CN114686608A - A rapid visual detection method for Actinobacillus pleuropneumoniae based on CRISPR-Cas12a - Google Patents

Abstract

The invention relates to a CRISPR-Cas12 a-based method for rapidly and visually detecting actinobacillus pleuropneumoniae. In particular to a kit, which comprises a pair of specific primers aiming at apxIV, and the primers are SEQ ID NO: 14 and 15, 16 and 17, 18 and 19, 20 and 21, or 26 and 27 (preferably SEQ ID NOs: 26 and 27), and the sequence of the product amplified against the above primer is SEQ ID NO: 3. 5, 6 or 8 (preferably SEQ ID NO: 8) for the rapid visual detection of A. pleuropneumoniae. The invention relates to a detection method based on RPA and a CRISPR/Cas12a system, which is characterized in that a Cas12a protein is utilized, a specific Recombinase Polymerase Amplification (RPA) primer and a CRISPR/Cas12a system probe are designed according to App specific gene apxIV, so that RPA specific target gene amplification is realized, a reporter gene is cut by the CRISPR/Cas12a system, a method for detecting pig actinobacillus pleuropneumoniae is established, and an RPA system and a CRISPR/Cas12a reaction system are optimized. The method is rapid, visual, specific and sensitive, and can be used for clinical detection.

Description

Rapid visual detection method of Actinobacillus pleuropneumoniae based on CRISPR-Cas12a

technical field

The invention relates to the field of medical detection methods, in particular to a rapid visual detection method for Actinobacillus pleuropneumoniae based on CRISPR-Cas12a.

Background technique

Porcine Contagious pleuropneumonia (PCP) is a respiratory disease characterized by hemorrhagic pleuritis and necrotizing pneumonia [1] caused by Actinobacillus pleuropneumoniae (APP) infection. In 1957, Pattison et al. discovered the disease for the first time in the United Kingdom. In 1987, the Harbin Veterinary Research Institute reported that the disease appeared in my country for the first time. Since then, the disease has been discovered in many provinces and cities. In 2006, a serological survey was conducted on large-scale pig farms in Luoyang, and the positive rate was 30.39%. It is recognized as one of the three major respiratory bacterial diseases that endanger the pig industry in the world. Acutely infected pigs can die within a few hours, and the mortality rate is extremely high, but most cases are latent infection. Recessive infected pigs are the carriers and spreaders of pathogens, and can become the outbreak source of pathogens under appropriate conditions, which has brought serious economic losses to the world pig industry. Therefore, establishing a quick and easy detection method is of great significance to the prevention and control of APP.

APP belongs to Actinobacteriaceae of Pasteuraceae, Gram-negative coccus. Up to now, APP has identified 18 serotypes through typing [2]. According to whether its growth requires V factor, it can be divided into biological type I and Biological type II, Actinobacillus pleuropneumoniae (APP) has multiple virulence factors, and hemolysin is one of the main virulence factors of APP pathogenesis. APP secretes four hemolysins, Apx I, Apx II, Apx III, and ApxIV. , each hemolysin has different pathogenicity, of which ApxIV can be secreted by all serotypes, which has been proved to be species-specific [3]. Therefore, ApxIVA gene can be used as a detection target for clinical diagnosis of APP infection.

In vitro nucleic acid amplification (NAA) has penetrated into various fields of life sciences since the advent of polymerase chain reaction (PCR) in 1983 [4]. By providing a temperature favorable for nucleic acid replication, the molecular weight of nucleic acid is amplified exponentially, which is more favorable for subsequent nucleic acid processing and detection. Although PCR has unprecedented pioneering progress, it is difficult to get out of the laboratory because of its high dependence on the PCR machine. Isothermal amplification technology has developed rapidly in recent years, among which loop-mediated isothermal amplification (LAMP) technology has been developed for the detection of animal pathogens. LAMP detection has high sensitivity, but the addition of multiple pairs of primers in the reaction reduces its reliability, which may produce False positive test results while requiring relatively high temperatures to initiate the reaction. Recombinase Polymerase Amplification (RPA) is a new constant temperature amplification technology developed by Niall Armes of ASM Science Co., Ltd. in 2006 [5], which can achieve specific nucleic acid sequences at a constant temperature of 37-42 °C. The amplification, and completes billions of DNA copies within 10 ~ 40min. RPA is an isothermal amplification technology, which requires low equipment and equipment, and only needs a constant temperature water bath to complete the reaction without precision instruments. It has developed rapidly in recent years.

