CN115141826B - RPA primer pair and application thereof, kit for visually detecting PCV4, application of kit and method for detecting PCV4 - Google Patents
RPA primer pair and application thereof, kit for visually detecting PCV4, application of kit and method for detecting PCV4 Download PDFInfo
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Abstract
The invention belongs to the field of visual detection of porcine circovirus type 4 nucleic acid, and particularly relates to an RPA primer pair and application thereof, a kit for visual detection of PCV4, application thereof and a method for visual detection of PCV 4; the RPA primer pair is a primer pair consisting of RPA F and RPA R, wherein the nucleotide sequence of the RPA F is shown as SEQ ID NO.1, and the nucleotide sequence of the RPA R is shown as SEQ ID NO. 2. The kit provided by the invention has the advantages of good specificity and low detection limit, and can rapidly and qualitatively detect PC V4. The method for judging and reading the kit is visual, and has no cross reaction with other poultry viruses. High-sensitivity and high-specificity detection of PCV4 can be realized. Can provide technical support for early detection and later epidemiological investigation of porcine circovirus type 4, and has guiding significance for prevention and control of the disease.
Description
Technical Field
The invention belongs to the field of visual detection of porcine circovirus type 4 nucleic acid, and particularly relates to detection of porcine circovirus type 4 nucleic acid, in particular to an RPA primer pair and application thereof, a kit for visual detection of PCV4, application thereof and a method for visual detection of PCV4.
Background
Accurate and rapid detection is an important means for reducing the spread of PCV4. Currently, virus isolation assays, conventional PCR, fluorescent quantitative PCR, and the like are commonly used for PCV4 assays. The common PCR has long detection time, and special equipment and technicians are required for virus separation and identification, fluorescent quantitative PCR and the like. The detection method based on the CRISP R-Cas13a system and the visual test strip is high in sensitivity, high in specificity and suitable for the requirements of on-site rapid detection and accurate detection, and provides powerful support for epidemiological investigation, grasp of propagation rules and diagnosis, prevention and control of epidemic diseases.
Disclosure of Invention
The invention aims at overcoming the defects of long PCV 4detection time, high specificity and low sensitivity in the prior art, and provides an RPA primer pair and application thereof, a kit for visually detecting PCV4 and application thereof and a method for visually detecting PCV4.
In order to achieve the above object, the first aspect of the present invention provides an RPA primer pair, which is a primer pair composed of an RPA F and an RPA R, wherein the nucleotide sequence of the RPA F is shown as SEQ ID No.1, and the nucleotide sequence of the RPA R is shown as SEQ ID No. 2.
The second aspect of the invention provides the application of the RPA primer pair in preparing the visual PCV4 for detection or diagnosis.
In a third aspect, the present invention provides a kit for visual detection of PCV4, said kit comprising: an RPA primer pair, a crRNA guide sequence and a target DNA fragment; the RPA primer pair is the RPA primer pair of claim 1.
Preferably, the nucleotide sequence of the crRNA guide sequence is shown in SEQ ID NO. 3.
Preferably, the target DNA fragment is obtained by amplifying the genomic DNA of interest by the RPA primer pair.
Preferably, the target DNA fragment is an amplified sequence of the Cap gene of PCV 4; the amplification sequence of the Cap gene is shown as SEQ ID No.4.
Preferably, the kit further contains at least one of Cas13a protein, NTP buffer mix, T7 RNA polymerasemix, RNase inhibitor, RNA Reporter, PCV4 plasmid standard, porcine circovirus 4 plasmid standard, and lateral flow test strip.
Preferably, the Cas13a protein is an LwCas13a nuclease.
In a fourth aspect, the present invention provides the use of a kit according to the third aspect as described above for the preparation of a visual detection or diagnostic test for PCV4.
