CN111850165A - RT-PCR detection method of hog cholera virus - Google Patents
RT-PCR detection method of hog cholera virus Download PDFInfo
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Abstract
The invention discloses a swine fever virus RT-PCR detection method, which comprises four parts, namely discovery of the repetitive characteristics of a nucleic acid sequence of a swine fever virus, design of a nucleic acid RT-PCR primer of the swine fever virus, an amplification reaction system, reaction conditions and judgment of a result; discovery of nucleic acid sequence repeat characteristics of hog cholera virus: in a nucleic acid sequence conservation region of the classical swine fever virus, a 10-base repetitive sequence exists, and 237 bases are separated between two repetitive sequences; the invention fully utilizes a section of characteristic base fragment repetition of a relative conservative region of the nucleic acid of the classical swine fever virus, amplifies 2 specific DNA bands of the classical swine fever virus with specific size by 1 RT-PCR reaction through ingenious primer design, converts the commonly regarded non-specific amplification of the method into specific amplification, greatly improves the specificity of the RT-PCR detection method of the classical swine fever virus, and has more accurate, intuitive and simple result judgment.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an RT-PCR detection method of hog cholera virus.
Background
At present, PCR (polymerase chain reaction) is a common biological nucleic acid detection technical method in the field of life science, and can be used for nucleic acid detection of various pathogenic microorganisms, so that the pathogen of the epidemic disease can be quickly and accurately diagnosed. However, the conventional PCR method designs 1 pair of primers (short oligonucleotide chains) at upstream and downstream positions for a relatively conserved region of a nucleic acid (DNA or RNA) sequence of a microorganism, performs PCR (or RT-PCR) amplification under specific reaction conditions, performs agarose gel electrophoresis on an amplification product, and observes whether the amplification product presents a DNA band with a specific size in the agarose gel by using a gel electrophoresis imager and a DNA Marker to determine whether a sample has a nucleic acid fragment of a specific microorganism, thereby determining whether the sample contains a specific microbial pathogen. However, due to the characteristics of the nucleic acid sequence of the sample to be detected, the unreasonable design of the primer, the non-optimized reaction conditions and reaction system, and the like, the method often causes non-specific amplification or interference of primer dimers, which affects the determination of the detection result. However, based on the discovery of the special characteristics of the nucleic acid sequence of the African swine fever virus, the invention amplifies two specific DNA bands of the swine fever virus by one RT-PCR reaction through ingenious primer design, converts originally regarded non-specific amplification into specific amplification, and greatly improves the specificity and the accuracy of RT-PCR detection of the swine fever virus.
Disclosure of Invention
In view of the above, the invention provides a novel RT-PCR detection method for classical swine fever viruses, which is based on the fact that two specific DNA bands of classical swine fever viruses are amplified by one RT-PCR reaction through ingenious primer design and the originally regarded 'non-specific amplification' is converted into specific amplification;
a CSFV RT-PCR detection method is characterized by comprising four parts, namely discovery of CSFV nucleic acid sequence repetitive characteristics, design of CSFV nucleic acid RT-PCR primers, amplification reaction system and reaction conditions, and judgment of results;
discovery of nucleic acid sequence repeat characteristics of hog cholera virus: in the conserved region of the nucleic acid sequence of classical swine fever virus, a 10-base sequence with sequence GGAGAATTCA was found, which repeats after 237 bases apart;
designing a swine fever virus nucleic acid RT-PCR primer: after the characteristics of the CSFV nucleic acid sequence are discovered, a 19-base upstream primer F:5-ATAACCGGCGGAGAATTCA-3 and a 19-base downstream primer R:5-ACCGTGCCTGCTGGTGTTG-3 are designed, 10 bases at the 3 'end of the upstream primer F are repetitive sequence fragments, 3 bases at the 5' end of the upstream primer F are the same as the upstream sites of two repetitive sequences to be annealed with the CSFV nucleic acid sequence, the rest 6 bases are designed to be 3 bases as the same as the upstream sites of the first repetitive sequence, and 3 bases as the upstream sites of the second repetitive sequence (the 3 bases can be designed to be degenerate bases), so that the upstream primer with the length of 19 bases can ensure that 16 bases have complementary sites (if the upstream primer is designed to be fully complementary) with the target position of the CSFV nucleic acid template, and the primers can be specifically degenerate at the CSFV nucleic acid repetitive sequence template, the PCR amplification reaction can start from the positions of two sections of repeated sequences at the upstream of the nucleic acid template of the classical swine fever virus at the same time and end at the downstream primer, and the downstream primer avoids a high homology region with the nucleic acid sequence of the bovine viral diarrhea mucosal disease virus, thereby ensuring the specificity of the detection method;
Amplification reaction system and reaction conditions: the amplification reaction system and the reaction conditions comprise a cDNA synthesis reaction system and a PCR reaction system, and the reaction conditions comprise temperature, time and cycle times;
the result determination includes gel electrophoresis and imaging of the RT-PCR product.
