CN112899407A - Method for detecting pseudorabies virus - Google Patents
Method for detecting pseudorabies virus Download PDFInfo
- Publication number
- CN112899407A CN112899407A CN202110401217.3A CN202110401217A CN112899407A CN 112899407 A CN112899407 A CN 112899407A CN 202110401217 A CN202110401217 A CN 202110401217A CN 112899407 A CN112899407 A CN 112899407A
- Authority
- CN
- China
- Prior art keywords
- pcr
- prv
- reaction
- annealing temperature
- nano
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000000137 annealing Methods 0.000 claims abstract description 50
- 230000003321 amplification Effects 0.000 claims abstract description 21
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 21
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 13
- 239000002773 nucleotide Substances 0.000 claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims description 63
- 238000001514 detection method Methods 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 13
- 238000012408 PCR amplification Methods 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 230000035945 sensitivity Effects 0.000 abstract description 16
- 108020004414 DNA Proteins 0.000 description 42
- 108090000623 proteins and genes Proteins 0.000 description 42
- 239000012634 fragment Substances 0.000 description 32
- 102100024209 CD177 antigen Human genes 0.000 description 19
- 101000980845 Homo sapiens CD177 antigen Proteins 0.000 description 19
- 238000001962 electrophoresis Methods 0.000 description 9
- 239000002299 complementary DNA Substances 0.000 description 8
- 238000005457 optimization Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 6
- 108020005202 Viral DNA Proteins 0.000 description 5
- 108020000999 Viral RNA Proteins 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000012154 double-distilled water Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 239000002105 nanoparticle Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 101150072564 gE gene Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 241000130076 Chicken astrovirus 1 Species 0.000 description 2
- 241000700586 Herpesviridae Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 230000003313 weakening effect Effects 0.000 description 2
- 244000061520 Angelica archangelica Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000725585 Chicken anemia virus Species 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 101710121417 Envelope glycoprotein Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 235000001287 Guettarda speciosa Nutrition 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 1
- 108010052762 Suid herpesvirus 1 glycoprotein E Proteins 0.000 description 1
- 102100021696 Syncytin-1 Human genes 0.000 description 1
- 101150024766 VP1 gene Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011852 carbon nanoparticle Substances 0.000 description 1
- 238000000658 coextraction Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 101150029683 gB gene Proteins 0.000 description 1
- 101150020597 gG gene Proteins 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 239000011787 zinc oxide Substances 0.000 description 1
Images
Classifications
-
- 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
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
-
- 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]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for detecting pseudorabies virus, which uses nano PCR to detect PRV, and adopts the nucleotide sequence shown in SEQ ID No.1-2 in a sequence table, the concentration of an upstream primer and a downstream primer is 0.1 mu mol/L, the annealing temperature is 50 ℃, the nucleotide sequence shown in SEQ ID No.3-4, the concentration of the upstream primer and the downstream primer is 1 mu mol/L, and the annealing temperature is 50 ℃ to detect, so that the method has the advantages of high sensitivity, strong specificity, loose amplification condition, capability of specifically detecting wild strains and the like.
Description
Technical Field
The application relates to the technical field of molecular biology, in particular to a method for detecting pseudorabies virus.
Background
Pseudorabies virus (PRV) belongs to the genus varicella (varicella) of the sub-family of Herpesviridae (Herpesviridae) α -Herpesviridae (Alphaerpesvirinae), and can infect various animals such as pigs, sheep, dogs, cows, cats, rabbits, and mice, which mainly cause symptoms such as fever, extreme itching (excluding pigs), and encephalomyelitis.
The nano PCR (nano-PCR) technology is to add nano materials (such as gold nanoparticles, silver nanoparticles, carbon nanoparticles, zinc oxide nanoparticles and the like) into a PCR reaction system so as to adjust and control PCR, achieve the effect that the special effect can not be achieved compared with the conventional PCR, and provide a new idea and technology for optimizing the conventional PCR. In 2005, Li H et al (Nanoparticle PCR: Nanogold-assisted PCR with enhanced specificity [ J ] Angelica chemical-international edition.2005,44:2-5) found that the activity and stability of DNA polymerase in the PCR technology can be improved, and the specific amplification effect of PCR can be significantly improved. Yan L et al (Mechanism of gold nanoparticle induced mutation in PCR amplification and specificity [ J ]. Chinese Science Bulletin,2013,58(036): 4593-. At present, scholars at home and abroad establish the identification and detection of various viruses by using the technology.