CRISPR-Cas technology is a newly emerging genome editing technology with strong target specificity. It uses its own RNA to pair with the target DNA to identify specific sequences. For double-stranded DNA, the CRISPR system requires additional recognition of a Protospacer Adjacent Motif (PAM) sequence on the target DNA. The PAM of Cas12a prefers AT-containing sequences, so by using a CRISPR system containing different gRNAs (guide RNA, which refers to sgRNA or crRNA), double-stranded DNA with a PAM sequence that matches the gRNA sequence can be recognized. After the cas12a protein recognizes the target, the enzymatic activity of its single-stranded DNA is activated, and indiscriminately cuts any single-stranded DNA in the system. The cutting speed is extremely fast, reaching more than 1000 molecules per second, and can last for several hours or more. Using this property, the light-emitting molecule can be linked by single-stranded DNA to an inhibitory molecule (referred to as a reporter sequence) that prevents the light-emitting molecule from emitting light. When Cas12a is activated by sequence-specific double-stranded DNA, it cleaves the single-stranded DNA that connects the light-emitting molecule and the inhibitory molecule, which removes the light-emitting inhibitory molecule and allows the light-emitting molecule to emit light, thereby detecting to the optical signal.

Based on this, the Doudna team established DETECTR, a CRISPR-based nucleic acid detection method. Zhang Feng's team combined CRISPR technology with lateral flow test strips to develop SHERLOCK technology, which can detect Zika virus (ZIKV) and dengue fever (DHF) within 2 hours.

SUMMARY OF THE INVENTION

The present disclosure provides the following technical solutions:

1. A kit comprising a pair of specific primer pairs for apxIV of SEQ ID NO: 14 and 15, SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, or SEQ ID NO: 16 and 17 ID NOs: 26 and 27 (preferably SEQ ID NOs: 26 and 27), and gRNAs with the sequence of SEQ ID NO: 3, 5, 6 or 8 (preferably SEQ ID NO: 8) for the products amplified by the above primers.

2. The kit of item 1, further comprising Cas12a protein.

3. The kit of any of the preceding, wherein the specific primers for apxIV are used in recombinase polymer amplification technology.

4. The kit of any of the foregoing, wherein the gRNA is used in CRISPR-Cas technology.

5. The kit according to any one of the preceding items, further comprising a ssDNA reporter molecule for subsequent fluorescence detection, preferably, the sequence of the ssDNA reporter molecule is SEQ ID NO:10.

6. The kit according to any one of the preceding items, further comprising a ssDNA reporter molecule for subsequent immunochromatographic detection, preferably, the sequence of the ssDNA reporter molecule is SEQ ID NO: 11.

7. The kit according to the preceding item 5 or 6, wherein the concentration of the ssDNA reporter molecule is 500 nM.

8. The kit of any one of the preceding items, wherein the concentration of the gRNA for subsequent immunochromatographic detection is 50 nM.

9. The kit of any preceding item, further comprising a detection buffer for subsequent immunochromatographic detection, wherein the detection buffer is 10% polyethylene glycol.

10. Specific primer pairs for apxIV of SEQ ID NO: 14 and 15, SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, or SEQ ID NO: 26 and 27 (preferably SEQ ID NOs: 26 and 27), and a gRNA with a sequence of SEQ ID NO: 3, 5, 6 or 8 (preferably SEQ ID NO: 8) for the products amplified by the above primers are prepared for rapid visual detection of pleuropneumonia Use of Actinobacteria in a Kit.

The primer pairs and gRNAs described above can be used in any combination.

Detailed description of the invention

The present disclosure establishes a rapid clinical detection method for Actinobacillus pleuropneumoniae (App) based on the recombinase polymerase amplification (Recombinase Polymerase Amplification, RPA) and the CRISPR/Cas12a system. Specifically, the inventors selected the conserved region of the specific gene apxIVA of the App, and designed and screened the probe gRNA of the CRISPR/Cas12a system in the conserved region according to the characteristics of the PAM sequence near the gRNA binding site, and based on the gRNA Design and screen specific RPA primers to achieve RPA-specific target gene amplification and CRISPR/Cas12a system cleavage of reporter genes, establish a method that can be used to detect Actinobacillus pleuropneumoniae, and analyze the RPA system The CRISPR/Cas12a reaction system was optimized, and the specificity and sensitivity of the method were evaluated, and 8 positive clinical samples and 5 negative samples were detected by this method. The bacilli have good specificity and have no cross-reaction to Haemophilus parasuis, Streptococcus suis, Salmonella, porcine circovirus, porcine pseudorabies virus, and porcine epidemic diarrhea virus. Get results in minutes and reduce dye contamination during gel staining.

