CN117947212A - Kit for multiple detection of four porcine diarrhea virus nucleic acids - Google Patents

Kit for multiple detection of four porcine diarrhea virus nucleic acids Download PDF

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CN117947212A
CN117947212A CN202311717282.2A CN202311717282A CN117947212A CN 117947212 A CN117947212 A CN 117947212A CN 202311717282 A CN202311717282 A CN 202311717282A CN 117947212 A CN117947212 A CN 117947212A
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黄柏成
吴越
曾梓菡
阚兴池
王鑫杰
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Zhejiang Lab
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Abstract

The invention discloses a kit for multiple detection of four porcine diarrhea virus nucleic acids, and relates to the technical field of molecular biological diagnosis. The PCR amplified DNA product and the clinical anal swab sample are selected as detection templates in the research, so that the aim of accurate detection can be achieved. The detection method can detect four kinds of virus nucleic acid with high sensitivity and high specificity, the detection lower limit can reach 1 multiplied by 10 1 copies/mu l, and the method has important production practical value. The detection method can overcome the defects of narrow detection coverage and relatively high detection cost of the existing detection method, and realizes single quadruple detection.

Description

Kit for multiple detection of four porcine diarrhea virus nucleic acids
Technical Field
The invention relates to the technical field of molecular biological diagnosis, in particular to a kit for multiple detection of nucleic acid of four porcine diarrhea viruses.
Background
Coronaviruses have the largest genome among single-stranded positive strand RNA viruses, including Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (Transmissible gastroenteritis virus, TGEV), porcine delta coronavirus (Porcine deltacoronavirus, PDCoV), and Porcine acute diarrhea syndrome coronavirus (Swine acute diarrhea syndrome coronavirus, SADS-CoV). Infection of newborn and lactating piglets with the above viruses can cause acute diarrhea, vomiting, dehydration, and diarrhea symptoms are highly similar and indistinguishable, causing significant economic losses to the pig farming industry, where SADS-CoV may pose public health threats. Therefore, the method has important significance for timely detection of the pathogens in clinic. In the existing diagnostic method, a multiple qRT-PCR method based on TaqMan probes is available, three viruses of PEDV, TGEV and PDCoV can be detected, and the method has higher accuracy, but has the defect of longer detection time in clinic. Therefore, the development of a novel method for multiple detection of the pig diarrhea pathogens is beneficial to enriching detection means of pig diarrhea diseases and promoting the development of pig breeding industry.
The CRISPR/Cas system is an adaptive immune system of prokaryotes that is used to resist invasion by foreign genetic elements present in phage or plasmids. Cas protein is an endonuclease that can recognize and target cleavage of foreign nucleic acids using crrnas corresponding to intermediate sequences in CRISPR sequences. Among them, cas12 and Cas13 systems of CRISPR are often applied in the field of nucleic acid detection, in particular in the field of disease-specific detection. Currently, cas12 a-based detection methods have great potential and are increasingly used for the diagnosis of infectious diseases. However, there is a large inter-species difference between different pathogens, so it is difficult to develop a Cas12a detection method in a standard manner, and it is necessary to optimize a detection system, such as a reaction volume, a Cas12a protein concentration, and the like, for different pathogens.
Detection methods developed based on Cas12a for single pathogens such as SADS-CoV or mycoplasma pneumoniae are now available. Although the detection method is sensitive and effective, only single pathogen infection can be detected, and in the pig industry, diseased pigs are likely to have mixed infection of multiple pathogens. The PCR method is used for diagnosing mixed infection in a laboratory, a large amount of detection consumables are needed, expensive scientific research instruments are seriously relied on, meanwhile, manpower with professional operation capability is needed, the detection time is long, and the method has certain limitation on portable application. If multiple pathogen infections can be detected in a single pass, detection time and cost would be greatly saved. The detection method based on isothermal amplification (RPA) and Cas12a has high sensitivity, is simple and convenient to operate, does not need to depend on a large-scale laboratory instrument, and has both sensitivity and convenience.