The PRV genome is a double-stranded linear DNA of about 150kb, with an average GC content of about 73%, and contains up to 70 genes, encoding about 100 proteins. Researchers have used PCR to identify wild strains of PRV. For example, Zhang Yue Yong et al (establishment of porcine pseudorabies virus nano PCR detection method [ J ]. Chinese animal quarantine, 2017,034(003): 102-. Maxingjie (establishment and evaluation of nano PCR and fluorescent quantitative PCR method for identifying porcine pseudorabies virus gE/gB/gG [ D ]. Chinese academy of agricultural sciences, 2014) and the like establish triple nano PCR aiming at gE/gB/gG gene of PRV, but the method takes 90 minutes to complete PCR reaction and has the defects of long time consumption, strict required annealing temperature and the like.
For the above reasons, it is desirable to provide a detection method capable of rapidly and efficiently detecting PRV virus.
Disclosure of Invention
The invention provides a method for detecting pseudorabies virus, which comprises the following steps:
(1) amplifying a DNA template;
(2) nano PCR amplification;
(3) and (5) detecting a PCR product.
Wherein, the primer pair sequence in the nano PCR amplification is selected from SEQ ID No.1-2, SEQ ID No.3-4 or SEQ ID No.5-6 in the sequence table.
The Nano PCR reaction system comprises 1 mu L of DNA template, 10 mu L of 2 XNano-QPCR buffer, 0.1-1 mu L of upstream and downstream primer mixed liquor (the final concentration is 0.1-1 mu mol/L) with the working concentration of 20 mu mol/L, 5U/uL of taq enzyme mix0.32 mu L and ddH in each 20uL reaction system2O supplementSufficient 20. mu.L.
The nano PCR reaction procedure is reaction at 95 ℃ for 3 min; reaction at 94 ℃ for 5s, annealing temperature of 50-57 ℃ for 5s, and reaction at 72 ℃ for 30s, and performing 20 cycles; the reaction is finished at 72 ℃ for 5min and 12 ℃.
Further, when the sequence of the primer pair is a nucleotide sequence shown as SEQ ID No.1-2 in the sequence table, the final concentration of the upstream primer and the downstream primer is 0.1 mu mol/L, and the annealing temperature is 50 ℃.
Further, when the sequence of the primer pair is a nucleotide sequence shown by SEQ ID No.3-4 in the sequence table, the final concentration of the upstream primer and the downstream primer is 1 mu mol/L, and the annealing temperature is 50 ℃.
The PCR product detection can adopt 1.2% agarose gel electrophoresis to verify the amplification result.
The beneficial effects of the invention include:
the inventor of the invention finds that the amplification of the target gene with high GC content by the nano PCR reaction solution is easier than that of the conventional PCR reaction solution; the optional annealing temperature range of the nano PCR reaction is loose. The PRV genome is double-stranded linear DNA with about 150kb, the average GC content is about 73 percent, the PRV genome contains up to 70 genes and codes about 100 proteins, wherein the gE gene codes envelope glycoprotein of the virus, key sites and regions of the protein are related to virulence and antigenicity and have certain conservation, therefore, the invention takes the gene as a target gene to carry out primer design, uses nano PCR to carry out PRV detection, and obtains three groups of primer pairs for amplification and suitable amplification reaction conditions thereof through a large number of experimental screening, in particular adopts a nucleotide sequence shown by SEQ ID No.1-2 in a sequence table, the final concentrations of an upstream primer and a downstream primer are 0.1 mu mol/L, the annealing temperature is 50 ℃, the PCR amplification reaction time is 45 minutes, the nucleotide sequence shown by SEQ ID No.3-4, the final concentrations of the upstream primer and the downstream primer are 1 mu mol/L, The annealing temperature is 50 ℃, and the PCR amplification reaction time is 45 minutes, so that the kit has the advantages of high sensitivity, strong specificity, loose amplification conditions, short time consumption and the like when detection is carried out. In addition, the primers designed according to the gE gene can detect the infection of the wild strains, distinguish and identify the wild strains, and have important significance for clinical detection.
