CN114686608A - 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 for 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-Cas12 a.

Background

Porcine Contagious Pleuropneumonia (PCP) is a respiratory disease characterized by hemorrhagic pleurisy and necrotizing pneumonia [1] caused by Actinobacillus pleuropneumoniae (APP) infection. In 1957, Pattison et al first discovered the disease in the United kingdom, in 1987, the Harbin veterinary institute first reported that the disease occurred in China, and thereafter, the disease was discovered in succession in many provinces and cities. In 2006, serology investigation was performed on a large-scale pig farm in the Luoyang region, the positive rate was 30.39%, in 2009, non-immune pig serum in Sichuan was detected, and the positive rate was 18.5%. Is one of three major respiratory bacterial diseases which are recognized in the world and endanger the pig industry, the acutely infected pigs can die within a few hours, the death rate is extremely high, but most cases show recessive infection. The recessive infection pig is a carrier and a transmitter of the pathogeny, can become a outbreak of the pathogeny under proper conditions, and brings serious economic loss to the world pig industry. Therefore, the establishment of a rapid and simple detection method has great significance for the prevention and control of APP.

APP belongs to the family Pasteurellaceae, actinobacillus, gram negative small ball bacillus, APP has already been identified 18 serotypes by typing [2], whether V factor is required for growth can be divided into biological I type and biological II type, actinobacillus pleuropneumoniae (APP) has a plurality of virulence factors, hemolysin is one of the main virulence factors causing APP diseases, APP secretes Apx I, Apx II, Apx III and ApxIV, four hemolysins are respectively contained, each hemolysin has different pathogenicity, wherein all serotypes of ApxIV can be secreted, species specificity is proved to have [3], therefore, ApxIVA gene can be used as a detection target point for APP infection clinical diagnosis.

In vitro nucleic acid amplification technology (NAA) has penetrated various fields of life sciences since the advent of the Polymerase Chain Reaction (PCR) in 1983 [4 ]. By providing a temperature that facilitates nucleic acid replication, the molecular weight of the nucleic acid is exponentially amplified, thereby facilitating subsequent nucleic acid processing and detection. Although PCR has unprecedented pioneering advances, it is difficult to move outside the laboratory due to the high dependence on PCR instruments. Isothermal amplification technology has been rapidly developed in recent years, among which loop-mediated isothermal amplification (LAMP) technology has been developed for animal pathogen detection, which has high detection sensitivity but has reduced reliability due to the addition of multiple pairs of primers in the reaction, may produce false positive detection results, and requires relatively high temperature to start the reaction. A Recombinase Polymerase Amplification (RPA) is a novel isothermal Amplification technology [5] developed by Niall arms of ASM science, Inc. in 2006, and can amplify a specific nucleic acid sequence at a constant temperature of 37-42 ℃ and complete billions of DNA copies within 10-40 min. RPA belongs to isothermal amplification technology, has low requirements on instruments and equipment, can complete reaction only by a constant-temperature water bath kettle, does not need precise instruments, and has rapid development in recent years.

The CRISPR-Cas technology is a newly emerged genome editing technology, has strong target specificity, and utilizes self RNA to perform sequence pairing with target DNA so as to recognize a specific sequence. For double-stranded DNA, the CRISPR system needs to additionally recognize a Protospacer Adjjacent Motif (PAM) sequence on the target DNA. PAM of Cas12a favors sequences containing AT, so double stranded DNA with PAM sequence and matching gRNA sequence can be recognized by using CRISPR system containing different grnas (guide RNA, which refers to guide sgRNA or crRNA). After the cas12a protein recognizes the target, the enzymatic activity of its single-stranded DNA is activated, and indiscriminate cutting is performed on any single-stranded DNA in the system, the cutting speed is extremely fast, reaching more than 1000 molecules per second, and the cutting can last for more than several hours. By virtue of this property, the luminescent molecule can be linked via single-stranded DNA to a suppressor molecule (referred to simply as a reporter sequence) which prevents the luminescent molecule from emitting light. When Cas12a is activated by sequence-specific double-stranded DNA, it cleaves the single-stranded DNA joining the light-emitting and the inhibiting molecules, which removes the light-emitting inhibiting molecule, allowing the light-emitting molecule to emit light, and thus detecting the light signal.

