CN115141826A - RPA primer pair and application thereof, kit for visual detection of PCV4, application thereof and PCV 4detection method - Google Patents

RPA primer pair and application thereof, kit for visual detection of PCV4, application thereof and PCV 4detection method Download PDF

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CN115141826A
CN115141826A CN202210617602.6A CN202210617602A CN115141826A CN 115141826 A CN115141826 A CN 115141826A CN 202210617602 A CN202210617602 A CN 202210617602A CN 115141826 A CN115141826 A CN 115141826A
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rpa
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CN115141826B (en
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王洁茹
殷冬冬
戴银
尹磊
刘林清
潘孝成
王重龙
沈学怀
胡晓苗
侯宏艳
赵瑞宏
周学利
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Institute of Animal Husbandry and Veterinary Medicine of Anhui Academy of Agricultural Sciences
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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 be used for quickly and qualitatively detecting the PC V4. The interpretation method of the kit is visual, and the kit has no cross reaction with other poultry viruses. The 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

RPA primer pair and application thereof, kit for visual detection of PCV4, application thereof and PCV 4detection method
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
Porcine Circovirus (PCV) is a non-enveloped circular DNA virus belonging to the family of circoviridae, the genus of circovirus. It is divided into four serotypes (PCV 1-4). PCV4 can cause diseases such as porcine respiratory disease syndrome, porcine dermatitis and nephrotic syndrome, myocarditis and sow dysgenesis. PCV4 is often clinically co-infected with other viruses, resulting in a significant increase in mortality (25% -40%) in sick pigs, and accurate monitoring is extremely important in order to prevent disease transmission and control viral transmission.
Is accurate rapid detection is an important means to reduce its propagation spread. Currently, virus isolation identification, conventional PCR, fluorescent quantitative PCR and the like are generally adopted for testing PCV4. The common PCR detection time is long, and professional equipment and technicians are needed for virus separation identification, fluorescent quantitative PCR and the like. The detection method based on the combination of the CRISP R-Cas13a system and the visual test strip has high sensitivity and specificity, meets the requirements of on-site rapid detection and accurate detection, and provides powerful support for epidemiological investigation, mastering of propagation rules and diagnosis and prevention and control of epidemic diseases.
Disclosure of Invention
The invention aims to provide an RPA primer pair and application thereof, a kit for visually detecting PCV4, application thereof and a method for visually detecting PCV4, aiming at the defects of long PCV 4detection time, high specialty and low sensitivity in the prior art.
In order to achieve the above object, a first aspect of the present invention provides an RPA primer pair, wherein the RPA primer pair is a primer pair composed 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 second aspect of the invention provides an application of the RPA primer pair in preparation of visual PCV4 for detection or diagnosis.
In a third aspect, the present invention provides a kit for visually detecting PCV4, the kit comprising: an RPA primer pair, a crRNA guide sequence and a target DNA fragment; the pair of RPA primers is the pair of RPA primers 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 amplification sequence of Cap gene of PCV 4; the amplification sequence of the Cap gene is shown as SEQ ID No.4.
Preferably, the kit further comprises at least one of a Cas13a protein, an NTP buffer mix, a T7 RNA polymerase mix, an RNase inhibitor, an RNA Reporter, a PCV4 plasmid standard, a porcine circovirus type 4 plasmid standard, and a lateral flow strip.
Preferably, the Cas13a protein is an LwCas13a nuclease.
In a fourth aspect, the invention provides the use of the kit of the third aspect for the preparation of a kit for detecting or diagnosing visual detection of PCV4.
