CN116891910A - African swine fever virus fluorescence PCR detection kit with anti-interference capability - Google Patents
African swine fever virus fluorescence PCR detection kit with anti-interference capability Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a fluorescence PCR detection kit with anti-interference capability for African swine fever virus, and relates to the technical field of animal quarantine. The detection kit provides a simple method for rapidly detecting African swine fever virus and obtaining an accurate detection result; the kit has the advantages of wide applicability, strong specificity, high sensitivity and the like.
Description
Technical Field
The invention relates to the technical field of animal quarantine, in particular to an African swine fever virus fluorescence PCR detection kit with anti-interference capability.
Background
The description of the background art to which the present invention pertains is merely for illustrating and facilitating understanding of the summary of the invention, and should not be construed as an explicit recognition or presumption by the applicant that the applicant regards the prior art as the filing date of the first filed application.
African Swine Fever (ASF) is a virulent infectious disease caused by infection of domestic pigs and various wild pigs with African Swine Fever Virus (ASFV), and the infection of virulent strains can lead to 100% mortality of animals, which poses a great threat and economic loss to the global pig industry. The disease is listed by the world animal health Organization (OIE) as an animal epidemic disease which must be reported, and is also an animal epidemic disease which is important to prevent in China.
At present, no effective vaccine and no treatment method exist for the disease, the prevention and control of the disease mainly depend on early virus detection, and the spread of epidemic diseases can be prevented only by timely and accurately and thoroughly killing and treating infected pigs. The current fluorescent PCR detection method becomes a common method for African swine fever virus, and is continuously popularized in units such as farms, slaughterhouses and the like in China. The detection range is also enlarged from the original pig breeding to the environment, factory entering and exiting personnel, articles, vehicles and the like, so that the pig breeding can be completely detected, epidemic diseases are prevented from occurring and diffusing in the farm comprehensively, the sample background for detecting PCR reaction is complex, such as humus in soil possibly existing in an environment sample, bile salt and complex polysaccharide in animal feces and iron ions in a vehicle sample often exist to inhibit the PCR reaction, and the detection result is abnormal: after the Ct value of the positive sample is greatly misplaced, the machine is seriously interfered to cause that the fluorescent signal cannot be read, false negative is generated, and the risk of missed detection is caused. Such detection errors greatly increase the risk of transmission of ASFV, resulting in economic losses. Therefore, a method for enhancing the anti-interference capability of a fluorescent quantitative PCR reaction system is developed to improve the detection signal-to-noise ratio and the fluorescence increment of the fluorescent quantitative PCR technology, eliminate false weak positive and false negative, and correctly judge the PCR result is important.
Disclosure of Invention
The invention aims to provide an African swine fever virus fluorescence PCR detection kit with anti-interference capability, and the kit is used for detection, so that a simple method is provided for accurately detecting a sample with a complex background. The kit has the advantages of wide applicability, strong specificity, high sensitivity and the like.
The technical aim of the invention is realized by the following technical scheme: the invention provides an African swine fever virus fluorescence PCR detection kit with anti-interference capability, which comprises a fluorescence PCR reaction solution, wherein a group of anti-interference compositions are added into an anti-interference 5xPC buffer solution used by the fluorescence PCR reaction solution, and the anti-interference compositions comprise 30% of trehalose, 0.5% of BSA, 35% of Brij and 0.5M of propylene glycol.
Further, the kit also comprises an upstream primer, a downstream primer and a probe; the sequences of the upstream and downstream primers and probes are as follows:
an upstream primer: ASFVF TCTTGCTCTRGATACGTTAATATGRC;
a downstream primer: ASFVR TCAAAGTTCTGCAGCTCTTACATAC;
and (3) probe: ASFVP 5'FAM-CACTGGGTTGGTATTCCTCCCGT-BHQ13'.
Further, one or more of fluorescent PCR reaction liquid, U-Taq enzyme, positive control and negative control are also included.
