CN113493864A - Triple fluorescent quantitative PCR detection kit for tick-borne African swine fever virus - Google Patents
Triple fluorescent quantitative PCR detection kit for tick-borne African swine fever virus Download PDFInfo
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Abstract
The invention provides a tick-borne African swine fever virus triple fluorescence quantitative PCR detection kit, which comprises PCR reaction liquid, hot-start Taq DNA polymerase, Buffer matched with probe-method fluorescence quantitative PCR, tick18S rRNA gene specific primers and fluorescent probes, African swine fever virus B646L gene specific primers and fluorescent probes, pig COX1 gene specific primers and fluorescent probes, ribozyme-free water, a reference substance and the like. The kit is a novel, rapid, highly sensitive and specific triple fluorescence quantitative PCR detection kit, and can simultaneously detect whether tick pests, African swine fever viruses and pig genomes exist in samples. The detection limits of the three genomes meet the actual requirements, the kit can be used for diagnosing tick-borne African swine fever viruses in a conventional laboratory, and the practicability and reliability of the kit are verified through detection of clinical samples.
Description
Technical Field
The invention belongs to biotechnology, relates to a tick-borne African swine fever virus triple fluorescence quantitative PCR detection kit and application, and provides a triple fluorescence quantitative PCR detection kit and a detection method capable of simultaneously detecting tick, African swine fever virus and pig genomes, and detection of three genomes can be completed in a short time. The African swine fever virus detection kit can provide scientific basis for host analysis of ticks and source tracing research of the African swine fever virus while detecting the African swine fever virus.
Background
Ticks are widely distributed in various regions of the world, and appropriate host blood suction needs to be found at each growth stage. The most serious hazard of ticks is that the various pathogens carried by their bodies can spread widely during blood sucking and host replacement. Tick pests can transmit various pathogens such as viruses, bacteria, protozoa and the like, and African swine fever virus is proved to be one of the pathogens, and the transmission route of the virus comprises the transmission through biological vectors such as tick pests. Therefore, the rapid and accurate diagnosis of whether the tick is carrying and transmitting the African swine fever virus is an important step for controlling and preventing the infectious disease.
African swine fever is an acute, virulent and highly contagious infectious disease caused by African swine fever virus, and seriously jeopardizes the global pig industry. The disease course is very short, and the death rate is very high. The world animal health Organization (OIE) ranks the animal epidemic disease as an animal epidemic disease which needs to be reported, and China ranks the animal epidemic disease as a type of animal epidemic disease and is also one of the most complex infectious diseases. At present, no effective vaccine or effective treatment measure exists for African swine fever, and the control can only depend on detection and killing.
In recent years, the degree of the hazard of African swine fever has been on the rise, and considerable diffusion and pollution surfaces are formed worldwide. The current prevention and control situation of China is still complex and severe, and the risk of foreign epidemic situation introduction is increased. The rapid and efficient detection of African swine fever virus is an important prerequisite for the prevention and control of disease epidemics. However, tick worms have important significance as storage hosts and biological vectors for African swine fever viruses, and their role in virus transmission is studied. The African swine fever virus detection method is concentrated on a conventional PCR technology and a fluorescent quantitative PCR technology, both of the conventional PCR technology and the fluorescent quantitative PCR technology are recommended by OIE, and the African swine fever virus detection method is high in sensitivity, strong in specificity, good in repeatability and suitable for detection of specific viruses. In order to solve the problems, the invention develops a novel, efficient, highly sensitive and specific triple fluorescence quantitative PCR detection kit, meets the application requirements of actual detection of tick-borne African swine fever viruses, and is expected to become a practical diagnostic tool for controlling and preventing African swine fever epidemic diseases.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a triple fluorescence quantitative PCR detection kit for tick-borne African swine fever viruses, which has good specificity, higher sensitivity and stability.
The kit of the present invention comprises: PCR reaction liquid, hot start Taq DNA polymerase, probe method fluorescent quantitative PCR matched Buffer, three pairs of specific primers and corresponding probes, water without ribozyme and reference substances (including standard substance positive control and negative control). Wherein the PCR reaction solution contains Mg2+Ions, PCR buffer solution, dNTPs mixture and the like. The reference substance is divided into a positive reference substance and a negative reference substance, the positive reference substance is a standard substance template with tick worm, African swine fever virus and pig genome DNA fragments, and the negative reference substance is water without ribozyme.
