CN116814859A - Primer probe composition, kit and method for identifying African swine fever virus genes I and II - Google Patents
Primer probe composition, kit and method for identifying African swine fever virus genes I and II Download PDFInfo
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Abstract
The invention relates to the technical field of molecular biology, in particular to a primer probe composition, a kit and a method for identifying African swine fever virus genes I and II. The primer probe composition for identifying the I type and II type nucleic acid of the African swine fever virus gene provided by the invention comprises a first group of primer probe compositions which are designed based on a nucleotide sequence shown as SEQ ID NO. 10; the second set of primer probe compositions was designed based on the nucleotide sequence shown as SEQ ID NO. 11; the third group of primer probe compositions are designed based on the nucleotide sequence shown as SEQ ID NO. 12; by adopting the primer composition for identification, the collected sample can be directly detected without extracting nucleic acid, so that the steps are simplified, the detection time is greatly shortened, the detection efficiency is remarkably improved, false negative and false positive of a detection result are avoided, the result is more accurate, and meanwhile, the high sensitivity and the high specificity are ensured.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a primer probe composition, a kit and a method for identifying African swine fever virus genes I and II.
Background
African swine fever virus (African swine fever virus, ASFV) is a member of the genus African swine fever virus of the family African swine fever virus, and is currently the only arbovirus DNA, and is transmitted mainly through contact between diseased pigs or pigs with viruses and contaminated articles thereof and susceptible pigs, and also through soft ticks. The genome of ASFV is double-stranded DNA virus, the genome size is 170-200kb, and 160 kinds of proteins can be encoded. ASFV has strict cell tropism and mainly infects macrophages and peripheral blood mononuclear cells of pigs. African swine fever (African swine fever, ASF) is a highly contagious, widely hemorrhagic swine virulent infectious disease caused by infection of pigs with African Swine Fever Virus (ASFV), and mortality is as high as 100%. All pigs were susceptible as no effective vaccine could be used. At present, biological safety prevention and control measures are mainly adopted for preventing and controlling the disease. It is reported that African swine fever virus not only has a high-pathogenicity gene type II strain, but also has a low-virulence gene type I, which clearly increases the difficulty in prevention and control of the disease. Based on the current epidemic situation, the design of a rapid, accurate and diagnostic kit for identifying the African swine fever virus gene type I and gene type II is of great social significance.
The existing virus nucleic acid detection methods mainly comprise the following steps: fluorescent quantitative PCR method, sequencing method, colloidal gold and chemiluminescent method. The fluorescence quantitative PCR method for detecting ASFV has the advantages of high sensitivity, high speed, high flux, strong universality, complete maturation, relatively short window period, direct target virus detection, and the like, and the degree of virus infection and the effect after the use can be judged through the relative abundance. However, the fluorescent quantitative PCR method detects viral nucleic acids, and samples thereof are mainly nasopharyngeal swab, anal swab, fecal sample, environmental sample swab, etc., and the samples need to be subjected to nucleic acid extraction, and it generally takes 4 to 5 hours from sample extraction to confirmation of detection results, which is inefficient, and cannot evaluate false negative and false positive samples.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a primer probe composition, a kit and a method for the African swine fever virus genes I and II, which have high accuracy, good specificity and high sensitivity.
For this purpose, the invention provides the following technical scheme:
a primer probe composition for identifying african swine fever virus genes type i and type ii comprising:
the first set of primer probe compositions was designed based on the nucleotide sequence shown as SEQ ID NO. 10;
the second set of primer probe compositions was designed based on the nucleotide sequence shown as SEQ ID NO. 11;
the third set of primer probe compositions was designed based on the nucleotide sequence shown as SEQ ID NO. 12.
Optionally, the method comprises the following steps:
the first set of primer probe compositions includes: the nucleotide sequence of the forward primer B646L-F is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer B646L-R is shown as SEQ ID NO. 2; and/or probe B646L-P, the nucleotide sequence of which is shown in SEQ ID NO. 3;
the second set of primer probe compositions includes: the nucleotide sequence of the forward primer MGF_505_1R-F is shown as SEQ ID NO.4, and the nucleotide sequence of the reverse primer MGF_505_1R-R is shown as SEQ ID NO. 5; and/or probe MGF_505_1R-P, the nucleotide sequence of which is shown as SEQ ID NO. 6;
the third set of primer probe compositions includes: the nucleotide sequence of the forward primer pRPS29-F is shown as SEQ ID NO.7, and the nucleotide sequence of the reverse primer pRPS29-R is shown as SEQ ID NO. 8; and/or probe pRPS29-P, the nucleotide sequence of which is shown in SEQ ID NO. 9.