Based on the method, the Doudna team establishes a nucleic acid detection method DETECTR based on CRISPR. By combining the CRISPR technology with a lateral flow test strip, a Zhang team develops the SHERELOCK technology, and Zika virus (ZIKV) and dengue fever (DHF) can be detected within 2 h.

Disclosure of Invention

The present disclosure provides the following technical solutions:

1. a kit comprising a pair of primers specific for apxIV and corresponding to 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 the sequence of the product amplified against the above primer is SEQ ID NO: 3. 5, 6 or 8 (preferably SEQ ID NO: 8).

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

3. The kit of any one of the preceding claims, wherein the specific primer for apxIV is 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 for a subsequent fluorescence detection method, preferably the sequence of the ssDNA reporter is SEQ ID NO: 10.

6. the kit of any preceding claim, further comprising a ssDNA reporter for a subsequent immunochromatographic assay, preferably the sequence of said ssDNA reporter is SEQ ID NO: 11.

7. the kit of any preceding item 5 or 6, wherein the concentration of the ssDNA reporter is 500 nM.

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

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

10. Specific primer pair for apxIV 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 the sequence of the product amplified against the above primer is SEQ ID NO: 3. 5, 6 or 8 (preferably SEQ ID NO: 8) in the preparation of a kit for the rapid visual detection of Actinobacillus pleuropneumoniae.

The primer pair and the gRNA described above may be used in any combination.

Detailed Description

The invention discloses a detection method for rapidly detecting porcine actinobacillus pleuropneumoniae (App) based on Recombinase Polymerase Amplification (RPA) and a CRISPR/Cas12a system. Specifically, the inventor selects a conserved region of App specific gene apxIVA, designs and screens probe gRNA of a CRISPR/Cas12a system in the conserved region according to the characteristic that PAM sequence is required near a gRNA binding site, designs and screens specific RPA primer according to the binding site of the gRNA to realize RPA specific target gene amplification and the cutting of reporter gene by the CRISPR/Cas12a system, establishes a method for detecting pig actinobacillus pleuropneumoniae, optimizes the RPA system and the CRISPR/Cas12a reaction system, evaluates the specificity and sensitivity of the method, and detects 8 positive clinical samples and 5 negative samples by using the method, and the result shows that: the detection method has the advantages that the actinobacillus pleuropneumoniae has good specificity, no cross reaction exists on haemophilus parasuis, streptococcus suis, salmonella, porcine circovirus, porcine pseudorabies virus and porcine epidemic diarrhea virus, the sensitivity is 10 copies/mu l, the detection result can be obtained within 30 minutes at the fastest speed, and the dye pollution generated during gel dyeing is reduced.

According to the application, the detection sensitivity of Cas12a is enhanced through RPA, and the lateral cleavage effect of CRISPR Cas12a is utilized, so that the application is sensitively and specifically detected by aiming at a conserved ApxIVA gene and combining immunochromatography, and expensive equipment and visualization are not needed.

The method combines RPA specific amplification with gRNA specific sequence identification, so that the enhanced Cas12a detection has more specificity; the detection is carried out at the temperature of 37 ℃, and the kit is suitable for laboratories or places lacking instruments and equipment such as a PCR instrument and the like; meanwhile, a fluorescence method (through blue light excitation and visual observation) or an immunochromatography method (direct naked eye observation) can be selected according to the existing conditions

Cas12a has high specificity and sensitivity for detecting actinobacillus pleuropneumoniae, and the detection limit is 10 copies/mu l. The sensitivity of the new method is similar to RT-PCR. Cas12a detection can be successfully applied to the rapid detection of actinobacillus pleuropneumoniae in clinical samples.

The method has the following characteristics:

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

Second, the enhanced Cas12a assay has stability. Although the efficiency of RPA is variable, influenced by factors such as the nucleotide sequence of the primer and the template, the low efficiency of RPA can be compensated by the cleavage activity of Cas12a, so that the enhanced Cas12a detection achieves satisfactory reaction activity.

