CN111763773B - Primer and probe for real-time fluorescent quantitative PCR detection of novel duck picornaviruses - Google Patents

Primer and probe for real-time fluorescent quantitative PCR detection of novel duck picornaviruses Download PDF

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CN111763773B
CN111763773B CN202010882043.2A CN202010882043A CN111763773B CN 111763773 B CN111763773 B CN 111763773B CN 202010882043 A CN202010882043 A CN 202010882043A CN 111763773 B CN111763773 B CN 111763773B
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陈翠腾
刘斌琼
陈珍
朱春华
蔡国漳
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Institute of Animal Husbandry and Veterinary of Fujian Academy of Agricultural Sciences
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Abstract

The invention relates to a primer and a probe for real-time fluorescence quantitative PCR detection of a novel duck micro ribonucleic acid virus, wherein the primer has the following sequence: the upstream primer DuMV-q-F:5'-GGATGGCTGGTGCAAAGGTT-3', downstream primer DuMV-q-R:5'-CTGTCCACTCCGCCACAATG-3'; the sequence of the probe is as follows: probe DuMV-q-probe:5'-TATGAAGCTCCCACCCCGCATTGTGCC-3', and its 5 '-end is labeled with a fluorescent reporter group FAM, and its 3' -end is labeled with Eclipse. The real-time fluorescence quantitative PCR detection method of the novel duck micro ribonucleic acid virus has the advantages of high sensitivity, good stability, strong specificity and good repeatability, and can detect 32.7 copies/. Mu.L at the lowest.

Description

Primer and probe for real-time fluorescent quantitative PCR detection of novel duck picornaviruses
Technical Field
The invention belongs to the field of animal infectious diseases, and particularly relates to a primer and a probe for real-time fluorescent quantitative PCR detection of a novel duck micro ribonucleic acid virus.
Background
The Real-time fluorescence quantitative PCR method (Real-time PCR) is to detect the PCR process in Real time through fluorescence signals in the PCR amplification process. Since the Ct value of a template and the initial copy number of the template have a linear relationship in the exponential phase of PCR amplification, the quantitative basis is established. Because of the shortcomings of conventional PCR, real-time qPCR has the advantages of simple operation, high sensitivity, good repeatability and the like, and has very rapid development, and has been involved in various fields of life science research, such as differential expression analysis of genes, SNP detection, allele detection, drug development, clinical diagnosis, transgenic research and the like. At present, according to the difference of fluorescent chemical substances used in real-time fluorescent quantitative PCR, the real-time fluorescent quantitative PCR technology mainly comprises two types of fluorescent dyes and fluorescent probes. Fluorescent probes are real-time fluorescent quantitative PCR techniques established based on the principle of fluorescence resonance energy transfer (fluorescence resonance energy transfer, FRET). The TaqMan probe is representative of hydrolysis probe, has high specificity to target sequence, and combines the specificity of primer, so that the specificity and accuracy of real-time fluorescent quantitative PCR technology are greatly improved, and oligonucleotide melting curve analysis is not needed after the reaction is finished, thereby shortening the experimental time. The TaqMan probe consists of 3 parts, wherein the 5 'end of the probe is marked with a fluorescent group, the middle part is a nucleotide sequence which can be specifically combined with a target gene, and the 3' end is marked with a quenching group.
The aquatic bird breeding amount in China is the first place in the world and is an important component part of the poultry industry, however, in recent years, a plurality of new infectious diseases are continuously generated in the aquatic bird industry in China, and the occurrence of the new infectious diseases has caused great economic loss for the aquatic bird industry in China and has become a key factor for severely restricting the healthy and stable development of the aquatic bird industry in China.