A fifth aspect of the present invention provides a method for visually detecting PCV4, the method comprising:
A. obtaining PCV4 genomic DNA from a tissue or serum sample to be tested;
B. PCV4 genomic DNA is used as a targeting site, an RPA amplification primer is designed according to the design principle of recombinase polymerase amplification technology RPA, and PCV4 genomic DNA is amplified through the RPA isothermal amplification technology to obtain PCV4 genomic DNA to be detected;
C. mixing and incubating PCV4 genomic DNA to be detected with the reagent provided in the kit of the fourth aspect according to the system;
diluting the detection product with the transverse flow test strip diluent, loading the diluted detection product onto a transverse flow test strip, standing for 3-5 minutes, and judging the result according to the chromogenic strip
The invention provides a visual PCV 4detection method based on an RPA-CRISPR-Cas13a system, which comprises the following steps of firstly carrying out RPA amplification and in-vitro transcription on double-stranded DNA of a sample to be detected to prepare target fragment single-stranded RNA, then guiding the CRISPR-Cas13a system to carry out recognition and combination on an RPA amplification product and cutting target single-stranded RNA to activate Cas13a nuclease under the mediation of a crRNA guide sequence, wherein the activated Cas1 a nucleic acid has RNA enzyme activity, a special RNA reporter molecule can be cut off, and a strip can appear on a test strip. And judging whether the target sequence exists or not according to the difference of the strip positions.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides a reagent for visually detecting porcine circovirus type 4 based on an RPA-CRISPR-Cas13a systemA kit and a method which have good specificity and a minimum detection limit of 1.0X 10 0 copy/uL, and can rapidly and qualitatively detect PCV4.
2. Compared with the traditional nucleic acid detection PCR technology, the kit disclosed by the invention is simple to prepare and apply, can be used for carrying out isothermal detection within 6 minutes at 37 ℃, is intuitive in interpretation method, and has no cross reaction with other poultry viruses. The high-sensitivity and high-specificity detection of PCV4 can be realized; the method can provide technical support for early detection and later epidemiological investigation of PCV4, and has guiding significance for prevention and control of the PCV4.
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FIG. 1 is a graph showing the results of the test of the RPA primer pair;
fig. 2 is a graph of the sensitivity test result of the 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 line and the C line are simultaneously colored to show that the T line is positive, the T line is not colored, and the C line is colored to show that the C line is negative;
FIG. 3 is a diagram showing the specificity test result of the CRISPR-Cas13a system lateral flow test strip established in the invention;
FIG. 4 is a graph showing the results of fluorescence quantitative PCR detection of 20 clinical samples;
FIG. 5 is a graph showing the results of detecting 10 simulated clinical samples by using the CRISPR-Cas13a system established in the present invention;
FIG. 6 shows CRISPR-Cas13a established by the invention the system detects 10 actual clinical sample result graphs.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Preparation example:
preparation of a standard plasmid containing a PCV4-Cap gene sequence:
1) The porcine circovirus type 4 (PCV 4) genome is extracted according to the instructions of the viral DNA/RNA co-extraction kit, and the obtained DNA is used as a template, and primers PCV4-F/R (PCV 4-F: AAACCT CACTTATCGCTGAC; PCV4-R: CAAGGTTCCCCGCACCTTT) to carry out PCR amplification on the target fragment of the standard product to obtain an amplification product; reaction system: 2 XTaq enzyme 10uL, upstream primer (1. Mu.M) 1uL, downstream primer (10. Mu.M) 1uL, cDNA template 1uL, ddH 2 O is supplemented to 20uL;
the amplification conditions were: reacting at 94 ℃ for 5min; then reacting at 94 ℃ for 30s, then reacting at 55 ℃ for 30s, and then reacting at 72 ℃ for 90s, and circulating the previous steps for 30 times; then reacting for 10min at 72 ℃;
2) Carrying out gel electrophoresis on the amplification product, cutting the gel, recovering and purifying a target fragment, connecting a pMD-19T cloning vector and transforming the cloning vector to escherichia coli DH5 alpha competent cells, screening positive cloning strains, carrying out amplification culture, extracting plasmids and sequencing, naming the positive plasmids with correct sequence identification as pMD-19T-PCV4, and storing the positive plasmids at-80 ℃ for later use;
3) pMD-19T-PCV4 concentration was determined and plasmid copy number was calculated, formula: copies = (a mount × 6.022 × 10) 23 )/(length×1×10 9 X 650), and the product is used as a standard substance (a standard plasmid containing PCV4-Cap gene sequence) of a CRISPR-Cas13a visual detection method.