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

Fourth, enhanced Cas12a detection was designed and implemented at 37 ℃ making it easy to use in poorly equipped laboratories or on-site.

In summary, the present disclosure establishes an APP visualized nucleic acid detection method based on CRISPR Cas12 a. The new method can provide an alternative tool for detecting APP, has low requirements on equipment, and is particularly suitable for resource-poor areas.

Drawings

FIG. 1 shows the fluorescence results of gRNA sequence screening of PCR products of target sequence conserved regions, wherein, a is the fluorescence detection result, and B is the detection result of an immunochromatography reagent strip.

Figure 2 shows the results of fluorescence screening for RPA primers to the gRNA binding site region.

FIG. 3 shows the results of reaction system optimization, wherein panel A is the results of the optimization for ssDNA reporter concentration in immunochromatography and panel B is the results of the optimization for ssDNA reporter concentration in fluorimetry; panel C is the optimization result for gRNA concentration in immunochromatography, and panel D is the optimization result for buffer in immunochromatography.

Fig. 4 shows the specific detection results of the CRISPR-Cas12a detection system for comparing signals of DNA samples of several pig pathogenic microorganisms with those of APP-containing genome samples, wherein, fig. a is the detection result of immunochromatography, fig. B is the detection result of fluorescence (naked eye observation), and fig. C is the detection result of fluorescence (dynamic detection).

Fig. 5 shows the sensitivity analysis result of the CRISPR-Cas12a detection system, wherein, fig. a is the immunochromatography detection result, fig. B is the fluorescence detection result (naked eye observation), and fig. C is the fluorescence detection result (dynamic detection).

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

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

Example 1:

1 materials and methods

The actinobacillus pleuropneumoniae used in the examples of the present invention,the genomic DNA of haemophilus parasuis, streptococcus suis, salmonella strains porcine circovirus, porcine pseudorabies virus and porcine epidemic diarrhea virus is provided by Harbin veterinary institute of Chinese academy of agricultural sciences. DNA extraction kit (Beijing Tiangen P302), RNA purification and recovery kit (Beijing Tiangen, DP421),

Basic (TwistDx, UK), Hiscript T7 Quick High Yield RNA Synthesis Kit (New England Biolabs), LbCas12a (New England Biolabs), Milenia HybriDetect 1 Diptic (TwistDx), centrifuge (eppendorf), thermostat water bath (fine macro), electrophoresis (Bio-Rad), vortexer (Vortex-Genie2), and nucleic acid detection imaging system (Bio-Rad), Nanodrop (IMPLEN), multifunctional enzyme reader (Perkin Elmer ENSPIRE), blue-light gel imager (Shanghai Processo), primers and reporter sequences were all synthesized by Guilin Kumai Tech, Inc.

2. Method of producing a composite material

2.1 Actinobacillus pleuropneumoniae target sequence selection and RPA amplification and gRNA design and amplification

1) Target selection: the ApxIVA protein has been shown to be specific for APP species and is commonly used clinically as a target for etiology detection, so the method selects 442bp of a conserved region in the ApxIVA gene, as detailed in SEQ ID NO: 1 as a target sequence.

2) gRNA design: since the lbcas12a needs to contain a PAM sequence (TTTN, N is any one nucleotide) near the binding site when specifically cleaving double-stranded DNA, the product after RPA amplification must contain the PAM sequence when being used as a template to be detected, and therefore, it is important to find an optimal region (a sequence that Cas12a can cleave) containing the PAM sequence in a target sequence. Combining the characteristic that the target to be detected needs to contain a PAM sequence and the length of an RPA amplification product has a larger adjustment range (100-300bp), a strategy of preferentially selecting a gRNA targeting site and preferentially selecting an RPA primer according to the region of the gRNA targeting site is adopted. Firstly, using CRISPR RGEN Tools, using the target (SEQ ID NO: 1) as a template, setting parameters: the length of gRNA is 20bp, score is larger than 66, 8 gRNA sequences meeting the parameters are selected as candidate gRNA sequences, and the candidate gRNA sequences are shown in SEQ ID NO: 2-9, the 8 grnas target different PAM sequences at different positions within the conserved region. And after the optimal gRNA sequence is screened out according to the fluorescence intensity, the optimal RPA primer is screened out according to the region where the binding site is designed.