The present disclosure enhances the detection sensitivity of Cas12a through RPA, and utilizes the lateral fission effect of CRISPR Cas12a, targeting the conserved ApxIVA gene and combining immunochromatography, to detect APP sensitively, specifically, without expensive equipment and visualization.

This method combines RPA-specific amplification with gRNA-specific sequence identification to make the enhanced Cas12a detection more specific; and the detection is carried out at 37 °C, which is suitable for laboratories or on-site lack of equipment such as PCR machines. Use; at the same time, fluorescence method (observation with the naked eye through blue light excitation) or immunochromatography (direct naked eye observation) can be selected according to the existing conditions.

Cas12a has high specificity and sensitivity in the detection of Actinobacillus pleuropneumoniae, with a detection limit of 10 copies/μl. The sensitivity of the new method is similar to RT-PCR. The Cas12a assay can be successfully applied to the rapid detection of Actinobacillus pleuropneumoniae in clinical samples.

This method has the following characteristics:

First, the enhanced Cas12a detection can not only be applied to the analysis of fluorescence detection samples, but also can be combined with immunochromatographic detection for visualization, which can be applied both in the laboratory and in the field.

Second, the enhanced Cas12a detection is stable. Although the efficiency of RPA is variable and affected by factors such as the nucleotide sequence of primers and templates, the lateral cleavage activity of Cas12a can compensate for the low efficiency of RPA, enabling the enhanced Cas12a detection to achieve satisfactory reactivity.

Third, combining RPA-specific amplification with gRNA-specific sequence identification makes enhanced Cas12a detection more specific.

Fourth, an enhanced Cas12a assay was designed and implemented at 37 °C, making it easy to use in poorly equipped laboratories or in situ.

In conclusion, the present disclosure establishes a CRISPR Cas12a-based APP visual nucleic acid detection method. This new method could provide an alternative tool for detecting APPs with less equipment requirements, especially in resource-poor areas.

Description of drawings

Figure 1 shows the fluorescence results of gRNA sequence screening of PCR products in the conserved region of the target sequence, wherein Figure A is the fluorescence detection result, and Figure B is the detection result of the immunochromatographic reagent strip.

Figure 2 shows the fluorescence results of RPA primer screening against the gRNA binding site region.

Figure 3 shows the optimization results of the reaction system, in which Figure A is the optimization result for the concentration of the ssDNA reporter molecule in the immunochromatography method, Figure B is the optimization result for the concentration of the ssDNA reporter molecule in the fluorescence method; Figure C is the optimization result of the immunochromatography method The optimization results for gRNA concentration in Figure D, and Figure D shows the buffer optimization results in immunochromatography.

Figure 4 shows the specific detection results of the CRISPR-Cas12a detection system comparing the signals of several domestic pig pathogenic microorganism DNA samples with those of APP-containing genome samples, in which Figure A is the detection result of immunochromatography, and Figure B is the detection result of fluorescence method (observed with naked eyes), Figure C is the result of fluorescence detection (dynamic detection).

Figure 5 shows the results of sensitivity analysis of the CRISPR-Cas12a detection system, in which Figure A is the detection result of immunochromatography, Figure B is the detection result of fluorescence method (observed with naked eyes), and Figure C is the detection result of fluorescence method (dynamic detection).

Figure 6 shows the detection results of positive and negative samples in the optimal reaction system, wherein Figure A is the immunochromatographic detection result of the positive sample, Figure B is the fluorescence detection result of the positive sample (observed with naked eyes), and Figure C is the fluorescence detection of the positive sample Results (dynamic detection), Figure D is the negative sample immunochromatographic detection result, Figure E is the negative sample fluorescence detection result (naked eye observation), and Figure F is the negative sample fluorescence detection result (dynamic detection).

