CN116287460A - RAA-CRISPR/Cas12a detection composition of PEAV, reaction device, test strip and kit - Google Patents
RAA-CRISPR/Cas12a detection composition of PEAV, reaction device, test strip and kit Download PDFInfo
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Abstract
The invention discloses a RAA-CRISPR/Cas12a detection composition, a reaction device, a test strip and a kit of PEAV, wherein a primer pair, a guide RNA (gRNA), a probe, a reaction device, a test strip and a kit of a PEAV nucleic acid rapid visual detection method based on the combination of RAA and CRISPR/Cas12a technology are designed, screened, optimized and finally obtained by taking an N gene of PEAV as a diagnostic target. The reaction device provided by the invention effectively avoids the problems of aerosol pollution and prolonged detection time caused by extremely easy amplicon uncapping transfer while realizing rapid, specific, sensitive and portable detection of PEAV, is suitable for on-site, rapid and visual detection of PEAV, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of animal epidemic disease detection, in particular to a RAA-CRISPR/Cas12a detection composition of PEAV, a reaction device, a test strip and a kit.
Background
In 2017, the newborn piglets in a pig farm in Guangdong province of China have severe diarrhea epidemic situation, the sick piglets have severe diarrhea, vomiting, dehydration and other main clinical symptoms, the death rate of the piglets within 5 days of age is up to 90%, and the lactating sows also have diarrhea symptoms; finally, a completely new porcine enterocoronavirus was first isolated from small intestine samples of diarrhea piglets by researchers and named as porcine entero A (alpha) coronavirus (Porcine Enteric Alphacoronavirus, PEAV). PEAV is a enveloped single-stranded positive-stranded RNA virus belonging to the order of the genus Picornaviridae (Nidovirales), the family Coronaviridae (Coronaviridae), and the genus alpha coronavirus (alpha coronavirus). The full length of the PEAV genome is about 27kb, and Open Reading Frames (ORFs) 1a and 1b, a fiber protein gene (S), an accessory protein gene ORF3, a envelope protein gene (E), a membrane protein gene (M), a nucleocapsid protein gene (N), and accessory protein genes NS7a and NS7b are sequentially located from the 5 'end to the 3' end, except for untranslated regions (Untranslated region, UTR) at the 5 'end and 3' end. PEAV has a potential zoonotic risk.
As a new porcine enteropathogenic coronavirus, the outbreak and transmission of PEAV have caused a major economic loss to the pig industry in China. Since the main clinical symptoms of PEAV infected pigs are very similar to important porcine diarrhea diseases such as porcine epidemic diarrhea and porcine butyl coronavirus infection, and are indistinguishable. Therefore, the development of a fast and reliable detection method is important for controlling the further spread and popularity of PEAV. Currently, various molecular biology and serology detection methods have been established and applied to diagnosis and detection of PEAV, including real-time RT-PCR based on TaqMan probes, microdroplet digital PCR, recombinase Polymerase Amplification (RPA), real-time fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP), microfluidic RT-LAMP chips, and indirect ELISA. Although the detection method plays an important role in detection and diagnosis of PEAV, the defects of complex operation, long detection time, excessive dependence on expensive instruments and equipment, skilled technicians and the like generally exist, the practical requirement of on-site rapid and visual detection of PEAV infected pigs and the requirement of epidemiological investigation are difficult to meet, and the method is difficult to popularize and use in laboratories with limited resources.
In recent years, CRISPR/Cas systems have been developed as an efficient gene editing technology and a new generation of nucleic acid molecule detection technology. However, nucleic acid detection by means of the CRISPR/Cas12a system alone is often difficult to achieve very high detection sensitivity and the detection time is long, for which rapid amplification enrichment of the target gene fragment prior to CRISPR/Cas12a detection is required. Recent studies have found that CRISPR/Cas12a has good technical complementarity with RAA isothermal amplification, which can significantly increase the detection sensitivity of CRISPR/Cas12a, whereas CRISPR/Cas12a technology can further increase the specificity of RAA amplification. Since the combination of isothermal amplification of nucleic acids with the CRISPR/Cas12a system, this combination technique has been known to be a powerful tool for detection of pathogen nucleic acids and related disease genes with its extremely high sensitivity, specificity and convenience. At present, RAA-CRISPR/Cas12a technology has been successfully applied to detection of important pathogens such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), african Swine Fever Virus (ASFV) and the like. However, the above-mentioned RAA-CRISPR/Cas12a detection method of pathogens generally adopts a two-step method, namely, firstly, RAA amplification is performed on pathogen nucleic acid, then CRISPR/Cas12a reagent is added into amplicon to perform enzyme digestion reaction by uncapping, and simple mixing of the RAA amplification and CRISPR/Cas12a reaction generally leads to rapid decrease of detection sensitivity, which is mainly because template DNA or RAA amplicon activates cis-cleavage activity of Cas12a enzyme, resulting in failure of template and amplicon by cis-cleavage; at the same time, the activated Cas12a "accessory cleavage" activity cleaves the single-stranded primer, resulting in a dramatic decrease in RAA amplification efficiency. Therefore, RAA amplification and CRISPR/Cas12a signal detection in the system are usually required to be carried out separately, but the step-by-step operation increases the complexity of operation on one hand, prolongs the whole detection time, and on the other hand, the uncapping operation is extremely easy to cause aerosol pollution, so that the method is unfavorable for clinical application.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Disclosure of Invention
The invention aims to provide a RAA-CRISPR/Cas12a detection composition, a reaction device, a test strip and a kit of PEAV, which can solve the problems of complex operation and long detection time of the existing detection method of PEAV.
