CN112575122A - Dual PCR primer group for rapidly detecting duck type 2 adenovirus and duck circovirus as well as detection method and application thereof - Google Patents
Dual PCR primer group for rapidly detecting duck type 2 adenovirus and duck circovirus as well as detection method and application thereof Download PDFInfo
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Abstract
The invention discloses a dual PCR primer group for rapidly detecting duck adenovirus type 2 and duck circovirus, a detection method and application thereof, wherein the dual PCR primer group consists of a primer for detecting DAdV-2 and a primer for detecting DuCV. The double PCR method for rapidly detecting DAdV-2 and DuCV by adopting the double PCR primer set realizes the simultaneous amplification of two viruses in the same reaction system, reduces the operation times and greatly avoids cross contamination. The method has important application value in the aspects of DAdV-2 and DuCV detection and identification, epidemic disease monitoring, epidemiological investigation and the like.
Description
Technical Field
The invention belongs to the technical field of avian virus detection. In particular to a dual PCR primer group for rapidly detecting duck type 2 adenovirus and duck circovirus as well as a detection method and application thereof.
Background
Duck adenovirus type 2 (DAdV-2) belongs to the genus of avian adenovirus of the family of adenoviridae, and is a linear double-stranded DNA virus. The disease mainly occurs in ducks of 20-30 days old, and the diseased ducks are mainly manifested by listlessness, depression, emaciation and light yellowish white excrement. The pathological changes are enlarged liver with white needle point-shaped dead center, enlarged kidney and spleen with congestion, and white punctate necrosis of pancreas. The disease was first epidemic in Muscovy ducks in France in 1977, and the Austrian scientist Ana Mark obtained the complete sequence of the virus by a high-throughput sequencing technology in 2014. In 2015, the first duck type-2 adenovirus in China is separated and identified by application of metagenomics such as Chenfeng and the like. The disease is widely spread in the areas with dense waterfowl culture, such as Guangdong, Fujian, Zhejiang and the like, and causes great economic loss to duck breeding industry at present.
Duckcircovirus (DuCV) is a new member of the circovirus genus of the circovirus family, DuCV is often latent infected and invades the immune system of ducks, so that the immune function of the bodies of the ducks is reduced, the resistance to various conditional pathogens is weakened, and the ducks are more easily attacked by other pathogenic factors. And once mixed infection (such as duck influenza virus, duck adenovirus, duck escherichia coli and the like) occurs, the death rate of the infected duck body is greatly improved. Meanwhile, DuCV can also cause the symptoms of disorder of duck feather, growth retardation, weight loss and the like, so that the circovirus is an important pathogenic microorganism and has no negligible harm to the breeding industry in China.
The two pathogens are DNA viruses, have certain similarity to clinically caused symptoms, are easy to generate mixed infection, and greatly increase the difficulty of clinical differential diagnosis. However, conventional diagnostic methods, such as animal experiments, immunology and virus isolation and culture, have the disadvantages of long diagnostic time, low sensitivity, complicated operation and the like in clinical diagnosis, and are not easy to rapidly diagnose the two diseases in clinical diagnosis. Therefore, it is urgently needed to establish a rapid, accurate and sensitive detection method for diagnosing and monitoring the two pathogens to ensure the healthy development of the duck breeding industry.
PCR is one of the detection methods commonly used in laboratories, and has the advantages of rapidness, sensitivity, low cost, simple operation and the like; the method can not only rapidly determine the virus species, but also detect early infection and latent infection of the virus. However, if a primer for amplifying a single virus is used to identify a sample to be tested during detection, multiple samples may need to be prepared and amplified for multiple times to determine the type of the infected virus, which not only needs to increase the detection times in actual operation, but also causes cross-infection. The double PCR is established on the basis of single PCR, and has the advantages that two pathogens can be simultaneously detected and identified through one-time PCR reaction, and the double PCR has important application value in clinic. At present, a double PCR detection method capable of simultaneously detecting duck circovirus and duck type 2 adenovirus is not reported at home and abroad.
