Streptococcus suis detection method based on RAA-CRISPR / cas12a
By combining RAA isothermal reaction and CRISPR/Cas12a system, a detection method of streptococci suis based on RAA-CRISPR/cas12a was developed, which solved the problem of low detection sensitivity in the prior art, and achieved efficient and accurate detection of streptococci suis, which was suitable for rapid detection of farms and complex environments.
Patent Information
- Application Number
- CN202510381116.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-06-06
AI Technical Summary
The prior art is difficult to provide an efficient and accurate detection method for Streptococcus suis, especially in farms or complex environments. Traditional isolation and culture and biochemical identification methods are time-consuming and labor-intensive and have low detection sensitivity.
Combined with RAA isothermal reaction and CRISPR/Cas12a system, the Streptococcus suis recN gene was selected as the target gene, and a Streptococcus suis detection method based on RAA-CRISPR/cas12a was developed. This method realizes nucleic acid amplification and endpoint detection through the design of RAA primers and crRNA, and has high sensitivity and high specificity.
This method can complete the RAA amplification reaction within 37°C and 10 minutes, and achieve fluorescence quantification through the CRISPR/Cas12a detection system, achieving a detection rate of 100.0%, and its sensitivity can reach 24 copies/μL. It is suitable for rapid and accurate detection of Streptococcus suis.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular biology, and in particular to a method for detecting Streptococcus suis based on RAA-CRISPR / cas12a. Background Art
[0002] Streptococcus suis ( Streptococcus suis , SS) is a Gram-positive coccus that is often found in the upper respiratory tract of pigs. It can cause swine sepsis, meningitis, etc. It is also a key zoonosis pathogen. SS can infect humans through wounds and respiratory tracts, causing suppurative arthritis, meningoencephalitis and endocarditis, etc., and can cause death in severe cases. While the bacteria endanger the development of the pig industry, it also poses a threat to public health and safety. Therefore, there are higher requirements and demands for timely and accurate monitoring of Streptococcus suis. At present, laboratory detection of Streptococcus suis mostly adopts traditional isolation culture and biochemical identification. Although SS is easy to identify from morphological characteristics in laboratory detection, the background bacteria in the farm environment and pig-derived animal products are often complex, and conventional pathogen identification methods are time-consuming and labor-intensive, and the detection sensitivity is not high. Therefore, the diagnosis of swine streptococcal disease requires the development of a field detection method for Streptococcus suis with strong specificity, fast accuracy and simple operation, which can be used as a monitoring tool for Streptococcus suis in farms or other complex environments.
[0003] Isothermal amplification technology has the potential to achieve detection in an environment with low resource allocation. The detection does not require strict temperature control and professional instruments, and can be carried out through a water bath and a heating block. At the same time, it is simple to operate and does not require a good sample loading environment, so non-professionals can quickly get started. Isothermal amplification technologies commonly used in detection include loop-mediated constant temperature amplification technology, rolling circle amplification technology, and recombinase-mediated isothermal nucleic acid amplification technology (Recombinase Aided Amplification, RAA). Among them, the RAA method only requires the design of a pair of primers, and the template does not need to be processed. The reaction at about 37°C for 15 minutes can complete the detection, which has a wider application prospect.
[0004] Depending on the purpose of detection, researchers will choose different target genes. The gdh gene has long been considered a reliable target gene for detecting Streptococcus suis due to its high conservation. However, in recent years, some researchers have found that the use of the gdh gene cannot distinguish between other streptococci (such as the original serotypes 20, 22, 26 and 32-34 of Streptococcus suis) and Streptococcus suis. Other target genes such as recN have been used for specific detection of Streptococcus suis. For example, based on the new SS classification (removing serotypes 20, 22, 26, 32, 33 and 34), Ishida et al. designed a new PCR based on the SS housekeeping gene recN sequence, which can effectively detect SS with a detection limit of 90 CFU / reaction. In 2015, Japanese scholars Arai et al. designed a LAMP method (LAMPss) based on the SSrecN gene. LAMPss can detect all serotypes of SS from raw pork samples with a detection limit of 5.4 CFU / reaction. In 2023, researchers used primers for the gdh gene to detect 11 positive cases in the identification of Streptococcus suis. After retesting with primers for the recN gene, they found that 27.2% of the strains were not actually Streptococcus suis. The recN gene may be more suitable as a target gene for Streptococcus suis detection for the identification of reclassified Streptococcus suis.