Further, the fluorescent PCR reaction solution comprises 1 volume concentration of 10 mu mol/L upstream primer, 1 volume concentration of 10 mu mol/L downstream primer, 0.3 volume concentration of 10 mu mol/L probe, 5 volumes of 5xPC buffer solution, 3 volumes concentration of 25mmol/L MgCl 2 3 volumes of dNTP Mix and 6.7 volumes of diethyl pyrocarbonate treatment water.
Further, the 5xPCR buffer comprises Tris-HCl with PH=9.0, 100mM KCl,5mM MgCl 2 0.05% TritonX-100,30% trehalose, 0.5% BSA,1% Brij35,0.5M propylene glycol.
Further, the U-Taq enzyme includes 0.4. Mu.L of Taq DNA polymerase and 0.1. Mu.L of UDG enzyme.
Further, the negative control is the water treated by the diethyl pyrocarbonate after being sterilized by high pressure, and the preparation method comprises the following steps: 1mL of diethyl pyrocarbonate is measured, added into double distilled water and the volume is fixed to 1L, so as to obtain diethyl pyrocarbonate solution with the volume concentration of 0.1 percent, the solution is stirred at room temperature for 12 hours, and steam sterilization is carried out at the high pressure of 121 ℃ for 15 minutes, thus obtaining the diethyl pyrocarbonate.
Furthermore, the positive control is an artificially synthesized African swine fever P72 gene fragment, and the gene sequence is as follows:
AATTCTCTTGCTCTGGATACGTTAATATGACCACTGGGTTGGTATTCCTCCCGTGGCTTCAAAGCAAAGGTAATCATCATCGCACCCGGATCATCGGGGGTTTTAATCGCATTGCCTCCGTAGTGGAAGGGTATGTAAGAGCTGCAGAACTTTGATGGA。
the invention also provides a test method of the African swine fever virus fluorescence PCR detection kit with anti-interference capability, which comprises the following steps:
s1: adding 20 mu L of fluorescent PCR reaction liquid and 0.5 mu L of U-Taq enzyme into a reaction tube, uniformly mixing, adding 5.0 mu L of sample nucleic acid to be detected, simultaneously setting positive and negative control tubes, covering a tube cap, centrifuging for 10s at 500g, and placing into a fluorescent PCR detector for fluorescent PCR reaction;
s2: the reaction conditions are as follows: a first stage, 50 ℃/2 minutes; the second stage is 95 ℃/3 minutes; 95 ℃/15 seconds, 60 ℃/30 seconds and 45 cycles in the third stage; fluorescence collection was performed at 60℃extension per cycle in the third stage using FAM channel read results.
In summary, the invention has the following beneficial effects:
1) The applicability is wide: the method is greatly helpful for improving the PCR performance, can detect various types of samples, has a weakening effect on PCR inhibitors in the samples, improves the interpretation accuracy of fluorescent PCR detection, and prevents false negative.
2) Sensitivity: because of adopting a specific fluorescent probe and a high-sensitivity fluorescent PCR instrument, the sensitivity of the fluorescent PCR method is 100-1000 times higher than that of the traditional PCR method; plasmid DNA of known copy number was tested. The results show that the sensitivity of the kit can reach 1 copy/. Mu.L.
3) Specific: because not only the specific primer is adopted, but also the specific probe is adopted, the specificity of the method is higher than that of the common PCR method; has no positive cross reaction with other confusing diseases including swine fever virus, epidemic diarrhea virus, blue ear disease virus, circovirus and pseudorabies virus, and shows that the specificity is good.
4) Stabilization: the repeatability test result shows that the stability of the established method is good; the repeated test was performed on plasmid DNA of known copy number at different concentrations with a coefficient of variation of less than 3%.
5) Is not easy to pollute: the UNG enzyme-dUTP anti-pollution system is used for fully sealing reaction, PCR post-treatment is not needed, and the risk of product pollution is greatly reduced while the operation is safe.
