Test kit for detecting African swine fever virus and method for detecting African swine fever virus

A technology of African swine fever virus and kit, applied in the field of biological detection, can solve the problems of reducing the accuracy of detection results, false positives, false negatives, etc., achieve broad market prospects and commercial value, easy operation, and short time-consuming effects

Inactive Publication Date: 2020-06-19

ZHEJIANG UNIV

8 Cites 3 Cited by

AI-Extracted Technical Summary

Problems solved by technology

In the prior art, wrong or inappropriate selection of positive control samples and internal reference gene samples cannot indicate the...

Method used

As can be seen from Fig. 1, in each group, the Ct mean value of β-Actin is maintained between 33.4-33.8, shows that experimental result stability and repeatability are good.

[0037] The invention provides a test kit for detecting African swine fever virus. The test kit uses the plasmid of the ASFV structural protein gene VP73 fragment as a positive control, and uses β-Actin as an internal reference control gene. The kit uses the β-Actin gene ...

Abstract

The invention provides a test kit for detecting African swine fever virus. The kit comprises a plasmid containing an ASFV structural protein gene VP73 fragment; an upstream primer of the VP73, a downstream primer of the VP73, an upstream primer of the beta-Actin and a downstream primer of the beta-Actin; a probe of VP73, a probe of beta-Actin. The test kit is easy and convenient to operate, low inprice and short in consumed time, has high sensitivity and specificity and has wide market prospects and commercial value. The invention also provides a method for detecting the African swine fever virus, which comprises the following steps: taking a plasmid containing an ASFV structural protein gene VP73 fragment as a positive control, and taking a porcine actin gene beta-Actin as an internal reference control gene; monitoring from the initial stage of detection, so that the whole virus detection process can be monitored, the effect of internal reference quality control is perfectly embodied, and the accuracy of the detection result is improved.

Application Domain

Microbiological testing/measurementDNA/RNA fragmentation

Technology Topic

Classical swine feverVirus detection +7

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Examples

Experimental program(2)
Effect test(1)

Example Embodiment

[0036] Example 1

[0037] The invention provides a kit for detecting African swine fever virus. The kit uses the plasmid of the ASFV structural protein gene VP73 fragment as a positive control, and β-Actin is used as an internal reference control gene. The kit uses β-Actin gene as an internal reference for quality control, which not only monitors the sample nucleic acid extraction process but also the gene amplification reaction process, which increases the accuracy of the detection results and reduces false negative results.

[0038] Specifically, a kit for detecting African swine fever virus includes: a plasmid containing the VP73 fragment of the ASFV structural protein gene, the upstream primer of VP73, the downstream primer of VP73, the upstream primer of β-Actin, the downstream primer of β-Actin, In some preferred modes, the kit also includes a probe for VP73 and a probe for β-Actin.

[0039] In this example, the plasmid containing the VP73 fragment of the ASFV structural protein gene is a freeze-dried dry plasmid powder. When in use, the plasmid solution can be obtained by adding Rnase Free Water to dissolve it.

[0040] The kit directly provides the experimenter with the upstream and downstream primers of VP73 and β-Actin, eliminating the need for the experimenter to design the primers themselves.

[0041] The upstream primer of VP73 is:

[0042] 5'-CGGATATGACTGGGACAACCA-3' (SEQ ID NO.: 1),

[0043] The downstream primers of VP73 are:

[0044] 5'-AGGGATCTACAAGCGTGTAAACG-3' (SEQ ID NO.: 2),

[0045] The probe of VP73 is:

[0046] 5'-FAM-ACACCTTTAGAGGGCG-TAMRA-3' (SEQ ID NO.: 3),

[0047] The upstream primer of β-Actin is:

[0048] 5'-CCAGTTCGCCATGGATGAC-3' (SEQ ID NO.: 4),

[0049] The downstream primers of β-Actin are:

[0050] 5'-ATGCCGGAGCCGTTGTC-3' (SEQ ID NO.: 5),

[0051] The probe for β-Actin is:

[0052] 5'-Cy5-TATTGCTGCGCTCGTG-BHQ2-3' (SEQ ID NO.: 6);

[0053] Among them, FAM and Cy5 in the VP73 probe and β-Actin probe are fluorescent groups, and TAMRA and BHQ2 are quenching groups.

[0054] The specific usage method of the kit is as follows: the plasmid solution containing the ASFV structural protein gene VP73 fragment is used as the positive control standard, and β-Actin is used as the internal reference control gene; the β-Actin gene is obtained by nucleic acid extraction in the sample to be tested.

[0055] (1) Take 10 μL of the sample to be tested and use a commercially available conventional nucleic acid extraction kit to extract the nucleic acid of the sample to be tested;

[0056] (2) Add RnaseFree Water to the dry plasmid powder containing the ASFV structural protein gene VP73 fragment provided in the kit to dissolve it to obtain a plasmid solution. The initial concentration of the plasmid solution is 10 6 copies/mL, diluted 1:10 to a concentration of 10 2 -10 6 copies/mL, the concentration is 10 6 copies/mL, 10 5 copies/mL, 10 4 copies/mL, 10 3 copies/mL, 10 2 copies/mL plasmid solution;

[0057] Use commercially available conventional quantitative PCR kits, use the nucleic acid extracted in step (1) and the plasmid in the above-mentioned plasmid solution as templates, and use the VP73 upstream and downstream primers provided in the kit as well as β-Actin upstream and downstream primers as templates. Primer, perform quantitative PCR reaction. Among them, in the quantitative PCR reaction, the experimenter can add the VP73 probe and β-Actin probe provided by this kit to carry out the quantitative PCR reaction by the probe method; it can also carry out the quantitative PCR reaction by the dye method.