Disclosure of Invention
Based on the defects in the prior art, the invention provides a CRISPR-based kit for multiplex detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine delta coronavirus and porcine acute diarrhea syndrome coronavirus nucleic acid. According to the Membrane (M) gene of PEDV, the Spike (S) gene of TGEV, the Membrane (M) gene of PDCoV and the Nucleocapsid (N) gene of SADS-CoV, specific RPA primers and crRNA are respectively designed, and a quadruple detection method for simultaneously detecting PEDV, TGEV, PDCoV and SADS-CoV based on RPA-CRISPR/Cas12a is developed, and the method has the advantages of being independent of a large instrument, relatively simple and convenient to operate, high in specificity and high in sensitivity (the lower detection limit of nucleic acid to be detected of each virus can reach 1X 10 1 copies/. Mu.l), relatively low in detection cost, beneficial to enriching technical means for detecting porcine diarrhea pathogens, and solving the problems of high detection efficiency due to more porcine diarrhea disease types.
In order to achieve the above purpose, the invention provides a target for PEDV, TGEV, PDCoV and SADS-CoV nucleic acid multiplex detection, wherein the target is a target combination consisting of four sequences, and the nucleotide sequences of the target combination are shown as SEQ ID NO.1 to SEQ ID NO. 4. The PCR amplification primer sequences of the target combinations are shown in SEQ ID NO.5 to SEQ ID NO. 12.
The invention also provides a primer combination for PEDV, TGEV, PDCoV and SADS-CoV nucleic acid multiplex detection, which comprises:
The PEDV specific RPA primer sequences are shown as SEQ ID NO.13 and SEQ ID NO.14,
TGEV specific RPA primer sequences are shown as SEQ ID NO.15 and SEQ ID NO.16, PDCoV specific RPA primer sequences are shown as SEQ ID NO.17 and SEQ ID NO.18,
SADS-CoV specific RPA primer sequences are shown as SEQ ID NO.19 and SEQ ID NO. 20.
The invention also provides a crRNA combination for use in PEDV, TGEV, PDCoV and SADS-CoV nucleic acid multiplex assays, the crRNA combination comprising:
the PEDV specific crRNA sequence is shown as SEQ ID NO.21,
TGEV specific crRNA sequence is shown as SEQ ID NO.22,
PDCoV the specific crRNA sequence is shown as SEQ ID NO.23,
SADS-CoV specific crRNA sequence is shown in SEQ ID NO. 24.
The invention also provides a kit for PEDV, TGEV, PDCoV and SADS-CoV nucleic acid multiplex detection, which comprises an isothermal amplification system, a detection system and an enzyme-labeled instrument fluorescence detection system;
The isothermal amplification system comprises the primer combination as isothermal amplification primer,
The detection system comprises the crRNA combination,
The enzyme-labeled instrument fluorescence detection system is used for detecting a reaction product generated by the detection system.
Preferably, the detection system further comprises a Cas12a protein, a ssDNA reporter system comprising ssDNA FQ reporter for fluorescent detection.
Specifically, ssDNAFQ reporter 'end-labeled fluorescent group is 6-FAM,3' end-labeled quenching group is BHQ-1 for fluorescence detection.
The invention also provides a method for multiplexing PEDV, TGEV, PDCoV and SADS-CoV nucleic acid detection, comprising the steps of:
(1) Extracting nucleic acid of a biological sample to be detected;
(2) RPA isothermal amplification and Cas12a detection: amplifying the biological sample nucleic acid obtained in the step (1) in an RPA isothermal amplification system by using the primer combination in the claim 2, carrying out Cas12a detection on the amplified product by using the crRNA combination in the claim 3, and reacting to obtain a Cas12a detection product;
(3) And (3) carrying out fluorescence signal detection on the detection product of the Cas12 a.
Preferably, the biological sample to be detected is a plasmid or a clinical anal swab sample.