3) Preparation of gRNA: with reference to the description of HiScribe T7 Quick High Yield RNA Synthesis Kit, the T7 promoter sequence was added as a template for in vitro transcription before the DNA sequence complementary to the gRNA (synthesized), reagents provided in the Kit were added, incubation was carried out overnight at 37 ℃ and the DNA template was digested with DNaseI, and the resulting gRNA was recovered using an RNA purification Kit. The gRNA concentration was determined by Nanodrop and was dispensed and frozen at-80 ℃.

4) ssDNA-reporter synthesis

After being combined with a target, the CRISPR-Cas12a protein can activate the accessory cutting activity of the protein, namely, the characteristic of indiscriminate cutting of any single-stranded DNA in a system is realized, the cut nucleotide sequence with a fluorescent group can emit fluorescence, and the fluorescence value in a reaction system can be detected through a fluorescence detection device, or the fluorescence value can be directly observed through naked eyes after being excited by blue light, or the immunochromatography detection can be carried out through a test strip. Respectively synthesizing single-stranded DNA sequences with 5 'ends modified by any one of FAM, FITC, RB200, TRITC, TET, PE, PI, AMCA, Att0425, PerCP, APC, Alexa Fluor 488, JOE, VIC, HEX, NED, Cy3, TAMRA, ROX, Texas red, Cy5, quaar 670, Cy5.5 and Cy7 and 3' ends modified by quenching groups for fluorescence detection; or single-stranded DNA with the 5 'end labeled with FAM and the 3' end modified with Biotin is used for immunochromatography detection. PAM of Cas12a is AT preference, and the single stranded DNA reporter contains AT least one of TTATT, TTTTA, aaaaat, ataat.

5) Determination of optimal gRNA

The selection of gRNA takes the target sequence amplified by PCR as a template. A 50 μ l PCR reaction containing 0.5 μ l app genomic DNA sample, denatured: 95 ℃ for 15 s; annealing at 55 ℃ for 15 s; extension: 72 ℃ for 30 s; the number of cycles: and 30s, obtaining a template for gRNA screening. PCR primers: a forward primer: 5'-TGGCACTGACGGTGATGATAATATC-3' (SEQ ID NO: 12), reverse primer: 5'-GGCCATCGACTCAACCATCTTCTCC-3' (SEQ ID NO: 13). 5uL target (i.e., the target gene product amplified by PCR as described above), 50nM gRNA (i.e., different gRNA synthesized by in vitro transcription), 50nM Cas12a (available from New England Biolabs), 500nM ssDNA-reporter, 1 XNEBuffer 2.1; reacting at 37 ℃ for 15-60 min. A gRNA with strong specificity and a corresponding detection area are preliminarily screened out by judging whether fluorescence can be excited or not in the fluorescence report experiment process and combining the fluorescence intensity.

6) RPA primer design: referring to the binding site of the optimal gRNA sequence obtained by the screening, in the upstream and downstream regions, an RPA Primer is designed by using Primer Premier 5.0, although a common PCR Primer can be used, the common PCR Primer is not optimal, the short Primer can reduce the recombination rate and influence the amplification speed and the detection sensitivity, therefore, the RPA Primer is longer than the common PCR Primer, but the product of the RPA reaction cannot exceed 500 bp. Based on the requirement of RPA amplification on the primer, the parameters are set as follows: the length of the primer is between 30 and 36bp, the length of the amplification product is between 100 and 200bp, the GC content is between 30 and 70 percent, and the Tm is more than 50 ℃. Preliminarily selecting 8 pairs of RPA primers meeting the parameters, and selecting the optimal RPA primer according to the fluorescence intensity, which is detailed in SEQ ID NO: 14-29.