Disclosure of Invention
Aiming at the problems, the invention provides a dual PCR primer group for rapidly detecting duck type 2 adenovirus and duck circovirus as well as a detection method and application thereof. The primer group solves the problems of complex operation and high cross-contamination risk of single PCR, can be used for differential diagnosis of the duck circovirus and duck type 2 adenovirus in the duck group, provides a reliable detection means for epidemiological investigation and prevention and control of the duck circovirus and duck type 2 adenovirus in the duck group, and has important clinical application value.
The invention is realized by the following technical scheme
A dual PCR primer set for rapid detection of DAdV-2 and DuCV, the dual PCR primer set consisting of a primer for detecting DAdV-2 and a primer for detecting DuCV,
the nucleotide sequence of the primer for detecting DAdV-2 is as follows:
the upstream primer F1: 5'-CGATGGAACGACAGTAAA-3'
The downstream primer R1: 5'-AAACGAAAAAGCAAGAGC-3'
The nucleotide sequence of the primer used for detecting DuCV is:
the upstream primer F2: 5'-GTGGTGGGACGGTTACTCGG-3'
The downstream primer R2: 5'-TTTATTGGGAACGGGAGGGT-3'
The application of the primer group in preparing a reagent for detecting DAdV-2 and DuCV.
A kit containing the double PCR primer set.
The method for simultaneously detecting DAdV-2 and DuCV by adopting the dual PCR primer set comprises the following steps:
(1) synthesizing a dual PCR primer group for detecting DAdV-2 and DuCV by a common method;
(2) extracting total DNA of a sample to be detected, and performing PCR amplification reaction by using the extracted total DNA as a template and the double PCR primer group synthesized in the step (1);
(3) and analyzing the PCR amplification product, and judging the result.
Further, the reaction system of the PCR amplification in the step (2) is as follows: the upstream primer F1, the downstream primer R1, the upstream primer F2 and the downstream primer R2 are respectively 0.5 mu L, and the final concentration of each primer is 0.4 pmol/mu L; 2 × Taq Master Mix enzyme 12.5 μ L; 1. mu.L of each DNA template; sterilized ddH2O is complemented to 25 mu L; the conditions of the PCR amplification reaction are as follows: pre-denaturation at 95 ℃ for 5 min; 90s at 94 ℃, 20s at 94 ℃, 30s at 54 ℃, 20s at 72 ℃, 30 cycles, and a final cycle with an extension of 5min at 72 ℃.
Further, the analyzing the PCR amplification product in the step (3) is to specifically perform agarose gel electrophoresis analysis on the PCR amplification product, and determine the type of the virus in the sample according to the electrophoresis result; when DAdV-2 exists in the sample, a band of about 598bp appears in an amplification product; when DuCV exists in a sample, a band of about 297bp appears in an amplification product; when the sample contains mixed infection of DAdV-2 and DuCV, two bands of about 598bp and 297bp appear in the amplification product.
Compared with the prior art, the invention has the following positive beneficial effects
The invention provides a dual PCR primer group capable of rapidly detecting two viruses of DAdV-2 and DuCV.A total genome of the two viruses of DAdV-2 and DuCV is analyzed in the early stage, the two pairs of primers are designed according to conserved gene regions of the two viruses, the annealing temperatures of the primer groups are similar, the sizes of amplified target fragments are approximate, and the two pairs of primers have no mutual interference so as to ensure that the primer groups are formed for use subsequently; in actual operation, the primer group only amplifies DAdV-2 and DuCV virus genomes without causing non-specific reaction, and has strong specificity;
the double PCR method for rapidly detecting the DAdV-2 virus and the DuCV virus can be used for carrying out PCR amplification on DNA obtained from the same sample as a template; judging the type of the virus infected by the sample to be detected through agarose gel electrophoresis according to the size of the amplified product, and realizing the simultaneous amplification of two viruses in the same reaction system;
the detection method is simple and convenient in actual operation, reduces the operation times, greatly avoids cross contamination, and saves the detection time and the reagent consumption. The established detection method aims at the gene amplification of the virus conserved region, so that the pertinence is strong, the sensitivity is high, and the minimum nucleic acid detection amount reaches pg level. The simple, economic, rapid and accurate inspection requirements can be met in clinical detection;
the double PCR method established by the invention realizes the identification and detection of two pathogens in the same reaction tube, and has important application value in the aspects of detection and identification of DAdV-2 and DuCV in animal-derived products, epidemic disease monitoring, epidemiological investigation and the like.