Drawings
FIG. 1 is a graph showing the comparison of the detection results of a fluorescent PCR reaction solution containing an anti-interference composition and a fluorescent PCR reaction solution without an anti-interference composition when a sample containing a PCR reaction inhibitor is detected in example 3 of the present invention;
FIG. 2 is a graph showing the result of a sensitivity test in example 4 of the present invention (African swine fever virus fluorescent PCR detection kit against various disturbances);
FIG. 3 is a graph showing the results of a specific assay in example 5 of the present invention (African swine fever virus fluorescent PCR detection kit against various interferences).
Detailed Description
The invention is described in further detail below with reference to fig. 1-3.
The preparation of the African swine fever virus PCR detection kit with the anti-interference capability comprises fluorescent PCR reaction liquid, U-Taq enzyme, negative control and positive control. The preparation method specifically comprises the following steps:
s1: preparing 5XPCR buffer solution of the anti-interference component;
Tris-HCl (pH 9.0) was weighed: 100mM, KCl:300mM, mgCl 2 6H2O:5mM, tritonX-100:0.05 percent of trehalose 30 percent, 0.5 percent of BSA, 35 percent of Brij35 percent, 0.5M of propylene glycol and DEPC treated water to 400mL, and the volume is fixed to 500mL. Filtering (0.45 μm) and sterilizing, and then preserving at 2-8 ℃ for standby.
S2: designing a primer and a probe;
referring to p72 gene sequences of 24 genotype reference strains of African swine fever virus and Chinese epidemic virus strains, a conserved region is screened, and a primer probe is designed as shown in table 1:
table 1: primer probe gene sequence
S3: preparing fluorescent PCR reaction liquid:
2.5mmol/L dNTP mix 3. Mu.L, upstream primer ASFVF (10. Mu. Mol/L) 1. Mu.L, downstream primer ASFVR (10. Mu. Mol/L) 1. Mu.L, probe ASFVP (10. Mu. Mol/L) 0.3. Mu.L, 5XPCR buffer 5. Mu.L, 25mmol/L MgCl 2 3 mu L of 0.1% DEPC treated water and 6.7 mu L of the water are mixed uniformly (whole-process sterile low temperature), and then filtered and sterilized by a 0.45 mu m filter membrane;
s4: positive control was prepared: cloning the African swine fever virus P72 gene by a conventional method, constructing a pMD20-T-P72 vector plasmid, and determining the sequence of the P72 gene in the plasmid. After the sequencing result is correct, converting the competent cells of the escherichia coli Top10, performing expansion culture, extracting plasmids, and diluting to serve as a positive control;
s5: preparation of negative control: 1mL of diethyl pyrocarbonate is measured, added into double distilled water and the volume is fixed to 1L, the final concentration is 0.1 percent, the mixture is stirred at room temperature for 12 hours, and the mixture is sterilized by steam at the high pressure of 121 ℃ for 15 minutes, namely a negative control;
s6: preparation of U-Taq enzyme: consists of 0.4. Mu.L of Taq DNA polymerase (5U/. Mu.L) and 0.1. Mu.L of UDG enzyme (2U/. Mu.L);
s7: optimizing reaction conditions: 20. Mu.L of fluorescent PCR reaction solution and 0.5. Mu.L of U-Taq enzyme were added to the reaction tube, and 5.0. Mu.L of the extracted nucleic acid was added thereto, while positive and negative control tubes were set. The tube cap is covered tightly, and the tube cap is centrifuged instantly and put into a fluorescent PCR detector. The reaction conditions are as follows: 95 ℃/3min;95 ℃/15s,60 ℃/30s for 45 cycles, fluorescence collection was performed at 60 ℃ extension for each cycle of the second stage;
s8: and (3) verifying a kit: (1) The kit can resist various fluorescent PCR reaction interferents, does not influence the sensitivity of PCR reaction, and has the function of improving the fluorescent PCR performance. (2) The positive control plasmid (104 copies/. Mu.L) is diluted 10 times, 100 times, 1000 times and 10000 times, and the positive control plasmid diluted by times (the copy numbers are 103, 102, 101 and 100 copies/. Mu.L in sequence) is detected according to the method in 6, so that the sensitivity of the kit is verified to still reach 1 copy/. Mu.L. (3) The specificity of the kit is verified by detecting classical swine fever virus nucleic acid, epidemic diarrhea virus nucleic acid, blue ear virus nucleic acid, circular ring virus nucleic acid and pseudorabies virus nucleic acid, and no positive cross reaction exists.