[0005] The CRISPR system is a promising nucleic acid detection tool, which is mainly driven by its effector proteins. Detection strategies based on CRISPR have been widely used in the detection of a variety of bacteria. In the study of Chen et al., an application for the detection of Pseudomonas aeruginosa confirmed the good compatibility between RAA and Cas12a systems, which can ensure the excellent performance of instant detection, high sensitivity and high specificity of the detection platform. Hao et al. combined recombinase polymerase amplification with CRISPR / Cas12a technology to establish a naked eye detection method for high-sensitivity detection of Haemophilus parasuis, which is more conducive to on-site detection than traditional PCR detection methods. The detection technology based on the CRISPR system has the advantages of high sensitivity, high specificity, rapidity and portability, and is suitable for the detection of pathogenic microorganisms. However, there is currently no universal detection method for Streptococcus suis based on this system.
[0006] So far, there is still a lack of more convenient and accurate detection methods for the accurate identification and detection of Streptococcus suis. Summary of the invention
[0007] The purpose of the present invention is to provide a method for detecting Streptococcus suis based on RAA-CRISPR / cas12a, so as to provide an efficient and accurate detection means for identifying Streptococcus suis.
[0008] In order to achieve the above-mentioned object, the present invention provides a RAA-CRISPR / cas12a-based Streptococcus suis detection system, comprising a RAA system and a CRISPR / Cas12a detection system, wherein the RAA system comprises upstream and downstream RAA primers whose nucleotide sequences are shown in SEQ ID NO.1 and 2; the CRISPR / Cas12a detection system comprises crRNA whose nucleotide sequence is shown in SEQ ID NO.6.
[0009] The Streptococcus suis detection system provided by the invention can be used for preparing products for detecting Streptococcus suis.
[0010] The present invention also provides a Streptococcus suis detection kit based on RAA-CRISPR / cas12a, which comprises the above-mentioned RAA system and CRISPR / Cas12a detection system.
[0011] The present invention also provides a method for detecting Streptococcus suis based on RAA-CRISPR / cas12a, comprising the following steps: (1) extracting DNA from Streptococcus suis, and performing RAA amplification reaction using upstream and downstream RAA primers with nucleotide sequences as shown in SEQ ID NOs. 1 and 2 to obtain RAA amplification products; (2) adding the RAA amplification product to the CRISPR / Cas12a detection system for reaction, wherein the crRNA nucleotide sequence of the CRISPR / Cas12a detection system is shown in SEQ ID NO.6; (3) Detecting the fluorescence changes in the CRISPR / Cas12a detection system and completing fluorescence quantification can achieve the detection of Streptococcus suis.
[0012] Preferably, the RAA amplification reaction conditions in the above detection method are 37°C, 10-30 min, more preferably 37°C, 10 min.
[0013] Preferably, the CRISPR / Cas12a detection system conditions in the above detection method are 37°C and 30 min.
[0014] The present invention has the following advantages: The present invention provides a method for detecting Streptococcus suis based on RAA-CRISPR / cas12a, provides a RAA primer set and crRNA sequence adapted for detecting Streptococcus suis, and a reaction system based on the combination of RAA-CRISPR / cas12a, which has been verified to have a detection rate of 100.0% for Streptococcus suis. The sensitivity of the RAA-CRISPR / Cas12a reaction provided by the present invention can reach 24 copies / μL, and Streptococcus suis can be efficiently and accurately detected and identified, thereby providing a new means for diagnosing Streptococcus suis, and having significant application potential in rapidly and accurately detecting Streptococcus suis in breeding and production. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is the result of multiple sequence alignment of the recN gene of Streptococcus suis in the present invention.