Specifically, in the step (2), the amplification conditions are 39 ℃ for 20 minutes; the Cas12a detection conditions were 37.5 ℃ for 15 minutes. And (3) detecting fluorescent signals by using a blue light glue irradiation instrument, wherein the detection wavelength is 470-520nm.
The specific experimental steps are as follows:
S1, screening a virus to be detected related to porcine diarrhea, which has serious clinical influence and a certain representativeness.
S2, synthesizing plasmids containing virus detection target fragments according to the conservation characteristics of the virus and gene sequences confirmed in the previous step so as to prepare a nucleic acid detection template.
S3, amplifying nucleic acid target judgment sequences of the four viruses by using a PCR method by using specific primers based on synthesized plasmids containing the nucleic acid sequences specific to the viruses to be detected, and obtaining the DNA template.
S4, designing and synthesizing specific crRNA based on detection of target fragments, and screening preferable crRNA through fluorescent signal characteristics.
S5, synthesizing and screening the optimal RPA primer combination of each virus according to the positions of the target nucleic acid sequence and the preferable crRNA
S6, constructing a quadruple RPA reaction system (one-tube method).
S7, performing CRISPR/Cas12a detection reaction, and detecting the specific nucleic acid sequences of the four viruses.
Preferably, the RPA Reaction system used in the present invention is 25. Mu.L, and specifically comprises Reaction buffer 10.0. Mu. L, ERA 5.0.0. Mu.L, upstream primer 1.4. Mu.L (10. Mu.M), downstream primer 1.4. Mu.L, agonist (MgOAc) 1.0. Mu. L, DNA template 2.0. Mu.L and ddH 2 O4.2. Mu.L, and the amplification conditions are incubation at 39℃for 20min.
Preferably, the assay sensitivity of the present study is 1.0X10 1 copies/. Mu.L.
Preferably, the instrument for detecting fluorescent signals used in the research is a blue light glue instrument, and the detection wavelength is 470-520nm.
The invention has the beneficial effects that:
1. the PCR amplified DNA product and the clinical anal swab sample are selected as detection templates in the research, so that the aim of accurate detection can be achieved. The detection method can detect PEDV, TGEV, PDCoV and SADS-CoV with high sensitivity and high specificity, the lower limit of detection can reach 1 multiplied by 10 1 copies/. Mu.l, and the production practical value is higher.
2. The RPA-CRISPR detection method can overcome the defect of higher cost in the existing method, and realizes single quadruple detection.
Drawings
FIG. 1 is a schematic diagram of a quadruple rapid detection method.
FIG. 2 shows the detection sensitivity results of PEDV, which indicates that p < 0.05.
FIG. 3 shows the detection sensitivity results of TGEV, which indicates p < 0.05.
FIG. 4 shows the detection sensitivity results PDCoV, which represents p < 0.05.
FIG. 5 shows the detection sensitivity results of SADS-CoV, which indicates p < 0.05.
FIG. 6 shows the fluorescence results of the quadruple RPA-CRISPR method for detecting the target gene of the nucleic acid in the plasmid sample.
Fig. 7 is a statistical analysis of RPA-CRISPR/Cas12a fluorescence detection results, representing p < 0.05.
FIG. 8 is a graph showing the results of positive detection of PEDV in clinical samples;
FIG. 9 is a graph showing the results of detecting TGEV positive in clinical samples;
FIG. 10 is a graph showing the results of positive detection PDCoV in a clinical sample;
FIG. 11 is a graph showing the results of detecting SADS-CoV as positive in a clinical sample;
FIG. 12 is a pUC57 plasmid map.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The general technical schematic diagram of the invention is shown in fig. 1, and comprises the following 4 parts: preparation of specific crRNA, nucleic acid release, isothermal amplification of nucleic acid and Cas12a detection, and fluorescence detection using a blue-light glue instrument.