Optionally, the 5 'ends of the probe B646L-P, the probe MGF_505_1R-P and the probe pRPS29-P are marked with fluorescent groups, and the 3' ends are marked with fluorescence quenching groups; the fluorescent groups of the probe B646L-P, the probe MGF_505_1R-P and the probe pRPS29-P are different;
optionally, the fluorophore comprises at least one of FAM, VIC, CY, ROX, HEX, JOE, NED, texas Red and CY 3;
optionally, the fluorescence quenching group includes at least one of TAMRA, BHQ-1, BHQ-2, and BHQ-3.
A detection kit for identifying the type I and type II African swine fever virus genes comprises the type I and type II nucleic acid detection primer probe composition of the African swine fever virus genes.
Optionally, the kit further comprises a sample collection liquid, wherein the sample collection liquid comprises sodium bicarbonate with the concentration range of 6-6.5g/L, sodium carbonate with the concentration range of 12-13g/L, tween-20 with the concentration range of 0.01-0.05v/v, cresol red with the concentration range of 0.01-0.05g/100ml, guanidine hydrochloride with the concentration range of 4-5mol/L and the balance of sterile water without enzyme.
Optionally, the kit also comprises a PCR reaction solution;
optionally, the kit also comprises a positive quality control substance, wherein the positive quality control substance is a nucleic acid molecule respectively containing target sequences with nucleotide sequences shown as SEQ ID No. 10-12;
optionally, the kit also comprises a negative control substance which is enzyme-free sterile water.
Optionally, the method further comprises a PCR reaction system, wherein the PCR reaction system is calculated by 20 mu L:
10 mu L of PCR reaction solution;
primer and probe mixed solution, wherein the concentration range of each primer is 0.1-1 mu M, and the concentration range of each probe is 0.1-1 mu M and 5 mu L;
and 5 mu L of a sample to be tested.
The primer probe composition for identifying the African swine fever virus genes I and II and/or the detection kit for identifying the African swine fever virus genes I and II are used for preparing products for identifying the African swine fever virus genes I and II.
A method for detecting nucleic acid of type i and type ii genes of african swine fever virus for non-disease diagnosis, comprising the steps of:
and taking a sample to be detected as a template, preparing a PCR reaction system by using the African swine fever virus gene I type and II type nucleic acid detection primer probe composition and/or the African swine fever virus gene I type and II type nucleic acid detection kit, and performing fluorescent quantitative PCR detection.
Alternatively, the reaction conditions for the fluorescent quantitative PCR detection are: pre-denaturation at 95 ℃ for 180 seconds; denaturation at 95℃for 5 seconds, annealing at 60℃for 15 seconds, 40 cycles total;
the qualitative method comprises the following steps: the channel corresponding to the fluorescent group of the probe in the first group of primers and the probe is a channel A; the channel corresponding to the fluorescent group of the probe in the second group of primers and the probe is a C channel; the channel corresponding to the fluorescent group of the probe in the third group of primers and the probe is a B channel; the judging method comprises the following steps:
if the Ct value of the sample to be tested in the A channel is less than or equal to 39, the Ct value in the B channel is less than 39, and the Ct value in the C channel is less than or equal to 39, the sample to be tested is positive in African swine fever virus gene type II, and type I is negative;
if the Ct value of the sample to be tested in the A channel is less than or equal to 39, the Ct value in the B channel is less than 39, and the Ct value in the C channel is not greater than 39, the sample to be tested is positive I type and negative II type of the African swine fever virus gene;
if the sample to be tested has no Ct value or Ct value more than 39 in the A channel, the Ct value in the B channel is less than 39, and the Ct value or Ct value in the C channel is more than 39, the sample to be tested is double negative of the African swine fever virus gene type I and type II;
if the sample to be tested has no Ct value or Ct value more than 39 in the A channel, ct value less than 39 in the B channel and Ct value less than or equal to 39 in the C channel, retesting is carried out;
if the sample to be tested has no Ct value in the A channel, the B channel and the C channel, retesting is carried out;
the quantitative detection method comprises the following steps:
establishing a standard curve: mixing the concentrations of the standard substances respectively comprising the nucleotide sequences shown in SEQ ID NO. 10-11, and diluting the obtained mixed standard substances to different concentrations; taking mixed standard substances with different concentrations as templates, performing fluorescence quantitative PCR detection, and drawing a standard curve by taking the concentration of each standard substance in the mixed standard substances as an abscissa and the corresponding Ct value as an ordinate after the detection is finished;
substituting the Ct value of the sample to be measured into the corresponding standard curve to obtain the quantitative result of the sample to be measured.
