CN111893218B - Primer and probe for real-time fluorescent quantitative PCR detection of duck hepatitis C virus - Google Patents

Primer and probe for real-time fluorescent quantitative PCR detection of duck hepatitis C virus Download PDF

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CN111893218B
CN111893218B CN202010882033.9A CN202010882033A CN111893218B CN 111893218 B CN111893218 B CN 111893218B CN 202010882033 A CN202010882033 A CN 202010882033A CN 111893218 B CN111893218 B CN 111893218B
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CN111893218A (en
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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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    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
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Abstract

The invention relates to a primer and a probe for real-time fluorescent quantitative PCR detection of duck hepatitis C virus, wherein the sequence of the primer is an upstream primer DuHCV-qF: 5'-GGTGTTCCTGTCACTGCTCG-3', downstream primer DuHCV-qR: 5'-TAGACGAACCAGGCTCAGCA-3', respectively; the sequence of the probe is as follows: probe DuHCV-qprobe: 5'-ACGAGCGACCTCCCAAAGGTGCTGT-3', wherein the 5 '-end is marked with a fluorescent reporter group FAM and the 3' -end is marked with Eclipse. The method for real-time fluorescent quantitative PCR detection of the duck hepatitis C virus, which is established by using the primers and the probe, has the advantages of high detection speed, high efficiency, accurate quantification, high sensitivity, strong specificity, good repeatability and capability of detecting 35.0 copies/. mu.L at least.

Description

Primer and probe for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of duck hepatitis C virus
Technical Field
The invention belongs to the field of poultry pathology, and particularly relates to a primer and a probe for real-time fluorescent quantitative PCR detection of duck hepatitis C virus.
Background
The Real-time fluorescent quantitative PCR method (Real-time PCR) is to detect the PCR process in Real time through a fluorescent signal in the PCR amplification process. In the exponential phase of PCR amplification, the Ct value of the template and the initial copy number of the template have a linear relationship, and therefore, the method becomes a basis for quantification. Due to the defects of the conventional PCR, the real-time qPCR has been developed rapidly due to its advantages of simple operation, high sensitivity, good repeatability, etc., and has been involved in various fields of life science research, such as gene differential expression analysis, SNP detection, allele detection, drug development, clinical diagnosis, transgenic research, etc. At present, the real-time fluorescence quantitative PCR technology mainly comprises two types of fluorescent dyes and fluorescent probes according to different fluorescent chemical substances used by the real-time fluorescence quantitative PCR. The fluorescent probe is a real-time fluorescent quantitative PCR technology established based on the principle of Fluorescence Resonance Energy Transfer (FRET). The TaqMan probe is a representative of a hydrolysis probe, has high specificity on a target sequence, and is combined with the specificity of a primer, so that the specificity and the accuracy of a real-time fluorescent quantitative PCR technology are greatly improved, oligonucleotide melting curve analysis is not needed after the reaction is finished, and the experiment time is shortened. The TaqMan probe consists of 3 parts, wherein a fluorescent group is marked at the 5 'end of the probe, a nucleotide sequence which can be specifically combined with a target gene is arranged in the middle of the probe, and a quenching group is marked at the 3' end of the probe.
The aquatic bird breeding amount of China is the first in the world and is an important component of poultry industry, however, in recent years, various new infectious diseases appear in the aquatic birds of China in succession without breaking off, and the occurrence of the new infectious diseases causes huge economic loss to the aquatic bird industry of China and becomes a key factor which seriously restricts the healthy and stable development of the aquatic bird industry of China.