To achieve the above object, the present invention provides a RAA-CRISPR/Cas12a detection composition of PEAV, comprising a primer pair, a guide RNA and a probe, wherein the primer pair is an upstream primer PEAV-N-F and a downstream primer PEAV-N-R for detecting PEAV N gene, the guide RNA is PEAV-N-gRNA, and the probe comprises a fluorescence quenching probe or a fluorescein probe, and the sequences are as follows:
the upstream primer PEAV-N-F:5'-AGTCACCTATCACTATGTAATGAAGGTCCCC-3' (SEQ ID NO. 1);
the downstream primer PEAV-N-R:5'-TAATTAATAATCTCATCCACCATCTCAACCT-3' (SEQ ID NO. 2);
guide RNA (PEAV-N-gRNA): 5'-UAAUUUCUACUAAGUGUAGAUUGAGCAAGUCUCGGCUUAC-3' (SEQ ID NO. 3);
fluorescence quenching probe ssDNA probe 1:5'-FAM-TTATTATT-BHQ1-3'; or alternatively
Fluorescent biotin probe ssDNA probe 2:5'-FAM-TTATTATT-Biotin-3';
the fluorescence quenching probe is characterized in that a1 st base T marks FAM luminescent groups from the 5' end, and an 8 th base T marks BHQ1 quenching groups; the fluorescent Biotin probe marks FAM luminous groups with 1 st base T from the 5' end, and the 8 th base T is subjected to Biotin (Biotin) modification.
The invention also provides a RAA-CRISPR/Cas12a detection reaction device of PEAV, which comprises an outer sleeve and an inner sleeve, wherein the inner sleeve is positioned inside the outer sleeve, the outer sleeve is used for adding a RAA reaction system, the inner sleeve is used for adding a CRISPR/Cas12a digestion system, and the CRISPR/Cas12a reaction system in the inner sleeve can be released into the outer sleeve through vortex.
The invention also provides a fluorescence method kit for RAA-CRISPR/Cas12a detection of PEAV, which is internally provided with the RAA-CRISPR/Cas12a detection reaction device of PEAV, the total reaction system of the fluorescence method kit comprises a 25 mu L RT-RAA amplification system and a 20 mu L CRISPR/Cas12a digestion system,
the RT-RAA amplification system comprises 14.70 mu L of reaction buffer A, 1.25 mu L of reaction buffer B, 1.00 mu L of 10 pmol/mu L of upstream primer PEAV-N-F (SEQ ID NO. 1), 1.00 mu L of 10 pmol/mu L of downstream primer PEAV-N-R (SEQ ID NO. 2), 1.00 mu L of sample RNA template to be detected and 6.05 mu L of nuclease-free double distilled water;
the CRISPR/Cas12a cleavage system included 1 pmol/. Mu.L Cas12a enzyme 2.00. Mu.L, 10 pmol/. Mu.L ssDNA probe 1 probe 2.00. Mu.L, 1 pmol/. Mu.L guide RNA (SEQ ID NO. 3) 2.00. Mu.L and buffer 14.00. Mu.L.
The invention also provides a fluorescence detection method of RAA-CRISPR/Cas12a of PEAV by using the fluorescence detection kit of RAA-CRISPR/Cas12a of PEAV, which comprises the following steps:
(1) Extracting RNA of a sample to be detected;
(2) Amplification of RT-RAA reaction System: taking the extracted RNA of the sample to be detected as a template, adding a reagent for detecting the RT-RAA amplification system of PEAV into an outer sleeve of a reaction device, simultaneously adding a reagent for detecting the CRISPR/Cas12a digestion system into an inner sleeve, and then carrying out nucleic acid amplification for 10min at a constant temperature of 37 ℃;
(3) After the amplification reaction is finished, releasing the CRISPR/Cas12a enzyme digestion system in the inner sleeve into the outer sleeve by vortex, utilizing the auxiliary cutting activity of the Cas12a enzyme to cut a fluorescence quenching probe, reacting for 10min at the constant temperature of 37 ℃, and observing fluorescence by using a portable Lan Guangyi with the wavelength of 440-460 nm.
The invention also provides a test strip method kit for RAA-CRISPR/Cas12a detection of PEAV, which is internally provided with the RAA-CRISPR/Cas12a detection reaction device of PEAV, wherein the total reaction system of the test strip kit comprises a 25 mu L RT-RAA amplification system, a 20 mu L CRISPR/Cas12a digestion system and a test strip;
the RT-RAA amplification system comprises 14.70 mu L of reaction buffer A, 1.25 mu L of reaction buffer B, 1.00 mu L of 10 pmol/mu L of upstream primer PEAV-N-F (SEQ ID NO. 1), 1.00 mu L of 10 pmol/mu L of downstream primer PEAV-N-R (SEQ ID NO. 2), 1.00 mu L of PEAV RNA template and 6.05 mu L of nuclease-free double distilled water;
the CRISPR/Cas12a cleavage system included 1 pmol/. Mu.L Cas12a enzyme 2.00. Mu.L, 10 pmol/. Mu.L ssDNA probe 2.00. Mu.L, 1 pmol/. Mu.L guide RNA (SEQ ID NO. 3) 2.00. Mu.L and buffer 14.00. Mu.L;
the test strip comprises a sample binding pad, a detection line and a quality control line, wherein the sample binding pad contains a colloidal gold-labeled mouse anti-FAM monoclonal antibody, the detection line contains a goat anti-mouse IgG antibody, and the quality control line contains streptavidin.
The invention also provides a test strip method kit for detecting RAA-CRISPR/Cas12a of PEAV, which comprises the following steps:
(1) Extracting RNA of a sample to be detected;
(2) Amplification of RT-RAA reaction System: taking the extracted RNA of the sample to be detected as a template, adding a reagent for detecting the RT-RAA amplification system of PEAV into an outer sleeve of a reaction device, simultaneously adding a reagent for detecting the CRISPR/Cas12a digestion system into an inner sleeve, and then carrying out nucleic acid amplification for 10min at a constant temperature of 37 ℃;
(3) After the amplification reaction is finished, releasing a CRISPR/Cas12a enzyme digestion system in the inner sleeve into the outer sleeve through vortex, utilizing the auxiliary cutting activity of Cas12a enzyme to cut a fluorescent biotin probe, reacting at the constant temperature of 37 ℃ for 10min, directly inserting the sample adding end of the colloidal gold test strip into a reaction solution, reacting at the room temperature for 1min, and observing the strip.