Drawings
FIG. 1 is a graph showing the temperature optimization results of PRV-1/2/3 fragment nanophase PCR and conventional PCR at 57-64 ℃, wherein FIG. 1A: electrophoresis diagram of PRV-1 fragment nano PCR; FIG. 1B: electrophoresis diagram of PRV-2 fragment nano PCR; FIG. 1C: electrophoresis diagram of PRV-3 fragment nano PCR; FIG. 1D: electrophoresis diagram of PRV-1 fragment conventional PCR; FIG. 1E: electrophoretogram of PRV-2 fragment conventional PCR; FIG. 1F: electrophoresis diagram of PRV-3 fragment conventional PCR; m: trans DNA Marker II; 1: 57.0 ℃; 2: 58.0 ℃; 3: 59.0 ℃; 4: 60.0 ℃; 5: 61.0 ℃; 6: 62.0 ℃; 7: 63.0 ℃; 8: 64.0 ℃; 9: and (5) negative control.
Detailed Description
The present invention will be further illustrated and described with reference to the following examples, but the examples described are only a part of the examples of the present invention, and not all of the examples. All other inventions and embodiments based on the present invention and obtained by a person of ordinary skill in the art without any creative effort belong to the protection scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Example 1 screening and optimization of PRV detection method
1 materials and methods
1.1 Strain and Positive cDNA Source
The chicken infectious anemia virus GXC060821 strain, the porcine pseudorabies virus PRV and the chicken astrovirus type 1 positive cDNA (derived from clinical samples) are preserved by Guangxi veterinary institute; extracting the DNA of CIAV and PRV viruses according to the instruction in the EasyPure Viral DNA/RNA Kit; the DNA and the chicken astrovirus type 1 positive cDNA template are stored at-20 ℃ for later use.
1.2 Primary reagents and instruments
The general amplification nano PCR kit is purchased from Shanghai Shantowen Biotech Co., Ltd, and the PCR instrument is purchased from the American Bio-rad Bole company; the DNA/RNA co-extraction Kit EasyPure Viral DNA/RNA Kit is purchased from Beijing Quanzijin Biotechnology Co., Ltd; pMD-18T, Premix TaqTM(TaKaRa TaqTMVersion 2.0plus dye) and
PCR Master Mix was purchased from Baoriri physicians & technology (Beijing) Ltd; NanoDrop2000 nucleic acid analyzer was purchased from Thermo Fisher Scientific; multicolor fluorescence imaging systems are available from BIO-RAD, USA.
1.3 design of primers
This study selected PRV with a high GC content (about 73%), CASTV-I with a low GC content (about 43%), and CIAV (about 55%) as the study subjects, and 12 pairs of primers were designed using the gE gene of PRV, the VP1 gene of CIAV, and the ORF1b gene of CASTV-I as the target genes, using MEGA 4.0 and Primer 4.0. As shown in Table 1, the primers were synthesized by Shenzhen, Inc., a Biotech gene technology of Huada.
TABLE 1 detection primers for CAStV-I, CIAV and PRV
1.4 preparation of Positive plasmid Standard
And amplifying corresponding target genes by using the synthesized CIAV, CAStV-I and PRV primer pairs respectively, connecting the amplified PCR positive product to a pMD-18T vector to construct a recombinant plasmid, sending the positive plasmid to Shenzhen Biotech company for sequencing, and using the plasmid with correct sequencing for a subsequent sensitivity test. The concentration of the positive recombinant plasmid is measured by a NanoDrop ND-2000 ultraviolet spectrophotometer, and the copy number is calculated according to the molecular weight and the concentration.
1.5 CAStV-I, CIAV and PRV reaction System optimization
1.5.1 CASTV-I, CIAV and PRV Nano PCR and conventional PCR reaction System and reaction procedure
20 μ L nano PCR reaction system: mu.L of cDNA/DNA template, 10. mu.L of 2 XNano-QPCR buffer, 0.1-1. mu.L of upstream and downstream primer mixture (working concentration of 20. mu. mol/L) (final concentration of 0.1-1. mu. mol/L), 0.32. mu.L of taq enzyme Mix (5U/uL), and ddH2O is complemented to 20 mu L; are set up simultaneouslyNegative control, no RNAase water as template added to the reaction tube.