According to the latest classification of the International Committee for viral Classification (International Committee on Taxonomy of Viruses), 4 virus genera were set under the Flaviviridae Family (Flaviviridae Family): flaviviruses (Flavivirus), 53 virus species, some of which are zoonotic pathogens such as epidemic encephalitis b virus, dengue virus; hepacivirus type c (hepacivirus), 14 virus species; pegivirus genus, 11 species; pestiviruses (pestiviruses) are 11 species, and are commonly exemplified by classical swine fever virus and bovine viral diarrhea virus. Hepatitis C Virus (HCV) is a single-stranded positive-stranded RNA virus belonging to the genus hepatitis C virus of the family flaviviridae, the virion of which is spherical and has a diameter of about 40-60nm, and the nucleocapsid is icosahedral in symmetry and surrounds a lipid-containing envelope with spikes. Duck hepatitis c virus (DuHCV) is a newly discovered hepatitis c virus from a duck group suffering from egg laying abnormality, the genome of the duck hepatitis c virus has the characteristics of general hepatitis c, is single-stranded positive-strand RNA, has the length of 11422bp, encodes an Open Reading Frame (ORF) with the length of 10824bp, and encodes a polyprotein with the length of 3607 amino acids.
However, no research report on a primer for performing TaqMan real-time fluorescent quantitative PCR detection on newly discovered duck hepatitis C virus (DuHCV) is found at present, and the establishment of the invention can fill the blank of related fields at home and abroad. The TaqMan real-time fluorescent quantitative PCR detection method established by the invention can be used for detecting and accurately quantifying the DuHCV prevalent in the duck group, lays a foundation for the development of new DuHCV molecular epidemiological investigation and subsequent scientific prevention and control of 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 fluorescent quantitative PCR detection of duck hepatitis C virus and a using method thereof. The method has the advantages of high sensitivity, good stability, strong specificity and good repeatability, can detect 35.0 copies/mu L at least, can detect and accurately quantify DuHCV (DuHCV) popular in ducks by using the TaqMan real-time fluorescence quantitative PCR detection method established by the primer and the probe, lays a foundation for developing new DuHCV molecular epidemiological investigation and subsequent scientific prevention and control related diseases in ducks, and has very important research significance.
The purpose of the invention is realized by the following technical scheme: a primer and a probe for real-time fluorescent quantitative PCR detection of duck hepatitis C virus are disclosed, wherein the primer sequence is as follows:
the upstream primer DuHCV-qF: 5'-GGTGTTCCTGTCACTGCTCG-3' the flow of the air in the air conditioner,
the downstream primer DuHCV-qR: 5'-TAGACGAACCAGGCTCAGCA-3', respectively;
the sequence of the probe is as follows:
probe DuHCV-qprobe: 5'-ACGAGCGACCTCCCAAAGGTGCTGT-3', and the 5 '-end thereof is labeled with a fluorescent reporter group FAM and the 3' -end thereof is labeled with Eclipse.
The real-time fluorescent quantitative PCR detection method of the primers and the probes for the duck hepatitis C virus comprises the following steps:
(1) extraction of nucleic acid and preparation of cDNA:
DuHCV nucleic acid RNA was extracted according to the Viral nucleic acid extraction Kit (EasyPure Viral DNA/RNA Kit). Using a reverse transcription kit (
Figure BDA0002654373700000031
One-Step gDNA Removal and cDNA Synthesis SuperMix) reverse transcribing the extracted RNA into cDNA for use.
(2) Construction of positive standard: using the prepared DuHCV nucleic acid cDNA as a template, the dna sequence of the dna fragment was amplified using the upstream primer DuHCV-1F: 5 '-TATATCTCGCACATCAGGTCGCT-3' and the downstream primer DuHCV-1R: 5 '-AGGACTGTTAGGAGGTGGTT-3' was amplified by PCR, and the expected amplified fragment size was 445 bp. Wherein the primer is synthesized by Biotechnology engineering (Shanghai) GmbH.