Example 1: establishment of a fluorescence PCR detection kit for African swine fever virus with resistance to various interferences:
on the basis of multiple sequence comparison, a probe primer group of African swine fever virus specificity is designed, a fluorescent PCR detection method is initially established, and optimization is carried out by applying a Taguchi method to find out primers, probes and Mg 2+ The optimal range of the concentration is correspondingly adjusted according to the optimal range, and finally the primer, the probe and the Mg are obtained 2+ To determine the optimum conditions for the detection method: the reaction system comprises 25 mu L of fluorescent PCR reaction liquid, U-Taq enzyme and a template. Wherein one reaction solution for detection consists of 1 volume concentration of 10 mu mol/L upstream primer, 1 volume concentration of 10 mu mol/L downstream primer, 0.3 volume concentration of 10 mu mol/L probe, 5 volumes of anti-interference 5XPCR buffer solution and 3 volumes concentration of 25mmol/L MgCl 2 3 volumes of dNTP Mix (dATP/dUTP/dCTP/dGTP mixture) and 6.7 volumes of diethyl pyrocarbonate treated water; one assay U-Taq enzyme consisted of 0.4. Mu.L of Taq DNA polymerase (5U/. Mu.L) and 0.1. Mu.L of UDG enzyme (2U/. Mu.L); finally determining the amplification condition of the detection method as a first stage, wherein the amplification condition is 50 ℃/2min; the second stage is 95 ℃/3 minutes; the third stage is 95 ℃/15sec,60 ℃/30sec for 45 cycles; fluorescence collection was performed at 60℃extension per cycle in the third stage.
After the fluorescent PCR reaction is finished, if the detection channels have no Ct value and no characteristic amplification curve, the sample has no African swine fever virus nucleic acid; if a characteristic amplification curve appears in the detection channel and the Ct value is less than or equal to 36.0, the sample only contains African swine fever virus nucleic acid. According to the detection method provided by the invention, the negative comparison has no Ct value and no amplification curve; the positive comparison shows a corresponding characteristic amplification curve, and Ct values are all less than or equal to 32; if the negative or positive control does not meet the above conditions, the experiment is regarded as ineffective. If Ct value is more than 36.0, and sample suggestion of typical amplification curve is repeated; and the repeated test shows that the result is positive, otherwise, the result is negative.
Example 2: application of anti-interference capability of anti-interference composition in African swine fever virus fluorescence PCR detection kit:
in the clinical application process of the African swine fever virus detection kit, various interferents such as humus and urea in soil, bile salts and complex polysaccharides in feces, heme and immunoglobulin in blood, iron ions in environmental samples and the like are frequently encountered in the samples. These interferents all affect the activity of the enzyme preparation during PCR amplification, and affect the judgment of PCR results, and the anti-interferents (shown in Table 2) of different combinations of the present invention: trehalose, BSA, brij35, propylene glycol were added to the PCR reaction buffer system and weak positive samples with interferents, including environmental swab samples containing fecal soil, vehicle swab samples containing rust components, blood samples containing anticoagulants, were detected at copy numbers < 100. Each weak positive sample was repeatedly tested 8 times using different combinations, and the anti-interference effect of different anti-interference compositions on PCR tested weak positive samples was compared, and the detection rates are shown in table 3. The results show that the different compositions of the invention have different anti-interference capability on different inhibitors, and the anti-interference capability is strongest in the combination containing all the compositions, so that all the weak positive samples containing the PCR inhibitor can be detected.