Example 1RPA primer design and amplification System determination
According to a PCV4-Cap gene sequence conserved region shown in SEQ ID NO.4, a series of primer pairs meeting the RPA primer design principle are designed and obtained, and are biosynthesized by Huada (PAGE purification); obtaining primer pairs RPA F and RPA R after a series of early detection and screening; wherein, the nucleotide sequence of RPA F is shown in SEQ ID NO.1, and the nucleotide sequence of RPA R is shown in SEQ ID NO. 2.
The amplified fragment size is 214bp;
amplification of RPA: 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 nucleic acid-free water (supplemented to 50. Mu.l) were mixed and reacted at 37 ℃ for 40 minutes to obtain an RPA amplification product.
Example 2
Synthesis of crRNA Probe
In vitro transcription is carried out to prepare designed crRNA (the nucleotide sequence of the crRNA is shown as SEQ ID NO. 3), and an accessory T7 promoter sequence is added at the 5' end of the crRNA. The two DNAs were annealed to form a duplex at a final concentration of 10. Mu.M, and in vitro transcription experiments were performed using the HiScribe T7 Quick 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 producing a composite material
Using LwCas13a nuclease (45 nM), crRNA probe (22.5 nM), RNA reporter (125 nM), RNase inhibitor (0.25 uL), NTP Buffer mix 2.5uL, and T7 polymerase (0.4 uL), detection Buffer was supplemented to 9uL and 1uL of RPA amplification product was added and incubated at 37 ℃ for 40 min. And diluting the detection product by 10 times by using the transverse flow test strip diluent, loading the diluted detection product on a transverse flow test strip, standing for 3-5 minutes, and observing the color development condition.
2. As a result, the
1. Assay sensitivity detection
pMD-19T-PCV4 standard plasmid (10) diluted in 10-fold gradient 8 copies/uL-×10 0 copies/uL) as template, ddH 2 O is used as a negative control, and the lowest concentration of a test line without obvious bands is used as the sensitivity of the detection method. The detection result is shown in FIG. 2, and the detection minimum detection limit can reach 1.0 × 10 0 copy/uL, which shows that the method has high detection sensitivity on porcine circovirus type 4.
2. Assay specificity detection
Genomic nucleic acids of PCV4 and PCV2, P CV3, pseudorabies virus (PRV), parvovirus (PPV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), japanese encephalitis B (JEV) and swine fever (CSFV) stored in the laboratory were extracted by a virus DNA/RNA co-extraction kit. And (3) carrying out a nucleic acid pre-amplification experiment by taking the viral genome as a template, carrying out CRISPR-Cas13a transverse flow test strip detection by applying the color development method established in the embodiment 3, and simultaneously setting a negative control to verify the specificity of the method. The detection result is shown in fig. 3, the color development of the test strip only appears on the test strip for detecting PCV4, the test strips for detecting other viruses are judged to be negative, and the result proves that the detection method established by the invention has good specificity.
3. Simulating clinical sample testing
Mixing PCV4 positive plasmids with different concentrations of serum of healthy pigs (samples No.1 to 5 are PCV4 positive plasmids: healthy pig serum =2, 1, 50, 1, 100, 1, 1000; the lowest virus titers were measured at different concentrations of PCV4 positive plasmid mixed with healthy pig serum (samples No. 6-10 were P CV4 positive plasmid: healthy pig serum =1, 1. The PCV4 fluorescent quantitative PCR method is constructed by a CRISPR-Cas13a lateral flow test strip detection method and a method referred to in the specification of "assessment of an SYBR Gre en-based real-time PCR assay for a radioactive circular type 4detection" published in Journal of visual Methods in 2020. The detection results are shown in fig. 4 (curves 1-10 correspond to the results of samples No. 1-10) and fig. 5, each sample test strip shows a positive strip, a negative result is established, and the matching degree with the fluorescent quantitative PCR detection result reaches 100%.