Amplification was performed using a 50. mu.L system recommended by PCR amplification reagents (2 XPCR Master reagent), in which 25. mu.L of 2 XPCR Master Mix reaction solution, and upstream/downstream primers (DuHCV-1F/DuHCV-1R) (primer concentration 10. mu. mol. L) -1 ) mu.L of each 2. mu. L, DuHCV nucleic acid cDNA template was supplemented with sterile deionized water to a final reaction volume of 50. mu.L. Mixing, performing PCR amplification under the conditions of pre-denaturation at 94 deg.C for 5min, circulating, denaturation at 94 deg.C for 30s, annealing at 53.5 deg.C for 30s, and extension at 72 deg.C for 40s, and final extension at 72 deg.C for 10min after 30 cycles.
After the PCR amplification reaction is finished, identifying the PCR product by using 1.0% agarose gel electrophoresis, and performing gel cutting recovery on the specific target fragment by using an agarose gel recovery kit. Cloning the target gene fragment onto pEASY-T1 vector according to pEASY-T1Simple Cloning Kit Cloning connection Kit instruction, randomly picking 8 single colonies, culturing for 14h in LB liquid culture medium with ampicillin resistance (the content is 100 mu g/mL), and extracting corresponding plasmid by using rapid plasmid miniextraction Kit. PCR identification is carried out on the extracted plasmid by using a primer (DuHCV-1F/DuHCV-1R) and conditions during PCR amplification, and the screened positive recombinant plasmid is sent to the company of bioengineering (Shanghai) and Limited for sequencing. After BLAST analysis, the positive recombinant plasmid that was expected from the experiment was used as the standard (P-DuHCV) in this study. After measuring the concentration with a spectrophotometer, the corresponding copy number was calculated to be 3.50X 10 8 Copy/. mu.L. After linearized enzyme digestion, the mixture is continuously diluted by 10 times to obtain a concentration of 3.50X 10 7 Copy/. mu.L to 3.50X 10 0 Copies/. mu.L were frozen at-20 ℃ until use.
(3) Optimizing the reaction conditions of TaqMan real-time fluorescent quantitative PCR:
preparing a 20 mu L real-time fluorescent quantitative PCR reaction system according to TaqMan instructions, optimizing different reaction conditions at different final concentrations of primers and probes, and determining the optimal reaction conditions of the established real-time fluorescent quantitative PCR method.
When the reaction system is optimized, the concentration of the primer, the concentration of the probe and the dosage of the primer and the probe are mainly optimized.
Optimizing the concentration of the primer: the concentrations of the upstream primer DuHCV-qF and the downstream primer DuHCV-qR are diluted to 2.5 mu mol/L, 5.0 mu mol/L, 10 mu mol/L and 20 mu mol/L respectively, and then the optimal concentrations of the upstream primer DuHCV-qF and the downstream primer DuHCV-qR are determined to be 10 mu mol/L through analysis and comparison of test results.
Optimizing the concentration of the probe: the concentration multiple ratio of the probe DuHCV-qprobe is diluted to 1.25. mu. mol/L, 2.5. mu. mol/L, 5.0. mu. mol/L, 10. mu. mol/L and then detected, and the optimum concentration of the probe DuHCV-qprobe is determined to be 5. mu. mol/L by analytical comparison of the test results.
When the annealing and extension temperatures are optimized, the annealing and extension temperatures are selected to be 53 ℃, 55 ℃, 57 ℃, 59 ℃, 61 ℃ and 63 ℃, and the optimal annealing and extension temperatures are both determined to be 59 ℃ through analysis and comparison of test results.
By the optimization method, the optimized TaqMan real-time fluorescent quantitative PCR optimal reaction system (20 mu L) is as follows: TaqMan qPCR Mix 10. mu.L, upstream/downstream primers (DuHCV-qF/DuHCV-qR, concentration 10. mu. mol/L each), 0.3. mu.L probe (DuHCV-qprobe, concentration 5. mu. mol/L), template 1. mu.L, and sterile deionized water was supplemented to a final volume of 20. mu.L. The optimized real-time fluorescent quantitative PCR method has the following optimal reaction conditions: pre-denaturation at 95 ℃ for 2 min; annealing and extension at 95 ℃ for 10s and 59 ℃ for 30s and 40 cycles.