The 18S rRNA gene specific primers and the fluorescent probe sequences for identifying the tick genome in the triple fluorescent quantitative PCR detection kit are as follows:
the base sequence of the upstream primer is shown as SEQ ID NO.1,
the base sequence of the downstream primer is shown as SEQ ID NO.2,
the base sequence of the fluorescent probe is shown as SEQ ID NO. 3;
the sequences of B646L gene specific primers and fluorescent probes for identifying African swine fever virus are as follows:
the base sequence of the upstream primer is shown as SEQ ID NO.4,
the base sequence of the downstream primer is shown as SEQ ID NO.5,
the base sequence of the fluorescent probe is shown as SEQ ID NO. 6;
the sequences of COX1 gene specific primers and fluorescent probes for identifying the pig genome are as follows:
the base sequence of the upstream primer is shown as SEQ ID NO.7,
the base sequence of the downstream primer is shown as SEQ ID NO.8,
the base sequence of the fluorescent probe is shown as SEQ ID NO. 9.
The specific sequence of the target gene of the positive standard substance is shown in a sequence table SEQ ID No. 10-12. The 18SrRNA gene segment for detecting the tick genome has a nucleotide sequence shown as SEQ ID No. 10; detecting that a B646L gene fragment of the African swine fever virus genome has a nucleotide sequence shown in SEQ ID No. 11; and the COX1 gene segment for detecting the pig genome has the nucleotide sequence shown in SEQ ID No. 12. Three target genes are amplified respectively and are connected into a pMD19-T vector to construct a recombinant plasmid which is used as a standard template.
The kit is stored at-20 ℃ and the times of repeated freeze thawing are reduced as much as possible.
The invention also aims to provide application of the kit in detecting tick pests, African swine fever virus and pig genomes, which is realized by the following steps:
the method comprises the following steps: using a white cloth dragging method to collect tick samples in field or picking tick samples from animals, and putting each tick into a 1.5ml sterile centrifuge tube;
step two: the DNA in the sample was extracted and purified according to the instructions of the genome extraction kit. Collected ticks were first washed with absolute ethanol and triturated, and 200. mu.l of buffer GA (or phosphate buffer) was added to make a cell suspension. Then digested for 3h by protease K water bath. Adding 200 μ l buffer solution GB, fully reversing and mixing, standing at 70 deg.C for 10min, and centrifuging briefly to remove water droplets on the inner wall of the tube cover; adding 200 μ l of anhydrous ethanol, shaking thoroughly, mixing for 15s, and centrifuging briefly to remove water drop on the inner wall of the tube cover; transferring the obtained solution and flocculent precipitate to an adsorption column CB3 in a collecting tube, centrifuging at 12000rpm for 30s, and pouring off waste liquid; adding 500 μ l buffer GD (ensuring that anhydrous ethanol is added before use), centrifuging at 12000rpm for 30s, and pouring off waste liquid; adding 600 μ l of rinsing solution PW (ensuring that anhydrous ethanol is added before use), centrifuging at 12000rpm for 30s, and pouring off waste liquid; repeating the previous step; centrifuging at 12000rpm for 2min, pouring off waste liquid, and standing adsorption column CB3 at room temperature for several minutes; transferring the adsorption column into a clean centrifuge tube, suspending and dripping 50 μ l of elution buffer TE into the center of the adsorption membrane, standing at room temperature for 2-5min, and centrifuging at 12000rpm for 2 min. Finally, eluting each part of extracted tick genomic DNA by using an elution buffer solution, and directly using the tick genomic DNA or freezing and storing the tick genomic DNA at the temperature of minus 20 ℃ for later use;
step three: and taking the genomic DNA of the tick sample extracted in the second step as a template to be detected, adding a plurality of designed pairs of primers and probes into the fluorescent quantitative PCR reaction solution, and performing triple fluorescent quantitative PCR amplification reaction in one reaction tube. The components and volumes of the mixed solution of the amplification reaction system are as follows: 10 μ l Probe Master Mix (containing Mg)2+Ions, dNTPs mixture, hot start Taq DNA polymerase and the like), 2 mul of template DNA, 0.8 mul of upstream and downstream primers for detecting 18S gene respectively, and 0.4 mul of probe; upstream and downstream primers for detecting African swine fever virus B646L gene are respectively 0.8 mul, and probe is 0.4 mul; the upstream and downstream primers for detecting COX1 gene were 0.8. mu.l each, 0.8. mu.l probe (the concentration of the primer and probe used was 10. mu.M), and 1.6. mu.l ribozyme-free water. The amplification reaction program of the reaction mixed solution in the operation of the fluorescent quantitative PCR instrument is as follows: carrying out contamination digestion at 37 ℃ for 2min, carrying out pre-denaturation at 95 ℃ for 30s, carrying out denaturation at 95 ℃ for 10s, carrying out annealing extension at 60 ℃ for 30s, and repeatedly and circularly carrying out the denaturation and annealing extension procedures for 40 times. Carrying out triple fluorescence quantitative PCR detection reaction under the optimal reaction mixture and program;
step four: all fluorescence detection channels of the fluorescence quantitative PCR instrument are selected to be opened, FAM, VIC and ROX are simultaneously tested for multiple reactions, a threshold value is set, and a Ct value is obtained through computer analysis. And (4) judging the result according to the obtained amplification curve and the read Ct value.
