CN114350850A - Multiplex detection kit for porcine fulminating infectious disease pathogens and application thereof - Google Patents
Multiplex detection kit for porcine fulminating infectious disease pathogens and application thereof Download PDFInfo
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Abstract
The invention discloses a multiple detection kit for pathogens of porcine fulminating infectious diseases and application thereof, wherein the kit comprises specific primers and fluorescent probes for amplifying 4 gene loci of African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus. Compared with the prior art, the invention has the following advantages: (1) the multiplex fluorescent quantitative PCR amplification technology is combined with the freeze drying technology, so that the simultaneous detection of multiple indexes of a single sample is realized, the experimental operation steps are simplified, the detection flux is improved, a new method is provided for the accurate detection of multiple indexes of the porcine fulminating infectious disease pathogen, and the urgent requirements of the rapid screening and the accurate detection of multiple indexes are met; (2) the detection areas are completely isolated and sealed in the detection process, and after the detection is finished, the sealing reagent is solidified, so that the outward diffusion of the amplification product in the whole detection process and after the detection is finished is effectively prevented, and the environmental pollution is prevented.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to detection of 4 porcine infectious disease pathogens, in particular to a porcine fulminating infectious disease pathogen multi-connected detection kit and application thereof.
Background
The swine acute and virulent infectious diseases have strong infectivity and morbidity and extremely high mortality rate. Such as severe epidemic diseases including African swine fever, foot and mouth disease, erysipelas, and lung plague.
The swine virulent infectious diseases pose serious threats to the swine breeding industry, for example, the virulent infectious diseases such as African swine fever, porcine reproductive and respiratory syndrome, classical swine fever, pseudorabies and the like pose great threats to the swine breeding industry, and also cause serious economic losses to the swine breeding industry. The porcine fulminant infectious disease has high infectivity, morbidity and lethality, is similar to the clinical symptoms of other non-fulminant infectious diseases, is not easy to distinguish the clinical symptoms, and is often mixed with other pathogens to infect, which brings great trouble to clinical diagnosis. At present, the laboratory diagnosis method mainly comprises pathogen direct smear microscopy observation, cell culture inspection, serum enzyme-linked immunoassay detection and nucleic acid detection technologies, wherein the microscopy and culture methods have the best specificity but poorer sensitivity, have high requirements on the collection, storage, transportation and experimental conditions of a sample, have long detection period, are extremely difficult to culture and cannot control the quality of part of pathogens, and have low detection rate on mixed infection; the immunological detection sensitivity is low, and the pathogen infection has a window period, so the diagnostic value of the porcine fulminant infectious disease with acute morbidity and high mortality is not great. Nucleic acid detection technology has been widely applied to the detection of pig disease related pathogens due to its high sensitivity and specificity, but the application of nucleic acid detection technology to pathogenic microorganisms is limited due to the problems of low detection flux and the need of cold chain transportation and low temperature storage of detection reagents. Therefore, the above methods have certain limitations in practical application.
Disclosure of Invention
The technical problem to be solved is as follows: in order to overcome the defects of the prior art, the method realizes the simultaneous detection of multiple indexes of a single sample, simplifies the experimental operation steps, improves the detection flux, meets the urgent requirements of the rapid screening and the accurate detection of the multiple indexes, and really realizes the integrated detection process of sample input and result output; in view of the above, the invention provides a multiple detection kit for swine fulminating infectious disease pathogens and application thereof.
The technical scheme is as follows: the kit comprises specific primers and fluorescent probes for amplifying 4 gene loci of African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus; wherein, the 4 gene loci are: VP72 gene of African swine fever virus, N gene of porcine reproductive and respiratory syndrome virus, 5' UTR gene of classical swine fever virus, gD gene of porcine pseudorabies virus; and a specific primer and a fluorescent probe of the pig reference gene ACTB.
Preferably, the specific primers and the fluorescent probe have the following sequences: ASFV, SEQ ID NO. 1-3; PRRSV, SEQ ID NO. 4-6; CSFV, SEQ ID NO. 7-9; PRV, SEQ ID NO. 10-12; sus, SEQ ID NO. 13-15.
TABLE 1 sequence Listing of specific primers and fluorescent probes
Preferably, the kit comprises a PCR reaction solution I and a PCR reaction solution II; wherein, the PCR reaction solution I comprises specific primers shown in SEQ ID NO.1-2, SEQ ID NO.4-5 and SEQ ID NO.13-14 and fluorescent probes shown in SEQ ID NO.3, SEQ ID NO.6 and SEQ ID NO. 15; the PCR reaction solution II comprises specific primers shown in SEQ ID NO.7-8 and SEQ ID NO.10-11 and fluorescent probes shown in SEQ ID NO.9 and SEQ ID NO. 12.