(4) Establishment of a Standard Curve
The optimized optimal reaction conditions of the TaqMan real-time fluorescent quantitative PCR method are adopted, and the optimal reaction conditions are different in plasmid concentration (3.50 multiplied by 10) 5 ～3.50×10 1 Copy/. mu.L) as a template, and performing real-time fluorescent quantitative PCR amplification reaction to obtain an amplification kinetic curve (see figure 1). And drawing a standard curve of the established real-time fluorescence PCR method by taking the common logarithm (lgC) of the initial copy number of the standard substance as an abscissa and taking the cycle number threshold (Ct value) as an ordinate.
As can be seen from the amplification kinetic curves of the standard substances with different concentrations, the established TaqMan real-time fluorescent quantitative PCR method is 3.50 multiplied by 10 5 ～3.50×10 1 The copy/. mu.L reaction range has good linear relation, and the correlation coefficient is 0.999. Taking the common logarithm of copy number (lgC) in each concentration standard template as an abscissa and the result of the appeared cycle number threshold (Ct value) as an ordinate, the equation of the standard curve (see figure 2) based on the real-time fluorescence quantitative PCR method for detecting DuHCV is-3.54X +36.86, which indicates that the established standard curve of the TaqMan real-time fluorescence quantitative PCR method has a good linear relationship.
The optimized optimal reaction conditions of the TaqMan real-time fluorescent quantitative PCR method are adopted, and the optimal reaction conditions are different in plasmid concentration (3.50 multiplied by 10) 2 ～3.50×10 0 Copy/. mu.L) as a template, and performing real-time fluorescent quantitative PCR amplification reaction to obtain the lowest detection limit of 35.0 copy/. mu.L (see figure 3).
The invention also provides application of the primer (DuHCV-qF/DuHCV-qR) and the probe (DuHCV-qprobe) in preparation of a kit for detecting duck hepatitis C virus.
Advantageous effects
The invention utilizes the primers and the probes for the real-time fluorescent quantitative PCR detection of the duck hepatitis C virus to detect the duck hepatitis C virus, and has the following advantages:
1. the detection is rapid and efficient: the detection method does not need to carry out conventional agarose gel electrophoresis detection, and the result can be judged by a program carried by a real-time fluorescent quantitative PCR machine after the reaction is finished.
2. The quantification is accurate: through preparing a standard substance and drawing a standard curve, the duck hepatitis C virus infection is directly judged according to the Ct value in a sample to be detected, and the infection degree of the duck hepatitis C virus infection can be accurately quantified.
3. The sensitivity is high: the minimum detection limit was 35.0 copies/. mu.L.
4. The specificity is strong: and common pathogens of waterfowls (such as duck hepatitis virus 1 (DHAV-1), duck hepatitis virus 3 (DHAV-3), duck Avian Influenza Virus (AIV), duck paramyxovirus 1 (APMV-1), Muscovy Duck Reovirus (MDRV) and novel duck reovirus (N-DRV)) have no reaction signals, and only a specific fluorescent signal appears when the waterfowl hepatitis virus C (DuHCV) is infected and detected.
5. The repeatability is good: the coefficient of variation in the group of the real-time fluorescent quantitative PCR detection method for DuHCV detection is 0.62-1.18%, and the coefficient of variation between the groups is 0.73-1.66%, which shows that the real-time quantitative PCR method established by the invention has good repeatability.
Drawings
FIG. 1 is an amplification curve of a PCR method for real-time fluorescent quantitative detection of duck hepatitis C virus; wherein, 1: the plasmid concentration was 3.50X 10 5 Copy/. mu.L; 2: the plasmid concentration was 3.50X 10 4 Copy/. mu.L; 3: the plasmid concentration was 3.50X 10 3 Copy/. mu.L; 4: the plasmid concentration was 3.50X 10 2 Copy/. mu.L; 5: the plasmid concentration was 3.50X 10 1 Copy/. mu.L.
