CN113604610B - Kit for synchronously detecting African swine fever virus and porcine pseudorabies virus - Google Patents
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Abstract
The invention relates to a kit for synchronously detecting African swine fever virus and porcine pseudorabies virus. The kit is a multiplex fluorescence quantitative PCR kit and comprises a primer pair for amplifying the African swine fever virus CD2v gene, the African swine fever virus P72 gene and the porcine pseudorabies virus EP0 gene and corresponding fluorescent probes, wherein the nucleotide sequences of the primer pair for amplifying the CD2v gene and the corresponding fluorescent probes, the primer pair for amplifying the P72 gene and the corresponding fluorescent probes and the primer pair for amplifying the EP0 and the corresponding fluorescent probes are respectively shown as SEQ ID NO. 1-9.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a kit for synchronously detecting African swine fever virus and porcine pseudorabies virus.
Background
In recent years, mixed infection with multiple pathogens has become a common phenomenon in pig raising. Swinery disease is often caused by two or more pathogens in combination, and diagnosis and control are delayed, so that high morbidity and high mortality of swinery are caused, and great economic loss is caused. In pig farms with multi-pathogen mixed infection, the clinical symptoms of pig diseases are complex, the case changes are atypical, the disease conditions are serious, the site diagnosis is difficult, and the general prevention and treatment measures are difficult to be effective. The detection of each virus may be performed by conventional diagnostic methods, such as viral isolation or serological detection. The virus separation detection method has the problems of complicated operation method, long diagnosis time, uncertain results caused by pollution of a sampling sample and the like, and the serological diagnosis method has various limitations in practical application due to the continuous occurrence of virus variants.
The current market nucleic acid detection kit mainly adopts a fluorescent quantitative PCR detection kit, and the general method adopted by the kit is Taqman probe method, which is consistent with the method required by the nucleic acid detection standard of various pathogens published in China. The Taqman probe method is to add a fluorescent labeled probe on the basis of conventional PCR to realize the quantitative function, and compared with the conventional PCR, the fluorescent quantitative PCR has a plurality of advantages. The method integrates the advantages of efficient nucleic acid amplification of the PCR technology, high specificity of the probe technology, high sensitivity and high precision quantification of the spectroscopic technology, and directly detects the change of fluorescent signals in the PCR process to obtain a quantification result. Compared with the traditional PCR technology, the method has the advantages of simple and convenient operation, rapidness and high efficiency. Secondly, the amplification is completed in a completely closed system, so that real-time monitoring is realized, and cross contamination is eliminated. Thirdly, gel electrophoresis is not needed, and the gel electrophoresis is only needed to be directly detected in the reaction tube through a special probe. Fourth, the quantitative result is accurate, and the reproducibility is good. Therefore, this technology is paid attention to by many researchers and is used in many fields.
Most commercial PCR kits are single PCR,1 kit can detect 1 pathogen only in 1 test, and 2 or more kits are needed for diagnosing the pathogen of the same pig disease material mixed with 2 or more pathogens and 2 or more PCR tests are carried out, so that time and labor are wasted, and the detection cost is high. Therefore, it is very useful to develop a method that can detect 2 or more pathogens simultaneously and has high sensitivity.
Disclosure of Invention
The invention provides a kit for synchronously detecting African swine fever virus and porcine pseudorabies virus to solve the technical problems, and the kit is used for simply, quickly and efficiently detecting the African swine fever virus and the porcine pseudorabies virus.