[0016] Figure 2 It is the fluorescence quantitative result of the combination detection of different RAA primers and crRNA sequences in the present invention.
[0017] Figure 3 These are the optimization results of different RAA nucleic acid amplification times in the present invention.
[0018] Figure 4 These are the results of temperature optimization for different RAA nucleic acid amplifications in the present invention.
[0019] Figure 5 This is the optimization result of different crRNA concentrations in the present invention.
[0020] Figure 6 The sensitivity of the method provided in the present invention is to detect the change in amplified fluorescence value.
[0021] Figure 7 These are the specific validation results of the method provided in the present invention in strains of different species. DETAILED DESCRIPTION
[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0023] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.
[0024] In order to provide a more convenient and accurate detection method for accurate identification and detection of Streptococcus suis, this study combined CRISPR / Cas12a and RAA technologies, selected the recN gene of Streptococcus suis as the target gene, and established a method for nucleic acid amplification based on RAA isothermal reaction, and then used the CRISPR / Cas12a system to perform endpoint detection on the RAA reaction product. This universal detection method can quickly and accurately detect Streptococcus suis in actual production and life, providing a new means for the prevention, control and diagnosis of Streptococcus suis.
[0025] Example 1 Primer Development for Identification of Streptococcus suis Some of the strains used in this example are shown in Table 1. The plasmid used is pUC57 plasmid, and the standard strain of Streptococcus suis BNCC357698 (ATCC43765) was purchased from the North Na Microbial and Cell Resource Collection and activated and preserved. Streptococcus suis was cultured in brain heart infusion broth (BHI) liquid medium (supplemented with 9.5% calf serum) and tryptone soy agar (TSA) plates (9.5% calf serum) at 37°C.
[0026] Table 1 Strains used in this experiment
[0027] 1. Analysis of the whole genome and recN gene of Streptococcus suis The DNA of the above-mentioned Streptococcus suis isolates was extracted according to the operating instructions of the Tiangen Bacterial Genomic DNA Extraction Kit and sent to the company for whole genome sequencing. The DNA quality was tested, and the qualified products began library construction and sequencing. The whole genome of Streptococcus suis was sequenced using the Illumina Hiseq 4000 platform (Pair end 150 bp), and the data volume for each strain was about 1G. All sequencing work was completed by Shanghai Sangon Biotechnology Co., Ltd.
[0028] Genome assembly and annotation: The raw data and filtered high-quality sequencing data (clean data) of Streptococcus suis isolates were downloaded. FastQCvO.11.7 was used to perform quality control and evaluation on the sequencing data. The quality-controlled raw genome data was uploaded to the SPAdesv3.13.1 sequence splicing software (https: / / github.com / ablab / spades) to obtain the strain genome assembly results. The prokaryotic genome annotation software RAST (http: / / rast.nmpdr.org / ) was used to annotate the assembled genome sequence.
[0029] The recN gene of Streptococcus suis was selected as the target gene for analysis and development. The sequence information of all isolated strains was compared with the sequence of Streptococcus suis downloaded from the NCBI website to determine the conservation of the recN gene in Streptococcus suis. At the same time, the recN gene of Streptococcus suis was compared with the recN of other Streptococci to determine the differences of the recN gene in different Streptococci. The multiple sequence alignment was completed using ClustalW (https: / / www.genome.jp / tools-bin / clustalw).
[0030] The results of whole genome sequencing data splicing and assembly of 158 strains of Streptococcus suis (including the strains in Table 1) preserved in the laboratory showed that the genome size of the 158 strains of Streptococcus suis ranged from 1.9 M to 2.5 M. The sequence information of the 158 strains of Streptococcus suis was compared with the sequence of Streptococcus suis downloaded from the NCBI website to determine the conservation of the recN gene in Streptococcus suis. At the same time, the recN gene of Streptococcus suis was compared with the recN of other Streptococcus species to determine the differences of the recN gene in different Streptococci.