Table 2: tamper-resistant material of different combinations
| Trehalose | BSA | Brij35 | Propylene glycol | |
| Combination 1 | - | - | - | - |
| Combination 2 | 30％ | 0.5％ | - | - |
| Combination 3 | 30％ | 0.5％ | - | 0.5M |
| Combination 4 | 30％ | 0.5％ | 1％ | - |
| Combination 5 | - | 0.5％ | 1％ | 0.5M |
| Combination 6 | 30％ | - | 1％ | 0.5M |
| Combination 7 | 30％ | 0.5％ | 1％ | 0.5M |
Table 3: detection rate of different combinations
| Detection rate of | Combination 1 | Combination 2 | Combination 3 | Combination 4 | Combination 5 | Combination 6 | Combination 7 |
| Environmental swab sample containing fecal soil | 1/8 | 2/8 | 4/8 | 3/8 | 6/8 | 4/8 | 8/8 |
| Vehicle swab sample containing rust component | 0/8 | 3/8 | 5/8 | 6/8 | 4/8 | 3/8 | 8/8 |
| Blood sample containing anticoagulant | 2/8 | 5/8 | 6/8 | 5/8 | 3/8 | 4/8 | 8/8 |
Example 3: effect of anti-interference composition on PCR reaction validation:
the optimal concentrations of trehalose, BSA, brij35 and propylene glycol in the determined anti-interference compositions are used for preparing a 5XPCR buffer solution with the anti-interference composition and a 5XPCR buffer solution without the anti-interference composition (the components are shown in table 4), and the stock solution and the 100-fold dilution sample of the African swine fever virus sample (comprising an environmental swab sample containing fecal soil, a vehicle swab sample containing rust components and a blood sample containing an anticoagulant) containing the PCR inhibitor are respectively detected according to the preparation and application methods of the reaction solution in the kit described in the patent. The experimental results are shown in fig. 1: when the fluorescent quantitative PCR reaction liquid without the anti-interference component detects the stock solution of the sample with the interference substance, the PCR amplification can be inhibited, and the normal amplification can be realized after 100 times dilution. When the fluorescent quantitative PCR reaction liquid with the anti-interference composition is added to detect the stock solution of the sample with the interference substances, the PCR amplification is not inhibited, and the normal amplification can be still realized after 100-time dilution, so that the performance of fluorescent PCR reaction detection can be improved by the addition of the anti-interference composition.
Table 4: 5XPCR buffer composition
Example 4: sensitivity test of African swine fever virus fluorescent PCR detection kit with anti-interference capability:
the african swine fever virus positive quality control (copy number 104 copies/. Mu.L) was diluted 1:10, 1:100, 1:1000, 1:10000, 1:100000 with DEPC treated water as a sensitivity quality control sample. The detection results of the kit for detecting and judging the diluted quality control products at 1:10, 1:100 and 1:1000 are positive, 1:10000 is positive, and 1:100000 is negative, which indicates that the detection limit of the kit can reach 1:10000, namely 100 copies/. Mu.L, as shown in figure 2.
Example 5: specificity test of African swine fever virus fluorescent PCR detection kit against various interferences:
the specificity of the kit is verified to have no positive cross reaction by detecting swine fever virus, epidemic diarrhea virus, blue-ear disease virus, circovirus, pseudorabies virus and healthy pig manure nucleic acid, and is shown in figure 3.
The present embodiment is only for explanation of the present invention and is not to be construed as limiting the present invention, and modifications to the present embodiment, which may not creatively contribute to the present invention as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present invention.