Compared with the prior art, the RAA-CRISPR/Cas12a detection composition, the reaction device, the test strip and the kit of the PEAV have the following beneficial effects:
the method has the advantages that the RAA-CRISPR/Cas12a primer, the guide RNA, the probe, the test strip and the kit are adopted to detect the PEAV, and the detection can be completed only by reacting for 20 minutes at the constant temperature of 37 ℃, so that the method has quick response, simple requirements on instruments, does not need a complex and expensive thermal cycler, greatly shortens the detection time, is suitable for instant detection (point of care testing, POCT), and can truly realize the detection of portable and quick visual PEAV nucleic acid; the RAA-CRISPR/Cas12a detection result can be rapidly visualized and interpreted through a portable Lan Guangyi or colloidal gold test strip, the invention has wide application prospect in the aspect of PEAV on-site rapid visual detection, is suitable for rapid detection of PEAV on a basic layer or on site, and strives for precious time for early prevention and control of PEAV infection.
Drawings
FIG. 1 is a diagram showing the whole gene sequence information of representative strains of all 59 porcine enterovirus A in GenBank database, and specific positions of optimal primers and guide RNA in N genes; the country of origin of the strain and the corresponding GenBank accession number are marked on the left side of the figure.
Fig. 2 is a specific flow chart of the RAA-CRISPR/Cas12a fluorescence detection method and the test strip detection method of the PEAV.
FIG. 3 shows a RAA-CRISPR/Cas12a reaction apparatus according to the present invention, wherein (A) is a reaction apparatus without adding a reaction reagent.
FIG. 4 shows the results of 2% agarose gel electrophoresis of RAA-CRISPR/Cas12a primer screening according to the present invention; wherein (A) the first round of screening uses randomly selected downstream primer R3 to screen the electrophoresis pattern of all 5 upstream primers F1-F5; (B) Screening an electrophoresis pattern of all 5 downstream primers R1-R5 by using the screened optimal upstream primer F3 in the first round of screening; (C) Screening the optimal downstream primer R1 screened in the first round in the second round of screening, and screening an electrophoresis pattern of the redesigned 16 upstream primers F3-1 to F3-16; (D) Screening an electrophoresis pattern of 15 redesigned downstream primers R1-1 to R1-15 by using the screened optimal upstream primer F3-7 in the second round of screening; m is DL500 DNA Marker; NTC is a negative control.
FIG. 5 is a schematic representation of the results of screening RAA-CRISPR/Cas12a guide RNA according to the present invention, curves 1-6 representing gRNA 1-gRNA 5 and negative control, respectively.
FIG. 6 is a fluorescent diagram and a test strip diagram of a specificity test of the RAA-CRISPR/Cas12a fluorescent method and the test strip method detection kit of the invention; wherein, (A) a fluorescence diagram of a specific analysis result of the RAA-CRISPR/Cas12a fluorescence method detection kit; (B) Test strip diagrams of specific analysis results of RAA-CRISPR/Cas12a test strip method detection kit, wherein 1-10 represent Porcine Enterovirus A (PEAV), porcine Epidemic Diarrhea Virus (PEDV), porcine butylcoronavirus (PDCoV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine pseudorabies virus (PRV), seuka Virus A (SVA), porcine circovirus type 2 (PCV 2) and negative control respectively.
FIG. 7 is a schematic representation of the analytical results of the sensitivity test of RAA-CRISPR/Cas12a fluorescence and dipstick detection kits according to the invention. Wherein, (A) RAA-CRISPR/Cas12a fluorescence method detects the fluorescence diagram of the sensibility analysis result of the kit; (B) Probability regression analysis graph of sensitivity analysis result of RAA-CRISPR/Cas12a fluorescence detection kit, wherein the detection limit of 95% confidence interval is 16.82 copies/mu L PEAV N gene plasmid; (C) A test strip diagram of the sensitivity analysis result of the RAA-CRISPR/Cas12a test strip method detection kit; (D) The probability regression analysis graph of the sensitivity analysis result of the RAA-CRISPR/Cas12a test strip method detection kit has the detection limit of 388.92 copies/mu L PEAV N gene plasmid of 95% confidence interval; (E) Schematic of the result of sensitivity analysis based on PEAV ORF1a real-time fluorescence quantitative RT-PCR; 1: 7.68X10 6 cobies/. Mu.L PEAV N gene plasmid; 2: 7.68X10 5 cobies/. Mu.L PEAV N gene plasmid; 3: 7.68X10 4 cobies/. Mu.L PEAV N gene plasmid; 4: 7.68X10 3 cobies/. Mu.L PEAV N gene plasmid; 5: 7.68X10 2 cobies/. Mu.L PEAV N gene plasmid; 6: 7.68X10 1 cobies/. Mu.L PEAV N gene plasmid; 7: 7.68X10 0 cobies/. Mu.L PEAV N gene plasmid; 8: 7.68X10 -1 copies/. Mu.L PEAV N gene plasmid; 9: a negative control; (F) Based on a probability regression analysis graph of PEAV ORF1a real-time fluorescence quantitative RT-PCR sensitivity analysis results, the detection limit of the 95% confidence interval is 149.74 copies/. Mu.L PEAV N gene plasmid.
Fig. 8 is a fluorescence and test strip diagram of the detection results of 175 clinical samples using the RAA-CRISPR/Cas12a fluorescence and test strip detection kit according to the present invention.
Detailed Description
The following detailed description of embodiments of the invention is, therefore, to be taken in conjunction with the accompanying drawings, and it is to be understood that the scope of the invention is not limited to the specific embodiments.
Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.
The RAA-CRISPR/Cas12a detection composition, the reaction device, the test strip and the kit of the PEAV according to the preferred embodiment of the invention.