The nano PCR reaction tube is placed in a PCR instrument, the annealing temperature is optimized according to the Tm values of different primers (the selected temperature range is 50-64 ℃), and the optimal annealing temperature is determined. PCR reaction procedure: 3min at 95 ℃; carrying out 20 cycles at 94 ℃ for 5s, annealing temperature for 5s and 72 ℃ for 30 s; 5min at 72 ℃ and finishing the reaction at 12 ℃. The amplification results were verified by 1.2% agarose gel electrophoresis using 6-8. mu.L of the PCR product.
20 μ L of conventional PCR reaction: mu.L cDNA template, 2 XPCR buffer 10. mu.L, upstream and downstream primer mixture (working concentration 20. mu. mol/L) 0.1-1. mu.L (final concentration of reaction 0.1-1. mu. mol/L), add ddH2O is complemented to 20 mu L; meanwhile, a negative control was set, and no RNAase water was added as a template to the reaction tube (note: target gene CASTV-1/2/3/4 (note: 1/2/3/4 represents the number of the primer pair, and the numbers in CIAV-1/2/3/4/5 and PRV-1/2/3 are as defined above and below) and PCR reaction solutions used for PCR reaction of CIAV-1/2/3/4/5 were TaKaRa TaqTMThe Version 2.0plus dye, PRV-1/2/3 fragment used
PCR Master Mix)。
The conventional PCR reaction tube is placed in a PCR instrument, the annealing temperature is optimized according to the Tm values of different primers (the temperature range is selected to be 50-64 ℃), and the optimal annealing temperature is determined. PCR reaction procedure: 5min at 95 ℃; carrying out 35 cycles of 94 ℃ for 1min, annealing temperature for 1min and 72 ℃ for 1 min; the reaction was terminated at 72 ℃ for 10min and 12 ℃. The amplification results were verified by 1.2% agarose gel electrophoresis using 6-8. mu.L of the PCR product.
1.6 comparison of the final concentration of different primers for NanPCR with conventional PCR sensitivity test
According to the reaction system of nano PCR and conventional PCR, the reaction system is configured according to the final concentration of different primers (1 mu mol/L, 0.5 mu mol/L and 0.1 mu mol/L respectively), and the prepared CAStV-I, CIAV and PRV total 12 recombinant plasmids are respectively diluted to 10 according to the calculated copy number in a gradient way6、105、104、103、102、101And 100Copies/. mu.L. And (3) comparing the sensitivity of the nano PCR and the conventional PCR method with different primer concentrations of the same pair of primers according to the reaction system and the optimal reaction temperature in the step 1.5, and taking the primer concentration with the highest sensitivity as the optimal primer concentration of the conventional PCR and the nano PCR.
2. Results
2.1 temperature optimization of different primers for PRV, CAStV and CIAV NanoPCR and conventional PCR
Selecting annealing temperature of 50-64 ℃, performing primer optimization on the PRV-1/2/3 fragment by using nano PCR, observing that the brightness of a band between 50-57 ℃ is not obviously different by naked eyes, obviously weakening the brightness of the band of the PRV-1/2 fragment at 64 ℃, gradually weakening the fragment of the PRV-3 fragment at 59-64 ℃, and selecting the lowest annealing temperature of 50 ℃ as the optimal annealing temperature of the PRV-1/2/3 fragment in order to improve the sensitivity of the PRV-1/2/3 fragment; during the conventional PCR amplification test, TaKaRa Taq was found to be causedTMSince Version 2.0plus dye cannot amplify the fragment of PRV-1/2, the fragment that can be efficiently amplified by using a fragment having a high GC content was used
The annealing temperature of the PCR Master Mix reagent is optimized, and the annealing temperatures of the PRV-1/2/3 fragments are determined to be 64 ℃, 63 ℃ and 63 ℃. The results of PRV-1/2/3 fragment nanophase PCR and conventional PCR temperature optimization are shown in FIG. 1 and Table 2.