The picornaviridae (Picornaviridae Family) virus is an important zoonotic agent that can cause subclinical infections or severe diseases from mild fever to the heart, liver and central nervous system in humans and animals. The picornaviruses are icosahedral symmetrical, spherical, single-stranded positive-strand RNA viruses with diameters of about 20-30nm and without envelope. The picornaviruses comprise 63 viruses, and commonly known are foot and mouth disease viruses (Aphthoviruses), enteroviruses (Enteroviruses), cardioviruses (cardioviruses), hepatoviruses (hepatoviruses), and the like. The length of the picornaviridae virus gene is generally 6.6kb to 9.8kb, the genome generally only contains one large open reading frame (open reading frame, ORF), the 3 'end is Poly A tail, the 5' end has VPg protein which is covalently bound with the DNA, and the genome RNA is infectious. In recent years, with the intensive research of virus metagenomics, pathogens belonging to the genus Megrivirus of the family micro ribonucleic acid are successively discovered from turkeys, chickens, pigeons and ducks. Genomic analysis revealed that the genomic structural features of the novel duck micro ribonucleic acid virus (DuMV) are similar to those of turkey-derived Megrivirus and chicken-derived Megrivirus, and that their polyproteins contain characteristic motifs of typical micro ribonucleic acid viruses. Genetic evolution analysis shows that the novel duck micro ribonucleic acid virus (DuMV) is in the same genetic evolution branch as the novel micro ribonucleic acid virus of turkeys, chickens and pigeons of the genus Megrivirus.
However, no research report on a primer for carrying out TaqMan real-time fluorescent quantitative PCR detection on a newly identified duck novel micro ribonucleic acid virus (DuMV) is currently seen, and the establishment of the invention can fill the blank in the related fields at home and abroad. The TaqMan real-time fluorescent quantitative PCR detection method established by the invention can detect and accurately quantify the popular DuMV in the duck group, lays a foundation for developing new DuMV molecular epidemiological investigation and subsequent scientific prevention and control related diseases in the duck group, and has very important research significance.
Disclosure of Invention
The invention aims to provide a primer and a probe for real-time fluorescence quantitative PCR detection of a novel duck picornavirus and a using method thereof. The method has the advantages of high sensitivity, good stability, strong specificity and good repeatability, can detect 32.7 copies/. Mu.L at the minimum, can detect and accurately quantify the DuMV popular in the duck group by using the TaqMan real-time fluorescence quantitative PCR detection method established by the primer and the probe of the invention, lays a foundation for developing new DuMV molecular epidemiological investigation and subsequent scientific prevention and control related diseases in the duck group, and has very important research significance.
The aim of the invention is realized by the following technical scheme: a primer and a probe for real-time fluorescence quantitative PCR detection of a novel duck micro ribonucleic acid virus, wherein the primer has the following sequence:
the upstream primer DuMV-q-F:5'-GGATGGCTGGTGCAAAGGTT-3' the number of the individual pieces of the plastic,
the downstream primer DuMV-q-R:5'-CTGTCCACTCCGCCACAATG-3';
the sequence of the probe is as follows:
probe DuMV-q-probe:5'-TATGAAGCTCCCACCCCGCATTGTGCC-3', and its 5 '-end is labeled with a fluorescent reporter group FAM, and its 3' -end is labeled with Eclipse.
The real-time fluorescence quantitative PCR detection method for the novel duck micro ribonucleic acid virus by using the primer and the probe comprises the following steps:
(1) Extraction of nucleic acid and preparation of cDNA:
DuMV nucleic acid RNA was extracted according to the viral nucleic acid extraction Kit (EasyPure Viral DNA/RNA Kit). Using reverse transcription kitOne-Step gDNA Removal and cDNA Synthesis SuperMix) reverse transcribes the extracted RNA into cDNA for later use.
(2) Construction of a positive standard:
the DuMV nucleic acid cDNA prepared was used as a template, using the upstream primer DuMV-F3:5 '-TGCCTCTGTCTTTCAAACATCT-3' and downstream primer DuMV-R3: the 5 '-TCCTGAATTTCTGACAAGAGACT-3' was amplified by PCR, and the expected amplified fragment size was 771bp. Wherein the primer is synthesized in the Shanghai Co., ltd.