Primer pair, guide RNA, probe, reaction device, test strip, kit and application thereof based on rapid visual detection method of PEAV nucleic acid by combining RAA and CRISPR/Cas12a technology. The N gene of PEAV is used as a diagnosis target, and a universal primer pair, a universal guide RNA and two different probes which can specifically detect PEAV nucleic acid are designed, screened, optimized and finally obtained. The primer pair, the probe, the reaction device and the RAA reagent are used for amplifying PEAV nucleic acid at the constant temperature of 37 ℃ for 10min, the RAA amplification product in the outer sleeve of the reaction device and the CRISPR/Cas12a system in the inner sleeve are uniformly mixed by vortex, the temperature is 37 ℃ for 10min, the probe is cut by the auxiliary cutting activity of Cas12a, and the detection result is visually interpreted by means of the portable Lan Guangyi or colloidal gold test strip. The detection method, the kit and the reaction device provided by the invention are suitable for on-site, rapid and visual detection of PEAV, and have wide application prospects.
The invention finally selects a region with the most conserved nucleotide sequence in the PEAV genome and located in N genes and with the length of about 231bp as a diagnosis target by downloading, comparing and analyzing the whole genome sequences of all 59 PEAV strains in a GenBank database, designs RAA primers, guide RNA, fluorescence quenching probes and fluorescence biotin probes, and successfully establishes two RAA-CRISPR/Cas12a detection methods capable of rapidly and visually detecting PEAV nucleic acid through multiple rounds of screening; the conservation of the designed primer and the guide RNA is far higher than that of other similar primers and guide RNA, so that the method is more suitable for the rapid and visual detection of PEAV. The RAA-CRISPR/Cas12a detection method based on the fluorescence quenching probe can visually judge the reaction product by means of a portable Lan Guangyi, and the RAA-CRISPR/Cas12a detection method based on the fluorescence biotin probe can visually judge by means of a colloidal gold test strip.
In order to avoid the problem that the traditional RAA-CRISPR/Cas12a stepwise detection needs to uncap and transfer RAA amplicon to perform CRISPR/Cas12a reaction so as to easily cause aerosol pollution, the invention also designs a convenient RAA-CRISPR/Cas12a reaction device, which consists of an outer sleeve and an inner sleeve, wherein the outer sleeve is used for adding a RAA reaction system, and the inner sleeve is used for adding a CRISPR/Cas12a reaction system, as shown in fig. 3 (A). In the initial detection stage, CRISPR/Cas12a and RAA reaction reagents can be respectively added into the inner sleeve and the outer sleeve, and after the RAA amplification in the outer sleeve is finished, the CRISPR/Cas12a reagent in the inner sleeve is released through vortex, so that one-step closed tube detection is realized, and aerosol pollution is effectively avoided; and the detection time is shortened, and the detection efficiency is improved.
The RAA-CRISPR/Cas12a detection composition of PEAV comprises a primer pair, a guide RNA and two probes, wherein the primer pair is an upstream primer PEAV-N-F and a downstream primer PEAV-N-R for detecting PEAV N genes, the guide RNA is PEAV-N-gRNA, the two probes comprise a fluorescence quenching probe (ssDNA probe 1) and a fluorescence biotin probe (ssDNA probe 2), and the two probes are respectively used with the following sequences:
the upstream primer PEAV-N-F:5'-AGTCACCTATCACTATGTAATGAAGGTCCCC-3' (SEQ ID NO. 1);
the downstream primer PEAV-N-R:5'-TAATTAATAATCTCATCCACCATCTCAACCT-3' (SEQ ID NO. 2);
guide RNA (PEAV-N-gRNA): 5'-UAAUUUCUACUAAGUGUAGAUUGAGCAAGUCUCGGCUUAC-3' (SEQ ID NO. 3);
fluorescence quenching probe (ssDNA probe 1): 5'-FAM-TTATTATT-BHQ1-3'; and
fluorescent biotin probe (ssDNA probe 2): 5'-FAM-TTATTATT-Biotin-3'.
A fluorescence quenching probe (ssDNA probe 1) T marks FAM luminescent groups from the 5' end at the 1 st base, and T marks BHQ1 quenching groups at the 8 th base; a fluorescent Biotin probe (ssDNA probe 2) marks FAM luminescent groups with a1 st base T from the 5' end, and the 8 th base T is subjected to Biotin (Biotin) modification.
The fluorescence method detection kit for PEAV comprises a RAA-CRISPR/Cas12a primer pair for detecting PEAV, a guide RNA, a fluorescence quenching probe, a reagent for reverse transcription recombinase mediated isothermal amplification and a CRISPR/Cas12a reaction system, wherein the reagent for reverse transcription recombinase mediated isothermal amplification comprises reverse transcriptase, recombinase, DNA polymerase and single-stranded DNA binding protein, and the CRISPR/Cas12a reaction system comprises Cas12a enzyme and buffer.
The total reaction system of the kit for detecting PEAV by using RAA-CRISPR/Cas12a fluorescence method is 45 mu L, wherein the RT-RAA amplification system is 25 mu L: comprises 14.70 mu L of reaction buffer A, 1.25 mu L of reaction buffer B, 10 pmol/. Mu.L of upstream primer PEAV-N-F1.00 mu L, 10 pmol/. Mu.L of downstream primer PEAV-N-R1.00 mu.L, 1.00 mu.L of RNA template of a sample to be detected and 6.05 mu L of nuclease-free double distilled water; reaction buffer A consisted of 100U/. Mu.L reverse transcriptase, 120 ng/. Mu.L recombinase, 30 ng/. Mu.L DNA polymerase, 800 ng/. Mu.L single-stranded DNA binding protein, reaction buffer B was 280mM magnesium acetate;
wherein the CRISPR/Cas12a cleavage system is 20 μl: comprises 1 pmol/. Mu.L Cas12a enzyme 2.00. Mu.L, 10 pmol/. Mu.L ssDNA probe 1 probe 2.00. Mu.L, 1 pmol/. Mu.L guide RNA 2.00. Mu.L and buffer 14.00. Mu.L; the buffer solution comprises 50mM NaCl, 10mM Tris-HCl, and 10mM MgCl 2 And 100. Mu.g/mL BSA.