The technical scheme for solving the technical problems is as follows: a kit for synchronously detecting African swine fever virus and porcine pseudorabies virus comprises a primer pair for respectively amplifying African swine fever virus CD2v gene, african swine fever virus P72 gene and porcine pseudorabies virus EP0 gene and corresponding fluorescent probes, wherein the primer pair for amplifying the CD2v gene is ASFV-CD2v-F and ASFV-CD2v-R, the corresponding fluorescent probes are ASFV-CD2v-P, the primer pair for amplifying the P72 gene is ASFV-P72-F and ASFV-P72-R, the corresponding fluorescent probes are ASFV-P72-P, the primer pair for amplifying the EP0 is PRV-EP0-F and PRV-EP0-R, the corresponding fluorescent probes are PRV-EP0-P, the primer pair for amplifying the CD2v-F, ASFV-CD2v-R, ASFV-CD2v-P, ASFV-P35-72-P78, the nucleotide sequence shown in SEQ ID No. 3, SEQ ID No. 3-P35-72-P, SEQ ID No. 0, SEQ ID No. 3 and SEQ ID No. 3-P8, SEQ ID No. 3 and SEQ ID No. 0-P3.
The beneficial effects of the invention are as follows: the kit utilizes different fluorescent dyes to mark specific probes of different pathogen detection targets, can detect African swine fever virus and porcine pseudorabies virus simultaneously, improves detection efficiency, designs two detection targets of CD2v and P72 aiming at the African swine fever virus, and improves detection reliability. The kit has the characteristics of high sensitivity and strong specificity, is suitable for scientific research and clinical application, and has good commercial application value.
On the basis of the technical scheme, the invention can be improved as follows.
Further, the 5 'ends of the fluorescent probes ASFV-CD2v-P, ASFV-P72-P and PRV-EP0-P are respectively marked with a fluorescent report group, and the 3' ends are respectively marked with a fluorescent quenching group.
Further, the fluorescent reporter group marked at the 5 'end of the fluorescent probe ASFV-CD2v-P is FAM, the fluorescent quencher group marked at the 3' end is BHQ1, the fluorescent reporter group marked at the 5 'end of the fluorescent probe ASFV-P72-P is ROX, the fluorescent quencher group marked at the 3' end is BHQ2, the fluorescent probe marked at the 5 'end of the fluorescent probe PRV-EP0-P is Cy5, and the fluorescent quencher group marked at the 3' end is BHQ3.
Further, the kit also comprises a positive control, wherein the positive control comprises a recombinant plasmid containing an African swine fever virus P72 gene, a recombinant plasmid containing an African swine fever virus CD2v gene and a recombinant plasmid containing a porcine pseudorabies virus EP0 gene.
Further, the kit also comprises multiple fluorescent quantitative PCR detection reagents and/or sample extraction reagents.
Drawings
FIG. 1 is an amplification plot of multiplex fluorescence quantitative PCR in example 2 of the present invention;
FIG. 2 is a graph showing multiplex fluorescence quantitative PCR amplification curves of CD2v gene at different Tm values in example 2 according to the present invention;
FIG. 3 is a graph showing multiplex fluorescence quantitative PCR amplification curves of the P72 gene at different Tm values in example 2 according to the present invention;
FIG. 4 is a graph showing multiplex fluorescence quantitative PCR amplification of EP0 gene at different Tm values in example 2 according to the present invention;
FIG. 5 is a graph of multiplex fluorescence quantitative PCR amplification of CD2v gene at different primer concentrations;
FIG. 6 is a graph showing multiplex fluorescence quantitative PCR amplification of the P72 gene at different primer concentrations;
FIG. 7 is a graph showing multiplex fluorescence quantitative PCR amplification of the EP0 gene at different primer concentrations;
FIG. 8 is a graph of multiplex fluorescence quantitative PCR amplification of CD2v gene at different fluorescent probe concentrations;
FIG. 9 is a graph showing multiplex fluorescence quantitative PCR amplification of the P72 gene at different fluorescent probe concentrations;
FIG. 10 is a graph of multiplex fluorescence quantitative PCR amplification of EP0 gene at different fluorescent probe concentrations;
FIG. 11 is a graph showing multiplex fluorescence quantitative PCR amplification of CD2v gene at different template concentrations in example 2;
FIG. 12 is a graph showing multiplex fluorescence quantitative PCR amplification of the P72 gene at different template concentrations in example 2;
FIG. 13 is a graph showing multiplex fluorescence quantitative PCR amplification of EP0 gene at different template concentrations in example 2;
FIG. 14 shows the results of specific detection by multiplex fluorescent quantitative PCR in example 2.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings and specific embodiments, the examples being provided for illustration only and not for the purpose of limiting the invention.