The preparation method of the RPA primer and the specific crRNA for screening the porcine diarrhea related virus to be detected comprises the following steps:
S1, screening a virus to be detected related to porcine diarrhea with serious clinical influence and a certain representativeness, and determining detection target fragments of four porcine diarrhea pathogens, namely an M gene of PEDV, an S gene of TGEV, an M gene of PDCoV and an N gene of SADS-CoV;
s2, synthesizing plasmids containing virus detection target fragments according to the conserved characteristics of the virus and gene sequences confirmed in the previous step so as to prepare a nucleic acid detection template;
s3, amplifying nucleic acid target judgment sequences of four viruses by using a PCR method by using specific primers based on synthesized plasmids containing the nucleic acid sequences of the specificity of the four viruses to be detected, so as to obtain a DNA template;
s4, designing and synthesizing specific crRNA based on detection of target fragments, and screening preferable crRNA through fluorescent signal characteristics.
S5, synthesizing and screening the optimal RPA primer combination of each virus according to the positions of the target nucleic acid sequence and the preferable crRNA
S6, constructing a quadruple RPA reaction system (one-tube method).
S7, performing CRISPR/Cas12a detection reaction, and detecting the specific nucleic acid sequences of the four viruses.
The method for PEDV, TGEV, PDCoV and SADS-CoV nucleic acid multiplex detection based on RPA and CRISPR/Cas12a comprises the following steps:
s1: preparation of specific crrnas. Designing crRNA containing Cas12a specificity aiming at the M gene of PEDV, the S gene of TGEV, the M gene of PDCoV and the N gene of SADS-CoV, constructing a vector, and performing in vitro transcription to obtain the crRNA;
s2: nucleic acid release. Releasing nucleic acid in the sample to be tested by using a nucleic acid quick release reagent;
S3: isothermal nucleic acid amplification and Cas12a detection. The S2 step sample was amplified in the RPA system using isothermal amplification primers (reaction at 39 ℃ C. For 20 min) and activated Cas12a cleavage probes were reacted at 37.5 ℃ C. For 15 min by the Cas12a recognizing the N gene-specific nucleic acid sequences of the M gene of PEDV, the S gene of TGEV, the M gene of PDCoV and SADS-CoV.
S4: and exciting the fluorescence of the reaction product by using a blue light gel cutting instrument, and judging the detection result by naked eyes.
Example 1
A multiplex detection method for PEDV, TGEV, PDCoV and SADS-CoV based on RPA-Cas12 a:
(1) Determining the pathogen to be detected related to pig diarrhea and its detection fragment
Through investigation, PEDV, TGEV, PDCoV and SADS-CoV which have serious influence on pig industry and are representative are selected as detection targets.
According to the conservation of gene sequences, the detection target fragments of the four porcine diarrhea pathogens determined by the invention are as follows:
The target gene fragment sequence of the PEDV M gene is shown as SEQ ID NO.1,
The sequence of the target gene fragment of the TGEV S gene is shown as SEQ ID NO.2,
The sequence of the target gene fragment of PDCoV M gene is shown as SEQ ID NO.3,
The sequence of the target gene fragment of the SADS-CoV N gene is shown as SEQ ID NO.4,
The above genes were synthesized by the division of biological engineering (Shanghai) and constructed on pUC57 vector (map shown in FIG. 12).
(2) PCR amplification to obtain 4 pathogenic DNA templates to be detected
The synthesized pUC57 plasmid containing four pathogenic target nucleic acid sequences is used as a template to carry out PCR amplification respectively, the primer amplification primers are shown in table 1, and the reaction system of each pathogenic PCR is 100 mu L, and specifically comprises: 1.0. Mu.L of template, 4.0. Mu.L of upstream PCR primer, 4.0. Mu.L of downstream primer, 50.0. Mu.L of 2X PHANTA FLASH MASTER Mix and 41.0. Mu.L of ddH 2 O. The PCR reaction procedure was as follows: denaturation at 98℃for 30s; then 98 ℃ for 10s,55 ℃ for 10s and 72 ℃ for 15s, and 32 cycles are carried out; finally, the temperature is 72 ℃ and the extension is 60 seconds. Adding 6×loading buffer into PCR product, electrophoresis with 1.5% agarose gel for 30min, cutting gel with gel recovery kit, recovering to obtain template DNA of 4 pathogens, and temporarily storing in-20deg.C refrigerator for subsequent RPA-CRISPR/Cas12a detection.