[0031] The results of multiple sequence alignment of Streptococcus suis recN gene are shown in Figure 1 As shown, (A) is a multiple sequence comparison of recN genes of Streptococcus suis and other Streptococci; (B) is a multiple sequence alignment of recN genes of Streptococcus suis. The results showed that the length of recN genes in different Streptococcus species ranged from 1662 bp to 1671 bp, among which the length of recN gene of Streptococcus suis was 1662 bp. By performing multiple sequence alignment of recN genes from 38 species of Streptococcus (see Figure 1 In (A), the results showed that the recN gene was highly diverse in different Streptococci. Multiple sequence alignment of 158 strains of S. suis and 156 S. suis sequences downloaded from NCBI showed that the recN gene was present and conserved in the species (see Figure 1 Therefore, the recN gene can be used as a target gene for identifying Streptococcus suis, and this gene has high specificity for Streptococcus suis.
[0032] 2. Design and synthesis of primers for recN gene for whole genome screening of S. suis 1. Select DNA repair protein recN gene (Gene ID: 8154275) as the target gene for Streptococcus suis detection. Based on the recN gene reference sequence of Streptococcus suis standard strain BNCC357698, combined with the recN gene sequences of 158 strains of Streptococcus suis that have completed second-generation sequencing in the laboratory and the target genes of 165 strains of Streptococcus suis retrieved from the NCBI gene library, sequence conservation analysis was performed (part of the results are shown in Figure 1 ), select the conserved region of the recN gene to design the primers of RAA and the DNA sequence to be transcribed by crRNA.
[0033] (1) Principles of crRNA design: The PAM site is TTTN, and the 20 bp sequence after the PAM site is selected as the crRNA complementary region. The crRNA sequence is designed according to the principle of DNA base complementary pairing. The CG content is 30%-70%. The position of the crRNA needs to avoid mutation sites, and a T7 promoter is added to the 3' end when synthesizing the sequence.
[0034] (2) RAA primer design principles: Design RAA upstream and downstream primers at both ends of the determined crRNA sequence. RAA primer design follows three principles: ① The length range is 30-50 bp, and it should not be too long or too short, otherwise it will affect the effect of nucleic acid amplification. ② The G+C content of the primer should be 30%-70%. ③ In order to achieve the best amplification effect, the product length should not be designed to be too long, preferably 100-300bp.
[0035] (3) Fluorescent reporter probe: The fluorescent reporter probe should be a sequence rich in T bases, with a length of 5-10 bases. If the length is too long, the fluorescent group and the quencher group will be too far apart, resulting in an excessively high background fluorescence signal.
[0036] Screening of RAA primers and crRNA Primers were designed and synthesized for the recN gene screened from the whole genome of Streptococcus suis, and the DNA repair protein recN gene (Gene ID: 8154275) was selected as the target gene for Streptococcus suis detection. Based on the recN gene reference sequence of the standard strain of Streptococcus suis BNCC357698 (ATCC43765), the recN gene sequences of 158 strains of Streptococcus suis that had been sequenced in the laboratory and the target genes of 165 strains of Streptococcus suis retrieved from the NCBI gene library were analyzed for sequence conservation, and the conservative regions of the recN gene were selected to design primers and crRNA for RAA. The information of RAA amplification primers is shown in Table 2, the information of crRNA sequences is shown in Table 3, and the sequence of fluorescent reporter probe is shown in Table 4.
[0037] Table 2 RAA amplification primer sequence information
[0038] Table 3 crRNA sequence information
[0039] Table 4 Fluorescent probe synthesis sequence information
[0040] 2. Screening of RAA primers for Streptococcus suis According to the RAA primer design principle, RAA upstream and downstream amplification primers were designed at both ends of the determined target sequence crRNA. The RAA reaction was carried out according to the instructions of the crowd-testing kit. The amplification reaction system is shown in Table 5. The specific operation steps are as follows.