Claims (9)
1. A fluorescence PCR detection kit with anti-interference capability for African swine fever virus is characterized in that: the anti-interference PCR reaction solution comprises a group of anti-interference compositions which comprise 30% of trehalose, 0.5% of BSA, 35% of Brij and 0.5M of propylene glycol, wherein the anti-interference 5xPC buffer solution is used in the fluorescent PCR reaction solution.
2. The African swine fever virus fluorescence PCR detection kit with anti-interference capability as claimed in claim 1, wherein the kit is characterized in that: the kit also comprises an upstream primer, a downstream primer and a probe; the sequences of the upstream and downstream primers and probes are as follows:
an upstream primer: ASFVF TCTTGCTCTRGATACGTTAATATGRC;
a downstream primer: ASFVR TCAAAGTTCTGCAGCTCTTACATAC;
and (3) probe: ASFVP 5'FAM-CACTGGGTTGGTATTCCTCCCGT-BHQ13'.
3. The anti-interference fluorescence PCR detection kit for African swine fever virus according to claim 1, wherein the kit comprises the following components: and one or more of fluorescent PCR reaction liquid, U-Taq enzyme, positive control and negative control are also included.
4. The African swine fever virus fluorescence PCR detection kit with anti-interference capability as claimed in claim 1, wherein the kit is characterized in that: the fluorescent PCR reaction solution comprises 1 volume concentration of 10 mu mol/L upstream primer, 1 volume concentration of 10 mu mol/L downstream primer, 0.3 volume concentration of 10 mu mol/L probe, 5 volumes of 5xPC buffer solution and 3 volumes concentration of 25mmol/L MgCl 2 3 volumes of dNTP Mix and 6.7 volumes of diethyl pyrocarbonate treatment water.
5. The anti-interference fluorescence PCR detection kit for African swine fever virus according to claim 4, wherein the kit is characterized in that: the 5xPC buffer comprises Tris-HCl with PH=9.0, 100mM KCl and 5mM MgCl 2 0.05% TritonX-100,30% trehalose, 0.5% BSA,1% Brij35,0.5M propylene glycol.
6. The African swine fever virus fluorescence PCR detection kit with anti-interference capability as claimed in claim 1, wherein the kit is characterized in that: the U-Taq enzyme includes 0.4. Mu.L of Taq DNA polymerase and 0.1. Mu.L of UDG enzyme.
7. The African swine fever virus fluorescence PCR detection kit with anti-interference capability as claimed in claim 1, wherein the kit is characterized in that: the negative control is autoclaved diethyl pyrocarbonate treated water, and the preparation method comprises the following steps: 1mL of diethyl pyrocarbonate is measured, added into double distilled water and the volume is fixed to 1L, so as to obtain diethyl pyrocarbonate solution with the volume concentration of 0.1 percent, the solution is stirred at room temperature for 12 hours, and steam sterilization is carried out at the high pressure of 121 ℃ for 15 minutes, thus obtaining the diethyl pyrocarbonate.
8. The African swine fever virus fluorescence PCR detection kit with anti-interference capability as claimed in claim 1, wherein the kit is characterized in that: the positive control is an artificially synthesized African swine fever P72 gene fragment.
9. The detection method of the African swine fever virus fluorescence PCR detection kit with anti-interference capability according to any one of claims 1-8, which is characterized in that: the method specifically comprises the following steps:
s1: adding 20 mu L of fluorescent PCR reaction liquid and 0.5 mu L of U-Taq enzyme into a reaction tube, uniformly mixing, adding 5.0 mu L of sample nucleic acid to be detected, simultaneously setting positive and negative control tubes, covering a tube cap, centrifuging for 10s at 500g, and placing into a fluorescent PCR detector for fluorescent PCR reaction;
s2: the reaction conditions are as follows: a first stage, 50 ℃/2 minutes; the second stage is 95 ℃/3 minutes; 95 ℃/15 seconds, 60 ℃/30 seconds and 45 cycles in the third stage; fluorescence collection was performed at 60℃extension per cycle in the third stage using FAM channel read results.
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