Amplification was performed using a 50. Mu.L system recommended for PCR amplification reagents (2 XPCR Master reagent) in which 25. Mu.L of 2 XPCR Master Mix reaction solution, the upstream/downstream primer (DuMV-F3/DuMV-R3) (primer concentration 10. Mu. Mol. L) -1 ) 1 μl of each 2 μ L, duMV nucleic acid cDNA template was added to the final reaction system with sterile deionized water at 50 μl. Mixing, performing PCR amplification under 94 deg.C for 5min, circulating, denaturing at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extending at 72 deg.C for 60s, and final extending at 72 deg.C for 10min after 30 cycles.
After the PCR amplification reaction is finished, the PCR product is identified by agarose gel electrophoresis of 1.0%, and the specific target fragment is subjected to gel cutting recovery by using an agarose gel recovery kit. Cloning the target gene fragment onto a pEASY-T1 vector according to the specification of a pEASY-T1 Simple Cloning Kit cloning connection kit, randomly picking 8 single colonies, culturing the single colonies in an ampicillin (100 mu g/mL) resistant LB liquid medium for 14h, and extracting corresponding plasmids by using a rapid plasmid small extraction kit. PCR amplificationThe extracted plasmid was identified by PCR using the primers (DuMV-F3/DuMV-R3) and conditions, and the screened positive recombinant plasmid was sent to the engineering (Shanghai) Co.Ltd for sequencing. After BLAST analysis, the positive recombinant plasmid, which meets the expectations of the experiment, was used as the standard (P-DuMV) for the study. After measuring its concentration by a spectrophotometer, the corresponding copy number was calculated to be 3.27×10 8 Copy/. Mu.L. After linearized enzyme cutting, continuous 10-fold ratio dilution is carried out to obtain the concentration of 3.27 multiplied by 10 7 Copy/. Mu.L to 3.27X10 0 Copy/. Mu.L, all frozen at-20deg.C for use.
(3) TaqMan real-time fluorescence quantitative PCR reaction condition optimization:
preparing a 20 mu L real-time fluorescent quantitative PCR reaction system according to a TaqMan instruction, optimizing different reaction conditions at different primer final concentrations, and determining the optimal reaction conditions of the established real-time fluorescent quantitative PCR method.
When the reaction system is optimized, primer concentration, probe concentration and the use amount of the primer and the probe are mainly optimized.
Primer concentration optimization: the concentrations of the upstream primer DuMV-q-F and the downstream primer DuMV-q-R are diluted to 2.5 mu mol/L, 5.0 mu mol/L, 10 mu mol/L and 20 mu mol/L respectively by doubling ratio, and the optimal concentration of the upstream primer DuMV-q-F and the downstream primer DuMV-q-R is determined to be 10 mu mol/L by analysis and comparison of test results.
Probe concentration optimization: the concentration ratio of the probe DuMV-q-probe was diluted to 2.5. Mu. Mol/L, 5.0. Mu. Mol/L, 10. Mu. Mol/L and 20. Mu. Mol/L, respectively, and the optimal concentration of the probe DuMV-q-probe was determined to be 10. Mu. Mol/L by analytical comparison of test results.
When the annealing and extension temperatures are optimized, the selected annealing and extension temperatures are 54 ℃, 56 ℃, 58 ℃,60 ℃, 62 ℃ and 64 ℃, and the optimal annealing and extension temperatures are determined to be 60 ℃ by the analysis and comparison of test results.
By the optimization method, the optimized TaqMan real-time fluorescence quantitative PCR optimal reaction system (20 mu L) is as follows: taqMan Mix 10. Mu.L, upstream/downstream primer (DuMV-q-F/DuMV-q-R, concentration of 10. Mu. Mol/L) 0.5. Mu.L, probe (DuMV-q-probe, concentration of 10. Mu. Mol/L) 1.0. Mu.L, template 1. Mu.L, and additional sterile deionized water to a final volume of 20. Mu.L. The optimized optimal reaction conditions of the real-time fluorescence quantitative PCR method are as follows: pre-denaturation at 95℃for 2min; the total of 30s,40 cycles of annealing and extension at 95℃for 10s and 60 ℃. After the cycle is completed, a corresponding dissolution curve is made.