As a further explanation of the invention, the result determination method for three genomes is that according to an amplification curve and a generated Ct value, when the Ct value is less than or equal to 32 cycles, the result can be determined to be positive; if Ct value is more than or equal to 38, the result is negative. Samples with Ct values between 32 and 38 were considered suspect and required reconfirmation.
As a further explanation of the invention, a positive control and a negative control should be set for each detection, the positive control uses the constructed standard plasmid as a template, and the negative control template is non-ribozyme water. The water is ribozyme-free water, and sterilized double distilled water can also be used.
As a further explanation of the present invention, the concentration of the primer and the probe used in the reaction mixture was 10. mu.M.
As a further illustration of the invention, the usage of each component of the mixed solution of the triple fluorescence quantitative PCR reaction is a selected proportion after a series of experiments are optimized. The detection of three genomes can be completed by meeting the following range conditions: mg in the reaction mixture2+The concentration of the dNTPs mixture is 1.0-3.0mM, the concentration of the dNTPs mixture is 20-200 mu M, the concentration of the upstream primer and the downstream primer is 0.1-0.5 mu M, the concentration of the probe is 0.1-0.5 mu M, and the concentration of Taq DNA polymerase is 0.5-5U/reaction.
As a further illustration of the invention, each tick test sample of the triple fluorescence quantitative PCR detection kit for the tick-borne African swine fever virus is provided with 3-4 parallel holes. For samples with questions, at least three replicates should be performed.
The kit comprises a specific primer and a fluorescent probe for detecting 18S rRNA of an identification gene of the tick, and the specific primer and the fluorescent probe can ensure that all ticks can be detected and are used as an internal reference of the whole system, so that a false positive result is avoided.
The kit comprises a specific primer and a fluorescent probe of an identification gene B646L for detecting the African swine fever virus, and experimental verification shows that the kit does not generate cross reaction with other important pathogens of pigs, including Classical Swine Fever Virus (CSFV), pseudorabies virus (PRV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus (PCV), and can be used as a method for specifically detecting the African swine fever virus. Meanwhile, the detection effect of the optimized system is slightly better than that of the detection method recommended by OIE.
The kit comprises specific primers and a fluorescent probe for singly detecting COX1 gene of pig species, and does not react on genome DNA of horses, cattle, sheep, dogs and cats. The specific primer and the fluorescent probe of the COX1 gene can be used for analyzing the internal relation between the parasitic stage of ticks and a pig as a host, and can be used for verifying whether other carrying hosts exist in the African swine fever virus or not, so that a proof is provided for future research. The method is used as a traceability system in the process of detecting the tick-borne African swine fever virus.