(3) Synthesis and screening of specific crrnas
Based on PAM motif sites containing "TTTN" within the detection target, preferably 4-5 crrnas per detection target fragment, the presence or absence of cross-reactivity between different crrnas and the pathogen template is tested in a matrix arrangement. The crRNA used in the invention is high-efficiency crRNA which is screened, and has no cross reaction with different templates. As shown in FIG. 6, the results demonstrate that multiple RPA products fluoresce upon cleavage of the side chains of four pathogen-specific crRNAs, respectively, and that the multiple RPA reaction system successfully amplifies the nucleic acid sequences of four viruses, indicating the high specificity of the crRNAs used in the present invention. The sequences of target gene fragments corresponding to the PEDV M gene, the TGEV S gene, the PDCoV M gene and the SADS-CoV N gene are shown in SEQ ID NO. 9-12, and specific crRNA information is shown in Table 1. crRNA was synthesized by gold sri biotechnology, inc.
(4) RPA primer synthesis and screening
Pre-amplification of porcine diarrhea pathogen nucleic acid was performed using reverse transcription isothermal amplification (RT-RPA) to perform Cas12a detection reactions.
According to the position of the detection target, 1-3 RPA primers are respectively designed at the upstream and downstream of the template, and the RPA amplification product is controlled within 200 bp. After the RPA primer is synthesized, the optimal primer combination mode is selected according to the fluorescence signal intensity after the RPA-Cas12a reaction. The RPA primer used in the invention is a preferable combination of upstream and downstream primers, and the sequence information is shown in Table 1.
The RPA-Cas12a detection is that firstly, a sample to be detected is added into an RPA reaction cavity, after the reaction is finished, isothermal amplification products are sucked into a Cas12a reaction chamber, and finally, all Cas12a detection reaction products are judged to detect the result through fluorescent signals, so that the detection is finished.
The present study utilizes an enzymatic recombinant isothermal amplification (Enzymatic Recombinase Amplification, ERA) technique with a reaction system of 25 μl, specifically comprising: reaction buffer 10. Mu.L, ERA 5. Mu.L, upstream primer (SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.11, 10. Mu.M) 1.4. Mu.L, downstream primer (SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.12, 10. Mu.M) 1.4. Mu.L, agonist (MgoAC) 1. Mu.L, DNA template 2. Mu.L (sample loading volume unchanged, different concentrations of gene copy number in different sample loading), ddH 2 O4.2. Mu.L, amplification conditions were 39℃for 20min.
Wherein, 4 pairs of RPA primers are diluted according to a certain proportion, and the specific application amount is 0.25 mu L,0.4 mu L,0.25 mu L and 0.5 mu L respectively according to actual conditions, and the total volume of the mixed RPA upstream and downstream primers is 1.4 mu L respectively, so as to construct a quadruple RPA reaction system.
(5) CRISPR/Cas12a reaction system
The CRISPR/Cas12a reaction system was 20. Mu.L, including ddH 2 O8.9. Mu.L, RNase inhibitor 0.1. Mu. L, buffer 3.1.1.2.0. Mu.L, ssDNA reporter 1.0. Mu. L, cas12a protein 1.0. Mu. L, crRNA (sequence shown in SEQ ID NO. 21-24) 2.0. Mu.L and RPA product 5.0. Mu.L. The reaction conditions were 37.5℃for 15min. Wherein, the 5 '-end labeled fluorescent group of ssDNAreporter is 6-FAM and the 3' -end labeled quenching group is BHQ1.