A fifth aspect of the present invention provides a method of visually inspecting PCV4, the method comprising:
A. obtaining PCV4 genomic DNA from a tissue or serum sample to be tested;
B. designing an RPA amplification primer by taking PCV4 genome DNA as a targeting site according to the design principle of an RPA (polymerase chain reaction) amplification technology, and amplifying PCV4 genome DNA by using an RPA isothermal amplification technology to obtain PCV4 genome DNA to be detected;
C. mixing and incubating PCV4 genomic DNA to be detected with reagents provided in the kit according to the fourth aspect;
diluting the detection product with a transverse flow test strip diluent, loading onto a transverse flow test strip, standing for 3-5 min, and judging according to the result of the color development strip
The method for visually detecting PCV4 based on the RPA-CRISPR-Cas13a system provided by the invention has the following steps that the target fragment single-stranded RNA is prepared by carrying out RPA amplification and in-vitro transcription on double-stranded DNA of a sample to be detected, then the CRISPR-Cas13a system is guided to carry out recognition and combination on an RPA amplification product and cut target single-stranded RNA to activate Cas13a nuclease under the mediation of a crRNA guiding sequence, the activated Cas13a nucleic acid has RNase activity, a special RNA reporter molecule can be cut off, and a strip appears on the reporter molecule. And judging whether the target sequence exists or not according to the difference of the positions of the bands.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a kit and a method for visually detecting porcine circovirus type 4 based on an RPA-CRISPR-Cas13a system, wherein the method has good specificity and the minimum detection limit of 1.0x100 copy/uL, and can rapidly and qualitatively detect PCV4.
2. Compared with the traditional nucleic acid detection PCR technology, the kit is simple to prepare and apply, can carry out isothermal detection within 6 0 minutes at 37 ℃, has visual interpretation method, and has no cross reaction with other poultry viruses. High-sensitivity and high-specificity detection of PCV4 can be realized; can provide technical support for early detection and later epidemiological investigation of PCV4, and has guiding significance for prevention and control of the disease.
Drawings
FIG. 1 is a graph of test results for RPA primer pairs;
fig. 2 is a graph of the sensitivity test results of a CRISPR-Cas13a system lateral flow test strip established in the present invention. T is a test line, C is a quality control line, the T line or the T and the C lines are simultaneously developed to be positive, and the T line is not developed and the C line is developed to be negative;
FIG. 3 is a graph of the results of a specificity test of a lateral flow test strip of a CRISPR-Cas13a system established in the present invention;
FIG. 4 is a graph showing the results of fluorescent quantitative PCR detection of 20 clinical samples;
FIG. 5 is a graph of results of detecting 10 simulated clinical samples using the CRISPR-Cas13a system established in the present invention;
fig. 6 is a graph of results of detection of 10 actual clinical samples using the CRISPR-Cas13a system established by the present invention.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Preparation example:
preparation of a Standard plasmid containing the PCV4-Cap Gene sequence:
1) Extracting porcine circovirus type 4 (PCV 4) genome according to the specification of the viral DNA/RNA co-extraction kit, using the obtained DNA as a template, using primers PCV4-F/R (PCV 4-F: AAACCT CACTTTATCGCTGAC; PCV4-R: CAAGGTTCTCCCGCACCTTT) carrying out PCR amplification on target fragments of the standard substance to obtain an amplification product; the reaction system: 2 XTaq enzyme 10uL, upstream primer (1 0. Mu.M) 1uL, downstream primer (10. Mu.M) 1uL, cDNA template 1uL, ddH 2O to 20uL;
the amplification conditions were: reacting at 94 ℃ for 5min; then reacting for 30s at 94 ℃, then reacting for 30s at 55 ℃ and then reacting for 90s at 72 ℃, and circulating the previous steps for 30 times; then reacting for 10min at 72 ℃;
2) Performing gel electrophoresis on the amplified product, cutting gel, recovering and purifying a target fragment, connecting a pMD-19T cloning vector, converting the target fragment into escherichia coli DH5 alpha competent cells, screening positive cloning strains, performing amplification culture, extracting plasmids, sequencing, naming the positive plasmids with correct sequence identification as pMD-19T-PCV4, and preserving at the temperature of-80 ℃ for later use;
3) The pMD-19T-PCV4 concentration was measured and plasmid copy number was calculated, formula was calculated: the samples= (a mount×6.022×10)/(length×1×10×9×650) was used as a standard (standard plasmid containing PCV4-Cap gene sequence) for CRISPR-Cas13a visual detection method.
Example 1RPA primer design and amplification System determination
According to the PCV4-Cap gene sequence conservation region shown in SEQ ID No.4, a series of primer pairs meeting the design principle of RPA primers are designed and obtained, and the primer pairs are synthesized by Huada biology (PAGE purification); after a series of early detection and screening, primer pairs RPA F and RPA R are obtained; wherein the nucleotide sequence of RPA F is shown as SEQ ID NO.1, and the nucleotide sequence of RPA R is shown as SEQ ID NO. 2.