The technical scheme of the invention has the following advantages:
1. the invention provides a probe composition for identifying African swine fever virus genes I and II, which comprises the following components: the first set of primer probe compositions was designed based on the nucleotide sequence shown as SEQ ID NO. 10; the second set of primer probe compositions was designed based on the nucleotide sequence shown as SEQ ID NO. 11; the third group of primer probe compositions are designed based on the nucleotide sequence shown as SEQ ID NO. 12; the invention discovers that the B646L gene is a relatively conserved gene and is a main target of ASFV detection at present through analyzing genome sequences of the African swine fever virus gene I type and the gene II type, and the MGF-505-1R gene only exists in a gene II type strain and is deleted in the gene I type strain, based on the fact, the invention provides a powerful tool for accurately preventing and controlling ASFV by designing a primer probe composition of the two target genes (the target gene corresponding to the nucleotide sequence shown as SEQ ID NO.10 is B646L, and the target gene corresponding to the nucleotide sequence shown as SEQ ID NO.11 is MGF_505_1R); furthermore, the invention also discovers that the gene sequence of the pig source sample housekeeping gene RPS29 (shown as SEQ No. 12) is commonly existing in the sample, the specificity is high and the conservation is high, the pig source sample housekeeping gene RPS29 is used as an endogenous internal standard design primer and a probe, the internal standard is used for monitoring the acquisition, transportation and extraction processes of the sample to be detected, the false negative and the false positive of the detection result are avoided, and if the pathogen genome is detected to have no fluorescent amplification signal, the DNA of the pig RPS29 gene is detected to have a fluorescent amplification signal; above, adopt above-mentioned scheme to distinguish, can avoid the detection result to appear false negative and false positive, the result is more accurate, sensitivity is high, specificity is high.
2. The invention provides a nucleic acid extraction-free detection primer composition for identifying African swine fever virus genes I and II, which comprises a first group of primer probe compositions, a second group of primer probe compositions and a third group of primer probe compositions; the first set of primer probe compositions includes: the nucleotide sequence of the forward primer B646L-F is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer B646L-R is shown as SEQ ID NO. 2; and/or probe B646L-P, the nucleotide sequence of which is shown in SEQ ID NO. 3; the second set of primer probe compositions includes: the nucleotide sequence of the forward primer MGF_505_1R-F is shown as SEQ ID NO.4, and the nucleotide sequence of the reverse primer MGF_505_1R-R is shown as SEQ ID NO. 5; and/or probe MGF_505_1R-P, the nucleotide sequence of which is shown as SEQ ID NO. 6; the third set of primer probe compositions includes: the nucleotide sequence of the forward primer pRPS29-F is shown as SEQ ID NO.7, and the nucleotide sequence of the reverse primer pRPS29-R is shown as SEQ ID NO. 8; and/or probe pRPS29-P, its nucleotide sequence is shown in SEQ ID NO. 9; by ingenious design of the amplification primers and the probes, the mutual interference between a plurality of primer pairs and corresponding detection probes is avoided, and the accuracy, sensitivity and specificity of detection results are ensured.