Further, the cDNA synthesis was carried out by pipetting 5ul of the extracted RNA solution of the sample to be tested into a PCR tube, adding 50p M downstream primer R, cooling in an ice-water bath for 5 minutes at 68 ℃, adding 2ul of 5 Xfirst strand buffer, 0.5ul of 0.1M DTT, 0.5ul of dNTP (10mM each), 0.25ul of RNAse inhibitor, and 0.5ul of reverse transcriptase (superscript III). The PCR reaction is carried out by sucking 3ul of the cDNA template and 34.5ul of enzyme-free water in a PCR instrument at 50 ℃ for 60 minutes, 75 ℃ for 10 minutes and 4 ℃ for storage, adding 5ul of 10 XPCR Buffer, 3ul of dNTP (10mM each), 1ul of upstream primer F, 1ul of downstream primer R and 2.5ul of Taq DNA polymerase, blowing the mixture evenly, carrying out pre-denaturation at 94 ℃ for 3 minutes, 94 ℃ for 50 seconds, 50 ℃ for 40 seconds, 72 ℃ for 30 seconds and 30 cycles, and carrying out storage at 72 ℃ for 10 minutes and 4 ℃ in the PCR instrument.
Further, the temperature reduction in the ice-water bath in the cDNA synthesis can be achieved by using the program in a PCR instrument: the samples were stored at 68 ℃ for 5 minutes and 4 ℃ instead of the above-mentioned samples.
Further, the electric pulse is obtained by taking a proper amount of the PCR amplification product and carrying out electrophoresis in 1% agarose gel, the buffer solution is 0.5 xTBE, the voltage is 100v 40 minutes, 2 bands with the sizes of 662bp and 425bp appear in gel imaging in the gel imaging and result judgment, the hog cholera virus nucleic acid is judged to be positive, and the hog cholera virus nucleic acid is judged to be negative otherwise.
By adopting the technical scheme, the method has the following beneficial effects:
the invention fully utilizes a section of characteristic base segment repetition of a relative conservative region of a nucleic acid sequence of the classical swine fever virus, amplifies specific DNA bands of 2 nucleic acids of the classical swine fever virus by 1 RT-PCR reaction through ingenious primer design, converts the original non-specific amplification into the specific amplification, has specific number of bands and specific size of the bands, eliminates the interference of nucleic acid amplification of other pathogenic microorganisms and organisms, and greatly improves the specificity of the RT-PCR detection method of the classical swine fever virus. Compared with the conventional RT-PCR method, the method has the advantages that no other components are added, the detection cost and the time cost are not increased, and only the ingenious primer design is adopted, so that a novel double-strip RT-PCR amplification result is generated, 2 amplification strips with specific sizes appear in the electrophoretic imaging, even the judgment can be made without expensive product sequencing, the result judgment is more visual and simple, and the judgment reliability is higher and more accurate than that of a single strip. The invention has strong practicability, novelty and innovation, and high popularization and application value.