[0058] The quantitative PCR reaction conditions are as follows:

[0059]

[0060] (4) After the reaction is completed, the amplification curve and dissolution curve of quantitative PCR are obtained, the amplification results are analyzed, and the concentration of ASFV nucleic acid in the sample to be tested is calculated, or the presence or absence of ASFV in the sample to be tested is directly judged. Specifically, 10 2 -10 6 The results of Ct value amplification of copies/mL plasmid solutions of different concentrations, take Ct as the abscissa and the plasmid concentration as the ordinate, generate a scatter plot, and fit the linear regression equation of the scatter plot, in the equation Substituting the Ct value of the sample to be tested, the concentration of ASFV nucleic acid in the sample to be tested (copies/mL) can be calculated.

Example Embodiment

[0061] Example 2

[0062] The present invention provides a method for detecting African swine fever virus, using the kit described above, using a plasmid solution containing the ASFV structural protein gene VP73 fragment as a positive control standard, and using pig actin gene β-Actin as an internal reference control gene , Including the following steps:

[0063] (1) Extract the nucleic acid of the sample to be tested;

[0064] (2) Use the nucleic acid of the sample to be tested and the plasmid containing the ASFV structural protein gene VP73 fragment as a template for gene amplification reaction;

[0065] (3) Analyze the amplification results.

[0066] In this method, a plasmid containing the ASFV structural protein gene VP73 fragment was selected as a positive control, and the pig actin gene β-Actin was used as an internal reference control gene. Monitoring from the initial stage of detection can monitor the entire virus detection process, which perfectly reflects the role of internal reference control genes and increases the accuracy of detection results.

[0067] When the test result of β-Actin is negative, it indicates that the test result is false negative, and the test result of the sample to be tested is not credible, and further verification is required. Therefore, this method can play an indicative role in the experimental result.

[0068] In some preferred methods, the concentration of plasmid in the positive control standard is 0-10 6 Copies/μL, this concentration can meet the requirement of detection and realize the function to be achieved by the present invention.

[0069] In some preferred modes, the gene amplification reaction is a fluorescent quantitative PCR reaction.

[0070] In some preferred modes, in the reaction system of the fluorescent quantitative PCR reaction, the upstream primer of VP73, the downstream primer of VP73, the upstream primer of β-Actin, the downstream primer of β-Actin; the probe of VP73, the probe of β-Actin The best concentration of the needle is 0.2-1μM, too high or too low primer concentration will affect the detection result.

[0071] In some preferred modes, the reaction steps of the quantitative PCR reaction are:

[0072]

[0073] The method will be described in detail with reference to specific embodiments.

[0074] (2.1) A detection method for African swine fever virus (ASFV)

[0075] Taking the rapid detection of African swine fever virus ASFV in the sample to be tested as an example, the virus detection method provided by the present invention is introduced in detail.

[0076] (1) Instrument reagents

[0077] Main instrument: fluorescence quantitative PCR instrument

[0078] Main reagents: nucleic acid extraction kit, quantitative PCR kit, plasmid containing VP73 fragment

[0079] (2) Primer design

[0080] When detecting African swine fever virus (ASFV), according to the sequence of ASFV structural protein gene VP73 and the sequence of porcine actin gene β-Actin, the upstream and downstream primers of VP73 and β-Actin, as well as the detection of VP73 and β-Actin The needle is as follows,

[0081] The upstream primer of VP73 is:

[0082] 5'-CGGATATGACTGGGACAACCA-3' (SEQ ID NO.: 1),

[0083] The downstream primers of VP73 are:

[0084] 5'-AGGGATCTACAAGCGTGTAAACG-3' (SEQ ID NO.: 2),

[0085] The probe of VP73 is:

[0086] 5'-FAM-ACACCTTTAGAGGGCG-TAMRA-3' (SEQ ID NO.: 3),

[0087] The upstream primer of β-Actin is:

[0088] 5'-CCAGTTCGCCATGGATGAC-3' (SEQ ID NO.: 4),

[0089] The downstream primers of β-Actin are:

[0090] 5'-ATGCCGGAGCCGTTGTC-3' (SEQ ID NO.: 5),

[0091] The probe for β-Actin is:

[0092] 5'-Cy5-TATTGCTGCGCTCGTG-BHQ2-3' (SEQ ID NO.: 6).

[0093] Among them, FAM and Cy5 in the probe primers of ASFV and β-Actin are fluorescent groups, and TAMRA and BHQ2 are quenching groups.

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Test kit for detecting African swine fever virus and method for detecting African swine fever virus

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Example Embodiment

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