Drawings
FIG. 1 is a graph showing different annealing temperature results of duck type 2 adenovirus;
m: marker 2000; lane 1: 51.9 ℃; lane 2: 53.8 ℃; lane 3: 56.1 ℃; lane 4: 58.0 ℃; lane 5: 59.2 ℃;
FIG. 2 is a graph showing the results of different annealing temperatures of duck adeno-associated virus;
m: marker 2000; lane 1: 50.5 ℃; lane 2: 53.1 ℃; lane 3: 54.9 ℃; lane 4: 56.4 ℃; lane 5: 57.4 ℃;
FIG. 3 is a graph showing the results of detection of DAdV-2 and DuCV duplex PCR under different primer concentrations;
m: marker 2000; lane 1: primer concentration 20 pmol/. mu.L; lane 2: primer concentration 10 pmol/. mu.L; lane 3: primer concentration 5 pmol/. mu.L; lane 4: primer concentration 1 pmol/. mu.L; lane 5: primer concentration 0.5 pmol/. mu.L;
FIG. 4 is a graph comparing the results of DAdV-2 and DuCV double PCR with single PCR; m: marker 2000; lane 1: the result of double PCR electrophoresis of DAdV-2 and DuCV; lane 2: single PCR electrophoresis result of DAdV-2; lane 3: DuCV Single PCR electrophoresis results;
FIG. 5 is a diagram showing the results of the specific electrophoresis of the primers for DAdV-2 and DuCV dual PCR;
m: marker 2000; lane 1: mixed nucleic acids of DAdV-2 and DuCV viruses; lane 2: (ii) a DAdV-2 viral nucleic acid; lane 3: DuCV viral nucleic acid; lane 4: duck tembusu virus nucleic acid; lane 5: duck viral hepatitis type i nucleic acid; lane 6: duck escherichia coli nucleic acid; lane 7: a listeria anatipestifer nucleic acid; lane 8: duck source avian pasteurella multocida nucleic acid; lane 9: duck reovirus nucleic acid; lane 10: duck plague virus nucleic acid; lane 11: duck parvovirus nucleic acid; lane 12: negative control;
FIG. 6 is a graph showing the results of the DAdV-2 and DuCV double PCR sensitivity tests;
m: marker 2000; lanes 1-5: mixed nucleic acid of DAdV-2 and DuCV 10-1～10-5And (5) dilution nucleic acid electrophoresis results.
Detailed Description
The present invention will be described in more detail with reference to the following embodiments in order to understand the technical solutions of the present invention, but the present invention is not limited to the scope of the present invention.
The samples used in the following examples are specified below: positive disease material, bacterial virus strain and clinical sample for test
Test positive disease material: the positive disease containing duck circovirus was identified and preserved by Shangqimeilan bioengineering GmbH.
Test strains: the duck adenovirus type 2, duck plague virus, duck viral hepatitis type I, duck reovirus, duck tembusu virus, duck parvovirus, duck colibacillus, riemerella anatipestifer and duck source avian pasteurella multocida are identified and stored by Shanqin Meilan bioengineering GmbH. Clinical samples are from suspected pathological materials of different provinces in 2019-2020, and the total amount is 83 parts.
Example 1
Establishment of a Dual PCR primer set for the Rapid detection of DAdV-2 and DuCV:
(1) primer design and Synthesis
According to the complete genome sequences of DAdV-2 and DuCV reported on GenBank, the genomes of different strains are aligned and analyzed, nucleic acid fragments which are conservative are screened, two pairs of specific primers aiming at the two viruses are designed by using Primer5.0 primer design software, and the two pairs of primers are analyzed on line through NCBI (https:// www.ncbi.nih.gov /). Two pairs of primers were finally screened, and as shown in Table 1, the primers were synthesized by Biotechnology (Shanghai) Ltd. Two pairs of primers can specifically amplify specific fragments of 598bp (DAdV-2) and 297bp (DuCV).
