CN112626279A - Real-time fluorescent quantitative PCR (polymerase chain reaction) primer and kit for detecting new duck type 4 adenovirus - Google Patents
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Abstract
The invention provides a real-time fluorescent quantitative PCR primer and a kit for detecting new duck type 4 adenovirus (duck adenovirus 4, DAdV-4), wherein the primer sequence is shown as SEQ ID NO.1-2, and the primer has high specificity and sensitivity. The method established by the invention can detect and quantify the new duck type 4 adenovirus infection in the duck flock, simplify the operation procedure and save the cost. After the real-time fluorescent quantitative PCR reaction is finished, the result can be directly judged by observing the amplification curve of the PCR and combining the melting curve peak value (Tm value), and the establishment of the invention can fill the blank of related fields at home and abroad.
Description
Technical Field
The invention belongs to the field of veterinary medicine, and particularly relates to a real-time fluorescent quantitative PCR primer and a kit for detecting new duck type 4 adenovirus (duck adenovirus 4, DAdV-4).
Background
Duck adenovirus type 4 (DAdV-4) is a novel duck adenovirus disease discovered in southern China in 2019. The nucleotide homology and the amino acid homology of main gene coding proteins of the virus genome are all lower than 80 percent with those of related genes of duck type 1 adenovirus, duck type 2 adenovirus and duck type 3 adenovirus which are found in the early stage of a duck group. Genetic evolutionary analysis shows that DAdV-4 belongs to avian adenovirus (Aviadenovirus) of Adenoviridae (Adenoviridae), but is in an independent genetic evolutionary branch and is named as duck adenovirus type 4 (DAdV-4). In 2020, the research team also confirmed that duck type 4 adenovirus (DAdV-4) infection (designated as DAdV-4-FJ001 strain, GenBank accession No. MW238795) existed in duck group in Fujian region, and the nucleotide homology between the determined DBP gene and the reference strain GD-2019 of duck type 4 adenovirus (GenBank accession No. MN733730) was 100%.
The Real-time fluorescent quantitative PCR method (Real time PCR) is a method for detecting the total amount of products after each Polymerase Chain Reaction (PCR) cycle by using fluorescent chemicals in a DNA amplification reaction. A method for quantitatively analyzing a specific DNA sequence in a sample to be detected by an internal reference method or an external reference method. The real-time fluorescence quantitative PCR is to detect the PCR process in real time through a fluorescence signal in the PCR amplification process. Since in the exponential phase of PCR amplification, there is a linear relationship between the Ct value of the template and the initial copy number of the template. However, no primer research report of a real-time fluorescent quantitative PCR detection method for duck type 4 adenovirus based on a saturated fluorescent dye EvaGreen is found at home and abroad at present, and the establishment of the invention can fill up the blank of research in related fields at home and abroad.
Disclosure of Invention
The invention aims to provide an EvaGreen real-time fluorescent quantitative PCR detection primer and a kit for detecting duck type 4 adenovirus, and establish a real-time fluorescent quantitative PCR detection method capable of detecting duck type 4 adenovirus.
The purpose of the invention is realized by the following technical scheme:
a real-time fluorescent quantitative PCR primer for detecting new duck type 4 adenovirus comprises the following primers:
DAdV-4-SYF1：5’-GCGATAGCGAAACTAGACCTATAC-3’，
DAdV-4-SYR1：5’-TAACTCCGATGCAAAGACGTAG-3’，
the size of the target fragment is 101 bp.
A real-time fluorescent quantitative PCR detection kit containing the primer for detecting the new duck type 4 adenovirus.
The EvaGreen real-time fluorescent quantitative PCR reaction system is as follows:
the real-time fluorescent quantitative PCR reaction system (20 μ L) is: EvaGreen Mix 10.0. mu.L, upstream/downstream primers (DAdV-4-SYF1, DAdV-4-SYR1) (10. mu. mol/L) 0.4. mu.L each, template 1. mu.L, made up to 20. mu.L with sterile double distilled water. The optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; 10s at 95 ℃, 30s at 55 ℃ and 30s at 72 ℃ for 40 cycles. After the reaction is finished, a melting curve is drawn.
And (4) judging a result: when a positive amplification signal is detected, and a single specific peak appears at a melting curve Tm of (85.76 +/-0.12) DEG C, the DAdV-4 is judged to be positive.
The invention provides an EvaGreen real-time fluorescent quantitative PCR detection primer and a kit for detecting duck type 4 adenovirus, which have the following advantages and effects:
1. and (3) detection: the method established by the invention can detect and quantify the new duck type 4 adenovirus infection in the duck flock, simplify the operation procedure and save the cost. After the real-time fluorescent quantitative PCR reaction is finished, the result can be directly judged by observing the amplification curve of the PCR in combination with the Tm value, and the establishment of the invention can fill the blank of related fields at home and abroad.