TABLE 212 GC content of target genes, average Tm value of amplification primers, annealing temperature Table for NanoPCR and conventional PCR
Annealing temperature optimization is carried out on 9 target genes including CAStV-1/2/3/4 and CIAV-1/2/3/4/5 by using nano PCR, the condition that the brightness of bands of the CAStV-2/3/4 target genes and the CIAV-1/2/3/4/58 target genes is not obviously different at 50-57 ℃ can be observed by naked eyes, the annealing temperature of 50 ℃ is selected as the optimal temperature of CAStV-2/3/4 and CIAV-1/2/3/4/5 fragments, and the optimal annealing temperature of the CAStV-1 fragment nano PCR is 58 ℃; the optimal annealing temperatures for conventional PCR of CAStV-1/2/3/4 and CIAV-1/2/3/4/59 target genes were 58, 55, 52, 50, 55, 50, 58, 56 and 55 ℃ respectively, wherein the target genes having no significant difference in fragment brightness between 50-57 ℃ were CastV-2/4 and CIAV-2 fragments respectively, and thus the optimal annealing temperatures for CAStV-2/4 and CIAV-2 fragments were selected at 50 ℃ and the results are shown in Table 2.
In conclusion, the nanometer PCR of 11 segments including PRV-1/2/3, CAStV-2/3/4 and CIAV-1/2/3/4/5 has no obvious difference in the brightness of the electrophoretic bands of the products when the annealing temperature is 50-57 ℃, and the proportion is 91.7% (11/12); the conventional PCR of 3 segments including CAStV-2/4 and CIAV-2 has no obvious difference in band brightness at 50-57 ℃, the ratio is 25.0% (3/12), and the rest 9 target genes all need strict annealing temperature; the above shows that the primer design principle of nano PCR is more loose, and the close annealing temperature is not required to be deliberately pursued in the PCR reaction process.
2.2 correlation between GC content of different target genes, Tm value of primer and annealing temperature for PRV, CAStV and CIAV
After determining the annealing temperatures of 12 pairs of primer nano PCR and conventional PCR (see Table 2), performing correlation coefficient analysis on the GC content of the target gene, the average Tm value of the primers and the annealing temperatures of the nano PCR and the conventional PCR to find that the annealing temperatures of the nano PCR of 12 target genes are not significantly related to the GC content of the target gene and the Tm value of the primers (P > 0.05); the annealing temperature of the conventional PCR was not significantly correlated with the Tm value of the primer (P >0.05), but was positively correlated with the GC content of the target gene (P <0.01) (results are shown in table 3).
TABLE 312 GC content of target genes, average Tm of amplification primers, annealing temperature Table for nanopatterned PCR and conventional PCR
Note: the significance test of the correlation coefficient shows that the difference of the significance test of the correlation coefficient is significant at the level of P <0.01, and the significance test of the correlation coefficient shows that the difference of the significance test of the correlation coefficient is significant at the level of P < 0.05
In the research, annealing temperature optimization is carried out on the nano PCR and the conventional PCR of 12 target genes in total, and the result shows that the PRV-1/2/3, CAStV-1/3 and CIAV-1/3/4/5 fragments can ensure that the conventional PCR can reach the best by strict annealing temperature; in addition to CAStV-1, the range of the nano PCR annealing temperature of the other 11 target genes in the research is relatively loose; in addition, the correlation coefficient analysis is carried out on the GC content of 12 target genes, the average Tm value of amplification primers and the annealing temperature of nano PCR and conventional PCR, and the result proves that the annealing temperature of the conventional PCR and the GC content of the target genes have a positive correlation (P <0.01) and have no correlation with the average Tm value of the amplification primers of the target genes; the annealing temperature of the nano PCR has no correlation with the GC content of the target gene and the average Tm value of the amplification primer. The above data indicate that the annealing temperature of the conventional PCR is only related to the GC content of the target gene, and the annealing temperature of the nano-PCR is not related to the GC content of the target gene and the average Tm value of the primers, which indicates that the requirement of the nano-PCR reaction on the annealing temperature is relatively loose.
2.3 comparison of sensitivity of the final concentration NanPCR with different primers for PRV, CAStV and CIAV and conventional PCR
The optimal primer concentrations of the reaction system are optimized according to the final concentrations of different primers (1 mu mol/L, 0.5 mu mol/L and 0.1 mu mol/L respectively), and the sensitivity among different primer concentrations is compared to determine the optimal primer concentrations of the nano PCR and the conventional PCR. The results are shown in Table 4. Through comparison, the sensitivity of the PRV-1 fragment of the nano PCR is 10 times higher than that of the conventional PCR under the condition that the final concentration of the primer is 0.1 mu mol/L; the sensitivity of the conventional PCR of the PRV-2 and PRV-3 fragments is 10 times higher than that of the nano PCR under the optimal primer concentration; the sensitivity of nanopcr and conventional PCR for the remaining 9 fragments was consistent.