(4) Establishment of a standard curve:
optimal reaction conditions of the optimized TaqMan real-time fluorescence quantitative PCR method are used for measuring the concentration of different plasmid (3.27 multiplied by 10) 6 ～3.27×10 2 Copy/. Mu.L) is used as a template, and a real-time fluorescent quantitative PCR amplification reaction is carried out to obtain an amplification kinetic curve (see FIG. 1). And drawing a standard curve of the established real-time fluorescence PCR method by taking the common logarithmic number (lgC) of the initial copy number of the standard product as an abscissa and taking a cycle number threshold (Ct value) as an ordinate.
As can be seen from the amplification kinetic curves of different concentration standards, the established TaqMan real-time fluorescent quantitative PCR method is 3.27×10 6 ～3.27×10 2 The copy/. Mu.L reaction range has a good linear relationship and the correlation coefficient is 1.00. The standard curve (see figure 2) Y= -3.36X+39.81 based on the detection DuMV real-time fluorescence quantitative PCR method is obtained by taking the common logarithm (lgC) of the copy number in each concentration standard template as the abscissa and taking the result of the occurring cycle number threshold (Ct value) as the ordinate, which shows that the established standard curve of the TaqMan real-time fluorescence quantitative PCR method has a good linear relationship.
Optimal reaction conditions of the optimized TaqMan real-time fluorescence quantitative PCR method are used for measuring the concentration of different plasmid (3.27 multiplied by 10) 3 ～3.27×10 0 Copy/. Mu.L) was used as a template for a real-time fluorescent quantitative PCR amplification reaction, resulting in a minimum detection limit of 32.7 copies/. Mu.L (see FIG. 3) according to the present invention.
The invention also provides application of the primer (the upstream primer DuMV-q-F and the downstream primer DuMV-q-R) and the probe (DuMV-q-probe) in preparation of a kit for detecting the novel duck micro ribonucleic acid virus.
The invention also provides a novel duck micro ribonucleic acid virus real-time fluorescence quantitative PCR detection kit, which comprises the primers (an upstream primer DuMV-q-F and a downstream primer DuMV-q-R) and a probe (DuMV-q-probe).
Advantageous effects
The invention uses the primer and the probe for the real-time fluorescence quantitative PCR detection of the novel duck micro ribonucleic acid virus to detect the novel duck micro ribonucleic acid virus, and has the following advantages:
1. and the detection is rapid and efficient: the detection method does not need to carry out conventional agarose gel electrophoresis detection, and can carry out result judgment through a program carried by a real-time fluorescent quantitative PCR machine after the reaction is finished.
2. The quantification is accurate: the method comprises the steps of preparing a standard substance, drawing a standard curve, directly judging the novel duck micro ribonucleic acid virus infection according to the Ct value in a sample to be detected, and accurately quantifying the infection degree.
3. The sensitivity is high: the minimum limit of detection was 32.7 copies/. Mu.L.
4. The specificity is strong: and common pathogens of waterfowl such as duck hepatitis virus 1 (DHAV-1), duck hepatitis virus 3 (DHAV-3), duck-origin Avian Influenza Virus (AIV), duck-origin avian paramyxovirus type 1 (APMV-1), muscovy Duck Reovirus (MDRV) and novel duck reovirus (N-DRV) have no response signals, and only fluorescent signals appear for detection of duck novel micro ribonucleic acid virus (DuMV) infection.
5. The repeatability is good: the built real-time fluorescence quantitative PCR detection method has the intra-group variation coefficient of 0.61-1.08% and the inter-group variation coefficient of 0.81-1.93% for DuMV detection, and shows that the built TaqMan real-time fluorescence quantitative PCR method has good repeatability.
Drawings
FIG. 1 is an amplification curve of a PCR method for quantitatively detecting duck novel picornaviruses by real-time fluorescence; wherein, 1: plasmid concentration was 3.27X10 6 Copy/. Mu.L; 2: plasmid concentration was 3.27X10 5 Copy/. Mu.L; 3: plasmid concentration was 3.27X10 4 Copy/. Mu.L; 4: plasmid concentration was 3.27X10 3 Copy/. Mu.L; 5: plasmid concentration was 3.27X10 2 Copy/. Mu.L.