3. A method for identifying African swine fever virus gene I type and II type nucleic acid extraction-free detection comprises the following steps: s1, performing virus lysis and nucleic acid purification on an African swine fever virus sample to obtain a nucleic acid extract; s2, preparing a PCR reaction system by using a nucleic acid extracting solution as a template and utilizing the primer composition, the probe composition and/or the extraction-free detection kit for identifying the African swine fever virus genes I and II, and performing fluorescent quantitative PCR detection; the detection principle of the method is as follows: at the beginning of a multiplex fluorescent quantitative PCR reaction using primer compositions and probe compositions and/or kits, the fluorescent quenching groups on the probes significantly reduce the fluorescence emitted by the fluorescent groups by Fluorescence Resonance Energy Transfer (FRET) when the probes remain intact. During PCR amplification, the primer and the probe are simultaneously bound to the template, and the binding position of the probe is positioned between the upstream primer and the downstream primer. When amplification extends to the point where the probe binds, taq enzyme uses 5 'exonuclease activity to cleave the fluorescent group attached to the 5' end of the probe from the probe, thereby causing it to fluoresce. The number of the detected fluorescent groups is proportional to the number of the PCR products, so that the number of the initial DNA templates can be calculated according to the fluorescence intensity in the PCR reaction system. The fluorescence quantitative PCR instrument automatically draws a real-time amplification curve according to the detected fluorescence signal, so that the qualitative detection of the African swine fever virus on the nucleic acid level is realized; the PCR reaction system established based on the primer probe composition realizes the qualitative detection of the African swine fever on the nucleic acid level, and the collected sample can be directly detected without extracting nucleic acid, so that the steps are simplified, the detection time is greatly shortened, the detection efficiency is remarkably improved, false negative and false positive of a detection result are avoided, the result is more accurate, and meanwhile, the high sensitivity and the high specificity are ensured.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a result of sensitivity detection of a plasmid standard whose sample to be tested is B646L in Experimental example 1 of the present invention;
FIG. 2 is a sensitivity test result of a plasmid standard whose sample to be tested is MGF_505_1R in Experimental example 1 of the present invention;
FIG. 3 is a result of detecting sensitivity of a plasmid standard whose sample to be tested is the internal standard gene pRPS29 in Experimental example 1 of the present invention;
FIG. 4 is a standard curve of a plasmid standard whose sample to be tested is B646L in Experimental example 1 of the present invention;
FIG. 5 is a standard curve of a plasmid standard with MGF_505_1R as the sample to be tested in Experimental example 1 of the present invention;
FIG. 6 is a standard curve of a plasmid standard whose sample to be tested is the internal standard gene pRPS29 in experimental example 1 of the present invention;
FIG. 7 shows the results of the test of a clinical specimen positive for the African swine fever virus genotype I in experimental example 2 of the present invention;
FIG. 8 shows the results of the test of a clinical specimen positive for the African swine fever virus gene type II in experimental example 2 of the present invention;
FIG. 9 shows the results of the test of the African swine fever virus-negative clinical samples of experimental example 2 of the present invention.
FIG. 10 shows the result of the specific detection in Experimental example 3 of the present invention.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
The preparation method of the mixed plasmid respectively comprising the B646L, MGF _505_1R and the pig RPS29 genes in the following examples adopts a conventional method, and specifically comprises the following steps:
the B646L, MGF _505_1R and pRPS29 genes are cloned to a pMD19 vector respectively, competent cells are transformed, plasmids are extracted, and the correct plasmids are identified as positive standard substances through sequencing. Measuring OD by ultraviolet spectrophotometry 260 The value was expressed as [ X (g/. Mu.L) DNA/DNA length (bp). Times.660 ]]×6.02×10 23 After converted to molar concentration =y (copies/. Mu.l)), diluted to 10 8 The copies/. Mu.L was stored at 20℃and diluted before use.
Example 1 primer composition and Probe composition
In order to ensure that the African swine fever virus genes I and II can be distinguished at the same time, the invention selects two genes B646L and MGF_505_1R-R in a virus genome as target sequences, designs specific primer probes (such as SEQ ID No. 1-6) for amplification, detects non-fluorescent amplification signals of pig genome DNA or other pathogen genomes, and detects fluorescent amplification signals of African swine fever virus genomes.
In order to eliminate false negative, false positive, pollution and extraction efficiency, the invention selects pig genome DNA (SEQ ID No. 12) as a target sequence, designs specific primer probes (SEQ ID No. 7-9) for amplification, detects a pathogen genome non-fluorescent amplification signal, and detects a pig gene pRPS29 fluorescent amplification signal.
Thus, this example provides a nucleic acid extraction-free detection primer composition and probe composition for identifying African swine fever virus genes type I and type II, comprising:
the first primer probe group comprises a forward primer B646L-F, a probe B646L-P and a reverse primer B646L-R, and the deoxynucleotide sequences of the first primer probe group are respectively shown in SEQ ID No. 1-3; wherein, the 5 'end of the probe B646L-P is marked with a fluorescent group FAM, and the 3' end is marked with a fluorescence quenching group BHQ-1;
the second primer probe group comprises a forward primer MGF_505_1R-F, a probe MGF_505_1R-P and a reverse primer MGF_505_1R-R, and the deoxynucleotide sequences of the second primer probe group are respectively shown as SEQ ID No. 4-6; wherein, the 5 'end of the probe B6R-P is marked with a fluorescent group CY5, and the 3' end is marked with a fluorescence quenching group BHQ-2;
the third primer probe group comprises a forward primer pRPS29-F, a probe pRPS29-P and a reverse primer pRPS29-R, and the deoxynucleotide sequences of the third primer probe group are respectively shown in SEQ ID No. 7-9; wherein, the 5 'end of the probe pRPS29-P is marked with a fluorescent group VIC, and the 3' end is marked with a fluorescent quenching group BHQ-2.