Example 1
In order to detect pathogenic bacteria of mixed infection of multiple pathogens in pig raising production, the inventor designs a kit for synchronously detecting African swine fever virus and porcine pseudorabies virus, and the kit is a multiplex fluorescence quantitative PCR kit for detecting the African swine fever virus CD2v gene, the African swine fever virus P72 gene and the porcine pseudorabies virus EP0 gene. The kit comprises a primer pair for amplifying the African swine fever virus CD2v gene, the African swine fever virus P72 gene and the porcine pseudorabies virus EP0 gene and corresponding fluorescent probes, wherein the primer pair for amplifying the CD2v gene ASFV-CD2v-F and ASFV-CD2v-R and corresponding fluorescent probes ASFV-CD2v-P are designed according to the gene sequences of the ASFV CD2v, the ASFV-P72 and the PRV-EP0, and the primer pair for amplifying the P72 gene ASFV-P72-F and ASFV-P72-R and corresponding fluorescent probes ASFV-P72-P and the primer pair for amplifying the EP0 is PRV-EP0-F and PRV-EP0-R and corresponding fluorescent probes PRV-EP0-P.
The specific method for designing the primer pair and the probe is as follows: the corresponding gene sequences disclosed in GenBank are downloaded locally for designing primers and probes, primer5.0 is used for analysis, stable primer dimer and hairpin structure are avoided to form among the primers, NCBI database is used for carrying out specificity and coverage analysis on all primer probe groups, the sequence specificity of the primer probe groups is ensured, and all genotypes of the two currently known viruses can be covered.
The nucleotide sequences of ASFV-CD2v-F, ASFV-CD2v-R, ASFV-CD2v-P, ASFV-P72-F, ASFV-P72-R, ASFV-P72-P, PRV-EP0-F, PRV-EP0-R and PRV-EP0-P are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively
ASFV-CD2v-F：gaagaaatagaaagtccaycrcc(SEQ ID NO:1)
ASFV-CD2v-R：gtaagggamatggkttgrgtg(SEQ ID NO:2)
ASFV-CD2v-P：ttgtgttgagggacgcatgtartaaat(SEQ ID NO:3)
ASFV-P72-F：ctgctcatggtatcaatcttatcga(SEQ ID NO:4)
ASFV-P72-R：gataccacaagatcrgccgt(SEQ ID NO:5)
ASFV-P72-P：ccacgggaggaataccaacccagtg(SEQ ID NO:6)
PRV-EP0-F：tcacctccatcctccaccac(SEQ ID NO:7)
PRV-EP0-R：ctgcccataragccagttgaag(SEQ ID NO:8)
PRV-EP0-P：ttcgtttgtggagacgcccgtgg(SEQ ID NO:9)
Fluorescent probes ASFV-CD2v-P, ASFV-P72-P and PRV-EP0-P are respectively marked with a fluorescent report group at the 5 'end and a fluorescent quenching group at the 3' end. The fluorescent reporter group marked at the 5 'end of the fluorescent probe ASFV-CD2v-P is FAM, the fluorescent quencher group marked at the 3' end is BHQ1, the fluorescent reporter group marked at the 5 'end of the fluorescent probe ASFV-P72-P is ROX, the fluorescent quencher group marked at the 3' end is BHQ2, the fluorescent probe marked at the 5 'end of the fluorescent probe PRV-EP0-P is Cy5, and the fluorescent quencher group marked at the 3' end is BHQ3.
In a preferred embodiment, the kit further comprises a positive control comprising a recombinant plasmid comprising the african swine fever virus P72 gene, a recombinant plasmid comprising the african swine fever virus CD2v gene, and a recombinant plasmid comprising the porcine pseudorabies virus EP0 gene.