The invention has the following good effects: (1) the triple fluorescence quantitative PCR detection kit and the detection method have good specificity. Can simultaneously detect the genomic DNA of tick pests, African swine fever virus and pigs, and has no cross reaction with other genes. The specificity of the primers and the probes is verified by common PCR and fluorescent quantitative PCR. The amplification curve and the standard curve generated by using the standard serial diluent as a template prove that the system has better stability. (2) The triple fluorescence quantitative PCR detection kit and the detection method have higher sensitivity. The lower detection limits of the recombinant plasmid standard products of the tick pests, the African swine fever virus and the pigs are 125.1 copy number/microliter, 517.9 copy number/microliter and 543.6 copy number/microliter respectively. The detection sensitivity of the single detection system of the African swine fever virus is slightly superior to that of the method recommended by the world animal health organization, and the actual detection needs are met. (3) The triple fluorescence quantitative PCR detection kit and the detection method have good repeatability. And (3) carrying out a repeatability analysis test on the standard template serial diluent in three independent machines on different dates through a triple fluorescence quantitative PCR amplification reaction. The range of Coefficient of Variation (CV) of the repeatability tests between the groups and between the groups is 0.05% to 2.75%, which indicates that the triple fluorescence quantitative PCR system has good repeatability. (4) The triple fluorescence quantitative PCR detection kit and the detection method have high efficiency and innovation. (5) The triple fluorescence quantitative PCR detection kit and the detection method are applied to 120 tick clinical samples collected in Zhejiang province. Each 5 ticks were set as one test sample, 24 in total, and three replicates were performed. The detection result is as follows: all the detection samples detect the signal of the tick genome, and the tick genome positive rate is 100.0 percent (24/24); only two samples detected the fluorescent signal of COX1 gene, so the positive rate of the pig genome was 8.3% (2/24). However, in this experiment, the African swine fever virus genome could not be detected, and the positive rate was 0% (0/24). The kit and the detection method have higher accuracy and practicability, and can be used for detecting the African swine fever virus in a pig blood clinical sample. In conclusion, the triple fluorescence quantitative PCR detection kit and the detection method provide a simple, convenient, efficient and high-reliability technical means for tick-borne African swine fever virus clinical detection and epidemiological investigation.
Drawings
FIG. 1 shows the electrophoretic verification of PCR products of the standard plasmid.
FIG. 2 PCR validation of three marker gene specific primers.
FIG. 3 shows the product electrophoresis verification of the triple fluorescence quantitative PCR detection system.
FIG. 4 shows a standard curve and an amplification curve of the triple detection system.
FIG. 5 clinical sample detection of triple fluorescent quantitative assay kit.
Detailed Description
The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. It should be understood that the examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
In this embodiment, a triple fluorescence quantitative PCR detection kit for tick-borne african swine fever virus with good specificity, high sensitivity and stability is provided, which includes the following steps:
1. material
LA Taq DNA polymerase reaction System, pMD19-T cloning System, DNA molecular weight Standard marker from Bao bioengineering (Dalian) Ltd, 2 × Master PCR mix reaction System from coastal protein technology Ltd, 2 × AceQ U+The Probe Master Mix reaction system is purchased from Nanjing Novovozan Biotechnology Co., Ltd, the blood/cell/tissue genome DNA extraction kit is purchased from TIANGEN Co., Ltd, the plasmid small-amount extraction kit and the gel recovery kit are purchased from Axygen Co., Ltd, common reagents such as ampicillin and the like are purchased from Shanghai Biotechnology engineering service Co., Ltd, the PCR instrument is a Proflex PCR instrument, and the fluorescent quantitative PCR instrument is a Burle CFX96 fluorescent quantitative PCR instrument.
2. Design and Synthesis of primer probes
Using the above-mentioned target gene sequence as a template, analysis and design of primers and probes were carried out using Primer Premier 5.0 software. The amplification primers were synthesized by Hangzhou Youkang Biotechnology Inc., and the probes were synthesized by general biosystems (Anhui) Inc.
3. Preparation and PCR (polymerase chain reaction) verification of three genome DNA (deoxyribonucleic acid) standard products
According to the genome extraction kit specification, extracting genome DNA by utilizing a tick sample stored in a laboratory and a muscle tissue of a domestic pig, and performing LA Taq DNA polymerase amplification on a PCR instrument by using corresponding standard substance upstream and downstream primers under the reaction conditions of pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 20s for 30 cycles, extension at 72 ℃ for 10min and storage at 4 ℃. After confirming the correct size of the band by 2% agarose electrophoresis of the PCR product, the band of interest was excised and recovered from the gel. The recovered product was inserted into pMD19-T vector using cloning system, transformed into E.coli, cultured in ampicillin-containing medium, and the positive clones were subjected to sequencing verification by sequencing department of KANGBIOLOGY TECHNOLOGY (Hangzhou) GmbH. Sequencing indicated that the insert was correct and the corresponding gel recovery product was adjusted to 10 with sterile deionized water11Copy number/microliter is the standard, and is used as the template diluent and positive control for subsequent experiments. Standard plasmid for ASFV was synthesized by the company. And the three plasmids were named pMD-19T-tick18S, pMD-19T-ASFVB646L and pMD-19T-pigCOX1, respectively. In addition, plasmid copy number was calculated by the following formula: n (ml) [6.02 × 10 ]23(copy number/mol). times.C (g/ml)](ii)/MW (g/mol), where C is the concentration and MW is the calculated average molecular weight (double-stranded DNA) calculated as the number of bases (B). times.660. By insertion in fragments in plasmidsThe number of bases to determine B.