FIG. 2 is a standard curve of PCR method for real-time fluorescence quantitative detection of duck hepatitis C virus.
FIG. 3 is a graph showing the results of the sensitivity test of the PCR method for real-time fluorescent quantitative detection of duck hepatitis C virus; wherein, 1: the concentration of the plasmid is 3.50X 10 2 Copy/. mu.L; 2 plasmid concentration of 3.50X 10 1 Copy/. mu.L; 3 plasmid concentration of 3.50X 10 0 Copy/. mu.L.
FIG. 4 is a diagram showing the results of the PCR method for real-time fluorescent quantitative determination of duck hepatitis C virus; wherein, 1: DuHCV; c: the test controls (DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV) were not effectively distinguished by eye.
Detailed Description
The 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
Pathogenic duck hepatitis C virus (DuHCV, strain H36a 8), duck hepatitis 1 virus (DHAV-1), duck hepatitis 3 virus (DHAV-3), duck Avian Influenza Virus (AIV), duck avian paramyxovirus 1 (APMV-1), Muscovy Duck Reovirus (MDRV) and novel duck reovirus (N-DRV) for the test are identified and stored by the animal husbandry and veterinary institute of agricultural and scientific institute of Fujian province.
1.2 primer design
According to the nucleotide sequence analysis and comparison results of duck hepatitis C virus (DuHCV) and other duck source flavivirus pathogenic genomes, a specific primer and a specific probe are designed by utilizing primer design software, wherein the primer sequence is as follows:
the upstream primer DuHCV-qF: 5'-GGTGTTCCTGTCACTGCTCG-3'
The downstream primer DuHCV-qR: 5'-TAGACGAACCAGGCTCAGCA-3'
The sequence of the probe is as follows:
probe DuHCV-qprobe: 5'-ACGAGCGACCTCCCAAAGGTGCTGT-3'
Wherein, the probe DuHCV-qprobe is labeled with a fluorescent reporter group FAM at the 5 '-end and Eclipse at the 3' -end;
all primers and probes were synthesized by Biotechnology engineering (Shanghai) GmbH and were in line with experimental expectations by primer-BLAST analysis.
1.3 extraction of nucleic acids and preparation of cDNA
Nucleic acid RNAs of DuHCV, DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV were extracted according to the Viral nucleic acid extraction Kit (easy pure Viral DNA/RNA Kit). Using a reverse transcription kit (
Figure BDA0002654373700000061
One-Step gDNA Removal and cDNA Synthesis SuperMix) reverse transcribing the extracted RNA into cDNA for use.
1.4 construction of Positive Standard
Primers were designed using Oligo 7 primer design software, with the upstream primer DuHCV-1F: 5 '-TATATCTCGCACATCAGGTCGCT-3', downstream primer DuHCV-1R: 5 '-AGGACTGTTAGGAGGTGGTT-3', the expected amplified fragment size is 445 bp. Amplification was performed using a 50. mu.L system recommended by PCR amplification reagents (2 XPCR Master reagent) with 25. mu.L of 2 XPCR Master Mix reaction solution, and upstream/downstream primers (DuHCV-1F/DuHCV-1R) (primer concentration 10. mu. mol. L) -1 ) mu.L of each 2. mu. L, DuHCV nucleic acid cDNA template was supplemented with sterile deionized water to the final reactionIs 50 μ L. Mixing uniformly, performing PCR amplification, performing cycle after the amplification condition is 94 ℃ pre-denaturation for 5min, performing 94 ℃ denaturation for 30s, 53.5 ℃ annealing for 30s, and 72 ℃ extension for 40s, and performing 72 ℃ final extension for 10min after 30 cycles.