During fluorescence detection, the Cas12a protein after PEDV, TGEV, PDCoV and SADS-CoV specific nucleic acid activation cleaves ssDNA FQ reporter, releasing the activating fluorophore, and fluorescence can be detected using a blue-light glue instrument. And detecting fluorescent signals by using a blue light glue detector, wherein the detection wavelength is 470-520nm.
The nucleic acid of the sample to be detected is incubated for 20min at 39 ℃ through an isothermal amplification reaction system, then 5 mu L of isothermal amplification reaction product is reacted for 15min at 37.5 ℃ through a CRISPR/Cas12a reaction system, and the instrument for detecting the fluorescent signal of the reaction product used in the research is a blue light gel instrument with the detection wavelength of 470-520nm.
The results in FIG. 6 confirm that the multiplex RPA products fluoresce upon side-chain cleavage of four specific crRNAs, respectively. The results of the study demonstrate that the multiplex RPA reaction system successfully amplifies nucleic acids of four viruses, including PEDV, TGEV, PDCoV and SADS-CoV.
FIG. 7 is a statistical analysis of the results of FIG. 6, and FIG. 6 represents the detection system of the present study, which contains the CRISPR components, the four-fold RPA upstream and downstream primers required for detection. Sterile water was added to the negative control group and the samples added to the test group were different plasmids to be tested. Different groupings are distinguished by specific crrnas. Both PEDV, TGEV, PDCoV and SADS-CoV groups showed a significant increase in fluorescence intensity compared to the control (NC) group. The results demonstrate that this study successfully constructs a multiplex assay for PEDV, TGEV, PDCoV and SADS-CoV nucleic acids.
Table 1: sequence information for RPA amplification, PCR amplification and crRNA synthesis in reactions
Example 2 detection sensitivity of RPA/CRISPR
The DNA template prepared in example 1 was gradient diluted to gene copy numbers of 1.0X10 6、1.0×105、1.0×104、1.0×103、1.0×102 and 1.0X10 1, respectively, and 0 copies (no template), and after detection by the RPA-CRISPP/Cas12a method, the detection sensitivity of the four pathogens was determined, respectively.
And respectively taking 2.0 mu L of DNA templates recovered by the gel, diluting to the lower limit of detection sensitivity, performing RPA amplification and CRISPR/Cas12a cutting, and finally judging and reading a fluorescence detection result by a blue-light gel analyzer.
The detection results are shown in figures 2-5, and figure 2 shows that the detection sensitivity of the detection method for PEDV can reach 1.0X10 1 copies/. Mu.L; FIG. 3 shows that the detection sensitivity of the detection method for TGEV can reach 1.0X10 1 copies/. Mu.L; FIG. 4 shows that the detection sensitivity of the detection method for PDCoV can reach 1.0X10. 10 1 copy/. Mu.L; FIG. 5 shows that the detection sensitivity of the detection method for SADS-CoV can reach 1.0X10 1 copies/. Mu.L. The above results demonstrate that the detection method has high sensitivity.
Example 3
Clinical sample detection: the clinical sample type is an anal swab. For sample processing, the EP tube containing the anal swab with 1.0mL PBS (pH 7.4) was centrifuged (1000 Xg, 1 min) first, after which 200. Mu.L of supernatant was aspirated into the new EP tube and RNA was extracted according to the kit (TIANGEN, DP 419) instructions.
Taking 2.0 mu L of RNA extraction product as a detection sample, carrying out RPA-CRISPR/Cas12a detection, and finally observing fluorescence by using a blue-light glue analyzer to judge the result.
The detection results are shown in figures 8-11, and figure 8 shows that the detection method detects that PEDV in a clinical sample is positive, and the other three pathogens are negative; FIG. 9 shows that the detection method detects that TGEV in clinical samples is positive and the other three pathogens are negative; FIG. 10 shows that the detection method detects PDCoV as positive in the clinical sample and the other three pathogens as negative; FIG. 11 shows that the detection method detects SADS-CoV as positive in clinical samples, and the other three pathogens are negative. The detection method is effective for detecting clinical samples.