The PCR verification result shows that the designed standard substance completely accords with the expectation through sequencing, and the target gene fragment sequences of three standard substances of tick worm, African swine fever virus and pig genome are respectively shown as SEQ ID No.10,11 and 12 in the sequence table. The results of the PCR and electrophoresis verification are shown in FIG. 1: lane M: DNA molecular weight Standard marker (100bp plus); lanes 1,4, 7: respectively taking the genomic DNA of tick, African swine fever virus and domestic pig as amplification strips of templates; lanes 2,5, 8: amplifying bands by taking the three constructed plasmid standards as templates; lanes 3,6, 9: and (5) a negative control product.
4. PCR validation of tick and pig genome specific primers
Muscle tissue or blood from several species of horses, cattle, sheep, dogs and cats were collected, genomic DNA was extracted according to the DNA extraction kit instructions and its concentration determined, and then adjusted with sterile deionized water to the appropriate concentration for use as a template for subsequent validation. Meanwhile, the genomic DNA of 10 tick samples stored in the parasite laboratory at Zhejiang university was extracted using the above method, and the species of the tick samples included Haematococcus, hard tick and Rhipicephalus. Genomic DNA from each sample was eluted from 50. mu.l buffer into 1.5ml sterile centrifuge tubes and stored at-20 ℃ for subsequent use.
The specificity of each pair of primers was verified using the obtained DNA. After 10. mu.l of 2 × Master PCR mix reaction solution was thawed in ice bath, 1. mu.l of each of the upstream and downstream primers was added to 1. mu.l of the DNA template to be detected, and then 7. mu.l of ribozyme-free water was added to make up to 20. mu.l. The prepared PCR reaction reagent tube is blown by a micropipette, evenly mixed, instantaneously centrifuged for 2s at high speed, placed on a PCR instrument and operated under the following reaction conditions: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, and extension at 72 deg.C for 20s for 30 cycles, extension at 72 deg.C for 10min, and storage at 4 deg.C. Then, 10. mu.l of the amplified product was subjected to 2% agarose electrophoresis to confirm the band size, and sequencing was performed. In the group A, genome DNAs of six species of pigs, horses, cattle, sheep, dogs and cats are used for verifying the specificity of the COX1 gene established in the experiment; in the group B, the specificity of the 18S rRNA primers is verified by using tick DNA stored in a laboratory and genome DNA of pigs, horses, cattle, sheep, dogs and cats; in the group C, several important swine pathogens are utilized to jointly verify the detection system of the African swine fever virus, and the part of experiment is completed with the assistance of the scientific and technical research institute of inspection and quarantine in Zhejiang province; the negative control used water without ribozyme as template.
The results of electrophoretic verification of the PCR products are shown in FIG. 2: group A: the primer of the constructed African swine fever virus detection system verifies that a lane M: DNA molecular weight Standard marker (100bp plus); lane a: the extracted african swine fever virus genome was used as template, lanes b-g: using the genomes of other swine pathogens as templates (including CSFV, PRV, PRRSV, PCV2, PCV3 and PCV4), respectively, and taking a lane h as a negative control; group B: validation of constructed COX1 identified gene primers lane M: DNA molecular weight Standard marker (100bp plus); lane 1: using the porcine genome as template, lanes 2-6: the genomes of the remaining species were used as templates, and lane 7 was a negative control; amplification verification was performed for each species using the same reference gene. Group C: verification of the constructed 18S rRNA gene primers, lane M: DNA molecular weight Standard marker (100bp plus); lane 0: using tick genome as template, lanes 1-6: lane 7 is a negative control using the genomes of the above species as templates.
5. Verification of triple fluorescent quantitative PCR detection kit
Reactions were performed on a Burley CFX96 fluorescent quantitative PCR instrument, USA, using the reaction conditions and reaction procedures provided in the 2 × Probe Master Mix instructions, according to the manufacturer's instructions. And carrying out fluorescent quantitative PCR amplification reaction on the three designed pairs of primers and the corresponding probes in the same reaction tube. The triple fluorescence quantitative reaction mixture included 10. mu.l Probe Master Mix, three pairs of primers and Probe combinations each having a concentration of 10. mu.M, 0.8. mu.l each, 5.6. mu.l of ribozyme-free water, and 2. mu.l of plasmid template. While the templates added in the set five groups are different,
only pMD-19T-tick18S standard plasmid of tick worm was added to the first group; only adding a standard plasmid pMD-19T-ASFVB646L of African swine fever virus in the second group as a template; the third group uses plasmid pMD-19T-pigCOX1 as a template; the fourth group of templates is the mixture of three plasmids in equal proportion; the fifth group was a negative control, using water without nuclease as template. And setting three parallel hole experiments for each group, and finally carrying out 2% agarose nucleic acid electrophoresis verification on reaction products of the fluorescent quantitative PCR to determine the size and the number of target bands.
