter with the concentration of 10. Mu. Mol/L;
3. Mu.L of the nucleic acid amplification product of step 2.
In the method, the reaction condition of the step 2 is 39 ℃ for 20min;
the reaction conditions in the step 3 are that the reaction is carried out for 30min at 37 ℃, and fluorescent signals are observed under a blue light.
Compared with the prior art, the invention has the outstanding characteristics that:
1) The detection limit of the RPA-CRISPR/Cas12a method provided by the invention on haemophilus parasuis is 10 higher than the lowest detection limit of common PCR 4 The sensitivity is good.
2) The method of the RPA-CRISPR/Cas12a has higher specificity for detecting haemophilus parasuis.
Drawings
FIG. 1 is a schematic diagram of the design of primers and gRNA in the reaction system according to the first embodiment;
FIG. 2A is a diagram showing the results of primer screening in example two;
FIG. 2B is a graph of the results of the gRNA screening of example two;
FIG. 2C is a graph of the results of optimization of the concentration of ssDNA reporter in example two;
FIG. 2D is a graph of optimized results for Cas12a protein concentration and gRNA concentration in the reaction system of example two;
FIG. 3A is a graph of fluorescence values for the specificity test of example three;
FIG. 3B is a photograph of a fluorescence of the specificity test of example three;
FIG. 4A is the results of a sensitivity test for HPS-5 DNA templates of example four at various dilutions;
FIG. 4B is the sensitivity results of conventional PCR with different dilution factors for the HPS-5 DNA template of example four;
FIG. 4C is a photograph of HPS-5 DNA template of example four with varying dilution factors;
FIG. 5A is a graph of the rapid detection of HPS serotypes by the RPA-CRISPR/Cas12a system of example five;
fig. 5B is a fluorescent photograph of the rapid detection of HPS serotypes by the RPA-CRISPR/Cas12a system of example five.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental materials and reagents used, unless otherwise specified, are those conventionally available commercially.
The nucleic acids of the clinical samples carried out in the present invention were all obtained from pig disease laboratory, animal health institute, academy of agricultural sciences, guangdong province.
Example one primer and Probe design
According to the method, an RPA primer (5 pairs) is designed according to the haemophilus parasuis OMPP2 gene in GenBank, a Protospacer Adjacent Motif (PAM) is found in an RPA amplified product by using software, gRNA (2 strips) which are complementary and paired with a target sequence base is designed according to the position of the PAM sequence, after the Cas12a is activated, the target DNA can be sheared, the auxiliary cleavage activity is also realized, any single-stranded DNA can be cut, and a ssDNA reporter molecule is required to be designed. The design pattern of the primers and the gRNA is shown in FIG. 1, and the nucleic acid sequences of the primers, the probes and the gRNA are shown in Table 1.
Example two amplification condition optimization
1. Screening of primers and gRNA
The effect of the primer on the RPA amplification efficiency is critical, while the gRNA cleavage efficiency is an important factor affecting Cas12a detection efficiency. Thus, we performed a test of the target gene (ompP 2) with 4 pairs of primers and 2 gRNA combinations, with a DNA concentration of 163 pg/. Mu.L for each reaction, respectively. The primer pairs and gRNA are shown in figure 1. To screen the optimal primer and gRNA combination for ompP2 gene, gRNA (1 # and 2 #) was used for 5 pairs of primers (F1R 1, F2R1, F3R3, F3R4 and F4R 4), respectively. The best performing primer pair (F1R 1) and gRNA (1#) were identified for subsequent experiments (FIGS. 2A and 2B).
2. ssDNA reporter, cas12a protein concentration and gRNA concentration optimization
The ssDNA reporter was optimized to a final concentration of 200nmol/L, 250nmol/L, 300nmol/L, 400nmol/L, 450nmol/L, and the optimal concentration was 450nmol/L as shown in FIG. 2C by observation of fluorescence intensity.
By optimizing the concentration of Cas12a and gRNA, using a real-time fluorescence quantitative PCR instrument, collecting fluorescence values of 465-510nm wavelength at 37 ℃ for 30min, referring to FIG. 2D, according to the fluorescence value results, the fluorescence value is maximum when the concentration of Cas12a protein is 100nmol/L, gRNA concentration and 50nmol/L, but the difference from the time when the concentration of Cas12a protein is 50nmol/L, gRNA concentration and 50nmol/L is not significant, and the concentration of Cas12a protein is 50nmol/L, gRNA concentration and 50nmol/L is selected as the test concentration in consideration of reagent cost.