[0041] Table 5 RAA reaction system
[0042] (1) Briefly centrifuge the unit tube containing the enzyme lyophilized powder and carefully open the lid. The purpose of this step is to avoid powder loss when opening the lid.
[0043] (2) Add 25 μL of A Buffer to the unit tube and invert thoroughly to mix.
[0044] (3) Place the enzyme-free PCR centrifuge tube on an ice box and set it to 40 °C. 2 Add O→A Buffer→F Primer→RPrimer→template→B Buffer in sequence, mix thoroughly, and centrifuge briefly.
[0045] (4) Place the centrifuge tube in the PCR instrument and set the reaction program to 37°C and 30 min.
[0046] (5) The amplified product was stored in a 4°C refrigerator for subsequent testing.
[0047] 3. Preparation of Streptococcus suis crRNA The company synthesized single-stranded DNA (see Table 3), and the experiment required RNA, which was completed through in vitro transcription. The preparation process is as follows: (1) Template preparation: The synthesized DNA is delivered in the form of dry powder and attached to the tube wall in the form of a very light dry film. Centrifuge at 12,000 rpm for 15 seconds and add the corresponding volume of DEPC-H to the tube wall. 2 O, vortex and mix well, then centrifuge instantly to obtain an annealing template with a concentration of 100 μM.
[0048] (2) Annealing: The T7 promoter primer is combined with the single-stranded DNA through annealing reaction, and the product is labeled as DNA mix. The annealing system and reaction procedure are shown in Table 6 and Table 7 respectively.
[0049] Table 6 Annealing system
[0050] Table 7 Annealing reaction program
[0051] (3) In vitro transcription: The annealed product was transcribed using the full-form gold high-efficiency in vitro transcription kit. The transcription system is shown in Table 8. The reaction procedure was 37°C and 2 h.
[0052] Table 8 Transcription system
[0053] (4) Removal of DNA: After the 2-hour incubation reaction, centrifuge at low speed to remove droplets on the tube wall. Add 1 μL DNaseI to the system and incubate at 37°C for 1 hour. The purpose of this step is to degrade the DNA template during the reaction, and the resulting product is used for subsequent purification.
[0054] (5) RNA purification: Follow the instructions of the column-based RNA rapid concentration and purification kit. The entire process is completed in a clean bench to avoid RNA enzyme contamination. After purification, take 2 μL of RNA for concentration measurement and quality inspection. The qualified RNA solution is packaged and stored at -80°C for subsequent experiments.
[0055] 4. Screening of Streptococcus suis crRNA The fluorescence value change results were displayed using a fluorescence quantitative PCR instrument, and the combination of RAA primers and crRNA sequences with significant differences in fluorescence values were selected for subsequent experiments. The reaction system is shown in Table 9, and the reaction procedure is 37°C, 30min.
[0056] Table 9 Cas12a reaction system
[0057] 3. Detection conditions and optimization of the recN gene of Streptococcus suis based on RAA-CRISPR / Cas12a 1. Screening of RAA primers and crRNA The RAA primers and crRNA of Streptococcus suis developed above were screened, and the fluorescence value of the FAM channel during the reaction was detected using a fluorescence quantitative PCR instrument, and the combination of RAA primers and crRNA with better peaks was selected for subsequent experiments. The sequence combination information of RAA primers and crRNA and the results of fluorescence quantitative PCR instrument are shown in Tables 10 and Figure 2 ,in, Figure 2 (A) Fluorescence values of G1-G5 group and its control group after Cas12a reaction for 10 min. Figure 2 (B) The fluorescence difference between the G1-G5 group and the control group at 10 min and 1 min of Cas12a reaction. NC indicates that there is no target DNA in the reaction system. It can be seen that Figure 2 (A) shows that among the five groups, the fluorescence values of G1, G2 and G3 at 10 min were not much different, and there was no significant difference between G4 and G5. Figure 2B in Figure 2 shows that G3 reached the plateau at 1 min, and the reaction rate was too fast, which was not conducive to the subsequent optimization of conditions. The background value of the control group of G2 was low. Therefore, G2 (i.e., RAA-F2 / R2 + crRNA2) was selected in this subsequent study.