The reaction result of the fluorescent quantitative PCR is as follows: only a single amplification curve appeared in the first three groups with single template added; three amplification curves are generated in the fourth group, and the curves all satisfy the similar S shape; while the fifth group did not produce fluorescence. The agarose electrophoresis results are shown in FIG. 3: lane M: DNA molecular weight Standard marker (100bp plus); lanes 1-5 are electrophoresis bands of nucleic acids of the corresponding group of fluorescent quantitative PCR reaction products, three lanes per group. The size and the number of the target bands are consistent with the expectation, and the success construction of the triple fluorescence quantitative PCR detection kit is proved.
6. Analysis and optimization of triple fluorescent quantitative PCR detection kit
Firstly, three single detection systems are identified, and a specific primer probe sequence combination is utilized to detect a corresponding standard template to carry out fluorescence quantitative PCR amplification reaction. Each of the standards prepared above was adjusted to a concentration of 1011Copy number/microliter, from 1011To 101A ten-fold gradient dilution was performed in copy number/microliter. Each diluted sample was used as a template for a single assay system, 10 dilutions were then analyzed and optimized with the corresponding primer probes on a berle CFX96 fluorescent quantitative PCR instrument, three replicates per template were run. The fluorescence quantitative PCR reaction system for each genome detection is 20 ul, which comprises 10 ul of Probe Master Mix, 2 ul of plasmid template, 0.8 ul of each primer and Probe with the concentration of 10 uM and 5.6 ul of ribozyme-free water. The amplification reaction program operated by the fluorescent quantitative PCR instrument is as follows: carrying out contamination digestion at 37 ℃ for 2min, carrying out pre-denaturation at 95 ℃ for 30s, carrying out denaturation at 95 ℃ for 10s, carrying out annealing extension at 60 ℃ for 30s, and repeatedly and circularly carrying out the denaturation and annealing extension procedures for 40 times. Drawing a standard curve of a single fluorescence quantitative PCR detection system, and calculating goodness of fit (R)2) And amplification efficiency (E). Meanwhile, the concentration ratio of the primers and the probes in the system is adjusted, so that a single system is optimal.
And adding the obtained primers and probes with the optimal ratio in each single detection system into a reaction tube to perform triple fluorescence quantitative PCR reaction. And simultaneously combining three standard substance plasmids in an equal ratio to prepare a standard substance of a triple system, carrying out ten-fold-ratio gradient dilution on the standard substance, and obtaining an amplification curve of the triple system by using the method. Drawing the standard curves of the three genome systems to be detected according to the amplification curves. Finally, the ratio of each component in the reagent kit is adjusted again, so that the triple fluorescence quantitative PCR detection system is optimized.
7. Standard and amplification curves for triple systems
A, B, C in FIG. 4 are the 3 standard curves generated by the triple detection system, each standard curve having a goodness of fit (R)2) Are all larger than 0.99, and the amplification efficiency (E) is between 90% and 110%. The triple system is proved to have good stability according to expected evaluation standards. In FIG. 4, D is the single fluorescent quantitative detection method of African swine fever virus constructed in this study, and a standard curve is generated by a fluorescent quantitative PCR instrument through experiments. In FIG. 4, E is a standard curve generated by a fluorescence quantitative PCR instrument for an experiment using the sequences and ratios of primers and probes recommended by the world animal health organization. The amplification curve generated by the triple system in step 6 is automatically generated by a fluorescent quantitative PCR instrument, as shown in fig. 4F: wherein, the red curve represents the signal intensity generated by the fluorescent group marking the pig COX1 gene segment; the green curve represents the signal generated by the fluorophore of the tick18S gene fragment; the blue curve represents the fluorophore signal for detection of the African swine fever virus genome.