In conclusion, the detection method based on RPA-CRISPR/Cas12a has high sensitivity and high specificity, can rapidly detect the pathogens of four porcine diarrhea diseases, enriches the detection means of the porcine diarrhea diseases, and has important significance for guiding the diagnosis of the diarrhea diseases in the production practice of live pig breeding. Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (10)

1. The target for multiplex detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine delta coronavirus and porcine acute diarrhea syndrome coronavirus nucleic acid is characterized by being a target combination consisting of four sequences, and the nucleotide sequence of each target is shown as SEQ ID NO. 1-SEQ ID NO. 4.
2. A primer combination for multiplex detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine delta coronavirus, and porcine acute diarrhea syndrome coronavirus nucleic acid, the primer combination comprising:
The specific RPA primer sequences of the porcine epidemic diarrhea virus are shown as SEQ ID NO.13 and SEQ ID NO.14,
The specificity RPA primer of the transmissible gastroenteritis virus is shown as SEQ ID NO.15 and SEQ ID NO.16,
The pig delta coronavirus specific RPA primer has a nuclear sequence shown as SEQ ID NO.17 and SEQ ID NO.18,
The sequences of the specific RPA primers of the porcine acute diarrhea syndrome coronavirus are shown as SEQ ID NO.19 and SEQ ID NO. 20.
3. A crRNA combination for multiplex detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine delta coronavirus, and porcine acute diarrhea syndrome coronavirus nucleic acid, the crRNA combination comprising:
The specific crRNA sequence of the porcine epidemic diarrhea virus is shown as SEQ ID NO.21,
The specificity crRNA sequence of the transmissible gastroenteritis virus is shown as SEQ ID NO.22,
The specific crRNA sequence of the porcine delta coronavirus is shown as SEQ ID NO.23,
The specific crRNA sequence of the porcine acute diarrhea syndrome coronavirus is shown as SEQ ID NO. 24.
4. The kit for multiplex detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine delta coronavirus and porcine acute diarrhea syndrome coronavirus nucleic acid is characterized by comprising an isothermal amplification system, a detection system and an enzyme-labeled instrument fluorescence detection system;
the isothermal amplification system comprising the primer combination according to claim 2 as isothermal amplification primer,
The assay system comprising the crRNA combination of claim 3,
The enzyme-labeled instrument fluorescence detection system is used for detecting detection reaction products generated by the detection system.
5. The kit of claim 4, wherein the detection system further comprises a Cas12a protein, a ssDNA reporter system comprising ssDNA FQ reporter for fluorescence detection.
6. The kit of claim 5, wherein the ssDNA FQ reporter 'end-labeled fluorescent group for fluorescence detection is 6-FAM and the 3' end-labeled quenching group is BHQ1.
7. A method for multiplex detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine delta coronavirus and porcine acute diarrhea syndrome coronavirus nucleic acid, comprising the steps of:
(1) Extracting nucleic acid of a biological sample to be detected;
(2) RPA isothermal amplification and Cas12a detection: amplifying the biological sample nucleic acid obtained in the step (1) in an RPA isothermal amplification system by using the primer combination in the claim 2, carrying out Cas12a detection on the amplified product by using the crRNA combination in the claim 3, and reacting to obtain a Cas12a detection product;
(3) And (3) carrying out fluorescence signal detection on the detection product of the Cas12 a.
8. The method of claim 7, wherein in step (1), the biological sample to be tested is a plasmid or a clinical anal swab sample.
9. The method of claim 7, wherein in step (2), the amplification conditions are 39 ℃ for 20 minutes; the Cas12a detection conditions were 37.5 ℃ for 15 minutes.
10. The method of claim 7, wherein the fluorescent signal is detected using a blue light glue detector at a wavelength of 470-520nm.
CN202311717282.2A 2023-12-13 2023-12-13 Kit for multiple detection of four porcine diarrhea virus nucleic acids Pending CN117947212A (en)

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