Examples three specificity assays
HPS-5 DNA template is used as positive control, ddH2O is used as negative control, and the specificity of the detection method is determined by carrying out specificity detection on HPS-5, streptococcus suis type 2, type 9, actinobacillus pleuropneumoniae, escherichia coli, salmonella, erysipelothrix rhusiopathiae and staphylococcus aureus by using an RPA-Cas12a detection method, and repeating each sample for 3 times.
The detection steps of the invention are specifically described by taking haemophilus parasuis as an example, and specifically comprise the following steps:
the reagents used in the invention are as follows: cas12a protein, NEB buffer 2.1, RPA amplification kit Twist Amp Basic, primer, gRNA and ddH 2 O, ssDNA reporter;
the specific test steps of the invention are as follows:
step 1: extracting DNA of a sample to be detected;
step 2: performing a nucleic acid amplification reaction on the DNA obtained in the step 1 by using an RPA primer group and a nucleic acid amplification reagent to obtain a nucleic acid amplification product;
step 3: mixing the gRNA, the probe and the Cas12a enzyme with the nucleic acid amplification product obtained in the step 2, and performing CRISPR/Cas12a reaction on the nucleic acid amplification product;
step 4: the detection result is determined by fluorescence detection.
In the above method, the system of step 2 is:
2.4. Mu.L of the upstream primer at a concentration of 10. Mu. Mol/L;
2.4. Mu.L of the downstream primer at a concentration of 10. Mu. Mol/L;
Primer Free Rehydration Buffer 29.5 μL；
11.2 mu L of deionized water;
mixing the above materials, adding into a freeze-drying tube, blowing, mixing, dividing into two parts, adding 1 μl of DNA template into each tube, mixing, and adding 1.25 μl of MgoAc with concentration of 280 mmol/L into each tube.
The system of the step 3 is as follows:
1.5. Mu.L of Cas12a enzyme at a concentration of 1. Mu. Mol/L;
gRNA at a concentration of 1. Mu. Mol/L, 1.5. Mu.L;
NEB buffer 2.1 mu L of 10 times unit;
1.35. Mu.L of ssDNA reporter with the concentration of 10. Mu. Mol/L;
3. Mu.L of the nucleic acid amplification product of step 2.
The reaction condition of the step 2 is 39 ℃ for 20min;
the reaction condition of the step 3 is that the reaction is carried out for 30min at 37 ℃, and fluorescent signals are observed.
Results: referring to FIG. 3B, only HPS-5 DNA template showed a distinct fluorescence reaction, whereas Streptococcus suis type 2, type 9, actinobacillus pleuropneumoniae, E.coli, salmonella, erysipelothrix suis, staphylococcus aureus did not show a fluorescence reaction. FIG. 3A shows the magnitude of the fluorescence values of the templates, it being seen that the fluorescence values of the HPS-5 DNA templates are most significant.
Example four sensitivity test
HPS-5 DNA template was subjected to ten-fold gradient dilution at an original concentration of 163 ng/. Mu.L, and the diluted template was subjected to reaction detection using the established method, while the diluted template was detected using the existing PCR method in the laboratory to compare the sensitivity of the two methods.
Results: referring to FIGS. 4A-4C, HPS-5 DNA template was subjected to ten-fold gradient dilutions at concentrations of 163 ng/. Mu.L, 16.3 ng/. Mu.L, 1.63 ng/. Mu.L, 163 pg/. Mu.L, 16.3 pg/. Mu.L, 1.63 pg/. Mu.L, 0.163 pg/. Mu.L, 16.3 fg/. Mu.L in this order; after using the RPA-Cas12a detection method, each sample was repeated at least 3 times, referring to fig. 4a,163 ng/μl, 16.3 ng/μl, 1.63 ng/μl, 163pg/μl, 16.3pg/μl, 1.63pg/μl all showed more pronounced fluorescence response, although the fluorescence intensity of 0.163pg/μl was weaker, fluorescence was also visible in comparison with negative versus photographic, so the sensitivity of the method was 0.163pg/μl; referring to FIG. 4B, the sensitivity of the conventional PCR detection method is only 1.63 ng/. Mu.L, which indicates that the sensitivity of the RPA-Cas12a detection method is 10000 times higher than that of the conventional PCR detection method. Fig. 4C shows fluorescence values at different concentrations.