4. Actual clinical sample testing
A DNA/RNA extraction kit is used for extracting a clinically collected serum sample genome as a detection template, then the template is subjected to isothermal amplification by adopting RPA to obtain an RPA amplification product, finally, 10 suspected diseased pig sera (No. 11-20 samples) collected from a diseased pig farm in Anhui partial area are detected by a CRISPR-Cas13a lateral flow test strip detection method and a fluorescent quantitative PCR method respectively, and the detection results are compared. The result shows that 10 suspected diseased pig serum samples collected from Anhui diseased pig farms are clinically detected by a fluorescent quantitative PCR and CRISPR-Cas13a lateral flow detection method respectively. The detection results are shown in fig. 4 (the curve 11-20 corresponds to the result of sample No. 11-20) and fig. 6, each sample test strip shows a positive strip, the negative result is true, and the coincidence degree with the fluorescent quantitative PCR detection result is 100%, which proves that the detection effects of the two methods are consistent.
To sum up: the invention discloses a porcine circovirus type 4 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 of the invention is different from the prior detection technology based on PCR technology, does not need a complex temperature control instrument in the whole reaction process, only needs isothermal detection at 37 ℃, can be used for fluorescent reading or visual reading, and has the lowest detection limit of 1.0 multiplied by 10 0 copy/uL, and no cross-reaction with other porcine viruses, the enhanced Cas13a assay may also play a role directly in viral detection of clinical samples. The invention can discover the pig infected with PCV4 earlier and more timely so as to carry out early treatment and accurate prevention and control.
While the invention has been described in further detail in connection with specific embodiments thereof, it will be understood that the invention is not limited thereto, and that various other modifications and substitutions may be made by those skilled in the art without departing from the spirit of the invention, which should be considered to be within the scope of the invention as defined by the appended claims.
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Claims (10)

1. An RPA primer pair, comprising: 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.
2. Use of the RPA primer pair of claim 1 for the preparation of a visual PCV4 assay or diagnosis.
3. A kit for visually detecting PCV4 is characterized in that: the kit comprises: an RPA primer pair, a crRNA guide sequence and a target DNA fragment;
the pair of RPA primers is the pair of RPA primers of claim 1.
4. The kit for visually detecting PCV4 according to claim 3, wherein: the nucleotide sequence of the crRNA guide sequence is shown as SEQ ID NO. 3.
5. The kit for visually detecting PCV4 according to claim 3, wherein: the target DNA fragment is obtained by amplifying the target genomic DNA through the RPA primer pair.
6. The kit for visually detecting PCV4 according to claim 5, wherein: the target DNA fragment is an amplification sequence of Cap gene of PCV 4; the amplification sequence of the Cap gene is shown as SEQ ID No.4.
7. The kit for visually detecting PCV4 according to claim 3, wherein: the kit further comprises at least one of Cas13a protein, NTP buffer mix, T7 RNA polymerase mix, RNase inhibitor, RNA Reporter, PCV4 plasmid standard, porcine circovirus type 4 plasmid standard and lateral flow test strip.
8. The kit for visually detecting PCV4 according to any one of claims 7, wherein: the Cas13a protein is an LwCas13a nuclease.
9. Use of a kit according to any one of claims 3 to 8 for the preparation of a test or diagnostic visual test for PCV4.
10. A method for visually detecting PCV4, comprising: the method comprises the following steps:
A. obtaining PCV4 genomic DNA from a tissue or serum sample to be tested;
B. PCV4 genomic DNA is used as a targeting site, an RPA amplification primer is designed according to the design principle of recombinase polymerase amplification technology RPA, and PCV4 genomic DNA is amplified through the RPA isothermal amplification technology to obtain PCV4 genomic DNA to be detected;
C. the PCV4 genomic DNA to be detected is ligated with any one of claims 3 to 8 the reagents provided in the kit are mixed and incubated according to a system; and diluting the detection product by using a transverse flow test strip diluent, loading the diluted detection product on a transverse flow test strip, standing for 3-5 minutes, and judging the result according to a color development strip.
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