After the PCR amplification reaction is finished, identifying the PCR product by using 1.0% agarose gel electrophoresis, and performing gel cutting recovery on the specific target fragment by using an agarose gel recovery kit. Cloning the target gene fragment onto pEASY-T1 vector according to pEASY-T1Simple Cloning Kit Cloning connection Kit instruction, randomly picking 8 single colonies, culturing for 14h in LB liquid culture medium with ampicillin resistance (the content is 100 mu g/mL), and extracting corresponding plasmid by using rapid plasmid miniextraction Kit. PCR identification is carried out on the extracted plasmid by using a primer (DuHCV-1F/DuHCV-1R) and conditions during PCR amplification, and the screened positive recombinant plasmid is sent to the company of bioengineering (Shanghai) and Limited for sequencing. After BLAST analysis, the positive recombinant plasmid that was expected from the experiment was used as the standard (P-DuHCV) in this study. After measuring the concentration with a spectrophotometer, the corresponding copy number was calculated to be 3.50X 10 8 Copies/. mu.L. After linearized enzyme digestion, the product is diluted by 10 times in succession to obtain a concentration of 3.50X 10 7 Copy/. mu.L to 3.50X 10 0 Copies/. mu.L were frozen at-20 ℃ until use.
1.5 TaqMan real-time fluorescent quantitative PCR reaction condition optimization
Preparing a 20 mu L real-time fluorescent quantitative PCR reaction system according to the TaqMan specification, optimizing different reaction conditions at different final concentrations of primers and probes, and determining the optimal reaction conditions of the established real-time fluorescent quantitative PCR method.
The optimized TaqMan real-time fluorescent quantitative PCR optimal reaction system (20 mu.L) is as follows: TaqMan qPCR Mix 10. mu.L, upstream/downstream primers (DuHCV-qF/DuHCV-qR, concentration 10. mu. mol/L each), 0.3. mu.L probe (DuHCV-qprobe, concentration 5. mu. mol/L), template 1. mu.L, and sterile deionized water was supplemented to a final volume of 20. mu.L. The optimized real-time fluorescent quantitative PCR method has the optimal reaction conditions that: pre-denaturation at 95 ℃ for 2 min; 95 ℃ for 10s, 59 ℃ for 30s, 40 cycles.
1.6 creation of Standard Curve
Optimized optimal reaction conditions of the TaqMan real-time fluorescent quantitative PCR method are adopted, and different plasmid concentrations (3.50 multiplied by 10) 5 ～3.50×10 1 Copy/. mu.L) as a template, and performing real-time fluorescent quantitative PCR amplification reaction to obtain an amplification kinetic curve (see figure 1). And drawing an established standard curve of the real-time fluorescent PCR method by taking the common logarithm (lgC) of the initial copy number of the standard substance as an abscissa and taking a cycle number threshold (Ct value) as an ordinate.
As can be seen from the amplification kinetic curves of the standard substances with different concentrations, the established TaqMan real-time fluorescence quantitative PCR method is 3.50 multiplied by 10 5 ～3.50×10 1 The copy/. mu.L reaction range has good linear relation, and the correlation coefficient is 0.999. Taking the common logarithm of copy number (lgC) in each concentration standard template as an abscissa and the result of the appearing cycle number threshold (Ct value) as an ordinate, the equation of the standard curve (see figure 2) based on the real-time fluorescent quantitative PCR method for detecting DuHCV is obtained to be-3.54X +36.86, which indicates that the established standard curve of the TaqMan real-time fluorescent quantitative PCR method has a good linear relationship.
1.7 sensitivity test
Optimized optimal reaction conditions of the TaqMan real-time fluorescent quantitative PCR method are adopted, and different plasmid concentrations (3.50 multiplied by 10) 2 ～3.50×10 0 Copy/. mu.L) as a template, performing real-time fluorescence quantitative PCR amplification reaction, and determining the lowest detection limit of the established real-time fluorescence quantitative PCR method.
The optimized TaqMan real-time fluorescent quantitative PCR method is used for detecting plasmids with different concentrations, and the result shows that (shown in figure 3) the lowest detection limit is 3.50 multiplied by 10 1 Copies/. mu.L (i.e., 35.0 copies/. mu.L).