2. 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. The nucleic acid extraction and result judgment only need 90min, and 96 sample detections can be simultaneously carried out at one time.
3. The quantification is accurate: by preparing a standard substance and drawing a standard curve, the duck type-4 adenovirus can be directly and accurately quantified according to the Ct value of the duck type-4 adenovirus in a sample to be detected.
4. The sensitivity is high: the duck type 4 adenovirus can detect 70.34 copies/mu L at the minimum.
5. The specificity is strong: no response signal is given to common infectious diseases in the duck group, such as (FAdV-4, DAdV-1, DAdV-2, DAdV-3, H9-AIV, DuCV, MDPV, DHAV-1 and DHAV-3), and only fluorescence signal appears when the fluorescence signal is detected for DAdV-4.
6. And performing real-time fluorescent quantitative PCR detection of DAdV-4 infection on 112 clinical detected duck-origin pathological materials, and detecting according to the established real-time fluorescent quantitative PCR system and conditions. As a result, it was found that there were 4 positive amplification signals indicating the presence of DAdV-4 infection in 4 test samples (concentrations of 5.205X 10, respectively)3Copy/. mu.L, 3.110X 103Copy/. mu.L, 2.902X 104Copies/. mu.L, and 8.727X 102Copy/. mu.L), the positive rate was 3.57%.
Drawings
FIG. 1 is an amplification curve of real-time fluorescent quantitative PCR method for duck type 4 adenovirus. Wherein, 1: 7.034X 105Copy/. mu.L; 2: 7.034X 104Copy/. mu.L; 3: 7.034X 103Copy/. mu.L; 4: 7.034X 102Copy/. mu.L; 5: 7.034X 101Copies/. mu.L.
FIG. 2 is a standard curve of real-time fluorescent quantitative PCR method for duck type 4 adenovirus.
FIG. 3 is a diagram showing the sensitivity detection result of the real-time fluorescent quantitative PCR method for duck type 4 adenovirus. Wherein, 1: 7.034X 102Copy/. mu.L; 2: 7.034 is prepared from101Copy/. mu.L; 3: 7.034X 100Copy/. mu.L; 4: and (5) negative control.
FIG. 4 is a graph showing the results of the specificity test of real-time fluorescent quantitative PCR. Wherein, 1: DAdV-4; controls: the test controls (e.g., FAdV-4, DAdV-1, DAdV-2, DAdV-3, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3) were indistinguishable to the naked eye due to the absence of a fluorescent signal.
FIG. 5 shows melting curves of real-time fluorescence quantitative PCR. Wherein, 1: DAdV-4; controls: the test controls (e.g., FAdV-4, DAdV-1, DAdV-2, DAdV-3, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3) are visually indistinguishable due to the absence of characteristic Tm.
Detailed Description
The following examples further illustrate the invention.
Example 1
1. Relevant test strains
Pathogenic avian adenovirus type 4 (FAdV-4), duck adenovirus type 1 (DAdV-1), duck adenovirus type 3 (DAdV-3), duck adenovirus type 4 (DAdV-4), duck source H9 subtype avian influenza virus (H9-AIV), duck circovirus (DuCV), duck parvovirus (MDPV), duck hepatitis viruses type 1 and 3 (DHAV-1, DHAV-3) for the test are identified and stored by animal husbandry and veterinary research institute of agricultural and scientific institute of Fujian province. Duck adenovirus type 2 (DAdV-2) DBP gene was synthesized by Biotechnology engineering (Shanghai) GmbH.
2. Primer design
The research selects the gene sequence characteristics of different host source adenoviridae member genes through analysis and comparison, determines the difference, and designs a primer aiming at the duck adenovirus type 4 (DAdV-4) gene characteristics:
DAdV-4-SYF1：5’-GCGATAGCGAAACTAGACCTATAC-3’，
DAdV-4-SYR1：5’-TAACTCCGATGCAAAGACGTAG-3’，
the size of the target fragment is 101 bp.
The primer sequences were subjected to Blast analysis (https:// www.ncbi.nlm.nih.gov/tools/primer-Blast/index. cgilink _ LOC ═ Blast hom e), all in line with experimental expectations.
The primers were all synthesized by Biotechnology engineering (Shanghai) GmbH.