7) RPA amplification system and procedure: according to

Basic product specification, RPA reaction system 50 μ L: the kit comprises 0.48 mu M of RPA upstream amplification primer, 0.48 mu M of RPA downstream amplification primer, 1 × Rehydration Buffer and 14mM MgOAc, 1 mu L of genomic DNA of a sample to be detected, and the balance of water for supplementing 50 mu L. RPA amplification procedure: keeping the temperature constant at 37-40 ℃ for reaction for 15 min.

2.2 cultivation of pathogenic bacteria and extraction of genome

Actinobacillus pleuropneumoniae strain S8 (provided by Harbin veterinary institute of Chinese academy of agricultural sciences) was streaked on a TNA (TSA containing 5% horse serum and 10. mu.g/ml NAD), cultured overnight at 37 ℃, a single colony was picked and cultured in TNB (TSB containing 5% horse serum and 10. mu.g/ml NAD) for 12 hours, the bacterial genome was extracted using a Tiangen bacterial genome DNA extraction kit, and quantification was performed using Nanodrop as the genome.

2.3 clinical sample genome extraction

The genome was extracted according to the blood and tissue sample genomic DNA extraction kit (DP304-03) and quantified using Nanodrop.

2.4 fluorescent detection of CRISPR-Cas12a in combination with RPA amplification

And (3) taking the RPA amplification target gene product, and adding the RPA amplification target gene product into a CRISPR-Cas12a reaction system. The 20uL final system contained 100ng of target-ApxIVA (i.e., RPA amplification target gene product), 50nM gRNA (i.e., gRNA against RPA amplification target gene product), 50nM Cas12a (available from New England Biolabs), 500nM ssDNA-reporter, 1 XNEBuffer, where the ssDNA-reporter used has the sequence 5 '-FAM-TTATT-TAMRA-3' (SEQ ID NO: 10). Reacting at 37 deg.C for 60min, and collecting FAM fluorescence every 1min at 485nm excitation wavelength and 535nm emission wavelength by using multifunctional microplate reader (Perkin Elmer ENSPIRE); or directly observing by naked eyes with a blue light gel imager after reacting for 15 min. The negative control was set to replace target-ApxIVA with deionized water.

2.5 immunochromatography detection of CRISPR-Cas12a in combination with RPA amplification

And taking the RPA amplification product, and adding the RPA amplification product into a CRISPR-Cas12a reaction system. The 20uL final system contained 100ng target-ApxIVA, 100nM gRNA, 50nM Cas12a, 500nM ssDNA-reporter, 1 XNEBuffer, at 37 ℃ for 60 min. The sequence used by ssDNA-reporter described herein is 5 '-FAM-TTATT-Biotin-3' (SEQ ID NO: 11). Mu.l of the reaction was transferred to 80. mu.l of detection buffer, and an immunochromatographic test strip (Milenia HybriDetect 1Dipstick, TwistDx Co.) was put in the solution, and the result was visually observed after incubation for 5 minutes. The negative control was set to replace target-ApxIVA with deionized water.

2.6 specificity assay

Common bacteria and viruses including actinobacillus pleuropneumoniae, haemophilus parasuis, streptococcus suis, salmonella, porcine circovirus, porcine pseudorabies virus and porcine epidemic diarrhea virus are detected by the detection method established in the embodiment, and a water-negative control is established. And judging the specificity of the detection method according to the detection result.

2.7 sensitivity test

Designing and amplifying PCR primers with homologous recombination sites according to ApxIVA conserved regions:

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

p2: 5'-CTGAATTCGAGCTCGGTACCGGCCATCGACTCAACCAT-3' (SEQ ID NO: 31), recovering the amplification product and ligating it to puT18 vector to obtain a plasmid containing a conserved region, determining the DNA concentration, calculating the copy number, and subjecting it to 10-fold serial dilution, setting up a water-negative control, and evaluating its sensitivity using an established detection method.

2.8 clinical sample testing

8 positive tissue samples and 5 negative samples of actinobacillus pleuropneumoniae confirmed by conventional PCR are selected and detected by an established PCR detection method, the genome of the actinobacillus pleuropneumoniae is used as a positive control, water is used as a negative control, and the detection results are compared. A primer AP-IVF (5'-ATACGGTTA ATGGCGGTAATG G-3') (SEQ ID NO: 32) and an AP-IVR (5'-ACCTGAGTGCTCACCAACG-3') (SEQ ID NO: 33) for identifying the actinobacillus pleuropneumoniae are selected for amplification by conventional PCR.