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

Example 1:

1 Materials and methods

Basic(英国TwistDx公司)、Hiscribe T7 Quick High Yield RNA SynthesisKit(New England Biolabs)、LbCas12a(New England Biolabs公司)、MileniaHybriDetect 1Dipstick(TwistDx)、离心机(eppendorf)、恒温水浴锅(精宏)、电泳仪(Bio-Rad)、涡旋仪(Vortex-Genie2)、及核酸检测成像系统(Bio-Rad)、Nanodrop(IMPLEN)、多功能酶标仪(Perkin Elmer ENSPIRE)、蓝光凝胶成像仪(上海生工)、引物及报告序列均由吉林省库美生物科技有限公司合成。Actinobacillus pleuropneumoniae, Haemophilus parasuis, Streptococcus suis, Salmonella strains porcine circovirus, porcine pseudorabies virus, and porcine epidemic diarrhea virus genomic DNA used in the examples of the present invention are provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences . DNA extraction kit (Beijing Tiangen P302), RNA purification and recovery kit (Beijing Tiangen, DP421),

Basic (TwistDx, UK), Hiscribe T7 Quick High Yield RNA SynthesisKit (New England Biolabs), LbCas12a (New England Biolabs), MileniaHybriDetect 1Dipstick (TwistDx), centrifuge (eppendorf), constant temperature water bath (Jinghong), electrophoresis apparatus (Bio-Rad), Vortex (Vortex-Genie2), Nucleic Acid Detection Imaging System (Bio-Rad), Nanodrop (IMPLEN), Multi-function Microplate Reader (Perkin Elmer ENSPIRE), Blue Gel Imager (Shanghai Bio-Rad) Worker), primers and reporter sequences were synthesized by Jilin Province Kumei Biotechnology Co., Ltd.

2. Method

2.1 Selection of A. pleuropneumoniae target sequence and RPA amplification and gRNA design and amplification

1) Target selection: ApxIVA protein has been proved to be specific to APP species and is often used as a target for pathogenic detection in clinic. Therefore, this method selects 442bp in the conserved region of ApxIVA gene, see SEQ ID NO: 1 for details, as the target sequence .

2) gRNA design: Since Lb Cas12a needs to contain a PAM sequence (TTTN, N is any nucleotide) near the binding site when it specifically cuts double-stranded DNA, the RPA-amplified product must be used as a template to be detected. Contains PAM sequence, therefore, it is very important to find the optimal region containing PAM sequence (the sequence that Cas12a can cut) in the target sequence. Combined with the characteristics that the target to be tested needs to contain the PAM sequence and the length of the RPA amplification product has a larger adjustment range (100-300bp), we first select the gRNA targeting site, and then select the RPA primer according to the region where the gRNA targeting site is located. Strategy. First, use CRISPR RGEN Tools, take the target (SEQ ID NO: 1) as the template, set the parameters: gRNA length is 20bp, score is greater than 66, select 8 gRNA sequences that meet the above parameters as candidate gRNA sequences, see SEQ ID for details ID NO: 2-9, these 8 gRNAs target different PAM sequences at different positions within the conserved region. After screening the optimal gRNA sequence according to the fluorescence intensity, the optimal RPA primer was then designed and screened according to the region of its binding site.

3) Preparation of gRNA: Referring to the instructions of HiScribe T7 Quick High Yield RNA Synthesis Kit, add the T7 promoter sequence as the template for in vitro transcription before the complementary DNA sequence (artificial synthesis) of the gRNA, and after adding the reagents provided in the kit, at 37 Incubate overnight at °C, digest the DNA template with DNaseI, and recover the resulting gRNA with an RNA purification kit. The gRNA concentration was determined with Nanodrop, aliquoted, and frozen at -80°C.

4) Synthesis of ssDNA-reporter (ssDNA reporter molecule)

The CRISPR-Cas12a protein can activate its accessory cleavage activity after binding to the target, that is, it can cut any single-stranded DNA in the system indiscriminately, and the cut nucleotide sequence with a fluorescent group can emit fluorescence, The fluorescence value in the reaction system can be detected by a fluorescence detection device, or directly observed with the naked eye after being excited by blue light, or detected by immunochromatography with a test strip. The 5' end was synthesized with FAM, FITC, RB200, TRITC, TET, PE, PI, AMCA, Att0425, PerCP, APC, Alexa Fluor 488, JOE, VIC, HEX, NED, Cy3, TAMRA, ROX, Texasred, Cy5, Any one of Quasar 670, Cy5.5 and Cy7 modified single-stranded DNA sequence modified with a quencher group at the 3' end for fluorescence detection; or labeled with FAM at the 5' end and modified with Biotin at the 3' end Single-stranded DNA was used for immunochromatographic detection. The PAM of Cas12a is AT-biased, and the single-stranded DNA reporter sequence contains at least one of TTATT, TTTTA, AAAAT, ATATAT.