In a preferred embodiment, the kit further comprises multiplex fluorescence PCR detection reagents and/or sample extraction reagents. The multiplex fluorescence PCR detection reagent comprises qPCRBuffer, dNTP and Taq DNA polymerase, and the sample extraction reagent is a nucleic acid extraction reagent.
Example 2A method for simultaneous detection of African swine fever Virus and porcine pseudorabies Virus
The detection of african swine fever virus and porcine pseudorabies virus was performed using the kit for simultaneous detection of african swine fever virus and porcine pseudorabies virus in example 1.
According toThe nucleic acid sequences of African Swine Fever Virus (ASFV) and porcine pseudorabies virus (PRV) disclosed in GenBank are respectively synthesized into plasmids containing CD2v, P72 and EP0 partial sequences, the 3 plasmids are subjected to linearization treatment by using restriction enzymes, are recovered after gel electrophoresis, the concentration of the 3 DNA fragments respectively recovered is measured by using a spectrophotometer, and the copy number of the 3 DNA fragments is obtained according to a general copy number conversion formula. Dilution of DNA fragments into 1X10 according to copy number gradient 6 copies/uL、1X10 5 copies/uL、1X10 4 copies/uL、1X10 3 copies/uL、1X10 2 copies/uL、1X10 1 copies/uL、1X10 0 The total of 7 gradients of cobies/uL, and multiplex fluorescence quantitative PCR was performed by using each gradient as a template
The concentrations of the synthesized primers and fluorescent probes were each diluted to a concentration of 50 uM.
The system of multiplex fluorescent quantitative PCR is as follows:
2XqPCRMixBuffer12.5uL, 0.1uL per primer and fluorescent probe (50 uM); template 10uL, the remainder DEPC-H 2 O was supplemented to 25uL.
Amplification conditions for multiplex fluorescent quantitative PCR were as follows:
pre-denaturation at 95℃for 1min;95 ℃ for 10s; and (5) collecting synchronous fluorescence signals at 59 ℃ for 40 seconds, and amplifying for 45 cycles.
FIG. 1 shows the amplification curves of multiplex fluorescence quantitative PCR under the above reaction system and reaction conditions using the kit of example 1 of the present invention, which proves that the kit can well distinguish three detection targets of CD2v, P72 and EP0 by applying the above reaction system and amplification conditions.
The concentration ranges of the primer, the fluorescent probe and the annealing temperature range are confirmed by experiments, wherein, the figures 2-4 are multiple fluorescent quantitative PCR amplification curves of three genes of CD2v, P72 and EP0 under different Tm values respectively, and the annealing temperature can be proved to obtain ideal Ct value at 59 ℃; FIGS. 5-7 are multiplex fluorescence quantitative PCR amplification curves of three genes CD2v, P72 and EP0 at different primer concentrations, respectively, demonstrating that the primer concentrations are all 0.2uM to obtain the ideal Ct value; FIGS. 8-10 are multiplex fluorescence quantitative PCR amplification curves for three genes CD2v, P72 and EP0 at different fluorescent probe concentrations, demonstrating that ideal Ct values can be obtained at fluorescent probe concentrations of 0.2 uM.
Kit sensitivity test:
multiplex fluorescence quantitative PCR was performed using the 7 gradient diluted DNA fragments as templates, respectively, according to the above system and amplification conditions, with 3 multiplex wells per gradient. As a result, as shown in FIGS. 11 to 13, when the viral nucleic acid fragment was 10 copies in the reaction system, an effective amplified signal was obtained.
Kit specificity test:
taking porcine parvovirus genome DNA, porcine circovirus-I genome DNA, porcine rhinomycoplasmas DNA, escherichia coli genome DNA and PK-15 cell genome DNA as reaction templates, taking ASFV-P72, ASFV-CD2v and PRV-EP0 plasmid mixture as positive reference, and DEPC-H 2 O is a blank control, and the detection is carried out by using the kit in the embodiment 1 of the invention, and the result is shown in figure 14, wherein the positive control has 3 amplification curves, and other reaction holes have no amplification curves, so that the kit has good specificity for detecting African swine fever virus and porcine pseudorabies virus.