As shown in fig. 2, the RAA-CRISPR/Cas12a fluorescence detection method of PEAV comprises the following steps:
(1) Extracting RNA of a sample to be detected;
(2) Amplification of RT-RAA reaction System: taking the extracted RNA of the sample to be detected as a template, adding a reagent for detecting the RT-RAA amplification system of PEAV into an outer sleeve of a reaction device, simultaneously adding a reagent for detecting the CRISPR/Cas12a digestion system into an inner sleeve, and then carrying out nucleic acid amplification for 10min at a constant temperature of 37 ℃;
(3) After the amplification reaction is finished, releasing the CRISPR/Cas12a enzyme digestion system in the inner sleeve into the outer sleeve by vortex, utilizing the auxiliary cutting activity of the Cas12a enzyme to cut a fluorescence quenching probe, reacting for 10min at the constant temperature of 37 ℃, and observing fluorescence by using a portable Lan Guangyi with the wavelength of 440-460 nm.
Result description and judgment: the visual result of the sample to be detected is colorless, and the sample is judged as negative; the visual result is green, and the sample is judged positive.
The RAA-CRISPR/Cas12a test strip method detection kit for PEAV comprises a RAA-CRISPR/Cas12a primer pair for detecting PEAV, a guide RNA and a fluorescein probe, a reverse transcription recombinase mediated isothermal amplification reagent, a CRISPR/Cas12a reaction system and a test strip; the test strip includes: sample binding pad, matter control line (C line) and detection line (T line), sample binding pad contains the monoclonal antibody of the mouse anti FAM of colloidal gold mark, and C line department contains streptavidin, and T line department contains goat anti mouse IgG antibody. The test strip sequentially comprises a sample pad, a binding pad, a Nitrocellulose (NC) membrane and absorbent paper from a sample adding end, wherein the NC membrane is provided with a C line and a T line.
As shown in fig. 2, the RAA-CRISPR/Cas12a test strip method detection method of PEAV comprises the following steps:
(1) Extracting RNA of a sample to be detected;
(2) Amplification of RT-RAA reaction System: taking the extracted RNA of the sample to be detected as a template, adding a reagent for detecting the RT-RAA amplification system of PEAV into an outer sleeve of a reaction device, simultaneously adding a reagent for detecting the CRISPR/Cas12a digestion system into an inner sleeve, and then carrying out nucleic acid amplification for 10min at a constant temperature of 37 ℃;
(3) After the amplification reaction is finished, releasing a CRISPR/Cas12a enzyme digestion system in the inner sleeve into the outer sleeve through vortex, utilizing the auxiliary cutting activity of Cas12a enzyme to cut a fluorescent biotin probe, reacting at the constant temperature of 37 ℃ for 10min, directly inserting the sample adding end of the colloidal gold test strip into a reaction solution, reacting at the room temperature for 1min, and observing the strip.
Result description and judgment: the positive result shows that two red stripes are generated, one is positioned on the T line, the other is positioned on the C line or only one red stripe positioned on the T line is generated, and no red stripe is generated on the C line; negative results only appear one red band on line C, while no red band appears on line T; if the red stripes are not appeared on the C line and the T line, the detection result is invalid.
The total reaction system of the RAA-CRISPR/Cas12a test strip method detection kit is 45 mu L, and the RT-RAA amplification system is 25 mu L: comprises 14.70 mu L of reaction buffer A, 1.25 mu L of reaction buffer B, 10 pmol/. Mu.L of upstream primer PEAV-N-F1.00 mu L, 10 pmol/. Mu.L of downstream primer PEAV-N-R1.00 mu.L, 1.00 mu.L of RNA template of a sample to be detected and 6.05 mu L of nuclease-free double distilled water; the CRISPR/Cas12a cleavage system was 20 μl: comprises 1 pmol/. Mu.L Cas12a enzyme 2.00. Mu.L, 10 pmol/. Mu.L ssDNAprobe 2 probe 2.00. Mu.L, 1 pmol/. Mu.L guide RNA 2.00. Mu.L and buffer 14.00. Mu.L.
Example 1 design and screening of RAA-CRISPR/Cas12a detection compositions for PEAV
And downloading the whole genome sequences of all 59 PEAV strains in a GenBank database (https:// www.ncbi.nlm.nih.gov /), comparing and analyzing the 59 PEAV whole genome sequences by using MEGA 11 software, and finally selecting a region with the most conserved nucleotide sequence in the PEAV genome and positioned in the N gene and with the length of about 231bp as a diagnosis target, and designing a primer pair and guide RNA of the RAA-CRISPR/Cas12 a. The RAA primer design follows the following basic principles: the length of the primer is more than or equal to 30bp, and is preferably between 30 and 35 bp; the length of the amplicon is not more than 500bp, and the optimal length is between 100 and 200 bp; GC content is more than 30% and less than 70%, preferably between 40% and 60%; preferably, the presence of many repeated short sequences in the primer is avoided; avoiding the direct formation of hairpin structure or primer dimer, etc. In order to ensure the RAA-CRISPR/Cas12a amplification efficiency, multiple rounds of screening of primers are required to obtain better primer combinations. The primer screening principle is as follows: the first screening is to screen all reverse primers by using a certain forward primer, select the optimal reverse primer to screen all forward primers, and finally screen a pair of good primers. If a high-sensitivity detection effect is desired, a second primer screening is required, and the strategy is to modify the optimal primer combination selected for the first time as follows: the position of the primer on the template is moved back and forth in the range of 1-3 bases or a plurality of bases are added and removed at the two ends of the primer, so that the screening effect is better than that of the primer combination obtained by the first screening. The guide RNA was targeted to the RAA amplicon region of the PEAV N gene and scored by on-line software Eukaryotic Pathogen CRISPR gRNA Design Tool (http:// grna. Ctegd. Uga. Edu /). According to the difference of the CRISPR/Cas12a fluorescence method and the test strip method detection principle, two different probes are respectively designed, wherein the 5 'end and the 3' end of the fluorescence quenching probe are respectively marked with a FAM fluorescent group and a BHQ1 quenching group, and the 5 'end and the 3' end of the fluorescence Biotin probe are respectively marked with a FAM fluorescent group and Biotin (Biotin).