Example 2:

results

1. Specific gRNA sequence screening

Using the target sequence conserved region amplified by PCR as a template, 5uL target (i.e., PCR amplification target gene conserved region product), 50nM gRNA (i.e., different gRNA synthesized by in vitro transcription), 50nM Cas12a (purchased from New England Biolabs), 500nM ssDNA-reporter, 1 × NEBuffer 2.1 (the components are 50mM NaCl, 10mM Tris-HCl, 10mM MgCl2, 100 μ g/ml BSA pH 7.9) were included in a 20uL screening system; the reaction was carried out at 37 ℃ for 60 min. A gRNA with strong specificity and a corresponding detection area are preliminarily screened out by judging whether fluorescence can be excited or not in the fluorescence report experiment process and combining the fluorescence intensity. DNA sequences of 8 candidate gRNAs are designed and synthesized according to a conserved region of a target gene, gRNAs are generated through in vitro transcription (see table I), and optimal gRNAs are simultaneously screened by a fluorescence method (figure 1A) and an immunochromatography method (figure 1B), and the results of the fluorescence method show that: seq-1, seq-2, seq-3, seq-4, seq-5 and seq-7 can reach peak value within 30 min. The immunochromatography result shows that: seq-2, seq-4, seq-5, seq-6, seq-7, seq-8 sequences all produce clear positive bands. Combining the results of the two methods, the SEQ-7(SEQ ID NO: 8) sequence has the fastest peak and has a clear positive strip, the SEQ-7(SEQ ID NO: 8) sequence is preferably used as gRNA for subsequent detection, and SEQ-2(SEQ ID NO: 3), SEQ-4(SEQ ID NO: 5) and SEQ-5(SEQ ID NO: 6) can also be used as alternative gRNA.

TABLE one gRNA candidate sequences

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

RPA primer screening

Based on the binding site (conserved region 318-: the primer sets Nos. 1, 2, 3, 4 and 7 are all capable of generating blue fluorescence when observed with the naked eye, but the primer set No. 7 has the strongest fluorescence intensity (see FIG. 2), and therefore, the pair of primers No. 7, 185-FR (i.e., 185-F and 185-R), are preferable as the primer for amplifying RPA for subsequent detection.

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

TABLE II RPA candidate primer sequences

Note: since the primer pairs nos. 1, 2, 3, 4, and 7 amplify products that cover the region to which gRNA seq-2, 4, 5, and 7 bind, as seen from the starting and terminating sites of the RPA product, those skilled in the art will understand that the RPA primer pairs nos. 1, 2, 3, 4, and 7 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 a better detection effect, the ssDNA reporter molecule concentration, the gRNA concentration, the detection buffer solution component and the like in the detection system are optimized. The results show that the detection line of the negative sample is the lightest and the false positive effect is the weakest in the immunochromatography method when the concentration of the ssDNA reporter molecule is 500nM (FIG. 3A), and the fluorescence intensity is the strongest in the fluorescence method when the concentration of the ssDNA reporter molecule is 500nM (FIG. 3B); the immunochromatography shows that the ideal effect can be achieved when the concentration of gRNA in the system is 50nM (FIG. 3C), and when the immunochromatography uses a reagent strip for final detection, the effect of buffer solution carried by the reagent strip when the reagent strip is purchased and 10% polyethylene glycol as the buffer solution are compared, and the result shows that: the negative sample detection line could completely disappear when 10% polyethylene glycol was used as a buffer (fig. 3D).

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

fluorescence detection: the 20uL final system contained 100ng target-ApxIVA, 50nM gRNA, 50nM Cas12a, 500nM ssDNA-reporter, 1 XNEBuffer.

And (3) immunochromatography detection: the 20uL final system contained 100ng target-ApxIVA, 50nM gRNA, 50nM Cas12a, 500nM ssDNA-reporter, 1 XNEBuffer.

The immunochromatography detection buffer solution is 10% polyethylene glycol.