Taking HPS bacterial solution cultured overnight, diluting with a doubling ratio, performing plate count, and adjusting bacteria to 1×10 according to bacterial solution concentration 9 cfu/mL, then 10-fold concentration gradient diluted to 1X 10 1 cfu/mL, detection is carried out by using an RPA-Cas12a method and a common PCR method respectively, and the result shows that the detection lower limit of the RPA-Cas12a is 1 multiplied by 10 1 cfu/mL, the detection limit of the common PCR is 1×10 4 cfu/mL。
Example five RPA-CRISPR/Cas12a system for rapid detection of HPS serotypes
Carrying out recovery on the HPS 11 strains, and then carrying out crude extraction on DNA, wherein the step of crude extraction of DNA comprises the following steps: taking 1ml of bacterial liquid 12000rmp at room temperature, centrifuging for 2min, discarding the supernatant, adding 1ml of PBS solution for resuspension, centrifuging for 1min at 12000rmp, discarding the supernatant, adding 100ul of enzyme-free water, using a metal bath at 100 ℃ for 5min, taking the supernatant, and measuring the concentration by using an enzyme-labeled instrument. And (3) carrying out amplification reaction on DNA templates of 11 strains according to the optimized RPA-CRISPR/Cas12a system, and after the reaction is finished, observing whether fluorescence appears or not under a gel imaging system, wherein each sample is repeated for at least 3 times.
Results: the bacterial genome was extracted by the water boiling method, and the extracted bacterial DNA was detected by RPA-CRISPR/Cas12a, referring to fig. 5A and 5B, which show fluorescence values and photographs, respectively, and the result shows that after the reaction of the crude DNA was completed, a fluorescent signal was observed in the gel imaging system, and a negative no fluorescent signal appeared. The detection method has better compatibility to the crude extracted bacterial DNA, greatly reduces the detection cost and the detection time, and can effectively detect 11 serotype clinical strains of HPS.
Comparative example 1
Referring to the above detection method, the primers and probes used are as follows in Table 2:
TABLE 2
| Nucleic acid sequence (5 '-3') | |
| RPA F2 | 5’- GGTGTATACTTTGGTCTTAAATATGTCAACG-3’（SEQ ID NO.4） |
| RPA R1 | 5’- AGTACCATAAGAGTAGTTTCCATACACGCCA-3’（SEQ ID NO.2） |
| gRNA 1 | 5’- UAAUUUCUACUAAGUGUAGAUUACAACACCAUGACCACCAUCAA-3’（SEQ ID NO.3） |
| Probe with a probe tip | 5'-FAM-TTATT-BHQ1-3' |
The detection sensitivity is as follows: the sensitivity of this method was 16.3 pg/. Mu.L and the primer was non-specifically cross-reactive with erysipelothrix rhusiopathiae.
Comparative example 2
Referring to the above detection method, the primers and probes used are shown in Table 3 below:
TABLE 3 Table 3
| Nucleic acid sequence (5 '-3') | |
| RPA F3 | 5’-TAAGAAAGACAAAGATGGTGTATACTTTGGT-3’（SEQ ID NO.5） |
| RPA R3 | 5’-TGTCTTTGTTCTTGATTAAACGACCTTCAA-3’（SEQ ID NO.6） |
| gRNA1 | 5’- UAAUUUCUACUAAGUGUAGAUUACAACACCAUGACCACCAUCAA-3’（SEQ ID NO.3） |
| Probe with a probe tip | 5'-FAM-TTATT-BHQ1-3' |
The detection sensitivity is as follows: the sensitivity of the method was 1.63 pg/. Mu.L.
Comparative example 3
Referring to the above detection method, the primers and probes used are as follows in Table 4:
TABLE 4 Table 4
| Nucleic acid sequence (5 '-3') | |
| RPA F3 | 5’-TAAGAAAGACAAAGATGGTGTATACTTTGGT-3’（SEQ ID NO.5） |
| RPA R4 | 5’-TTGTAAGTACCATAAGAGTAGTTTCCATAC-3’（SEQ ID NO.7） |
| gRNA1 | 5’-UAAUUUCUACUAAGUGUAGAUUACAACACCAUGACCACCAUCAA-3’（SEQ ID NO.3） |
| Probe with a probe tip | 5'-FAM-TTATT-BHQ1-3' |
The detection sensitivity is as follows: the sensitivity of the method was 1.63 pg/. Mu.L.