FIG. 2 is a standard curve of PCR method for real-time fluorescent quantitative detection of duck novel picornaviruses.
FIG. 3 is a graph showing the result of a sensitivity test of a PCR method for quantitatively detecting a novel duck picornavirus by real-time fluorescence, wherein 1: plasmid concentration was 3.27X10 3 Copy/. Mu.L; 2: plasmid concentration was 3.27X10 2 Copy/. Mu.L; 3: plasmid concentration was 3.27X10 1 Copy/. Mu.L; 4: plasmid concentration was 3.27X10 0 Copy/. Mu.L.
FIG. 4 is a diagram of the specific detection result of the PCR method for quantitatively detecting the novel duck picornavirus by real-time fluorescence; wherein, 1: duMV; c: test controls (DHAV-1, DHAV-3, AIV, APMV-1, MDRV, and N-DRV) were not distinguishable by the naked eye.
Detailed Description
The present invention is described in detail below with reference to the drawings and examples of the specification:
example 1:
1. materials and methods
1.1 strains and strains
The pathogenic duck novel micro ribonucleic acid virus (DuMV, M19g24 strain), duck type 1 hepatitis virus (DHAV-1), duck type 3 hepatitis virus (DHAV-3), duck-derived Avian Influenza Virus (AIV), duck-derived avian type 1 paramyxovirus (APMV-1), muscovy Duck Reovirus (MDRV) and novel duck reovirus (N-DRV) for the test are all identified and stored by the institute of livestock and veterinary at the national academy of agricultural sciences of Fujian province.
1.2 primer design
According to the analysis and comparison result of the nucleotide sequence of the novel duck micro ribonucleic acid virus (DuMV), designing a specific primer by using primer design software, wherein the primer sequence is as follows:
the upstream primer DuMV-q-F:5'-GGATGGCTGGTGCAAAGGTT-3'
The downstream primer DuMV-q-R:5'-CTGTCCACTCCGCCACAATG-3'
The probe sequence is as follows:
DuMV-q-probe：5’-TATGAAGCTCCCACCCCGCATTGTGCC-3’
wherein, the 5 '-end of the probe DuMV-qprobe is marked with a fluorescent reporter group FAM, and the 3' -end of the probe is marked with Eclipse;
all primers and probes were synthesized by the company Shanghai, inc. of Biotechnology, inc. according to experimental expectations by primer-BLAST analysis.
1.3 extraction of nucleic acids and preparation of cDNA
DuMV, DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV nucleic acid RNA was extracted according to a viral nucleic acid extraction Kit (EasyPure Viral DNA/RNA Kit). Using reverse transcription kitOne-Step gDNA Removal and cDNA Synthesis SuperMix) reverse transcribes the extracted RNA into cDNA for later use.
1.4 construction of Positive standards
Primers were designed using Oligo 7 primer design software, the upstream primer DuMV-F3:5 '-TGCCTCTGTCTTTCAAACATCT-3', downstream primer DuMV-R3:5 '-TCCTGAATTTCTGACAAGAGACT-3', the expected amplified fragment size is 771bp. Amplification was performed using a 50. Mu.L system recommended for PCR amplification reagents (2 XPCR Master reagent) in which 25. Mu.L of 2 XPCR Master Mix reaction solution, the upstream/downstream primer (DuMV-F3/DuMV-R3) (primer concentration 10. Mu. Mol. L) -1 ) 1 μl of each 2 μ L, duMV nucleic acid cDNA template was added to the final reaction system with sterile deionized water at 50 μl. Mixing, performing PCR amplification under 94 deg.C for 5min, circulating, denaturing at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extending at 72 deg.C for 60s, and final extending at 72 deg.C for 10min after 30 cycles.