According to the basic principles of RAA primer, guide RNA and probe design, 5 first round screening upstream primers (F1-F5), 5 first round screening downstream primers (R1-R5), 16 second round screening upstream primers (F3-1-F3-16), 15 second round screening downstream primers (R1-R1-15), 5 guide RNA (gRNA 1-gRNA 5) and 2 probes (ssDNA probe 1 and ssDNA probe 1) were designed, respectively. All primer, guide RNA and probe sequence information are shown in Table 1, and their relative positions in the N gene are shown in FIG. 1.
Table 1 RAA candidate primers, guide RNA and probes designed
(1) Preparing 25 mu L RT-RAA amplification system, adding nuclease-free double distilled water as template into negative control, and adding 10 into positive control 3 TCID 50 An RNA template of a PEAV strain;
(2) RT-RAA amplification and product analysis
The reaction conditions were set as: and (3) after the reaction solution is instantaneously and centrifugally mixed, placing the mixture into a constant-temperature water bath kettle at 37 ℃ for amplification of 10 min.
Amplification product analysis: taking 20 mu L of amplified product, performing 2% agarose gel electrophoresis, and observing the result under ultraviolet rays;
quality control standard: the negative control has no amplified band, and the positive control has specific amplified band, so that the test result is effective, otherwise, the test result is ineffective;
result description and judgment: the sample to be detected has no amplified strip, and the sample is judged as negative; specific amplification bands appear, and the sample is judged positive.
The primer screening steps were as follows: the first round of screening strategy is to screen all 5 upstream primers F1-F5 with randomly selected downstream primer R3, wherein F3 is shown as the optimal upstream primer; all 5 downstream primers R1-R5 were then screened using F3, where R1 was shown to be the optimal downstream primer, and therefore the optimal primer combination for the first screening was F3/R1. If it is desired to further increase the detection sensitivity of the primer combination, it is necessary to redesign the primers around the optimal primer combination F3/R1 selected in the first round, and to perform the second round of screening on the primers. Around the primers F3 and R1, a step of 1-3 bases is moved back and forth or a plurality of bases are added or deleted at two ends of the primers, thereby redesigning 16 new upstream primers and 15 new downstream primers, and a second round of screening is performed on the redesigned primers together with F3/R1 by adopting the same strategy as the first round of screening, and as shown in FIG. 4, the upstream primers F3-7 and the downstream primers R1-2 are finally determined as the optimal primer combination through the second round of screening.
(3) Preparing 20 mu L of CRISPR/Cas12a enzyme digestion system by a fluorescence method, adding 5.00 mu L of RAA amplification product into a reaction system, and carrying out instantaneous centrifugation and uniform mixing.
Setting reaction conditions: placing the reaction tube in a CFX96 Bio-Rad real-time fluorescence PCR instrument, reacting for 30min at 37 ℃, and collecting fluorescence signals;
quality control standard: the negative control has no amplification curve, and the positive control has a specific amplification curve, so that the test result is effective, otherwise, the test result is ineffective;
result description and judgment: judging that the sample to be detected has no amplification curve and the sample is negative; and (5) a specific amplification curve appears, and the sample is judged to be positive. Wherein the earliest peak of the curve is determined to be the optimal guide RNA.
As shown in fig. 5, curve 2 corresponds to the highest peak of gRNA2, the shortest time used, and was ultimately selected as the optimal guide RNA for RAA-CRISPR/Cas12 a.
After the design and screening of the primers and the guide RNA, the optimal upstream primer F3-7, the optimal downstream primer R1-2 and the optimal guide RNA gRNA2 for the detection of the PEAV nucleic acid RAA-CRISPR/Cas12a fluorescence method and the test strip method are finally determined. For convenience of description herein, we rename F3-7 to PEAV-N-F, R1-2 to PEAV-N-R, and gRNA2 to PEAV-N-gRNA.
Example 2 specific analysis of RAA-CRISPR/Cas12a fluorescence method and test strip method detection kit of PEAV
The RAA-CRISPR/Cas12a detection was performed according to the sample loading method described above using nucleic acids of enterovirus A (PEAV), porcine Epidemic Diarrhea Virus (PEDV), porcine butylcoronavirus (PDCoV), porcine transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine pseudorabies virus (PRV), seeca Virus A (SVA), porcine circovirus type 2 (PCV 2) as templates, respectively, and setting a negative control. As shown in fig. 6, other virus and negative control visual detection results were negative except for the visualized green fluorescence and test strip positive results of PEAV RNA templates. The result shows that the RAA-CRISPR/Cas12a fluorescence method and the test strip method detection kit can realize specific detection of PEAV nucleic acid and do not cross react with other important porcine viruses.
Example 3 sensitivity analysis of PEAV RAA-CRISPR/Cas12a fluorescence method and test strip method detection kit
By setting 8 groups of pEASY-Blunt-N plasmids containing full-length PEAV N genes with different concentrations, amplification is carried out under the RAA-CRISPR/Cas12a optimum condition, and sensitivity analysis is carried out, wherein the specific steps are as follows:
pEASY-Blunt-N plasmid (concentration 7.68X10) 6 copies/μL～7.68×10 - 1 copies/. Mu.L) is used as a template, nucleic acid amplification and Cas12a digestion are carried out under the RAA-CRISPR/Cas12a optimal condition, and no-template control is set. Meanwhile, real-time fluorescent quantitative RT-PCR was performed on gradient-fold diluted plasmids using primer pairs (PEAV-F and PEAV-R) and TaqMan fluorescent probes (PEAV-P) as described in published literature (Pan Z., et al, virus, 2020, 11:707-718), and the detection and alignment were performed at ordinary times.