[0058] Table 10 Different RAA-crRNA combinations
[0059] 2. Optimization of RAA optimal reaction conditions According to the fluorescence increment in the above results, one of the five nucleic acid amplification time gradients (10, 15, 20, 25, and 30 min) was selected as the RAA reaction time in this method. The optimization results of RAA nucleic acid amplification time are shown in Figure 2. Figure 3 As shown in the figure, (A) is the difference between the fluorescence value at 10 min and the fluorescence value at 1 min, (B) is the change in fluorescence value during the Cas12a reaction, and NC indicates that there is no target DNA in the reaction system. It can be seen that the reactions of the five time gradients all reached the plateau at 10 cycles, and the results were not much different. Considering the time cost of the detection method, 10 min was obtained as the optimal time for nucleic acid amplification.
[0060] Similarly, according to the fluorescence increment of the above results, one of the five nucleic acid amplification temperatures (36, 37, 38, 39, 40°C) was selected as the reaction temperature applicable to this method. The RAA nucleic acid amplification temperature optimization results are shown in Figure 4 As shown, (A) in the figure is the difference between the fluorescence value at 10 min and the fluorescence value at 1 min, (B) is the change in fluorescence value during the Cas12a reaction, and NC indicates that there is no target DNA in the reaction system. It can be seen that the RAA nucleic acid reaction temperature is a wide range, which is significant at 36°C to 40°C. After comparing the fluorescence increment, it is found that the RAA reaction temperature is best at 37°C.
[0061] 3. Optimization of the optimal reaction conditions for Cas12a reaction According to the fluorescence increment in the above results, one of the five crRNA concentration gradients was selected as the crRNA reaction concentration in this method. Figure 5 As shown, (A) in the figure is the difference between the fluorescence value at 10 min and the fluorescence value at 1 min, (B) is the change in fluorescence value during the Cas12a reaction, and NC indicates that there is no target DNA in the reaction system. It can be seen that when the crRNA concentration is 150 ng / μL, the Cas12a reaction peak effect is the best, which is determined as the sample loading concentration of this method.
[0062] 4. Verification of sensitivity test using plasmid as standard The plasmid was used as a standard for sensitivity testing. The glycerol bacteria containing the target gene provided by the company were revived in LB solid medium containing ampicillin resistance, and the plasmid was extracted according to the operating instructions of the Tiangen bacterial plasmid extraction kit. The concentration and quality of the plasmid were tested with a NanoDrop2000 ultra-micro spectrophotometer, and the plasmid concentration in the obtained solution was 117ng / μL.
[0063] Calculation of plasmid copy number: The original size of the plasmid is 2710 bp, and the length of the inserted specific sequence is 1662 bp. According to the following formula, the plasmid copy number is calculated to be 2.44×10 10 copies / μL.
[0064]
[0065] Dilution of plasmid standard: dilute the plasmid solution 10 times with sterile water, shake and mix, and centrifuge instantly to obtain 10 9 ~10 0 There are 10 gradients in total, which are stored in a -20 ℃ refrigerator for later use.
[0066] When conducting sensitivity tests, the concentration of 2.44×10 0 -2.44×10 5 A total of 6 gradients of plasmid solutions were used as templates. The results of sensitivity detection of amplification fluorescence value changes are shown in Figure 6 As shown in the figure, 5-0 is the index of each concentration gradient. The results show that the 6 gradient dilution concentrations show obvious reaction gradients on the instrument. For plasmid standards, the sensitivity of the RAA-CRISPR / Cas12a reaction based on the recN gene of Streptococcus suis can reach 24 copies / μL.