And calculating the detection limit of each detection system through each standard curve, and comparing and evaluating the sensitivity of the triple fluorescence quantitative PCR detection system with that of a single detection system. The results are shown in the table below, and the sensitivity of detecting the African swine fever virus in the triple detection system is approximately equal to the sensitivity of the method recommended by the world animal health organization, so that the method meets the actual requirements. The kit has relatively high sensitivity for genome detection of ticks and pigs, and can be used as a triple fluorescent quantitative PCR detection kit. In addition, for reproducibility analysis, serial dilutions of standard template were tested for reproducibility analysis in three separate machines on different dates by triple fluorescent quantitative PCR amplification reactions. The results show that: the range of Coefficient of Variation (CV) of the repeatability tests between the groups and between the groups is 0.05% to 2.75%, which indicates that the triple fluorescence quantitative PCR system has good repeatability.
8. Application of triple fluorescence quantitative detection kit
The method is the same as the step 6, and finally the mixed solution of the amplification reaction system is determined to be composed of the following components through a series of optimization experiments of the mixed solution in the system: 10 μ l Probe Master Mix (containing Mg)2+Ions, dNTPs mixture, hot start Taq DNA polymerase and the like), 2 mul of template DNA, 0.8 mul of upstream and downstream primers for detecting 18S gene respectively, and 0.4 mul of probe; upstream and downstream primers for detecting African swine fever virus B646L gene are respectively 0.8 mul, and probe is 0.4 mul; the upstream and downstream primers for detecting COX1 gene were 0.8. mu.l each, 0.8. mu.l probe, and 1.6. mu.l ribozyme-free water. During the period from 8 to 11 months in 2020, 120 ticks were collected in Hangzhou and Ningbo cities of Zhejiang province. Every five sterile centrifuge tubes with the volume of 1.5ml are collected, and 24 parts of tick genome is extracted after being cleaned according to the genome extraction kit specification. And carrying out fluorescent quantitative PCR reaction according to the consumption of the reaction mixed solution obtained by the optimized experiment.
See figure 5 for results: the same significance is obtained for the amplification curve of FIG. 4 automatically generated by the above-mentioned instrument. The green curve represents the amplification curve of the target fragment of the 18S rRNA gene of the tick, and the Ct values are all about 20. All the detected samples have tick genomes, the positive rate is 100.0 percent (24/24), and the successful extraction of the sample genome DNA is also proved. The red curve represents the detection result of the pig COX1 gene fragment, the Ct value generated by two groups of detection samples is about 30, the two groups of detection samples are considered as positive samples, and the other groups of detection samples are negative. The genomic DNA of the pig exists in only two samples of 24 tick samples, and the positive rate is detected to be 8.3% (2/24); the blue curve represents the fluorescence signal for detecting the African swine fever virus genome, and the African swine fever virus genome cannot be detected, and the positive rate is 0% (0/24).
The invention is described by combining the best embodiment, and the triple fluorescence quantitative detection kit can also be applied to detecting African swine fever virus in clinical pig blood disease materials. However, after reading the above description of the present invention, one skilled in the art can make various changes and modifications to the present invention, and such equivalents fall within the scope of the appended claims of the present application.
Sequence listing
<110> Zhejiang university
<120> tick-borne African swine fever virus triple fluorescence quantitative PCR detection kit
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<170> SIPOSequenceListing 1.0
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gcctgagtta ctcaaatgac tcgg 24
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<211> 24
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<213> tick (all attributes)
<400> 2
gctctcaatc tgtcaatcct ccca 24
<210> 3
<211> 24
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<213> tick (all attributes)
<400> 3
ctccactcct ggtggtgccc ttcc 24
<210> 4
<211> 25
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<213> African swine fever virus (African swine fever virus)
<400> 4
ctgctcatgg tatcaatctt atcga 25
<210> 5
<211> 22
<212> DNA
<213> African swine fever virus (African swine fever virus)