3. Establishment of real-time fluorescent quantitative PCR detection method
3.1 nucleic acid extraction and cDNA Synthesis
DNA of nucleic acid of DAdV-4(DAdV-4-FJ001 strain, GenBank accession No. MW238795) was extracted using EasyPure Viral DNA/RNA Kit from Beijing Quanji Biotechnology Ltd, according to the protocol described in the specification. And simultaneously extracting nucleic acid DNA (FAdV-4, DAdV-1, DAdV-3, DuCV and MDPV without reverse transcription) or nucleic acid RNA (H9-AIV, DHAV-1 and DHAV-3) of the test control according to the method of the kit, and reversely transcribing the extracted nucleic acid RNA into cDNA by using a FastKing one-step method to remove the first strand synthesis premixed reagent of the genomic cDNA. All are stored at-20 ℃ for standby.
3.2 construction of standards
According to the characteristics of the identified genome sequence of the DAdV-4(DAdV-4-FJ001 strain), a specific primer is designed by utilizing primer design software Oligo (version v7.37), and the sequence of the primer is as follows: DAdV-4-2F: 5'-AACACGATCTGGCCTGGACT-3' and DAdV-4-2R: 5'-AGGGTTTGCGCTTTCTAC-3', for amplifying about 491bp DAdV-4 gene fragment, the primers were synthesized by Biotechnology engineering (Shanghai) GmbH.
PCR was performed using the DNA of DAdV-4(DAdV-4-FJ001 strain) as a template, and amplification was performed using a 50. mu.L system recommended by PCR amplification reagents (2 XPCR Master MIX), in which 25. mu.L of 2 XPCR Master MIX reaction solution, upstream and downstream primers (DAdV-4-2F and DAdV-4-2R) (primer concentration 10. mu. mol. L)-1) Mu. L, DNA 1 each, 1. mu.L, 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 95 deg.C for 5min, circulating, denaturation at 94 deg.C for 50s, annealing at 54 deg.C for 30s, extension at 72 deg.C for 45s, and final extension at 72 deg.C for 7min after 35 cycles.
After the PCR reaction is finished, identifying the PCR product by using 1.5% agarose gel electrophoresis, and performing gel cutting and recovery on the specific target fragment by using an agarose gel recovery kit. The DAdV-4 gene fragment was cloned into pEASY-T1 vector according to pEASY-T1 Simple Cloning Kit instructions, 8 single colonies were randomly picked up and cultured in ampicillin (content 100. mu.g/mL) resistant LB liquid medium for 14h, and then the corresponding plasmid was extracted using fast plasmid mini-extraction Kit. The extracted plasmids are subjected to PCR identification by using primers (DAdV-4-2F and DAdV-4-2R) and conditions during PCR amplification, and the screened positive recombinant plasmids are sent to the company of bioengineering (Shanghai) and Limited for sequencing. The sequencing result is verified by BLAST analysis on NCBI, and the positive recombinant plasmid which is in line with the experimental expectation is used as a positive standard (T-D4) of the real-time fluorescent quantitative PCR, and the nucleotide homology of the positive recombinant plasmid (plasmid T-D4) and the nucleotide homology of a duck type 4 adenovirus reference strain GD-2019 strain (GenBank accession No. MN733730) are 100%.
Measuring the concentration of positive standard (T-D4) with a micro-nucleic acid analyzer, and calculating its copy number to be 7.034 × 107Copies/. mu.L, were serially diluted 10-fold and the plasmid contents were 7.034X 10, respectively6～7.034×100Copying/microliter, subpackaging and storing at-80 ℃ for later use.
3.3 optimization of real-time fluorescent quantitative PCR reaction conditions
Taking a positive standard substance (T-D4) as a template, carrying out real-time fluorescence quantitative PCR reaction at different annealing temperatures (54-64 ℃) and concentrations (2.5-20 mu mol/L) of primers (DAdV-4-SYF1 and DAdV-4-SYR1), optimizing reaction conditions, and screening out optimal conditions (when a cycle threshold (Ct value) is small and a fluorescence value delta Rn is large).
The optimal reaction system (20. mu.L) is optimized as follows: EvaGreen Mix 10.0. mu.L, upstream/downstream primers (DAdV-4-SYF1, DAdV-4-SYR1) (10. mu. mol/L) 0.4. mu.L each, template 1. mu.L, made up to 20. mu.L with sterile double distilled water. The optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; 10s at 95 ℃, 30s at 55 ℃ and 30s at 72 ℃ for 40 cycles. After the reaction is finished, a melting curve is drawn.
And (4) judging a result: when a positive amplification signal is detected, and a single specific peak appears at a melting curve Tm of (85.76 +/-0.12) DEG C, the DAdV-4 is judged to be positive.
Using optimized reaction conditions at 7.034X 105～7.034×101Copy/. mu.L was used as template to obtain an amplification kinetics curve (see FIG. 1).