Example 2 kit
This example provides a kit for the hands-free detection of nucleic acids identifying the african swine fever virus genes type i and type ii comprising the primer composition and probe composition of example 1, comprising the following separately packaged reagents:
TABLE 1 composition of reagents
Example 3
This example provides a kit for the hands-free detection of nucleic acids identifying the african swine fever virus genes type i and type ii comprising the primer composition and probe composition of example 1, comprising the following separately packaged reagents:
TABLE 2 composition of reagents
Example 4
This example provides a kit for the hands-free detection of nucleic acids identifying the african swine fever virus genes type i and type ii comprising the primer composition and probe composition of example 1, comprising the following separately packaged reagents:
TABLE 3 composition of reagents
Example 5 authentication method
This example provides a method for the identification of african swine fever virus genes type i and type ii nucleic acid extraction-free assays using example 2, comprising the steps of:
sample type: including whole blood or serum, pharyngeal swabs, and other suspicious specimens.
Sample collection: sample collection is carried out according to the requirements of a manual for collecting microorganism samples;
sample preservation: the specimens should be refrigerated (2 to 8deg.C or frozen (-20deg.C or lower) and detected as soon as possible within one week, specimens that can be detected can be stored at 4deg.C, specimens that cannot be detected can be stored at-20deg.C for a short period, and preserved below-70deg.C (after collection > 60 days) for a long period, whole blood is refrigerated (2 to 8deg.C) before plasma separation, and then stored at-20deg.C or below.
Sample processing: the sample to be tested is treated with the sample treatment liquid in the kit (the sample specimen is immersed in the sample treatment liquid to gently rotate the specimen for 5 times for about 1 minute), and then nucleic acid extraction is performed using a commercially available viral nucleic acid extraction kit (the method is performed with reference to the nucleic acid extraction kit instructions), and the extracted nucleic acid sample is used for the next reaction. The positive control and the negative control are not involved in extraction and are directly used as templates.
Nucleic acid detection: real-time fluorescent quantitative PCR (qPCR) method for detecting African swine fever virus nucleic acid (performed in PCR laboratory).
(1) Preparation of a reaction system: mu.L of sample DNA was added to each well at 15. Mu.L, and a total of 20. Mu.L; the PCR reaction system was formulated as in table 4: for each PCR reaction, except for the detection sample, a DNA-free control (sterile and enzyme-free water is used for replacing DNA), a negative control and a positive control should be added; all samples, including the control, should be countersunk.
TABLE 4 PCR reaction System
| Reaction mixture composition | Amount of each sample to be tested |
| PCR reaction solution | 10μL |
| Primer and probe mixed solution | 5μL |
| Test sample or control | 5μL |
| Total volume of | 20μL |
(2) Sample adding: each reaction tube was filled with 5. Mu.L of nucleic acid or control, and the tube was capped and placed on a fluorescent quantitative PCR detector.
(3) Amplification parameter settings were set as in table 5:
TABLE 5
(4) The instrument detection channel selection, namely selecting FAM, VIC, CY three channels to collect real-time fluorescent signals by using a fluorescent quantitative PCR detector, and collecting specific setting methods at 58 ℃ refer to the instruction book of each instrument.
(5) And setting a result analysis threshold value, namely taking 3-10 or 6-15 cycles of fluorescent signals from a base line when the result analysis is carried out by using a fluorescent quantitative PCR detector, wherein the threshold value setting principle is that a threshold value line just exceeds the highest point of an amplification curve of a normal negative control product, and the threshold value can be adjusted according to the noise condition of the instrument.
Determination of nucleic acid detection results
(1) Quality control of the fluorescent quantitative PCR reaction system is shown in table 6:
TABLE 6
(2) The result was determined as shown in table 7:
TABLE 7
All positive and in-gray (suspicious) samples should be repeatedly tested or confirmed by sequence analysis. Note that: when the sample is only a throat swab or serum or plasma, and the detection result is negative, the negative diagnosis should be carefully made in combination with clinical symptoms.