Table 412 results of sensitivity comparison between nanopattern PCR and conventional PCR of target genes at different primer concentrations
Note: the sensitivity result under different final concentrations of the primers is shown in copy/. mu.L, and the bold font is the lowest detection concentration of the target gene
The PRV whole genome is a genome with high GC content, and the GC content of the whole gene is up to 73 percent. The research compares the sensitivity of PRV same target gene of nano PCR and conventional PCR, in the test process, the conventional PCR reaction solution can not amplify the PRV-1/2 fragment with longer fragment, after the special PCR reaction solution with high-efficiency amplification and high GC content target gene is replaced, the PRV-1/2 fragment can be successfully amplified in the conventional PCR reaction, and the PRV-1/2/3 fragment can be amplified only when the temperature reaches above 60 ℃; compared with PRV nano PCR, PRV-1/2/3 can be amplified at 50-57 ℃ annealing temperature, and the requirement on annealing temperature is not high, so that nano PCR has more advantages than conventional PCR reaction solution in the amplification of longer target gene with high GC content.
In conclusion, for the target gene PRV gene with high GC content, the sensitivity of the nano PCR method is basically consistent with that of the conventional PCR method under the optimal reaction condition, and the nano PCR reaction solution is easier to amplify and has more advantages than the conventional PCR reaction solution; the optional annealing temperature range of the nano PCR reaction is loose.
Embodiment 2PRV detection method
The method comprises the following steps:
(1) preparation of template DNA
PRV virus DNA can be extracted according to the instructions in the EasyPure Viral DNA/RNA Kit.
(2) Nano PCR reaction
Reaction system (20 μ L): mu.L of cDNA/DNA template, 10. mu.L of 2 XNano-QPCR buffer, 0.1. mu.L of upstream and downstream primer mixture (final primer concentration 0.1. mu. mol/L) with working concentration 20. mu. mol/L, 0.32. mu.L of taq enzyme Mix (5U/uL), plus ddH2O make up to 20. mu.L.
The primer combination is PRV-1F/PRV-1R.
PCR reaction procedure: 3min at 95 ℃; carrying out 20 cycles of 94 ℃ for 5s, 50 ℃ for 5s and 72 ℃ for 30 s; 5min at 72 ℃ and finishing the reaction at 12 ℃.
(3) PCR product detection
The amplification results can be verified by electrophoresis in a 1.2% agarose gel.
Embodiment 3PRV detection method
The method comprises the following steps:
(1) preparation of template DNA
PRV virus DNA can be extracted according to the instructions in the EasyPure Viral DNA/RNA Kit.
(2) Nano PCR reaction
Reaction system (20 μ L): mu.L of cDNA/DNA template, 10. mu.L of 2 XNano-QPCR buffer, 1. mu.L of upstream and downstream primer mixture (final primer concentration of 1. mu. mol/L) with working concentration of 20. mu. mol/L, 0.32. mu.L of taq enzyme Mix (5U/uL), and ddH2O make up to 20. mu.L.
The primer combination is PRV-2F/PRV-2R.
PCR reaction procedure: 3min at 95 ℃; carrying out 20 cycles of 94 ℃ for 5s, 50 ℃ for 5s and 72 ℃ for 30 s; 5min at 72 ℃ and finishing the reaction at 12 ℃.
(3) PCR product detection
The amplification results can be verified by electrophoresis in a 1.2% agarose gel.
The method comprises the following steps:
(1) preparation of template DNA
PRV virus DNA can be extracted according to the instructions in the EasyPure Viral DNA/RNA Kit.
(2) Nano PCR reaction
Reaction system (20 μ L): mu.L of cDNA/DNA template, 10. mu.L of 2 XNano-QPCR buffer, 0.1-1. mu.L of upstream and downstream primer mixture (final concentration of primer is 0.1-1. mu. mol/L), 0.32. mu.L of taq enzyme Mix (5U/uL), and ddH2O make up to 20. mu.L.
The primer combination is selected from PRV-1F/PRV-1R, PRV-2F/PRV-2R or PRV-3F/PRV-3R.