The size of the amplified fragment is 214bp;
RPA amplification: RPA-F240 pM, RPA-R240 pM, amplification buffer+enzyme Mix (25. Mu.l), mgOAc solution (2.5. Mu.l), nucleic acid template (1. Mu.l) and non-nucleic acid water (supplementary volume to 50. Mu.l) were mixed and reacted at 37℃for 40 minutes to obtain an RPA amplification product.
Example 2
Synthesis of crRNA probes
The designed crRNA (the nucleotide sequence of the crRNA is shown as SEQ ID NO. 3) is prepared by in vitro transcription, and an accessory T7 promoter sequence is added at the 5' end of the crRNA. The two DNA fragments were annealed to form a double strand at a final concentration of 10. Mu.M, and an in vitro transcription experiment was performed using HiScribe T7Quick High Yield RNA Synthesis kit (New England Bi olabs). crRNA was purified using RNAXP magnetic beads and product concentration was determined by Qubit.
Example 3
Establishment and application of RPA-CRISPR-Cas13a method
1. Method of
Using LwCas13a nuclease (45 nM), crRNA probe (22.5 nM), RNA reporter (125 nM), RNaseinunder (0.25 uL), NTP Buffer mix 2.5uL, and T7 polymerase (0.4 uL), use detection Buffer to supplement 9uL and add 1uL of RPA amplification product and incubate at 37℃for 40 minutes. And diluting the detection product by 10 times by using a transverse flow test strip diluent, loading the diluted detection product onto a transverse flow test strip, standing for 3-5 minutes, and observing the color development condition.
2. Results
1. Analytical sensitivity detection
Sensitivity detection was performed using a 10-fold gradient diluted pMD-19T-PCV4 standard plasmid (10 8 copies/uL-. Times.10 0 copies/uL) as a template, ddH 2O as a negative control, and the lowest concentration of no distinct band on the test line as the sensitivity of the detection method. As shown in the detection result figure 2, the detection minimum limit can reach 1.0x100copy/uL, which proves that the detection sensitivity of the method of the invention on porcine circovirus type 4 is high.
2. Assay specific detection
Genomic nucleic acids of PCV4 and PCV2, P CV3, pseudorabies virus (PRV), parvovirus (PPV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), japanese Encephalitis (JEV) and swine fever (CSFV) preserved in the laboratory were extracted using a viral DNA/RNA co-extraction kit. Nucleic acid pre-amplification experiments are carried out by taking the viral genome as a template, CRISPR-Cas13a lateral flow test strip detection is carried out by applying the chromogenic method established in the embodiment 3, and meanwhile, negative control is set, so that the specificity of the method is verified. As shown in FIG. 3, the test strip for detecting PCV4 only has the color development of the test strip, and the test strips for detecting other viruses are all judged to be negative, so that the test method established by the invention has good specificity.
3. Simulated clinical sample detection
Mixing PCV4 positive plasmids with healthy pig serum with different concentrations (PCV 4 positive plasmids are respectively 1-5 samples, namely healthy pig serum=2:1, 10:1, 50:1, 100:1 and 1000:1), and detecting the tolerance of the invention to serum; the lowest viral titers were detected by mixing PCV4 positive plasmids with healthy pig serum at different concentrations (samples No. 6-10P CV4 positive plasmids: healthy pig serum = 1:10, 1:50, 1:100, 1:1000, 1:10000, respectively). PCV4 fluorescence quantitative PCR method is respectively used for detection by CRISPR-Cas13a lateral flow test strip, and the detection method is constructed by the text of Establishment of an SYBR Gre en-based real-time PCR assay for porcinecircovirustype 4detection published in Journal of Virological Methods in 2020. The detection results are shown in fig. 4 (the results of the samples 1-10 corresponding to the curves 1-10) and fig. 5, each sample test strip shows positive strips, the negative result is established, and the coincidence degree with the fluorescent quantitative PCR detection result is 100%.