Preferably, the final concentration of the specific primer and the fluorescent probe in the amplification system is: 0.2. mu.M for SEQ ID NO.1, 0.2. mu.M for SEQ ID NO.2, 0.2. mu.M for SEQ ID NO.3, 0.2. mu.M for SEQ ID NO.4, 0.2. mu.M for SEQ ID NO.6, 0.12. mu.M for SEQ ID NO.7, 0.12. mu.M for SEQ ID NO.8, 0.12. mu.M for SEQ ID NO.9, 0.12. mu.M for SEQ ID NO.10, 0.12. mu.M for SEQ ID NO.11, 0.12. mu.M for SEQ ID NO.12, 0.12. mu.M for SEQ ID NO.13, 0.12. mu.M for SEQ ID NO.14 and 0.12. mu.M for SEQ ID NO. 15.
Preferably, the kit comprises: dNTP 0.2mM, Taq enzyme 2.5U, RTase 1.25U, RNase Inhibitor 1.25U, MgCl2Was 4 mM.
Preferably, the 5 ' end of SEQ ID NO.3 is labeled with fluorochrome 6-FAM, the 5 ' end of SEQ ID NO.6 is labeled with fluorochrome HEX, the 5 ' end of SEQ ID NO.15 is labeled with fluorochrome CY5, the 5 ' end of SEQ ID NO.9 is labeled with fluorochrome 6-FAM, and the 5 ' end of SEQ ID NO.12 is labeled with fluorochrome HEX.
The kit is applied to the simultaneous detection of African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus aiming at a single sample.
Preferably, the detection sensitivity of the kit to African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus reaches 500 copies/reaction.
Preferably, the specific method for simultaneously detecting the African swine fever virus, the porcine reproductive and respiratory syndrome virus, the classical swine fever virus and the porcine pseudorabies virus aiming at a single sample comprises the following steps: adding a sample to be detected into a sample hole of a nucleic acid extraction shell, obtaining purified nucleic acid in the sample after the sample passes through the whole nucleic acid extraction process, transferring the obtained purified nucleic acid into a nucleic acid detection shell filled with a detection freeze-drying reagent, re-dissolving the freeze-drying reagent into a complete nucleic acid amplification system after the freeze-drying reagent is contacted with the liquid purified nucleic acid, finally, carrying out sample detection by adding a sealing reagent, and automatically judging a detection result by a detection instrument after the whole nucleic acid amplification process is finished.
Has the advantages that: (1) the kit combines a multiple fluorescence quantitative PCR amplification technology with a freeze drying technology, realizes simultaneous detection of multiple indexes of a single sample, simplifies experimental operation steps, improves detection flux, provides a new method for accurate detection of multiple indexes of swine fulminating infectious disease pathogens, and meets urgent requirements of rapid screening and accurate detection of multiple indexes; (2) the application method of the kit is simple, and the detection can be carried out only by placing the extraction reagent and the detection reagent which is lyophilized in advance in corresponding areas and adding the sample; (3) the detection areas of the kit are completely isolated and sealed from each other in the detection process, and after the detection is finished, the sealing reagent is solidified, so that the outward diffusion of the amplification product in the whole detection process and after the detection is finished is effectively prevented, and the environmental pollution is prevented; (4) the kit can realize the detection of 4 common porcine fulminating infectious disease pathogens in one device within about 2 hours.
Drawings
FIG. 1 is a schematic structural diagram of a detection device applied to the kit of the present invention.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
The pig fulminating infectious disease pathogen multi-connected detection kit comprises: the kit comprises specific primers and fluorescent probes for amplifying 4 gene loci of African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus; wherein, the 4 gene loci are: VP72 gene of African swine fever virus, N gene of porcine reproductive and respiratory syndrome virus, 5' UTR gene of classical swine fever virus, gD gene of porcine pseudorabies virus; and a specific primer and a fluorescent probe of the pig reference gene ACTB.
The specific primers and the fluorescent probe have the following sequences: ASFV, SEQ ID NO. 1-3; PRRSV, SEQ ID NO. 4-6; CSFV, SEQ ID NO. 7-9; PRV, SEQ ID NO. 10-12; sus, SEQ ID NO. 13-15.