<400> 5
ccgataccac aagatcrgcc gt 22
<210> 6
<211> 23
<212> DNA
<213> African swine fever virus (African swine fever virus)
<400> 6
ccacgggagg aataccaacc cag 23
<210> 7
<211> 26
<212> DNA
<213> pig (Sus scrofa)
<400> 7
cccgagcata ctttacatct gctaca 26
<210> 8
<211> 30
<212> DNA
<213> pig (Sus scrofa)
<400> 8
atgtctaggg aggaattagc tagtacaatg 30
<210> 9
<211> 25
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<213> pig (Sus scrofa)
<400> 9
ccgccgtgca gggtagctaa tcaac 25
<210> 10
<211> 200
<212> DNA
<213> tick (all attributes)
<400> 10
gcctgagtta ctcaaatgac tcggcgggca gcttccggga aaccaaagta tttgggttcc 60
gggggaagta tggttgcaaa gctgaaactt aaaggaattg acggaagggc accaccagga 120
gtggagcctg cggcttaatt tgactcaaca cgggaaaact tacccggccc ggacactggg 180
<210> 11
<211> 252
<212> DNA
<213> African swine fever virus (African swine fever virus)
<400> 11
ctgctcatgg tatcaatctt atcgataagt ttccatcaaa gttctgcagc tcttacatac 60
ccttccacta cggaggcaat gcaattaaaa cccccgatga tccgggtgcg atgatgatta 120
cctttgcttt gaagccacgg gaggaatacc aacccagtgg tcatattaac gtatccagag 180
caagagaatt ttatattagt tgggacacgg attacgtggg gtctatcact acggctgatc 240
<210> 12
<211> 193
<212> DNA
<213> pig (Sus scrofa)
<400> 12
cccgagcata ctttacatct gctacaataa tcattgctat tcccactgga gtaaaagtat 60
ttagttgatt agctaccctg cacggcggca atattaaatg atcacccgca atactatgag 120
ctctgggctt catcttccta ttcaccgtag gaggtctaac gggcattgta ctagctaatt 180
Claims (6)
1. The triple fluorescence quantitative PCR detection kit for tick-borne African swine fever virus is characterized by comprising PCR reaction liquid, hot-start Taq DNA polymerase, Buffer matched with probe-method fluorescence quantitative PCR, tick18S rRNA gene specific primers and fluorescent probes, African swine fever virus B646L gene specific primers and fluorescent probes, pig COX1 gene specific primers and fluorescent probes, ribozyme-free water and a reference substance, wherein the PCR reaction liquid contains Mg2+Ions, PCR buffer solution and dNTPs mixture;
wherein the sequences of the 18S rRNA gene specific primers and the fluorescent probe are as follows:
the base sequence of the upstream primer is shown as SEQ ID NO. 1;
the base sequence of the downstream primer is shown as SEQ ID NO. 2;
the base sequence of the fluorescent probe is shown as SEQ ID NO. 3;
wherein the sequences of the B646L gene specific primers and the fluorescent probe are as follows:
the base sequence of the upstream primer is shown as SEQ ID NO. 4;
the base sequence of the downstream primer is shown as SEQ ID NO. 5;
the base sequence of the fluorescent probe is shown as SEQ ID NO. 6;
the sequences of the COX1 gene specific primer and the fluorescent probe are as follows:
the base sequence of the upstream primer is shown as SEQ ID NO. 7;
the base sequence of the downstream primer is shown as SEQ ID NO. 8;
the base sequence of the fluorescent probe is shown as SEQ ID NO. 9.
2. The kit according to claim 1, wherein the control is divided into a positive control and a negative control, the positive control is a standard template with tick, African swine fever virus and pig genome DNA fragments, and the negative control is water without ribozyme.
3. The kit of claim 1, wherein the kit is stored at-20 ℃ to reduce repeated freezing and thawing.
4. Use of the kit according to claim 1 for the detection of ticks, african swine fever virus and swine genomes.
5. The use of claim 4, wherein the positive control plasmid used for detection is a recombinant standard plasmid constructed by amplifying three target genes respectively, including the 18S rRNA gene, the B646L gene and the COX1 gene, into a pMD19-T vector to construct a recombinant plasmid used as a standard template. The negative control in the assay was the use of water without ribozyme as template.
6. The use according to claim 4, characterized by the fact that it is achieved by the following steps:
the method comprises the following steps: collecting ticks in field or picking ticks from animals;
step two: cleaning and mashing the collected ticks, and extracting and purifying DNA in a sample by using a genome extraction kit;
step three: taking the DNA in the second step as a template, adding designed primers and probes into the fluorescent quantitative PCR reaction solution, performing triple fluorescent quantitative PCR detection reaction by using chemically modified hot start Taq DNA polymerase in cooperation with the optimal Buffer optimized for fluorescent quantitative PCR under the optimal reaction mixture and program;
step four: all fluorescence detection channels of the fluorescence quantitative PCR instrument are selected to be open, FAM, VIC and ROX are simultaneously tested for multiple reactions, a threshold value is set, and a Ct value is obtained through computer analysis.
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