[0067] 5. Specificity test of detection method In order to verify the specificity of this method, the standard strain of Streptococcus suis BNCC357698 and other streptococci closely related to Streptococcus suis and strains frequently colonized in pig nasal swabs were selected for testing. A total of three types of strains were selected for the specificity test of the detection method, including ① other streptococci closely related to Streptococcus suis, ② non-Streptococcus bacterial strains colonized in the pig nasal cavity, and ③ common bacterial strains in pig herds. The specific test strains include Streptococcus parasuis, Streptococcus suis, Streptococcus oralis, Streptococcus multi-animal, Proteus, Atropha viridis, Lactobacillus gossypii, Escherichia coli, and Enterococci isolated and preserved in this laboratory; as well as the standard strains of Staphylococcus aureus and Salmonella enteritidis preserved in this experiment. The verification results are as follows: Figure 7As shown, it can be seen that the method provided by the present invention can distinguish closely related streptococci from other common bacteria in the pig nasal cavity, has good specificity, and can be used as a tool for detecting swine streptococci.
[0068] 4. Comparison and validation of the isothermal detection method of recN gene of Streptococcus suis and the PCR detection method The streptococci isolated in this study (including Streptococcus suis and other streptococci) and 5 non-streptococcal strains in the laboratory strain library were selected for coincidence detection. The isothermal detection method of Streptococcus suis recN gene established in this study obtained a 100.0% detection rate of Streptococcus suis, and the detection results were consistent with the results of whole genome sequencing; the coincidence rate of the common PCR method (target gene gdh) was 97.5%. The time, professional equipment and cost required for the two methods were compared, and the results are shown in Table 11. In comprehensive comparison, the cost of the RAA-CRISPR / Cas12a detection method (10 yuan / piece) is slightly higher than that of the PCR detection method (3 yuan / piece), but the RAA-CRISPR / Cas12a detection method takes less time, and the detection can be completed in less than 30 minutes, and no professional equipment is required, which is more conducive to on-site detection. Therefore, the method established in this study has accurate results, saves more time, and can accurately and precisely detect and identify Streptococcus suis.
[0069] Table 11 Comparison and verification results
[0070] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.
Claims
1. A Streptococcus suis detection system based on RAA-CRISPR / cas12a, characterized in that: Including RAA system and CRISPR / Cas12a detection system: The RAA system comprises upstream and downstream RAA primers having nucleotide sequences as shown in SEQ ID NOs. 1 and 2; The CRISPR / Cas12a detection system comprises a crRNA having a nucleotide sequence as shown in SEQ ID NO.
6.
2. Use of the Streptococcus suis detection system as claimed in claim 1 in preparing a product for detecting Streptococcus suis.
3. A Streptococcus suis detection kit based on RAA-CRISPR / cas12a, characterized in that: The kit comprises the RAA system and CRISPR / Cas12a detection system as described in claim 1.
4. A method for detecting Streptococcus suis based on RAA-CRISPR / cas12a, characterized in that: The following steps are included: Extracting DNA of Streptococcus suis, performing RAA amplification reaction using upstream and downstream RAA primers with nucleotide sequences as shown in SEQ ID NO. 1 and 2, and obtaining RAA amplification products; The RAA amplification product is added to the CRISPR / Cas12a detection system for reaction, wherein the crRNA nucleotide sequence of the CRISPR / Cas12a detection system is shown in SEQ ID NO.6; Detect the fluorescence changes in the CRISPR / Cas12a detection system to complete fluorescence quantification.
5. The detection method according to claim 4, characterized in that: The conditions of the RAA amplification reaction are 37°C and 10-30 min.
6. The detection method according to claim 5, characterized in that: The conditions of the RAA amplification reaction are 37° C. and 10 min.
7. The detection method according to claim 4, characterized in that: The conditions of the CRISPR / Cas12a detection system are 37°C and 30 min.