The kit for synchronously detecting the African swine fever virus and the porcine pseudorabies virus in the embodiment 1 of the invention utilizes different fluorescent dyes to mark the specific probes of different pathogen detection targets, can detect the African swine fever virus and the porcine pseudorabies virus simultaneously, improves the detection efficiency, designs two detection targets of CD2v and P72 aiming at the African swine fever virus, and improves the detection reliability. The kit has the characteristics of high sensitivity and strong specificity, is suitable for scientific research and clinical application, and has good commercial application value.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
SEQUENCE LISTING
<110> institute of martial arts virus of national academy of sciences
<120> a kit for simultaneous detection of African swine fever virus and porcine pseudorabies virus
<130> 20230411
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<170> PatentIn version 3.3
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Claims (5)
1. A kit for synchronously detecting African swine fever virus and porcine pseudorabies virus is characterized in that the kit is a multiplex fluorescent quantitative PCR kit and comprises a primer pair for respectively amplifying African swine fever virus CD2v gene, african swine fever virus P72 gene and porcine pseudorabies virus EP0 gene and a corresponding fluorescent probe, wherein the primer pair for amplifying the CD2v gene is ASFV-CD2v-F and ASFV-CD2v-R, the corresponding fluorescent probe is ASFV-CD2v-P, the primer pair for amplifying the P72 gene is ASFV-P72-F and ASFV-P72-R, the corresponding fluorescent probe is ASFV-P72-P, the primer pair for amplifying EP0 is PRV-EP0-F and PRV-EP0-R, the corresponding fluorescent probe is ASFV-CD2v-F, ASFV-CD2 v-37P, ASFV-P F, ASFV-P72-P8, the nucleotide sequence of which is shown in SEQ ID No. 3, SEQ ID No. 7-P72-P8, SEQ ID No. 3 and SEQ ID No. 3-P8-P3.
2. The kit for synchronously detecting african swine fever virus and porcine pseudorabies virus according to claim 1, wherein the fluorescent probes ASFV-CD2v-P, ASFV-P72-P and PRV-EP0-P are each labeled with a fluorescent reporter group at the 5 'end and a fluorescent quenching group at the 3' end.
3. The kit for synchronously detecting african swine fever virus and porcine pseudorabies virus according to claim 2, wherein the fluorescent reporter group marked at the 5 'end of the fluorescent probe ASFV-CD2v-P is FAM, the fluorescent quencher group marked at the 3' end is BHQ1, the fluorescent reporter group marked at the 5 'end of the fluorescent probe ASFV-P72-P is ROX, the fluorescent quencher group marked at the 3' end is BHQ2, the fluorescent probe marked at the 5 'end of the fluorescent probe PRV-EP0-P is Cy5, and the fluorescent quencher group marked at the 3' end is BHQ3.
4. The kit for synchronously detecting african swine fever virus and porcine pseudorabies virus according to claim 2, further comprising a positive control comprising a recombinant plasmid containing african swine fever virus P72 gene, a recombinant plasmid containing african swine fever virus CD2v gene and a recombinant plasmid containing porcine pseudorabies virus EP0 gene.
5. The kit for simultaneous detection of african swine fever virus and porcine pseudorabies virus according to claim 4, further comprising multiplex fluorescent quantitative PCR detection reagents and/or sample extraction reagents.
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| Identification of the molecular determinants for nuclear import of PRV EP0;Mingsheng Cai等;Biol Chem.;第400卷(第10期);第1385-1394页 * |
| Molecular cloning and characterization of pseudorabies virus EP0 gene;Mei-Li Li等;Indian J Biochem Biophys;第51卷(第2期);第100-114页 * |
| 一步法RT-PCR检测人狂犬病毒初探;孙来晶等;中国法医学杂志;第23卷(第6期);全文 * |
| 猪伪狂犬病病毒新型检测方法研究进展;杨志刚等;黑龙江畜牧兽医(第621期);第34-38页 * |
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