1.8 specificity test
And (3) performing real-time fluorescent quantitative PCR amplification reaction by using the optimized optimal reaction conditions of the TaqMan real-time fluorescent quantitative PCR method, taking DuHCV as a positive control, performing reverse transcription on nucleic acid RNA extracted from common duck pathogens such as DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV into cDNA as a template, and evaluating the specificity of the established real-time fluorescent quantitative PCR method.
As can be seen from the amplification curve (see figure 4), the established real-time fluorescent quantitative PCR method only has positive amplification signals for DuHCV detection, and has no positive amplification fluorescent signals for other common pathogens of waterfowl (such as DHAV-1, DHAV-3, AIV, APMV-1, MDRV and N-DRV), which indicates that the established real-time fluorescent quantitative PCR method has strong specificity.
1.9 coefficient of variation determination
Optimized optimal reaction conditions of the TaqMan real-time fluorescent quantitative PCR method are adopted, and different plasmid concentrations (3.50 multiplied by 10) 6 Copy/. mu.L, 3.50X 10 4 Copy/. mu.L, 3.50X 10 2 Copy/. mu.L) as template. The content of each standard substance is repeated for 3 times, and the intra-group (intra-group) variation coefficient is calculated. Respectively subpackaging the above standard substances, storing at-20 deg.C, taking out every 7d, detecting with optimized real-time fluorescence quantitative PCR method for 3 times, and calculating inter-group (inter-group) variation coefficient.
The standard substances with different dilutions are respectively subjected to the repeatability tests in groups and between groups, and the results show that (shown in table 1) the coefficient of variation in the groups is 0.62-1.18%, and the coefficient of variation between the groups is 0.73-1.66%, which indicates that the real-time quantitative PCR method established by the research has good repeatability.
TABLE 1 Intra-and inter-group coefficient of variation for real-time fluorescent quantitative PCR
Figure BDA0002654373700000091
2. Clinical sample testing
According to the method, 79 parts of clinical delivery duck raw materials are treated 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 the DuHCV infection is detected by using an optimized TaqMan real-time fluorescence quantitative PCR method. As a result, 8 DuHCV infections are detected to be positive (Ct values are 19.05, 22.11, 30.98, 16.81, 23.41, 29.65, 26.61 and 20.18 respectively), and the positive rate is 10.13%, which indicates that the established method can be used for molecular epidemiological investigation and subsequent pathogenic mechanism related research of DuHCV.
Sequence listing
<110> animal husbandry and veterinary institute of agricultural academy of sciences of Fujian province
<120> primers and probes for real-time fluorescent quantitative PCR detection of duck hepatitis C virus
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<170> SIPOSequenceListing 1.0
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<213> Artificial sequence (Artificial sequence)
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ggtgttcctg tcactgctcg 20
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tagacgaacc aggctcagca 20
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acgagcgacc tcccaaaggt gctgt 25

Claims (3)

1. A primer and a probe for real-time fluorescent quantitative PCR detection of duck hepatitis C virus are characterized in that: the primer sequences are as follows:
the upstream primer DuHCV-qF: 5'-GGTGTTCCTGTCACTGCTCG-3' the flow of the air in the air conditioner,
the downstream primer DuHCV-qR: 5'-TAGACGAACCAGGCTCAGCA-3';
the sequence of the probe is as follows:
probe DuHCV-qprobe: 5'-ACGAGCGACCTCCCAAAGGTGCTGT-3', and the 5 '-end is labeled with a fluorescent reporter group FAM and the 3' -end is labeled with Eclipse.
2. Use of the primers and probes of claim 1 in the preparation of a kit for detecting duck hepatitis c virus.
3. A real-time fluorescent quantitative PCR detection kit for duck hepatitis C virus is characterized in that: the kit comprises the primer and the probe of claim 1.
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