4. Actual clinical sample detection
And (3) extracting a clinically collected serum sample genome by using a DNA/RNA extraction kit as a detection template, carrying out isothermal amplification on the template by adopting RPA to obtain an RPA amplification product, and finally detecting the serum (sample No. 11-20) of 10 suspected pigs collected from a pig farm in part of Anhui by using a CRISPR-Cas13a lateral flow test strip detection method and a fluorescent quantitative PCR method respectively, and comparing detection results. The result shows that 10 suspected disease pig serum samples collected from Anhui disease pig farms are clinically detected by a fluorescence quantitative PCR and CRISPR-Cas13a lateral flow detection method respectively. The detection results are shown in fig. 4 (the results of the 11-20 samples corresponding to the curves 11-20) and fig. 6, each sample test strip shows positive strips, the negative result is established, the coincidence degree with the fluorescence quantitative PCR detection result reaches 100%, and the detection effects of the two methods are consistent.
To sum up: the invention discloses a porcine circovirus 4-type nucleic acid CRISPR-Cas13a system, an RPA primer pair and crRNA. The invention establishes a visual, sensitive and specific porcine circovirus type 4 rapid clinical detection system based on a CRISPR-Cas13a diagnosis platform based on a designed RPA primer pair and specific crRNA. The detection system is different from the traditional detection technology based on the PCR technology, does not need a complicated temperature control instrument in the whole reaction process, only needs to carry out isothermal detection at 37 ℃, can be used for fluorescent reading or visual reading, has the lowest detection limit of 1.0 multiplied by 100 copy/uL, does not have cross reaction with other porcine viruses, and can directly play a role in the virus detection of clinical samples by the enhanced Cas13a detection. The invention can discover pigs infected with PCV4 earlier and more timely so as to perform early treatment and accurate prevention and control.
The foregoing is a further elaboration of the present invention in connection with the detailed description, and it is not intended that the invention be limited to the specific embodiments shown, but rather that a number of simple deductions or substitutions be made by one of ordinary skill in the art without departing from the spirit of the invention, should be considered as falling within the scope of the invention as defined in the appended claims.
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Sequence listing
<110> institute of livestock and veterinary at the academy of agricultural sciences of Anhui province
<120> RPA primer pair and application thereof, kit for visual detection of PCV4, application thereof and method for detection of PCV4
<140> 2022106176026
<141> 2022-06-01
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 59
<212> DNA
<213> porcine circovirus type 4 primer 1 (Porcine Circovirus Primer 1)
<400> 1
gaaattaata cgactcacta tagggttttt cccttccccc acatagtctc catccagtt 59
<210> 2
<211> 34
<212> DNA
<213> porcine circovirus type 4 primer 2 (Porcine Circovirus Primer 2)
<400> 2
gccctcttgg aacgttggac attacgattt caaa 34
<210> 3
<211> 89
<212> DNA
<213> porcine circovirus type 4 primer 3 (Porcine Circovirus Primer 3)
<400> 3
gaaattaata cgactcacta taggggattt agactacccc aaaaacgaag gggactaaaa 60
cttacagcct cccatttgca tattaccgg 89
<210> 4
<211> 307
<212> DNA
<213> porcine circovirus type 4 (Porcine Circovirus Type 4)
<400> 4
tttggagtga aatagcgact gtgtctagca atattggtga aaccatgcct gctgctgtgg 60
tttgccagga catcataagt ttggtttttc ccttccccca catagtctcc atccagttgt 120
atagcagtgc tagagtaagt cctattactg ttaatgccat ttagtggcag aaattcgact 180
ttgacctttc tgatccggta atatgcaaat gggaggctgt aaaggtttac gatcgttccc 240
ggtccttttg ggataaagtc cttcagtttg aaatcgtaat gtccaacgtt ccaagagggc 300
gtggaaa 307
Claims (3)
1. A kit for visual detection of PCV4, characterized in that: the kit comprises: an RPA primer pair, a crRNA guide sequence and a target DNA fragment;
the RPA primer pair is a primer pair consisting of RPA F and RPA R, wherein the nucleotide sequence of the RPA F is shown as SEQ ID NO.1, and the nucleotide sequence of the RPA R is shown as SEQ ID NO. 2;
the nucleotide sequence of the crRNA guide sequence is shown as SEQ ID NO. 3;
the target DNA fragment is obtained by amplifying target genomic DNA through the RPA primer pair;
the target DNA fragment is an amplified sequence of a Cap gene of PCV 4; the amplification sequence of the Cap gene is shown as SEQ ID No.4;
the kit further comprises a Cas13a protein, which Cas13a protein is an LwCas13a nuclease.