As shown in FIG. 7 (A), when the concentration of pEASY-Blunt-N plasmid was 7.68X10 × 6 copies/μL～7.68×10 0 When the concentration of pEASY-Blunt-N plasmid is 7.68X10, the detection results of RAA-CRISPR/Cas12a fluorescence method are positive -1 When the probes are at the concentration of mu L, the detection result of the RAA-CRISPR/Cas12a fluorescence method is negative, which indicates that the RAA-CRISPR/Cas12a fluorescence method can detect 7.68X10 0 The pepies/. Mu.L pEASY-Blunt-N plasmid. As shown in FIG. 7 (B), by performing probability regression analysis on 8 independent RAA-CRISPR/Cas12a fluorescence detection results, it was confirmed that RAA-CRISPR ++when the confidence was set to 95%The detection sensitivity of the Cas12a fluorescence method to PEAV is 16.82 copies/. Mu.L pEASY-Blunt-N plasmid template.
As shown in FIG. 7 (C), when the concentration of pEASY-Blunt-N plasmid was 7.68X10 × 6 copies/μL～7.68×10 1 When the copies/. Mu.L, the detection result of the RAA-CRISPR/Cas12a test strip method is positive, and when the concentration of pEASY-Blunt-N plasmid is 7.68X10% 0 copies/. Mu.L and 7.68X10 -1 When the probes are at the concentration of mu L, the detection results of the RAA-CRISPR/Cas12a test strip method are all negative, which indicates that the RAA-CRISPR/Cas12a test strip method can detect 7.68X10 1 The pepies/. Mu.L pEASY-Blunt-N plasmid. As shown in FIG. 7 (D), by performing probability regression analysis on the detection results of 8 independent RAA-CRISPR/Cas12a test strip methods, when the confidence is set to be 95%, the detection sensitivity of the RAA-CRISPR/Cas12a test strip method to PEAV is 388.92 copies/. Mu.L pEASY-Blunt-N plasmid template.
As shown in FIG. 7 (E), when the concentration of pEASY-Blunt-N plasmid was 7.68X10 × 6 copies/μL～7.68×10 0 The detection results of real-time fluorescence quantitative RT-PCR were positive at the values of copies/. Mu.L, and the concentration of pEASY-Blunt-N plasmid was 7.68X10 × -1 When the probes are at the concentration of mu L, the detection result of the real-time fluorescent quantitative RT-PCR is negative, which indicates that the real-time fluorescent quantitative RT-PCR can detect 7.68X10 0 The pepies/. Mu.L pEASY-Blunt-N plasmid. As shown in FIG. 7 (F), by performing probability regression analysis on 8 independent real-time fluorescent quantitative RT-PCR detection results, it was confirmed that the detection sensitivity of the real-time fluorescent quantitative RT-PCR to PEAV was 16.82 copies/. Mu.L pEASY-Blunt-N plasmid template when the confidence was set to 95%.
The combination of the primer pair, the guide RNA and the probe designed by the invention can ensure the sensitivity during detection.
Example 4 detection application to actual samples
Samples such as intestinal tissues, anus swabs and serum of the pig with clinical diarrhea and the PEAV manual toxicity attack are collected for 175 parts (comprising 40 parts of intestinal tissues, 30 parts of serum and 105 parts of anus swabs), RNA of 175 parts of clinical samples is extracted according to RNA extraction reagent specifications, and the extracted RNA is stored at-80 ℃ (repeated freezing and thawing is not more than 3 times).
And (3) detecting the extracted RNA by using a RAA-CRISPR/Cas12a fluorescence method and a test strip method according to the sample adding method. Meanwhile, real-time fluorescent quantitative RT-PCR was performed on sample RNA using the primer pairs (PEAV-F and PEAV-R) and TaqMan fluorescent probes (PEAV-P) described in published literature (Pan Z., et al, virus, 2020, 11:707-718) for routine detection and alignment.
The results showed that 175 clinical samples were tested by RAA-CRISPR/Cas12a fluorescence, 124 positive, as shown in table 2 and fig. 8; detecting by RAA-CRISPR/Cas12a test strip method, wherein positive 116 parts; the detection is carried out by a real-time fluorescence quantitative RT-PCR method, wherein 119 parts of the detection are positive. The detection coincidence rate of the RAA-CRISPR/Cas12a fluorescence method and real-time fluorescence quantitative R T-PCR on 175 samples is 97.1 percent (170/175), the Kappa value is 0.933, and P is less than 0.001; the detection coincidence rate of the RA A-CRISPR/Cas12a test strip method and the real-time fluorescence quantitative RT-PCR on 175 samples is 98.3 percent (172/175), the Kappa value is 0.986, and the P is less than 0.001.
TABLE 2 clinical sample detection results
In conclusion, the method can realize rapid, specific, sensitive, visual and convenient detection of PEAV. The method has the advantages of quick response, simple requirement on instruments, no need of complex and expensive thermal cycling reaction instrument, suitability for carrying out instant detection on PEAV at a base layer or on site, and capability of truly realizing portable and quick nucleic acid detection.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (6)
1. A RAA-CRISPR/Cas12a detection composition for PEAV, comprising a primer pair, a guide RNA and a probe, wherein the primer pair is an upstream primer PEAV-N-F and a downstream primer PEAV-N-R for detecting PEAV N gene, the guide RNA is PEAV-N-gRNA, and the probe comprises a fluorescence quenching probe ssDNA probe 1 or a fluorescein probe ssDNA probe 2, the sequences of which are as follows:
the upstream primer PEAV-N-F:5'-AGTCACCTATCACTATGTAATGAAGGTCCCC-3' (SEQ ID NO. 1);
the downstream primer PEAV-N-R:5'-TAATTAATAATCTCATCCACCATCTCAACCT-3' (SEQ ID NO. 2);
PEAV-N-gRNA：5’-UAAUUUCUACUAAGUGUAGAUUGAGCAAGUCUCGGCUUAC-3’(SEQ ID NO.3)；
fluorescence quenching probe ssDNA probe 1:5'-FAM-TTATTATT-BHQ1-3'; or alternatively
Fluorescent biotin probe ssDNA probe 2:5'-FAM-TTATTATT-Biotin-3';
the fluorescence quenching probe is characterized in that a1 st base T marks FAM luminescent groups from the 5' end, and an 8 th base T marks BHQ1 quenching groups; the fluorescent Biotin probe marks FAM luminous groups with 1 st base T from the 5' end, and the 8 th base T is modified by Biotin.