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FIG. 1 is a diagram showing gel imaging and result determination according to the present invention;
FIG. 2 is a schematic representation of the position of the listed repeat sequence GGAGAATTCA and primers in the nucleic acid sequence of the Shibata Swine fever Virus strain.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
Example 1:
the RT-PCR detection method for CSFV as shown in FIG. 1-FIG. 2 comprises four steps, which are respectively the discovery of CSFV nucleic acid sequence repeat characteristics, the design of CSFV nucleic acid RT-PCR primers, the amplification reaction system and reaction conditions, and the judgment of the result;
discovery of nucleic acid sequence repeat characteristics of hog cholera virus: in the conserved region of the nucleic acid sequence of classical swine fever virus, a 10-base sequence with sequence GGAGAATTCA was found, which repeats after 237 bases apart;
designing a swine fever virus nucleic acid RT-PCR primer: after the characteristics of the CSFV nucleic acid sequence are discovered, a 19-base upstream primer F:5-ATAACCGGCGGAGAATTCA-3 and a 19-base downstream primer R:5-ACCGTGCCTGCTGGTGTTG-3 are designed, 10 bases at the 3 'end of the upstream primer F are repetitive sequence fragments, 3 bases at the 5' end of the upstream primer F are the same as the upstream sites of two repetitive sequences to be annealed with the CSFV nucleic acid sequence, the rest 6 bases are designed to be 3 bases which are the same as the upstream site of the first repetitive sequence, and 3 bases which are the same as the upstream site of the second repetitive sequence (the 3 bases can be designed to be degenerate bases), so that the upstream primer with the length of 19 bases can ensure that 16 bases have complementary sites with the target position of the CSFV nucleic acid template, and ensure that the primers can be specifically annealed with the CSFV nucleic acid repetitive sequence template, the PCR amplification reaction can start from two repeated sequence sites at the upstream of the nucleic acid template of the classical swine fever virus at the same time and end at the downstream primer, and the sequence design of the downstream primer avoids the non-specific reaction of the bovine viral diarrhea mucosal virus which is highly homologous with the classical swine fever virus, thereby ensuring the specificity of the RT-PCR detection method of the classical swine fever virus;
Amplification reaction system and reaction conditions: the amplification reaction system and the reaction conditions comprise a cDNA synthesis reaction system and a PCR reaction system, and the reaction conditions comprise temperature, time and cycle times;
the result determination includes gel electrophoresis and imaging of the RT-PCR product.
Further, the cDNA synthesis was carried out by pipetting 5ul of the extracted RNA solution of the sample to be tested into a PCR tube, adding 50p M downstream primer R, cooling in an ice-water bath for 5 minutes at 68 ℃, adding 2ul of 5 Xfirst strand buffer, 0.5ul of 0.1M DTT, 0.5ul of dNTP (10mM each), 0.25ul of RNAse inhibitor, and 0.5ul of reverse transcriptase (superscript III). The samples were stored in a PCR apparatus at 50 ℃ for 60 minutes, 75 ℃ for 10 minutes and 4 ℃.
Further, the temperature reduction in the ice-water bath in the cDNA synthesis can be achieved by using the program in a PCR instrument: the samples were stored at 68 ℃ for 5 minutes and 4 ℃ instead of the above-mentioned samples.
Further, the PCR reaction was carried out by pipetting 3ul of the above cDNA template and 34.5ul of enzyme-free water, adding 5ul of 10 XPCR Buffer, 3ul of dNTP (10mM each), 1ul of the upstream primer F, 1ul of the downstream primer R, and 2.5ul of Taq DNA polymerase, pipetting the mixture uniformly, pre-denaturing at 94 ℃ for 3 minutes, at 94 ℃ for 50 seconds, at 50 ℃ for 40 seconds, at 72 ℃ for 30 seconds, for 30 cycles, at 72 ℃ for 10 minutes, and preserving at 4 ℃.
And further, the electric pulse is obtained by taking a proper amount of the PCR amplification product to carry out electrophoresis in 1% agarose gel, the buffer solution is 0.5 xTBE, the voltage is 100v 40 min, 2 bands with the sizes of 662bp and 425bp appear in the gel imaging in the result judgment, the hog cholera virus nucleic acid is judged to be positive, and the hog cholera virus nucleic acid is judged to be negative otherwise.
The invention is carried out against the way that the common PCR (or RT-PCR) method is that a pair of primers amplifies 1 strip, the 1 pair of primers of the invention amplifies 2 specific strips, the common PCR method is changed into the common PCR method that the non-specific amplification is regarded as the specific amplification, and the design thought of the method has enough novelty. The method is characterized in that 10 bp base repeats are found in a conservative region of a hog cholera virus genome sequence with the length of 12.3kb for the first time, a pair of primers is designed by utilizing the characteristic for the first time, PCR amplification reaction starts at 2 places (the resetting point of 10 bp bases) of a nucleic acid template of a detected sample and ends at 1 place (the downstream primer), 2 specific bands are amplified through 1 reaction, and a detection result is judged by combining a characteristic 'double band' shown by gel imaging, so that the method has sufficient innovation. Based on the existing mature PCR detection technology, the RT-PCR detection method for the classical swine fever virus is combined with new discovery and is supplemented with ingenious primer design, so that the specificity of the RT-PCR detection method for the classical swine fever virus is greatly improved, compared with the conventional RT-PCR method, no other component is added, the detection cost and the time cost are not increased, even the judgment can be made without expensive product sequencing, the result judgment is more visual and simple, the judgment reliability is higher and more accurate than that of a single-strip judgment, and the RT-PCR detection method for the classical swine fever virus has stronger practicability and high popularization and application values.