4. Specificity detection

To assess the specificity of Cas12a immunochromatography and fluorescence detection, we tested a variety of bacteria and viruses including haemophilus parasuis, streptococcus suis, salmonella, porcine circovirus, porcine pseudorabies virus, porcine epidemic diarrhea virus. The results show that: nucleic acid samples were tested by immunochromatography, visualization by fluorescence (visual observation, statistics of fluorescence), and the results showed that immunochromatography test strips for 6 pathogens other than A. pleuropneumoniae were negative (FIGS. 4A-4C).

5. Sensitivity detection

To analyze the sensitivity of the enhanced immunochromatographic detection of Cas12a, we tested the 10-fold serial dilutions of the plasmid containing the conserved region using immunochromatographic assay (fig. 5A), fluorescence (macroscopic, fig. 5B; fluorescence statistics, fig. 5C), and the results indicated that: the detection limit of the fluorescence method and the immunochromatography method is 10 copies/mu l.

6. Clinical sample testing

In order to further confirm the application effect of the visualization method in clinical samples, genome extraction is performed on lung tissue samples (hailbine veterinary institute of chinese academy of agricultural sciences) infected with actinobacillus pleuropneumoniae from different pig farms, 8 positive tissue samples and 5 negative tissue samples of actinobacillus pleuropneumoniae confirmed by a conventional PCR method are selected, the method of embodiment 1 is used for detecting the positive tissue samples and the negative tissue samples by combining with the optimal reaction system described in embodiment 2, and the immunochromatographic detection result shows that: positive samples except for negative control, the immunochromatographic test strips were all positive (fig. 6A); the negative samples were negative in the immunochromatographic test strips except for the positive control (FIG. 6D). The observation result of the naked eye method shows that: the positive samples all fluoresced green except for the negative control (FIG. 6B), and none of the negative samples (FIG. 6E). The fluorescence detection result shows that: the fluorescence values of the positive samples can reach over 12000, the fluorescence values of the negative controls are below 2000 (FIG. 6C), the fluorescence values of the negative samples are all below 2000 except the fluorescence values of the positive controls (FIG. 6F). The detection result of the method is consistent with that of the PCR method.

The results prove that the detection method can be used for rapidly detecting actinobacillus pleuropneumoniae in clinical samples, different detection methods can be adopted by combining local experimental conditions and the number of detected samples, when the detection method is observed by naked eyes and operated by a fluorescence detector, the detection process can be completed within 30min under the condition that a sample genome is prepared in advance in a laboratory, and the detection result can be obtained within 75min when the detection is carried out by a reagent strip, compared with the conventional PCR detection method (amplification is carried out for 60min, and agarose gel electrophoresis is used for detecting for 30min) in the laboratory, the method disclosed by the invention can realize rapid visual detection and reduce dye pollution generated during gel dyeing.

The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only examples of the present invention, and should not be construed as limiting the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Citations

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Claims (10)

1. A kit comprising a pair of primers specific for apxIV and corresponding to 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 the sequence of the product amplified against the above primer is SEQ ID NO: 3. 5, 6 or 8 (preferably SEQ ID NO: 8).

2. The kit of claim 1, further comprising a Cas12a protein.

3. The kit of any preceding claim, wherein the specific primer for apxIV is 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 according to any of the preceding claims, further comprising a ssDNA reporter molecule for a subsequent fluorescence detection method, preferably the sequence of said ssDNA reporter molecule is SEQ ID NO: 10.

6. the kit according to any of the preceding claims, further comprising a ssDNA reporter for a subsequent immunochromatographic assay, preferably the sequence of said ssDNA reporter is SEQ ID NO: 11.

7. the kit of claim 5 or 6, wherein the concentration of ssDNA reporter is 500 nM.

8. The kit of any preceding claim, wherein the concentration of gRNA for subsequent immunochromatographic assays is 50 nM.

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

10. Specific primer pair for apxIV 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 the sequence of the product amplified against the above primer is SEQ ID NO: 3. 5, 6 or 8 (preferably SEQ ID NO: 8) in the preparation of a kit for the rapid visual detection of Actinobacillus pleuropneumoniae.

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