5) Determination of optimal gRNA

The screening of gRNA uses the target sequence amplified by PCR as the template. In a 50 μl PCR reaction system containing 0.5 μl APP genomic DNA sample, after denaturation: 95°C, 15s; annealing, 55°C, 15s; extension: 72°C, 30s; cycle number: 30s, a template for gRNA screening was obtained. PCR primers: forward primer: 5'-TGGCACTGACGGTGATGATAATATC-3' (SEQ ID NO: 12), reverse primer: 5'-GGCCATCGACTCAACCATCTTCTCC-3' (SEQ ID NO: 13). In the 20uL screening gRNA system, 5ul target (ie, the target gene product amplified by the above PCR), 50nM gRNA (ie, different gRNAs synthesized by in vitro transcription), 50nM Cas12a (purchased from New England Biolabs), 500nM ssDNA- reporter, 1×NEBuffer 2.1; react at 37°C for 15-60min. A specific gRNA and the corresponding detection area were preliminarily screened by whether the fluorescence could be excited during the fluorescent reporter experiment and combined with the fluorescence intensity.

6) RPA primer design: with reference to the binding site of the optimal gRNA sequence obtained by the above screening, in its upstream and downstream regions, use Primer Premier 5.0 to design RPA primers. Although common PCR primers can be used, they are not optimal. Shorter ones will reduce the recombination rate and affect the amplification speed and detection sensitivity. Therefore, the RPA primers are longer than the general PCR primers, but the product of the RPA reaction cannot exceed 500bp. Based on the primer requirements for RPA amplification, the set parameters are: primer length between 30-36 bp, amplification product length between 100-200 bp, GC content between 30-70%, Tm>50°C. Eight pairs of RPA primers satisfying the above parameters were preliminarily selected, and the optimal RPA primers were selected according to the fluorescence intensity, see SEQ ID NOs: 14-29 for details.

Basic产品说明书，RPA反应体系50μL：其中包括RPA上游扩增引物0.48μM，RPA下游扩增引物0.48μM，1×Rehydration Buffer，14mM MgOAc，待检测样本基因组DNA 1μL，其余用水补足50μL。RPA扩增程序：恒温37-40℃反应15min。7) RPA amplification system and procedures: according to

Basic product manual, RPA reaction system 50 μL: including RPA upstream amplification primer 0.48 μM, RPA downstream amplification primer 0.48 μM, 1× Rehydration Buffer, 14 mM MgOAc, 1 μL of genomic DNA of the sample to be tested, and the rest with water to make up 50 μL. RPA amplification procedure: reaction at constant temperature of 37-40°C for 15min.

2.2 Pathogenic bacteria culture and genome extraction

Take Actinobacillus pleuropneumoniae strain S8 (provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences), streak on TNA (TSA containing 5% horse serum, 10 μg/mL NAD) plate, culture at 37 °C overnight, pick a single colony in The cells were cultured in TNB (TSB containing 5% horse serum and 10 μg/mL NAD) for 12 h, and the bacterial genome was extracted using a DNA extraction kit for bacterial genome of Rhizoctonia, and the genome was quantified using Nanodrop.

2.3 Genome extraction from clinical samples

Genomes were extracted according to the Blood and Tissue Sample Genomic DNA Extraction Kit (DP304-03) and quantified using Nanodrop for genomes.

2.4 CRISPR-Cas12a combined with RPA amplification for fluorescence detection

The RPA amplified target gene product was taken and added to the CRISPR-Cas12a reaction system. 20uL final system contains 100ng target-ApxIVA (ie, target gene product amplified by RPA), 50nM gRNA (ie, gRNA targeting gene product amplified by RPA), 50nM Cas12a (purchased from New England Biolabs), 500nM ssDNA-reporter , 1×NEBuffer, the sequence used in the ssDNA-reporter described here is 5'-FAM-TTATT-TAMRA-3' (SEQ ID NO: 10). The reaction was carried out at 37 °C for 60 min, and the FAM fluorescence was collected at 485 nm excitation wavelength and 535 nm emission wavelength by a multifunctional microplate reader (Perkin Elmer ENSPIRE) at 1 min intervals; or directly after the reaction for 15 min with a blue light gel imager. Observed. The negative control was set by replacing target-ApxIVA with deionized water.