Comparative example 4
Referring to the above detection method, the primers and probes used are shown in Table 5 below:
TABLE 5
| Nucleic acid sequence (5 '-3') | |
| RPA F3 | 5’-TAAGAAAGACAAAGATGGTGTATACTTTGGT-3’（SEQ ID NO.5） |
| RPA R3 | 5’-TGTCTTTGTTCTTGATTAAACGACCTTCAA-3’（SEQ ID NO.6） |
| gRNA2 | 5’-UAAUUUCUACUAAGUGUAGAUAUGUUACUCCAAAAUCUGGCGUG-3’（SEQ ID NO.8） |
| Probe with a probe tip | 5'-FAM-TTATT-BHQ1-3' |
The detection sensitivity is as follows: the sensitivity of the method was 163 pg/. Mu.L.
Comparative example 5
Referring to the above detection method, the primers and probes used are as follows in Table 6:
TABLE 6
| Nucleic acid sequence (5 '-3') | |
| RPA F3 | 5’-TAAGAAAGACAAAGATGGTGTATACTTTGGT-3’（SEQ ID NO.5） |
| RPA R4 | 5’-TTGTAAGTACCATAAGAGTAGTTTCCATAC-3’（SEQ ID NO.7） |
| gRNA2 | 5’-UAAUUUCUACUAAGUGUAGAUAUGUUACUCCAAAAUCUGGCGUG-3（SEQ ID NO.8） |
| Probe with a probe tip | 5'-FAM-TTATT-BHQ1-3' |
The detection sensitivity is as follows: the sensitivity of this method was 16.3 pg/. Mu.L.
Comparative example 6
Referring to the above detection method, the primers and probes used are as follows in Table 7:
TABLE 7
| Nucleic acid sequence (5 '-3') | |
| RPA F4 | 5’- GTAGCTGTTGATGGTGGTCATGGTGTTGTAAA-3’（SEQ ID NO.9） |
| RPA R4 | 5’-TTGTAAGTACCATAAGAGTAGTTTCCATAC-3’（SEQ ID NO.7） |
| gRNA2 | 5’-UAAUUUCUACUAAGUGUAGAUAUGUUACUCCAAAAUCUGGCGUG-3（SEQ ID NO.8） |
| Probe with a probe tip | 5'-FAM-TTATT-BHQ1-3' |
The detection sensitivity is as follows: the sensitivity of this method was 16.3 pg/. Mu.L.
In comparative examples 1-6, 5 sets of screened upstream and downstream primers (RPA F1-4 and RPA R1-4) and 2 sets of gRNAs (gRNA 1 and gRNA 2) were used. Through the use, the primers and the gRNA can effectively detect the target gene, but the target gene can reach the degree of the order of small points of sensitivity, and the result of the invention can be obtained only through the combination of RPA F1R1 and gRNA1 through repeated optimization.
Analysis of results:
1. according to the invention, the cost and the test effect are comprehensively considered, and the concentration of the ssDNA reporter molecule is 450nmol/L, and the concentration of the Cas12a protein is 50nmol/L, gRNA and 50nmol/L are taken as the optimal test concentration.
2. The invention has stronger specificity for detecting haemophilus parasuis, and is negative for detecting common swine bacterial pathogens such as streptococcus suis type 2, type 9, actinobacillus pleuropneumoniae, escherichia coli, salmonella, erysipelothrix rhusiopathiae and staphylococcus aureus.
3. The sensitivity of the invention to HPS detection is 0.163 pg/. Mu.L, and is 10000 times higher than that of common PCR.
4. The detection method has better compatibility to the crude extracted bacterial DNA, and greatly reduces the detection cost and the detection time.
5. The invention can effectively detect 11 serotypes of HPS clinical strains.
The foregoing examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (2)
1. An RPA-CRISPR/Cas12a primer group, gRNA and a probe for detecting haemophilus parasuis, which is characterized by comprising an RPA primer group, a gRNA and a probe;
the RPA primer group comprises an upstream primer and a downstream primer shown as SEQ ID NO.1 and SEQ ID NO. 2;
the sequence of the gRNA is shown as SEQ ID NO. 3;
the structure of the sequence of the probe is 5'-FAM-TTATT-BHQ1-3';
the 5-end and the 3-end of the probe are respectively a fluorescent group and a quenching group.
2. A kit comprising the RPA-CRISPR/Cas12a primer set, gRNA and probe for detecting haemophilus parasuis of claim 1.
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