After the PCR amplification reaction is finished, the PCR product is identified by agarose gel electrophoresis of 1.0%, and the specific target fragment is subjected to gel cutting recovery by using an agarose gel recovery kit. Cloning the target gene fragment onto a pEASY-T1 vector according to the specification of a pEASY-T1 Simple Cloning Kit cloning connection kit, randomly picking 8 single colonies, culturing the single colonies in an ampicillin (100 mu g/mL) resistant LB liquid medium for 14h, and extracting corresponding plasmids by using a rapid plasmid small extraction kit. The extracted plasmid was identified by PCR using the primers (DuMV-F3/DuMV-R3) and conditions at the time of PCR amplification, and the screened positive recombinant plasmid was sent to the division of biological engineering (Shanghai) for sequencing. After BLAST analysis, it meets the expectations of experimentsAs a standard for this study (P-DuMV). After measuring its concentration by a spectrophotometer, the corresponding copy number was calculated to be 3.27×10 8 Copy/. Mu.L. After linearized enzyme cutting, continuous 10-fold ratio dilution is carried out to obtain the concentration of 3.27 multiplied by 10 7 Copy/. Mu.L to 3.27X10 0 Copy/. Mu.L, all frozen at-20deg.C for use.
1.5TaqMan real-time fluorescence quantitative PCR reaction condition optimization
Preparing a 20 mu L real-time fluorescent quantitative PCR reaction system according to a TaqMan instruction, optimizing different reaction conditions at different primer final concentrations, and determining the optimal reaction conditions of the established real-time fluorescent quantitative PCR method.
The optimized TaqMan real-time fluorescence quantitative PCR optimal reaction system (20 mu L) is as follows: taqMan Mix 10. Mu.L, upstream/downstream primer (DuMV-q-F/DuMV-q-R, concentration of 10. Mu. Mol/L) 0.5. Mu.L, probe (DuMV-q-probe, concentration of 10. Mu. Mol/L) 1.0. Mu.L template 1. Mu.L, and additional sterile deionized water to a final volume of 20. Mu.L. The optimized optimal reaction conditions of the real-time fluorescence quantitative PCR method are as follows: pre-denaturation at 95℃for 2min;95℃10s,60℃30s,40 cycles. After the cycle is completed, a corresponding dissolution curve is made.
1.6 establishment of standard curve
Optimal reaction conditions of the optimized TaqMan real-time fluorescence quantitative PCR method are used for measuring the concentration of different plasmid (3.27 multiplied by 10) 6 ～3.27×10 2 Copy/. Mu.L) is used as a template, and a real-time fluorescent quantitative PCR amplification reaction is carried out to obtain an amplification kinetic curve (see FIG. 1). And drawing a standard curve of the established real-time fluorescence PCR method by taking the common logarithmic number (lgC) of the initial copy number of the standard product as an abscissa and taking a cycle number threshold (Ct value) as an ordinate.
As can be seen from the amplification kinetic curves of different concentration standards, the established TaqMan real-time fluorescent quantitative PCR method is 3.27×10 6 ～3.27×10 2 The copy/. Mu.L reaction range has a good linear relationship and the correlation coefficient is 1.00. The common logarithm (lgC) of the copy number in each concentration standard template is taken as an abscissa, and the result of the threshold value (Ct value) of the cycle number appearing is taken as an ordinate, so that the real-time fluorescence quantitative PCR method based on detection of DuMV is obtainedY= -3.36X+39.81 (see FIG. 2), indicating that the established standard curve of the TaqMan real-time fluorescent quantitative PCR method has a good linear relationship.
1.7 sensitivity test
Optimal reaction conditions of the optimized TaqMan real-time fluorescence quantitative PCR method are used for measuring the concentration of different plasmid (3.27 multiplied by 10) 3 ～3.27×10 0 Copy/. Mu.L) is used as a template, a real-time fluorescent quantitative PCR amplification reaction is carried out, and the lowest detection limit of the established real-time fluorescent quantitative PCR method is determined.
The optimized TaqMan real-time fluorescence quantitative PCR method is used for detecting plasmids with different concentrations, and the result shows that the lowest detection limit is 3.27 multiplied by 10 (see figure 3) 1 Copy/. Mu.L (i.e., 32.7 copies/. Mu.L).