TABLE 1 sequence Listing of specific primers and fluorescent probes
The kit comprises a PCR reaction solution I and a PCR reaction solution II; wherein, the PCR reaction solution I comprises specific primers shown in SEQ ID NO.1-2, SEQ ID NO.4-5 and SEQ ID NO.13-14 and fluorescent probes shown in SEQ ID NO.3, SEQ ID NO.6 and SEQ ID NO. 15; the PCR reaction solution II comprises specific primers shown in SEQ ID NO.7-8 and SEQ ID NO.10-11 and fluorescent probes shown in SEQ ID NO.9 and SEQ ID NO. 12.
The final concentration of the specific primer and the fluorescent probe in an amplification system is as follows: 0.2. mu.M for SEQ ID NO.1, 0.2. mu.M for SEQ ID NO.2, 0.2. mu.M for SEQ ID NO.3, 0.2. mu.M for SEQ ID NO.4, 0.2. mu.M for SEQ ID NO.6, 0.12. mu.M for SEQ ID NO.7, 0.12. mu.M for SEQ ID NO.8, 0.12. mu.M for SEQ ID NO.9, 0.12. mu.M for SEQ ID NO.10, 0.12. mu.M for SEQ ID NO.11, 0.12. mu.M for SEQ ID NO.12, 0.12. mu.M for SEQ ID NO.13, 0.12. mu.M for SEQ ID NO.14 and 0.12. mu.M for SEQ ID NO. 15.
The kit comprises: dNTP 0.2mM, Taq enzyme 2.5U, RTase 1.25U, RNase Inhibitor 1.25U, MgCl2Was 4 mM.
The 5 ' end of SEQ ID NO.3 is marked by fluorescent dye 6-FAM, the 5 ' end of SEQ ID NO.6 is marked by fluorescent dye HEX, the 5 ' end of SEQ ID NO.15 is marked by fluorescent dye CY5, the 5 ' end of SEQ ID NO.9 is marked by fluorescent dye 6-FAM, and the 5 ' end of SEQ ID NO.12 is marked by fluorescent dye HEX.
Example 2 specificity test
(1) The detection device comprises 2 parts, one part is an extraction reagent, the extraction reagent with corresponding volume is canned according to requirements, and plastic package is carried out; the other part is a detection reagent which adopts a freeze-dried ball form and mainly comprises a primer and Mg2+dNTPs, polymerase, reverse transcriptase and the like, and the components are prepared according to a certain proportion and then are freeze-dried. The freeze-drying procedure includes pre-freezing at-45-50 deg.C, primary drying, and secondary drying. After the part is taken out of the compartment, the part is combined with the reagent extraction part and is stored in a vacuum pumping way.
(2) Taking out the detection device, adding a plasmid containing an African swine fever virus VP72 gene sequence, a pre-family porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain), a zilu swine fever live vaccine, a pre-family pseudorabies live vaccine (HB-98 strain), a Havy porcine transmissible gastroenteritis/porcine epidemic diarrhea/porcine rotavirus (G5 type) triple live vaccine, a pre-family porcine parvo inactivated vaccine, a pre-family porcine encephalitis B live vaccine, a midge porcine circovirus type 2 inactivated vaccine, a pre-family porcine mycoplasma pneumonia inactivated vaccine and a pre-family porcine influenza virus H1N1 subtype inactivated vaccine into a sample hole of the detection device, placing the sample hole into an instrument, and then performing nucleic acid extraction and detection, wherein the detection result is shown in Table 2.
TABLE 2 results of specific detection
In table 2, + indicates a positive detection result; negative results.
The results in Table 2 show that the kit can specifically detect the African swine fever virus, the porcine reproductive and respiratory syndrome virus, the classical swine fever virus and the porcine pseudorabies virus without cross reaction with other pathogenic microorganisms, such as the porcine transmissible gastroenteritis virus, the porcine epidemic diarrhea virus, the porcine rotavirus, the porcine parvovirus, the porcine Japanese encephalitis virus, the porcine circovirus type 2, the porcine mycoplasma pneumoniae and the porcine influenza virus.
Example 3 sensitivity detection assay
The plasmid DNA containing respective targets of African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus is diluted by sterilized purified water to a certain copy number ratio diluent, and then is respectively added to a detection hole of a detection device for detection.
The detection result shows that the detection sensitivity of the detection method to the African swine fever virus, the porcine reproductive and respiratory syndrome virus, the classical swine fever virus and the porcine pseudorabies virus is 500 copies/reaction.