2.5 CRISPR-Cas12a combined with RPA amplification for immunochromatographic detection

Take the RPA amplification product and add it to the CRISPR-Cas12a reaction system. The 20uL final system contains 100ng target-ApxIVA, 100nM gRNA, 50nM Cas12a, 500nM ssDNA-reporter, 1×NEBuffer, and react at 37℃ for 60min. The sequence used for the ssDNA-reporter described here is 5'-FAM-TTATT-Biotin-3' (SEQ ID NO: 11). The above 20 μl reaction system was transferred to 80 μl detection buffer, and then immunochromatographic test strips (MileniaHybriDetect 1 Dipstick, TwistDx Company) were put into the solution, and the results were observed with naked eyes after 5 minutes of incubation. The negative control was set by replacing target-ApxIVA with deionized water.

2.6 Specificity experiments

Using the established detection method in this example, common bacteria including Actinobacillus pleuropneumoniae, Haemophilus parasuis, Streptococcus suis, Salmonella, porcine circovirus, porcine pseudorabies virus, and porcine epidemic diarrhea virus were detected. and viruses were tested, and water was set up as a negative control. The specificity of the detection method is judged according to the detection results.

2.7 Sensitivity experiment

PCR primers for amplification with homologous recombination sites were designed according to the conserved regions of ApxIVA:

P1: 5'-GCTTGCATGCCTGCAGTGGCACTGACGGTGAT-3' (SEQ ID NO: 30)

P2: 5'-CTGAATTCGAGCTCGGTACCGGCCATCGACTCAACCAT-3' (SEQ ID NO: 31), the amplified product was recovered, and ligated to the puT18 vector to obtain a plasmid containing the conserved region, the DNA concentration was determined, the copy number was calculated, and the 10-fold serial dilution, set up water as a negative control, and use the established detection method to evaluate its sensitivity.

2.8 Clinical sample inspection

8 cases of Actinobacillus pleuropneumoniae positive tissue samples and 5 cases of negative samples confirmed by conventional PCR were selected, and the established PCR detection methods were used to detect them. Compare the detection results. The identification primers of Actinobacillus pleuropneumoniae were selected by conventional PCR, AP-IVF (5'-ATACGGTTA ATGGCGGTAATG G-3') (SEQ ID NO: 32), AP-IVR (5'-ACCTGAGTGCTCACCAACG-3') (SEQ ID NO: 33) ) to expand.

Example 2:

result

1. Screening of specific gRNA sequences

Using the conserved region of the target sequence amplified by PCR as a template, 5ul target (that is, the product of the conserved region of the target gene amplified by PCR), 50nM gRNA (that is, different gRNAs synthesized by in vitro transcription), 50nM Cas12a were included in the 20uL screening system (purchased from New England Biolabs), 500nM ssDNA-reporter, 1×NEBuffer 2.1 (its components are 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100μg/ml BSA pH 7.9); 37°C for 60min. A specific gRNA and the corresponding detection area were preliminarily screened by whether the fluorescence could be excited during the fluorescent reporter experiment and combined with the fluorescence intensity. The DNA sequences of 8 candidate gRNAs were designed and synthesized based on the conserved regions of the target genes, and the gRNAs were generated by in vitro transcription (see Table 1), and the optimal gRNAs were simultaneously screened by fluorescence method (Fig. , the fluorescence method results show that: seq-1, seq-2, seq-3, seq-4, seq-5, seq-7 can reach the peak within 30min. The results of immunochromatography showed that: seq-2, seq-4, seq-5, seq-6, seq-7, seq-8 sequences could produce clear positive bands. Combining the results of the two methods, the seq-7 (SEQ ID NO: 8) sequence peaked the fastest and had a clear positive band. The seq-7 (SEQ ID NO: 8) sequence was preferred as the gRNA for subsequent detection, while the seq- 2 (SEQ ID NO:3), seq-4 (SEQ ID NO:5), seq-5 (SEQ ID NO:6) can also be used as alternative gRNAs.