Having thus described the basic principles and principal features of the invention, it will be appreciated by those skilled in the art that the invention is not limited by the embodiments described above, which are given by way of illustration only, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents.
Claims (5)
1. A CSFV RT-PCR detection method is characterized by comprising four parts, namely discovery of CSFV nucleic acid sequence repetitive characteristics, design of CSFV nucleic acid RT-PCR primers, amplification reaction system and reaction conditions, and result judgment;
discovery of nucleic acid sequence repeat characteristics of hog cholera virus: in the conserved region of the nucleic acid sequence of classical swine fever virus, a 10-base sequence with sequence GGAGAATTCA was found, which repeats after 237 bases apart;
designing a swine fever virus nucleic acid RT-PCR primer: after the characteristics of the CSFV nucleic acid sequence are discovered, a 19-base upstream primer F:5-ATAACCGGCGGAGAATTCA-3 and a 19-base downstream primer R:5-ACCGTGCCTGCTGGTGTTG-3 are designed, 10 bases at the 3 'end of the upstream primer F are repetitive sequence fragments, 3 bases at the 5' end of the upstream primer F are the same as the upstream sites of two repetitive sequences to be annealed with the CSFV nucleic acid sequence, the rest 6 bases are designed to be 3 bases as the same as the upstream sites of the first repetitive sequence, and 3 bases as the upstream sites of the second repetitive sequence (the 3 bases can be designed to be degenerate bases), so that the upstream primer with the length of 19 bases can ensure that 16 bases have complementary sites (if the upstream primer is designed to be fully complementary) with the target position of the CSFV nucleic acid template, and the primers can be specifically degenerate at the CSFV nucleic acid repetitive sequence template, the PCR amplification reaction can start from the sites of two upstream repeated sequences of the nucleic acid template of the classical swine fever virus and end at the downstream primer, and the downstream primer avoids a high homology region with the nucleic acid sequence of the bovine viral diarrhea mucosal disease virus, so that the specificity of the RT-PCR detection method of the classical swine fever virus is ensured;
Amplification reaction system and reaction conditions: the amplification reaction system and the reaction conditions comprise a cDNA synthesis reaction system and a PCR reaction system, and the reaction conditions comprise temperature, time and cycle times;
the result determination includes gel electrophoresis and imaging of the RT-PCR product.
2. The RT-PCR detection method for swine fever virus according to claim 1, wherein the cDNA synthesis comprises the steps of sucking 5ul of the extracted RNA solution of the sample to be detected into a PCR tube, adding 50p M downstream primer R, cooling in an ice water bath for 5 minutes at 68 ℃, adding 2ul of 5 x first strand buffer, 0.5ul of 0.1M DTT, 0.5ul of dNTP (10mM each), 0.25ul of RNAse inhibitor, and 0.5ul of reverse transcriptase (superscript III), and storing in a PCR instrument at 50 ℃ for 60 minutes, 75 ℃ for 10 minutes, and 4 ℃.
3. The method for detecting classical swine fever virus according to claim 2, wherein the cooling in an ice water bath during the cDNA synthesis is performed by using a PCR instrument with the following procedures: the samples were stored at 68 ℃ for 5 minutes and 4 ℃ instead of the above-mentioned samples.
4. The method for detecting classical swine fever virus double-strip RT-PCR as defined in claim 1, wherein the PCR reaction is performed by sucking 3ul of the cDNA synthesis reaction product of claim 2 or claim 3, 34.5ul of enzyme-free water, adding 5ul of 10 XPCR Buffer, 3ul of dNTP (10mM each), 1ul of the upstream primer F, 1ul of the downstream primer R, and 2.5ul of Taq DNA polymerase, pipetting the mixture evenly, pre-denaturing at 94 ℃ for 3 minutes, at 94 ℃ for 50 seconds, at 50 ℃ for 40 seconds, at 72 ℃ for 30 seconds, for 30 cycles, at 72 ℃ for 10 minutes, and storing at 4 ℃.
5. The method for detecting Classical Swine Fever Virus (CSFV) according to claim 1, wherein the electrical pulse is obtained by taking a proper amount of the PCR amplification product of claim 4 and performing electrophoresis in 1% agarose gel, the buffer solution is 0.5 XTBE, the voltage is 100v 40 min, 2 bands with the size of 662bp and 425bp appear in gel imaging during the result determination, the CSFV nucleic acid is determined to be positive, otherwise, the CSFV nucleic acid is determined to be negative.
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| CN114703317A (en) * | 2020-11-17 | 2022-07-05 | 广东壹号地方猪研究院有限公司 | Diagnosis method of hog cholera virus |
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