Sequence listing
<110> Ci Zhi Da science Co., Ltd
Multiplex detection kit for <120> swine fulminating infectious disease pathogens and application thereof
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Claims (9)
1. The kit is characterized by comprising specific primers and fluorescent probes for amplifying 4 gene loci of African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus; wherein, the 4 gene loci are: VP72 gene of African swine fever virus, N gene of porcine reproductive and respiratory syndrome virus, 5' UTR gene of classical swine fever virus, gD gene of porcine pseudorabies virus; and a specific primer and a fluorescent probe of the pig reference gene ACTB.
2. The multiple detection kit for the swine fulminating infectious disease pathogen according to claim 1, wherein the specific primers and the fluorescent probe have the following sequences: ASFV, SEQ ID NO. 1-3; PRRSV, SEQ ID NO. 4-6; CSFV, SEQ ID NO. 7-9; PRV, SEQ ID NO. 10-12; sus, SEQ ID NO. 13-15.
3. The multiple detection kit for the swine fulminating infectious disease pathogen according to claim 1, wherein the kit comprises a PCR reaction solution I and a PCR reaction solution II; wherein, the PCR reaction solution I comprises specific primers shown in SEQ ID NO.1-2, SEQ ID NO.4-5 and SEQ ID NO.13-14 and fluorescent probes shown in SEQ ID NO.3, SEQ ID NO.6 and SEQ ID NO. 15; the PCR reaction solution II comprises specific primers shown in SEQ ID NO.7-8 and SEQ ID NO.10-11 and fluorescent probes shown in SEQ ID NO.9 and SEQ ID NO. 12.
4. The multiple detection kit for the swine fulminating infectious disease pathogen according to claim 1, wherein the final concentrations of the specific primers and the fluorescent probe in the amplification system are as follows: 0.2. mu.M for SEQ ID NO.1, 0.2. mu.M for SEQ ID NO.2, 0.2. mu.M for SEQ ID NO.3, 0.2. mu.M for SEQ ID NO.4, 0.2. mu.M for SEQ ID NO.6, 0.12. mu.M for SEQ ID NO.7, 0.12. mu.M for SEQ ID NO.8, 0.12. mu.M for SEQ ID NO.9, 0.12. mu.M for SEQ ID NO.10, 0.12. mu.M for SEQ ID NO.11, 0.12. mu.M for SEQ ID NO.12, 0.12. mu.M for SEQ ID NO.13, 0.12. mu.M for SEQ ID NO.14 and 0.12. mu.M for SEQ ID NO. 15.
5. The multiple detection kit for the porcine fulminating infectious disease pathogen according to claim 1, wherein the kit comprises: dNTP 0.2mM, Taq enzyme 2.5U, RTase 50U, RNase Inhibitor 10U, MgCl2Was 4 mM.
6. The multiple detection kit for the pathogens of swine fulminating infectious diseases, according to claim 1, wherein the 5 ' end of SEQ ID No.3 is labeled with a fluorescent dye 6-FAM, the 5 ' end of SEQ ID No.6 is labeled with a fluorescent dye HEX, the 5 ' end of SEQ ID No.15 is labeled with a fluorescent dye CY5, the 5 ' end of SEQ ID No.9 is labeled with a fluorescent dye 6-FAM, and the 5 ' end of SEQ ID No.12 is labeled with a fluorescent dye HEX.
7. Use of a kit according to any one of claims 1 to 6 for the simultaneous detection of African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus in a single sample.
8. The use according to claim 7, wherein the kit has a detection sensitivity of 500 copies/reaction for African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus.
9. The use according to claim 7, wherein the specific method for simultaneously detecting African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus in a single sample is as follows: adding a sample to be detected into a sample hole of a nucleic acid extraction shell, obtaining purified nucleic acid in the sample after the sample passes through the whole nucleic acid extraction process, transferring the obtained purified nucleic acid into a nucleic acid detection shell filled with a detection freeze-drying reagent, re-dissolving the freeze-drying reagent into a complete nucleic acid amplification system after the freeze-drying reagent is contacted with the liquid purified nucleic acid, finally, carrying out sample detection by adding a sealing reagent, and automatically judging a detection result by a detection instrument after the whole nucleic acid amplification process is finished.
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| CN116355744A (en) * | 2023-05-29 | 2023-06-30 | 哈尔滨瀚邦医疗科技有限公司 | Detection kit for synchronously detecting various porcine infectious disease viruses and application |
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