The RAA-CRISPR/Cas12a detection reaction device for PEAV is characterized by comprising an outer sleeve and an inner sleeve, wherein the inner sleeve is positioned inside the outer sleeve, the outer sleeve is used for adding a RAA reaction system, the inner sleeve is used for adding a CRISPR/Cas12a digestion system, and the CRISPR/Cas12a reaction system in the inner sleeve can be released into the outer sleeve through vortex.
A fluorescence method kit for RAA-CRISPR/Cas12a detection of PEAV, characterized in that the RAA-CRISPR/Cas12a detection reaction device of PEAV according to claim 2 is built in, and the total reaction system of the fluorescence method kit comprises 25 μl RT-RAA amplification system and 20 μl CRISPR/Cas12a cleavage system;
the RT-RAA amplification system comprises 14.70 mu L of reaction buffer A, 1.25 mu L of reaction buffer B, 1.00 mu L of 10 pmol/mu L of upstream primer PEAV-N-F (SEQ ID NO. 1), 1.00 mu L of 10 pmol/mu L of downstream primer PEAV-N-R (SEQ ID NO. 2), 1.00 mu L of sample RNA template to be detected and 6.05 mu L of nuclease-free double distilled water; the reaction buffer A consists of 100U/. Mu.L of reverse transcriptase, 120 ng/. Mu.L of recombinase, 30 ng/. Mu.L of DNA polymerase and 800 ng/. Mu.L of single-stranded DNA binding protein, and the reaction buffer B is 280mM magnesium acetate;
the CRISPR/Cas12a cleavage system included 1 pmol/. Mu.L Cas12a enzyme 2.00. Mu.L, 10 pmol/. Mu.L ssDNA probe 1 probe 2.00. Mu.L, 1 pmol/. Mu.L guide RNA (SEQ ID NO. 3) 2.00. Mu.L and buffer 14.00. Mu.L; the buffer solution comprises 50mM NaCl, 10mM Tris-HCl and 10mM MgCl 2 And 100. Mu.g/mL BSA.
4. A method of PEAV RAA-CRISPR/Cas12a fluorescence detection using the PEAV RAA-CRISPR/Cas12a fluorescence kit of claim 3, comprising the steps of:
(1) Extracting RNA of a sample to be detected;
(2) Amplification of RT-RAA reaction System: taking the extracted RNA of the sample to be detected as a template, adding a reagent for detecting the RT-RAA amplification system of PEAV into the outer sleeve of the detection reaction device, simultaneously adding a reagent for detecting the CRISPR/Cas12a digestion system into the inner sleeve, and then carrying out nucleic acid amplification, and carrying out isothermal amplification for 10min at 37 ℃;
(3) After the amplification reaction is finished, releasing the CRISPR/Cas12a enzyme digestion system in the inner sleeve into the outer sleeve by vortex, utilizing the auxiliary cutting activity of the Cas12a enzyme to cut a fluorescence quenching probe, reacting for 10min at the constant temperature of 37 ℃, and observing fluorescence by using a portable Lan Guangyi with the wavelength of 440-460 nm.
A test strip method kit for detecting RAA-CRISPR/Cas12a of PEAV, which is characterized in that a RAA-CRISPR/Cas12a detection reaction device of PEAV according to claim 2 is built in, and the total reaction system of the test strip method kit comprises a 25 mu L RT-RAA amplification system, a 20 mu L CRISPR/Cas12a digestion system and a test strip;
the RT-RAA amplification system comprises 14.70 mu L of reaction buffer A, 1.25 mu L of reaction buffer B, 1.00 mu L of 10 pmol/mu L of upstream primer PEAV-N-F (SEQ ID NO. 1), 1.00 mu L of 10 pmol/mu L of downstream primer PEAV-N-R (SEQ ID NO. 2), 1.00 mu L of sample RNA template to be detected and 6.05 mu L of nuclease-free double distilled water;
the CRISPR/Cas12a cleavage system included 1 pmol/. Mu.L Cas12a enzyme 2.00. Mu.L, 10 pmol/. Mu.L ssDNA probe 2.00. Mu.L, 1 pmol/. Mu.L guide RNA (SEQ ID NO. 3) 2.00. Mu.L and buffer 14.00. Mu.L;
the test strip comprises a sample binding pad, a detection line and a quality control line, wherein the sample binding pad contains a colloidal gold-labeled mouse anti-FAM monoclonal antibody, the detection line contains a goat anti-mouse IgG antibody, and the quality control line contains streptavidin.
6. A RAA-CRISPR/Cas12a test strip detection method for PEAV using the RAA-CRISPR/Cas12a test strip kit for PEAV of claim 5, comprising the steps of:
(1) Extracting RNA of a sample to be detected;
(2) Amplification of RT-RAA reaction System: taking the extracted RNA of the sample to be detected as a template, adding a reagent for detecting the RT-RAA amplification system of PEAV into the outer sleeve of the reaction device, simultaneously adding a reagent for detecting the CRISPR/Cas12a digestion system into the inner sleeve, and then carrying out nucleic acid amplification, and carrying out isothermal amplification for 10min at 37 ℃;
(3) After the amplification reaction is finished, releasing a CRISPR/Cas12a enzyme digestion system in the inner sleeve into the outer sleeve through vortex, utilizing the auxiliary cutting activity of Cas12a enzyme to cut a fluorescent biotin probe, reacting at the constant temperature of 37 ℃ for 10min, directly inserting the sample adding end of a colloidal gold test strip into a reaction solution, reacting at the room temperature for 1min, and observing the strip.
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