With the common logarithm of copy number (lgC) in each concentration standard template as the abscissa and the threshold value of cycle number (Ct value) as the ordinate, the slope of the obtained standard curve (as shown in FIG. 2) is-3.390, the Y-axis intercept is 35.97, the correlation coefficient is 1.00, and the amplification efficiency is 0.97, which is in line with the experimental expectation.
3.4 sensitive assay
Using optimized reaction conditions at 7.034X 102～7.034×100Copy/. mu.L is used as template to obtain the lowest detection limit of real-time fluorescence quantitative PCR. As can be seen from FIG. 3, the lowest detection limit of the established real-time fluorescent quantitative PCR method is 7.034X 101Copies/. mu.L (i.e., 70.34 copies/. mu.L).
3.5 specific detection
No response signal was observed against common infectious diseases in the duck population (e.g., FAdV-4, DAdV-1, DAdV-2, DAdV-3, H9-AIV, DuCV, MDPV, DHAV-1, DHAV-3), and only fluorescence signal was detected for DAdV-4 (FIG. 4).
As can be seen from fig. 5, DAdV-4 was determined to be positive when a single specific peak appeared at Tm ═ 85.76 ± 0.12 ℃. No specific dissolution curve peak is seen for other pathogens (such as FAdV-4, DAdV-1, DAdV-2, DAdV-3, H9-AIV, DuCV, MDPV, DHAV-1 and DHAV-3), and the established real-time fluorescence quantitative PCR method is strong in specificity.
3.6 repeatability test
The established real-time fluorescent quantitative PCR detection method has the intra-group variation coefficient of 0.57-1.06 percent and the inter-group variation coefficient of 0.66-1.87 percent when carrying out DAdV-4 detection, and shows that the established real-time fluorescent quantitative PCR detection method has good repeatability.
TABLE 1 determination of coefficient of variation for real-time fluorescent quantitative PCR method
4. Clinical application
And performing real-time fluorescent quantitative PCR detection of DAdV-4 infection on 112 clinical detected duck-origin pathological materials, and detecting according to the established real-time fluorescent quantitative PCR system and conditions. As a result, it was found that there were 4 positive amplification signals indicating the presence of DAdV-4 infection (concentration) in the 4 test samplesDegree of 5.205 × 103Copy/. mu.L, 3.110X 103Copy/. mu.L, 2.902X 104Copies/. mu.L, and 8.727X 102Copy/. mu.L), the positive rate was 3.57%.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Sequence listing
<110> animal husbandry and veterinary institute of agricultural academy of sciences of Fujian province
<120> real-time fluorescent quantitative PCR primer and kit for detecting new duck type 4 adenovirus
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Claims (2)
1. A real-time fluorescent quantitative PCR primer for detecting new duck type 4 adenovirus is characterized in that: the primers are as follows:
DAdV-4-SYF1：5’-GCGATAGCGAAACTAGACCTATAC-3’，
DAdV-4-SYR1：5’-TAACTCCGATGCAAAGACGTAG-3’，
the size of the target fragment is 101 bp.
2. A real-time fluorescent quantitative PCR detection kit for detecting duck type 4 adenovirus, which contains the primer of claim 1.
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Citations (5)
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|---|---|---|---|---|
| US20140010834A1 (en) * | 2011-03-16 | 2014-01-09 | University Of Guelph | Non-pathogenic serotype 4 fowl adenovirus (fadv-4) and viral vector thereof |
| US20160199484A1 (en) * | 2013-08-19 | 2016-07-14 | Veterinarmedizinische Universität Wien | Fowl Adenovirus Vaccine |
| CN107058634A (en) * | 2017-06-13 | 2017-08-18 | 福建省农业科学院畜牧兽医研究所 | The type of Ana 1 aviadenovirus 2 and Ana 1 aviadenovirus A type double PCR detection primers and kit |
| CN111926116A (en) * | 2020-08-12 | 2020-11-13 | 广东省农业科学院动物卫生研究所 | Primer and probe for rapidly and quantitatively detecting duck adenovirus type 4, detection method and application thereof |
| CN112048570A (en) * | 2020-08-12 | 2020-12-08 | 广东省农业科学院动物卫生研究所 | PCR primer for detecting duck adenovirus type 4 and detection method and application thereof |
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| CN111926116A (en) * | 2020-08-12 | 2020-11-13 | 广东省农业科学院动物卫生研究所 | Primer and probe for rapidly and quantitatively detecting duck adenovirus type 4, detection method and application thereof |
| CN112048570A (en) * | 2020-08-12 | 2020-12-08 | 广东省农业科学院动物卫生研究所 | PCR primer for detecting duck adenovirus type 4 and detection method and application thereof |
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Application publication date: 20210409 |