1.8 specificity test
And (3) performing real-time fluorescence quantitative PCR amplification reaction on nucleic acid RNA extracted from common pathogens of duck groups, such as DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV, by using the optimized optimal reaction conditions of the TaqMan real-time fluorescence quantitative PCR method and taking DuMV as a positive control, and evaluating the specificity of the established real-time fluorescence quantitative PCR method.
As can be seen from the amplification curve (see FIG. 4), the established real-time fluorescent quantitative PCR method only has positive amplification signals for DuMV detection, but has no positive amplification fluorescent signals for other common waterfowl pathogens (such as DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV), and the established real-time fluorescent quantitative PCR method has strong specificity.
1.9 coefficient of variation determination
Optimal reaction conditions of the optimized TaqMan real-time fluorescence quantitative PCR method are used for measuring the concentration of different plasmid (3.27 multiplied by 10) 6 Copy/. Mu.L, 3.27X10 4 Copy/. Mu.L, 3.27X10 2 Copy/. Mu.L) was used as template for detection. The content of each standard was repeated 3 times, and the intra-group (intra-group) variation coefficient was calculated. And respectively subpackaging the standard substances, then storing at-20 ℃, taking out every 7d, detecting by using an optimized real-time fluorescence quantitative PCR method for 3 times, and calculating an inter-group variation coefficient.
The repeated tests of the standard substances with different dilutions in groups and between groups are respectively carried out, and the results show (see table 1) that the variation coefficient in the groups is 0.61-1.08%, and the variation coefficient between groups is 0.81-1.93%, which shows that the real-time fluorescence quantitative PCR method of TaqMan established in the study has good repeated performance.
TABLE 1 coefficients of intra-and inter-group variation for real-time fluorescent quantitative PCR
2. Clinical sample detection
According to 82 clinical detection duck source diseases, the method is carried out according to a conventional method, corresponding nucleic acid RNA is extracted by using a virus nucleic acid extraction Kit EasyPure Viral DNA/RNA Kit and then is reversely transcribed into cDNA, and DuMV infection is detected by using an optimized TaqMan real-time fluorescent quantitative PCR method. As a result, 11 parts of DuMV are detected as positive (Ct values of 28.01, 18.39, 20.31, 30.22, 19.89, 21.24, 26.01, 19.33, 30.08, 22.55, 24.67, respectively) and the positive rate is 13.41%, indicating that the established method can be used for molecular epidemiological investigation of DuMV and subsequent development of pathogenesis-related studies.
Sequence listing
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tatgaagctc ccaccccgca ttgtgcc 27

Claims (1)

1. A novel duck micro ribonucleic acid virus real-time fluorescence quantitative PCR detection kit is characterized in that: the kit comprises a primer and a probe for real-time fluorescence quantitative PCR detection of the novel duck picornavirus, wherein the primer has the following sequence:
the upstream primer DuMV-q-F:5'-GGATGGCTGGTGCAAAGGTT-3' the number of the individual pieces of the plastic,
the downstream primer DuMV-q-R:5'-CTGTCCACTCCGCCACAATG-3';
the sequence of the probe is as follows:
probe DuMV-q-probe:5'-TATGAAGCTCCCACCCCGCATTGTGCC-3', and its 5 '-end is labeled with a fluorescent reporter group FAM, and its 3' -end is labeled with Eclipse;
the reaction system of the kit 20. Mu.L is as follows: taqMan Mix 10. Mu.L, upstream primer DuMV-q-F0.5. Mu.L at a concentration of 10. Mu. Mol/L, downstream primer DuMV-q-R0.5. Mu.L at a concentration of 10. Mu. Mol/L, probe DuMV-q-probe 1.0. Mu.L at a concentration of 10. Mu. Mol/L, template 1. Mu.L, additional sterile deionized water to a final volume of 20. Mu.L;
the reaction conditions are as follows: pre-denaturation at 95℃for 2min; 95. 10s at 60 ℃ and 30s for 40 cycles of annealing and extension; after the cycle is completed, a corresponding dissolution curve is made.
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CN112458213A (en) * 2020-12-16 2021-03-09 福建省农业科学院畜牧兽医研究所 Novel loop-mediated isothermal amplification detection primer group and kit for duck picornaviruses

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