Table 1 gRNA candidate sequences

Note: "Starting position" in the table refers to the position of the first base of PAM in the conserved region (SEQ ID NO: 1) of ApxIVA gene

2. RPA primer screening

According to the binding site of the optimal gRNA (SEQ ID NO: 8) (318-341 bp in the conserved region), 8 pairs of candidate RPA primers (see Table 2) were designed and synthesized under the condition that the design requirements of RPA primers were met. The best RPA primers were screened by the RPA and CRISPER-Cas methods and fluorescence method in Example 1. The results showed that the primer combinations of Nos. 1, 2, 3, 4, and 7 could all produce blue fluorescence by naked eyes, but No. 7 The primer combination of 185-FR (ie, 185-F and 185-R) has the strongest fluorescence intensity (see Figure 2). Therefore, the primer pair 185-FR (ie, 185-F and 185-R) is preferably used as the primer for RPA amplification for subsequent detection.

The target sequence of the RPA primer is SEQ ID NO:1.

Table 2 RPA candidate primer sequences

Note: From the start and end sites of the RPA product, it can be seen that the products amplified by primer pairs No. 1, 2, 3, 4, and 7 cover the regions bound by gRNA seq-2, 4, 5, and 7. , so those skilled in the art can understand that these RPA primer pairs and gRNA seq-2, 4, 5, and 7 can be used in any combination for fluorescence and immunochromatography.

3. Reaction system optimization

In order to obtain better detection effect, we optimized the ssDNA reporter concentration, gRNA concentration and detection buffer components in the detection system. The results showed that when the concentration of ssDNA reporter molecule was 500nM by immunochromatography, the detection line of negative samples was the shallowest and the false positive effect was the weakest (Figure 3A). 3B); immunochromatography showed that the ideal effect could still be achieved when the concentration of gRNA in the system was 50nM (Fig. 3C). When using the reagent strip for the final detection, the immunochromatography method compared the buffer that came with the reagent strip when purchased. and the effect of 10% polyethylene glycol as the buffer, the results show that the detection line of the negative sample can completely disappear when 10% polyethylene glycol is used as the buffer (Fig. 3D).

Therefore, the optimal CRISPR-Cas12a reaction system for detecting A. pleuropneumoniae based on the combination of RPA and CRISPR/Cas12a in the embodiment of the present invention is:

Fluorescence detection: 20uL final system contains 100ng target-ApxIVA, 50nM gRNA, 50nM Cas12a, 500nM ssDNA-reporter, 1×NEBuffer.

Immunochromatographic detection: 20uL final system contains 100ng targe-ApxIVA, 50nM gRNA, 50nMCas12a, 500nM ssDNA-reporter, 1×NEBuffer.

The immunochromatographic detection buffer was 10% polyethylene glycol.

4. Specific detection

To evaluate the specificity of Cas12a immunochromatographic and fluorometric detection, we tested a variety of Bacteria and viruses were tested. The results showed that the nucleic acid samples were detected by immunochromatographic assay and fluorescence method (visual method, fluorescence value statistics), and the immunochromatographic detection bands of other 6 pathogens except Actinobacillus pleuropneumoniae were negative. (FIGS. 4A-4C).

5. Sensitivity testing

To analyze the sensitivity of the enhanced Cas12a immunochromatographic detection, we tested the above-mentioned 10-fold serial dilutions containing conservative The results show that the detection limit of fluorescence method and immunochromatography method are both 10copies/μl.

6. Clinical sample detection

In order to further confirm the application effect of this visualization method in clinical samples, genome extraction was performed on lung tissue samples (Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) infected with Actinobacillus pleuropneumoniae from different pig farms, and the selection was confirmed by conventional PCR method. 8 cases of Actinobacillus pleuropneumoniae positive tissue samples and 5 cases of negative tissue samples were detected by the method of Example 1 combined with the best reaction system described in Example 2, and the results of immunochromatography showed that the positive samples were in addition to Except for the negative control, the immunochromatographic detection bands were all positive (Fig. 6A); except for the positive control, the remaining immunochromatographic detection bands of the negative samples were negative (Fig. 6D). The results of naked eye observation showed that, except for the negative control, the positive samples all showed green fluorescence (Fig. 6B), and the negative samples did not emit light (Fig. 6E). The fluorescence detection results showed that the fluorescence values of the positive samples were all above 12000, and the negative control values were below 2000 (Figure 6C). In the negative samples, except for the positive control fluorescence values above 12000, the fluorescence values of the remaining samples were all below 2000 (Figure 6F). ). The detection result of this method is consistent with the detection result of PCR method.