PCR reaction procedure: 3min at 95 ℃; carrying out 20 cycles of 94 ℃ for 5s, 50 ℃ for 5s and 72 ℃ for 30 s; 5min at 72 ℃ and finishing the reaction at 12 ℃.
(3) PCR product detection
The amplification results can be verified by electrophoresis in a 1.2% agarose gel.
Sequence listing
<110> Guangxi Zhuang nationality autonomous region veterinary research institute
<120> a method for detecting pseudorabies virus
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgggccgtgt tctttgt 17
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcgccgtcgt agtagtcctc 20
<210> 3
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cgggccgtgt tctttgt 17
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggtagatgca gggctcgtac 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gggctgtacg tgctcgtgat 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tcgccgtcgt agtagtcctc 20
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atggattgga cycgcttyga tggc 24
<210> 8
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
agcagcagca taytcctcga cgcagt 26
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ggattggacc cgctttgatg 20
<210> 10
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tggagaattg cccagaagga tt 22
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tggcacaatt ccaaaagcac tc 22
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ttcacccaca tcccaaagac a 21
<210> 13
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cgcatccggc agattaga 18
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gtcatcccac agaacgag 18
<210> 15
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gcctaattgc tggctgccgc tagata 26
<210> 16
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ccatgggtat ggcctttgyc tgttac 26
<210> 17
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
aatgatgatg cagcccacgg actctt 26
<210> 18
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cgtcatcggt tgttcgcctt ttcg 24
<210> 19
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
cggaggagac agcggtatcg tagac 25
<210> 20
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tagcggcagc cagcaattag gc 22
<210> 21
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
aaatgagacc cgacgagc 18
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ttacccagtt gccagacc 18
<210> 23
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
cagctccttg ccactaag 18
<210> 24
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gtgtcccatg accaccaa 18
Claims (10)
1. A method of detecting pseudorabies virus, the method comprising the steps of: (1) amplifying a DNA template; (2) nano PCR amplification; (3) and PCR product detection, characterized in that the primer pair sequence in the nano PCR amplification is selected from SEQ ID No.1-2, SEQ ID No.3-4 or SEQ ID No.5-6 in the sequence table.
2. The method for detecting pseudorabies virus of claim 1, wherein the Nano PCR reaction system comprises 1. mu.L of DNA template, 10. mu.L of 2 XNano-QPCR buffer, final concentration of upstream and downstream primers of 0.1-1. mu. mol/L, 5U/uL taq enzyme mix of 0.32. mu.L, and ddH for each 20uL of reaction system2O make up to 20. mu.L.
3. The method of detecting pseudorabies virus according to claim 2, wherein said nanopcr reaction procedure is a reaction at 95 ℃ for 3 min; reaction at 94 ℃ for 5s, annealing temperature of 50-57 ℃ for 5s, and reaction at 72 ℃ for 30s, and performing 20 cycles; the reaction is finished at 72 ℃ for 5min and 12 ℃.
4. The method for detecting pseudorabies virus of claim 3, wherein the sequence of said primer pair is the nucleotide sequence shown in SEQ ID No.1-2 of the sequence Listing.
5. The method of claim 4, wherein the final concentration of the primers is 0.1 μmol/L.
6. The method of detecting pseudorabies virus of claim 4 or 5, wherein said annealing temperature is 50 ℃.
7. The method for detecting pseudorabies virus of claim 3, wherein the sequence of said primer pair is the nucleotide sequence shown in SEQ ID No.3-4 of the sequence Listing.