2. The kit for visual detection of PCV4 according to claim 1, wherein: the kit also contains at least one of NTP buffer mix, T7 RNA polymerase mix, RNase inhibitor, RNA Reporter, PCV4 plasmid standard, porcine circovirus 4 plasmid standard and transverse flow test strip.
3. Use of the kit of any one of claims 1-2 for the preparation of a reagent for detecting or diagnosing PCV4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210617602.6A CN115141826B (en) | 2022-06-01 | 2022-06-01 | RPA primer pair and application thereof, kit for visually detecting PCV4, application of kit and method for detecting PCV4 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210617602.6A CN115141826B (en) | 2022-06-01 | 2022-06-01 | RPA primer pair and application thereof, kit for visually detecting PCV4, application of kit and method for detecting PCV4 |
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| Publication Number | Publication Date |
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| CN115141826A CN115141826A (en) | 2022-10-04 |
| CN115141826B true CN115141826B (en) | 2024-01-05 |
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| CN202210617602.6A Active CN115141826B (en) | 2022-06-01 | 2022-06-01 | RPA primer pair and application thereof, kit for visually detecting PCV4, application of kit and method for detecting PCV4 |
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| CN (1) | CN115141826B (en) |
Citations (6)
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|---|---|---|---|---|
| CN111826472A (en) * | 2020-09-03 | 2020-10-27 | 扬州大学 | Primer probe group, kit and iPCR method for detecting porcine circovirus type 4 |
| CN112941237A (en) * | 2021-03-25 | 2021-06-11 | 中国人民解放军军事科学院军事医学研究院 | CRISPR nucleic acid detection kit for specifically detecting H7N9 avian influenza A virus |
| KR20210130612A (en) * | 2020-04-22 | 2021-11-01 | 김성천 | Method and apparatus for target nucleic acid detection by simply extracting and analyzing nucleic acid |
| CN113774166A (en) * | 2021-09-13 | 2021-12-10 | 青岛农业大学 | Porcine circovirus type 2, type 3 and type 4 on-site rapid high-sensitivity differential diagnosis kit and use method thereof |
| WO2022033607A2 (en) * | 2020-08-10 | 2022-02-17 | 苏州顶点生物医药有限公司 | Magnetic bead technology system for amplifying signal of nucleic acid detection based on crispr technology, and use thereof |
| CN114381552A (en) * | 2021-12-31 | 2022-04-22 | 安徽农业大学 | Primer, probe and detection method for detecting porcine circovirus type 4 based on CRISPR-Cas12a |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551800B (en) * | 2018-06-03 | 2022-08-19 | 上海吐露港生物科技有限公司 | Application of high-temperature-resistant Cas protein, and detection method and kit of target nucleic acid molecule |
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2022
- 2022-06-01 CN CN202210617602.6A patent/CN115141826B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20210130612A (en) * | 2020-04-22 | 2021-11-01 | 김성천 | Method and apparatus for target nucleic acid detection by simply extracting and analyzing nucleic acid |
| WO2022033607A2 (en) * | 2020-08-10 | 2022-02-17 | 苏州顶点生物医药有限公司 | Magnetic bead technology system for amplifying signal of nucleic acid detection based on crispr technology, and use thereof |
| CN111826472A (en) * | 2020-09-03 | 2020-10-27 | 扬州大学 | Primer probe group, kit and iPCR method for detecting porcine circovirus type 4 |
| CN112941237A (en) * | 2021-03-25 | 2021-06-11 | 中国人民解放军军事科学院军事医学研究院 | CRISPR nucleic acid detection kit for specifically detecting H7N9 avian influenza A virus |
| CN113774166A (en) * | 2021-09-13 | 2021-12-10 | 青岛农业大学 | Porcine circovirus type 2, type 3 and type 4 on-site rapid high-sensitivity differential diagnosis kit and use method thereof |
| CN114381552A (en) * | 2021-12-31 | 2022-04-22 | 安徽农业大学 | Primer, probe and detection method for detecting porcine circovirus type 4 based on CRISPR-Cas12a |
Non-Patent Citations (1)
| Title |
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| Rapid Detection of Genotype II African Swine Fever Virus Using CRISPR Cas13a-Based Lateral Flow Strip;Wei Ning等;Viruses;第14卷;第1-12页 * |
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| Publication number | Publication date |
|---|---|
| CN115141826A (en) | 2022-10-04 |
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