The quantitative detection method comprises the following steps:
establishing a standard curve: mixing the concentrations of the standard substances respectively comprising the nucleotide sequences shown in SEQ ID NO. 10-11, and diluting the obtained mixed standard substances to different concentrations; taking mixed standard substances with different concentrations as templates, performing fluorescence quantitative PCR detection, and drawing a standard curve by taking the concentration of each standard substance in the mixed standard substances as an abscissa and the corresponding Ct value as an ordinate after the detection is finished;
substituting the Ct value of the sample to be measured into the corresponding standard curve to obtain the quantitative result of the sample to be measured.
Experimental example 1 sensitivity
The sample to be tested was selected from plasmid standards containing B646L or MGF_505_1R or pRPS29 using DEPC water (RNase-Free H 2 O) was diluted 10-fold in a gradient with a concentration of 1X 10 respectively 6 、1×10 5 、1×10 4 、1×10 3 、1×10 2 、1×10 1 、1×10 0 The detection results of the samples/μl were positive as shown in fig. 1-3, and the lowest detection limit of either B646L or mgf_505_1r was 10 samples/μl. Then, a standard curve was drawn as in example 5, and the result was shown as the standard curve correlation coefficient R of FIG. 4-FIG. 6, B646L or MGF_505_1R 2 All greater than 0.99, standard curve correlation coefficient R of pRPS29 2 And the detection effect is stable when the detection effect is larger than 0.98.
Experimental example 2 detection of clinical samples
Sample nucleic acids which are clinically diagnosed as positive and negative of the African swine fever virus genes I and II are respectively detected by the method of the embodiment 5, and the detection results are as shown in figures 7-9 and are consistent with clinical results, so that the method has better accuracy.
Experimental example 3 specificity experiments
The positive nucleic acids confirmed to be Classical Swine Fever Virus (CSFV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine Parvovirus (PPV), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV 2) and porcine circovirus type 3 (PCV 3) are used as templates, and detected by the method of the embodiment 5 of the present invention, as shown in FIG. 10, the system has no amplification except the signal of the internal reference VIC channel, which indicates that the method has good specificity and has no cross reaction with other common infectious disease virus antigens.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. A primer probe composition for identifying african swine fever virus genes type i and type ii, comprising:
the first set of primer probe compositions was designed based on the nucleotide sequence shown as SEQ ID NO. 10;
the second set of primer probe compositions was designed based on the nucleotide sequence shown as SEQ ID NO. 11;
the third set of primer probe compositions was designed based on the nucleotide sequence shown as SEQ ID NO. 12.
2. The probe composition for identifying african swine fever virus genes type i and type ii according to claim 1, comprising:
the first set of primer probe compositions includes: the nucleotide sequence of the forward primer B646L-F is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer B646L-R is shown as SEQ ID NO. 2; and/or probe B646L-P, the nucleotide sequence of which is shown in SEQ ID NO. 3;
the second set of primer probe compositions includes: the nucleotide sequence of the forward primer MGF_505_1R-F is shown as SEQ ID NO.4, and the nucleotide sequence of the reverse primer MGF_505_1R-R is shown as SEQ ID NO. 5; and/or probe MGF_505_1R-P, the nucleotide sequence of which is shown as SEQ ID NO. 6;
the third set of primer probe compositions includes: the nucleotide sequence of the forward primer pRPS29-F is shown as SEQ ID NO.7, and the nucleotide sequence of the reverse primer pRPS29-R is shown as SEQ ID NO. 8; and/or probe pRPS29-P, the nucleotide sequence of which is shown in SEQ ID NO. 9.
3. The probe composition for identifying african swine fever virus genes type i and type ii according to claim 2, wherein the probe B646L-P, the probe mgf_505_1r-P and the probe prs 29-P are labeled with a fluorescent group at the 5 'end and a fluorescence quenching group at the 3' end; the fluorescent groups of the probe B646L-P, the probe MGF_505_1R-P and the probe pRPS29-P are different;
optionally, the fluorophore comprises at least one of FAM, VIC, CY, ROX, HEX, JOE, NED, texas Red and CY 3;
optionally, the fluorescence quenching group includes at least one of TAMRA, BHQ-1, BHQ-2, and BHQ-3.