The above results prove that this detection method can be used for the rapid detection of Actinobacillus pleuropneumoniae in clinical samples. Different detection methods can be adopted in combination with the experimental conditions and the number of detection samples. Under the condition of pre-prepared sample genome in the laboratory, the detection process can be completed within 30 minutes, and the detection results can also be obtained within 75 minutes when detected by reagent strips. Compared with the laboratory conventional PCR detection method (amplification 60 minutes, agar Glucose gel electrophoresis detection 30min), the method of the present invention can realize rapid visual detection, and reduce the dye pollution generated during gel staining.

The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above-mentioned specific embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principle of the present invention, any modifications, equivalent replacements, improvements, etc. made should be included within the protection scope of the present invention.

Citation

1 Guo Kun, Gao Xiaona, Luo Junrong, Wang Tiancheng, Guo Xiaoshu, Guo Xiaoquan, Hu Guoliang, Heilongjiang Animal Husbandry and Veterinary Liu J: Research Progress on Actinobacillus pleuropneumoniae 2017(2): 59-62

2Bosse JT, Li Y, Fernandez Crespo R, Lacouture S, Gottschalk M, Sarkozi R, Fodor L, Casas Amoribieta M, Angen O, Nedbalcova K et al: Comparative sequence analysis of the capsular polysaccharide loci of Actinobacillus pleuropneumoniae serovars 1-18, and development of two multiplex PCRs forcomprehensive capsule typing. Vet Microbiol 2018, 220: 83-89.

3 Huang Hongliang, Zhou Rui, Chen Meiling, Liu Jianjie, Xu Xiaojuan, Chinese Journal of Biological Engineering Chen J: Cloning and expression of apxIVA gene of Actinobacillus pleuropneumoniae toxin and establishment of indirect ELISA method 2005, 21(2): 294-299

4 Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H: Specificenzymatic amplification of DNA in vitro: the polymerase chain reaction. ColdSpring Harb Symp Quant Biol 1986, 51 Pt 1:263-273.

5 Piepenburg O, Williams CH, Stemple DL, Armes NA: DNA detection using recombination proteins. PLoS Biol 2006, 4(7):e204

6Li SY, Cheng QX, Liu JK, Nie XQ, Zhao GP, Wang J: CRISPR-Cas12a has bothcis-and trans-cleavage activities on single-stranded DNA. Cell Res 2018, 28(4): 491-493.

Claims (10)

1. A kit comprising a pair of specific primer pairs for apxIV of SEQ ID NO: 14 and 15, SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, or SEQ ID NO: 16 and 17 ID NOs: 26 and 27 (preferably SEQ ID NOs: 26 and 27), and gRNAs with the sequence of SEQ ID NO: 3, 5, 6 or 8 (preferably SEQ ID NO: 8) for the products amplified by the above primers. 2. The kit of claim 1, further comprising Cas12a protein. 3. The kit of any preceding claim, wherein the specific primers for apxIV are used in a recombinase polymer amplification technique. 4. The kit of any preceding claim, wherein the gRNA is used in CRISPR-Cas technology. 5. The kit of any preceding claim, further comprising a ssDNA reporter molecule for subsequent fluorescence detection, preferably, the sequence of the ssDNA reporter molecule is SEQ ID NO:10. 6. The kit of any preceding claim, further comprising an ssDNA reporter molecule for subsequent immunochromatographic detection, preferably, the sequence of the ssDNA reporter molecule is SEQ ID NO:11. 7. The kit of claim 5 or 6, wherein the concentration of the ssDNA reporter molecule is 500 nM. 8. The kit of any preceding claim, wherein the concentration of gRNA for subsequent immunochromatographic detection is 50 nM. 9. The kit of any preceding claim, further comprising a detection buffer for subsequent immunochromatographic detection, wherein the detection buffer is 10% polyethylene glycol. 10. Specific primer pairs for apxIV of SEQ ID NO: 14 and 15, SEQ ID NO: 16 and 17, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, or SEQ ID NO: 26 and 27 ( Preferably SEQ ID NOs: 26 and 27), and gRNAs with sequences of SEQ ID NO: 3, 5, 6 or 8 (preferably SEQ ID NO: 8) for the products amplified by the above primers are prepared for rapid visual detection of the pleura Use of the kit for Actinobacillus pneumoniae.

Images