8. The method of claim 7, wherein the final concentration of the primers is 1 μmol/L.
9. The method of detecting pseudorabies virus of claim 7 or 8, wherein said annealing temperature is 50 ℃.
10. The method of claim 1, wherein the PCR product detection is performed by 1.2% agarose gel electrophoresis to verify the amplification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110401217.3A CN112899407A (en) | 2021-04-14 | 2021-04-14 | Method for detecting pseudorabies virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110401217.3A CN112899407A (en) | 2021-04-14 | 2021-04-14 | Method for detecting pseudorabies virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112899407A true CN112899407A (en) | 2021-06-04 |
Family
ID=76110422
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110401217.3A Pending CN112899407A (en) | 2021-04-14 | 2021-04-14 | Method for detecting pseudorabies virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112899407A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224995A (en) * | 2013-04-10 | 2013-07-31 | 中国农业科学院哈尔滨兽医研究所 | Nanometer PCR detection kit for rapidly identifying and diagnosing virulent virus and attenuated virus of porcine pseudorabies virus, and applications thereof |
| CN105506175A (en) * | 2015-06-12 | 2016-04-20 | 邓小红 | Mutiplex PCR detection kit for detecting PRRSV, PCV2, PRV and CSFV |
-
2021
- 2021-04-14 CN CN202110401217.3A patent/CN112899407A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224995A (en) * | 2013-04-10 | 2013-07-31 | 中国农业科学院哈尔滨兽医研究所 | Nanometer PCR detection kit for rapidly identifying and diagnosing virulent virus and attenuated virus of porcine pseudorabies virus, and applications thereof |
| CN105506175A (en) * | 2015-06-12 | 2016-04-20 | 邓小红 | Mutiplex PCR detection kit for detecting PRRSV, PCV2, PRV and CSFV |
Non-Patent Citations (3)
| Title |
|---|
| YAKUNLUO等: "Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 张民秀等: "PRV和CAstV-Ⅰ及CIAV纳米PCR检测方法与常规PCR检测方法敏感度的比较", 《中国兽医科学》 * |
| 马兴杰等: "鉴别猪伪狂犬病病毒强弱毒的高效纳米PCR检测试剂盒及初步应用", 《养猪》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111996191A (en) | Primer group and kit for simultaneously identifying African swine fever wild strain and gene deletion strain based on multiple qPCR technology | |
| CN111020062A (en) | Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strain and gene deletion strain | |
| CN106957927A (en) | African swine fever fluorescence PCR detection reagent, African swine fever fluorescence PCR detection reagent kit and its application | |
| CN110777220A (en) | Primer group, probe, RPA test strip kit and identification method | |
| CN112795706A (en) | Fluorescent probe primer group and kit for African swine fever virus P72 gene and application of fluorescent probe primer group and kit | |
| CN111254225A (en) | Mandarin fish frog virus and rhabdovirus double PCR detection kit and detection method | |
| Tong et al. | Rapid detection of Decapod iridescent virus 1 (DIV1) by recombinase polymerase amplification | |
| CN111118215A (en) | Fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting capripoxvirus virus | |
| CN113046489B (en) | Multiple RT-PCR primer group for detecting porcine astrovirus, kit and application thereof | |
| CN108676917B (en) | Dual nano PCR detection method for identifying CCoVI and CCoVII | |
| CN112831609A (en) | PCR primer, kit and method for detecting African swine fever virus MGF-505-1R gene | |
| CN113174446A (en) | One-step double RT-PCR detection method for bovine viral diarrhea virus typing | |
| CN108998575B (en) | Establishment of double PCR detection method for chicken parvovirus and chicken newcastle disease virus | |
| CN112899407A (en) | Method for detecting pseudorabies virus | |
| CN112941240B (en) | Primer pair, kit and method for detecting goose astrovirus and goose goblet virus | |
| Kumar et al. | A novel HRM-based gap-qRT-PCR for identification and quantitation of the vaccine and field strain (s) of lumpy skin disease virus | |
| CN112626278B (en) | Primer and probe for identifying canine distemper virus wild strain and vaccine strain and application | |
| CN113755565A (en) | Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying African swine fever wild viruses and vaccine strains | |
| CN111719020B (en) | Kit, primer and probe for detecting bovine rotavirus | |
| CN108866245B (en) | Establishment of triple PCR detection method for chicken parvovirus, avian influenza virus and newcastle disease virus | |
| CN112831608A (en) | Primer for detecting goat endemic intranasal tumor virus and application of primer in HRM detection reagent | |
| CN113718044A (en) | Primer, probe and kit for fluorescent quantitative detection of swine mycoplasma, mycoplasma microformis and new mycoplasma haemophilus | |
| CN111518953A (en) | Primer group, kit and method for double nano PCR detection of H7 and N2 subtype avian influenza virus | |
| CN111004868A (en) | Fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting goat intranasal tumor virus | |
| CN111321246A (en) | Canine distemper virus nano PCR detection kit and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210604 |
|
| RJ01 | Rejection of invention patent application after publication |