4. A test kit for identifying african swine fever virus genes type i and type ii, comprising the nucleic acid test primer probe composition for african swine fever virus genes type i and type ii of any one of claims 1 to 3.
5. The kit for identifying type I and type II African swine fever virus genes according to claim 4, further comprising a sample collection liquid, wherein the sample collection liquid comprises sodium bicarbonate in a concentration range of 6-6.5g/L, sodium carbonate in a concentration range of 12-13g/L, tween-20 in a concentration range of 0.01-0.05v/v, cresol red in a concentration range of 0.01-0.05g/100ml, guanidine hydrochloride in a concentration range of 4-5mol/L, and the balance being sterile water without enzymes.
6. The kit for identifying african swine fever virus genes type i and type ii according to claim 4 or 5, further comprising a PCR reaction solution;
optionally, the kit also comprises a positive quality control substance, wherein the positive quality control substance is a nucleic acid molecule respectively containing target sequences with nucleotide sequences shown as SEQ ID No. 10-12;
optionally, the kit also comprises a negative control substance which is enzyme-free sterile water.
7. The kit for identifying the type I and type II African swine fever virus genes according to claim 6, further comprising a PCR reaction system of 20. Mu.L:
10 mu L of PCR reaction solution;
primer and probe mixed solution, wherein the concentration range of each primer is 0.1-1 mu M, and the concentration range of each probe is 0.1-1 mu M and 5 mu L;
and 5 mu L of a sample to be tested.
8. Use of the primer probe composition for identifying african swine fever virus genes type i and type ii according to any one of claims 1 to 3 and/or the detection kit for identifying african swine fever virus genes type i and type ii according to any one of claims 4 to 7 for preparing a product for identifying african swine fever virus genes type i and type ii.
9. A method for detecting nucleic acid of type i and type ii genes of african swine fever virus for non-disease diagnosis, comprising the steps of:
using a sample to be detected as a template, preparing a PCR reaction system by using the African swine fever virus gene I and II nucleic acid detection primer probe composition according to any one of claims 1-3 and/or the African swine fever virus gene I and II nucleic acid detection kit according to any one of claims 4-7, and performing fluorescence quantitative PCR detection.
10. The method for detecting the nucleic acid of the type I and the type II of the African swine fever virus gene according to claim 9, wherein the reaction conditions of the fluorescent quantitative PCR detection are as follows: pre-denaturation at 95 ℃ for 180 seconds; denaturation at 95℃for 5 seconds, annealing at 60℃for 15 seconds, 40 cycles total;
the qualitative method comprises the following steps: the channel corresponding to the fluorescent group of the probe in the first group of primers and the probe is a channel A; the channel corresponding to the fluorescent group of the probe in the second group of primers and the probe is a C channel; the channel corresponding to the fluorescent group of the probe in the third group of primers and the probe is a B channel; the judging method comprises the following steps:
if the Ct value of the sample to be tested in the A channel is less than or equal to 39, the Ct value in the B channel is less than 39, and the Ct value in the C channel is less than or equal to 39, the sample to be tested is positive in African swine fever virus gene type II, and type I is negative;
if the Ct value of the sample to be tested in the A channel is less than or equal to 39, the Ct value in the B channel is less than 39, and the Ct value in the C channel is not greater than 39, the sample to be tested is positive I type and negative II type of the African swine fever virus gene;
if the sample to be tested has no Ct value or Ct value more than 39 in the A channel, the Ct value in the B channel is less than 39, and the Ct value or Ct value in the C channel is more than 39, the sample to be tested is double negative of the African swine fever virus gene type I and type II; i
If the sample to be tested has no Ct value or Ct value more than 39 in the A channel, ct value less than 39 in the B channel and Ct value less than or equal to 39 in the C channel, retesting is carried out;
if the sample to be tested has no Ct value in the A channel, the B channel and the C channel, retesting is carried out;
the quantitative detection method comprises the following steps:
establishing a standard curve: mixing the concentrations of the standard substances respectively comprising the nucleotide sequences shown in SEQ ID NO. 10-11, and diluting the obtained mixed standard substances to different concentrations; taking mixed standard substances with different concentrations as templates, performing fluorescence quantitative PCR detection, and drawing a standard curve by taking the concentration of each standard substance in the mixed standard substances as an abscissa and the corresponding Ct value as an ordinate after the detection is finished;
substituting the Ct value of the sample to be measured into the corresponding standard curve to obtain the quantitative result of the sample to be measured.
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