TABLE 1 Dual PCR primer set information sequences
(2) Extraction of nucleic acid from test sample
Extracting nucleic acid of a sample to be detected: selecting suspected pathogenic animals, aseptically collecting about 5g of visceral tissues (heart, liver and spleen), shearing the pathological tissues with surgical scissors on an ultraclean operation table, diluting with PBS according to a ratio of 5:1(W/V), placing at-70 ℃ for repeated freeze thawing for three times, extracting nucleic acid of the sample to be detected and the control strain according to a Virus genome extraction Kit (TIANAmp Virus DNA/RNA Kit), and storing the extracted nucleic acid at-70 ℃ for later use.
Reverse transcription reaction conditions: keeping the temperature at 50 ℃ for 30min, and keeping the temperature at 70 ℃ for 5 min; the resulting cDNA was stored at-20 ℃ for PCR amplification. Reverse transcription PCR system, as shown in Table 2.
TABLE 2 reverse transcription PCR System
(3) Single PCR amplification and amplification sequence verification
DNA of DAdV-2 and DuCV stored at-70 ℃ was used as a template for single PCR amplification using 2 pairs of specific primers designed in example 1. The single PCR reaction was amplified according to the 25uL amplification system of the kit, as shown in Table 3. The reaction conditions are 94 ℃ for 90s, 94 ℃ for 20s, 50-60 ℃ for 30s, 72 ℃ for 20s, 30 cycles, and the final cycle is extended for 5min at 72 ℃. The PCR product was detected by 2.0% TAE agarose gel electrophoresis. As shown in FIGS. 1 and 2, the PCR product was purified to obtain the target gene by a purification kit and sequenced by Biotechnology engineering (Shanghai) Co., Ltd. The sequencing results were aligned online by NCBI (https:// www.ncbi.nih.gov /) as DAdV-2 and DuCV viruses.
TABLE 3 PCR reaction System
(4) Establishment of Dual PCR primer set
Optimizing a double PCR reaction system and reaction conditions: under the condition of unchanged reaction conditions (94 ℃ 90s, 94 ℃ 20s, 54 ℃ 30s, 72 ℃ 20s, 30 cycles, and the last cycle is extended for 5min at 72 ℃), DNA extracted by DAdV-2 and DuCV with the same concentration is mixed to be used as a double PCR template, and the double PCR reaction conditions are optimized.
And (3) primer concentration screening: the reaction system used was 2 XTaq Master Mix 12.5. mu.l, two pairs of upstream and downstream primers 0.5. mu.l each, DNA template 1. mu.l each, ddH2O, complementing 25 mu l; the results of PCR amplification and primer concentration screening were performed by selecting 5 different primer concentration gradients of 20 pmol/. mu.L, 10 pmol/. mu.L, 5 pmol/. mu.L, 1 pmol/. mu.L, and 0.5 pmol/. mu.L in total, as shown in FIG. 3 (the size and brightness of the target band can be used as an index for determining the result of amplification of primer concentration; different primer concentrations are used; the results are shown in FIG. 3The primer concentration can specifically amplify a target band, but with the reduction of the primer concentration, the probability of the combination of the primer and the template gene is relatively reduced, the brightness of the target band is gradually reduced, and the target band is gradually reduced; but still amplified the gene of interest at a lower concentration of 0.5 pmol/. mu.L of primer).
Screening the volume of the reaction system: under the condition that the primer concentration was 10 pmol/. mu.L, reaction systems of 12.5. mu.L, 25. mu.L and 50. mu.L were selected, respectively, for amplification. Considering economy and simplicity of operation, the final dual PCR reaction system is selected as follows: 2 XTaq Master Mix 12.5. mu.l, two pairs of upstream and downstream primers 0.5. mu.l each (10 pmol/. mu.L), cDNA templates 1. mu.l each, ddH2Make up to 25. mu.L of O.
The double PCR amplification of DAdV-2 and DuCV is carried out by utilizing the optimized conditions, and the electrophoresis result shows that the double PCR can simultaneously amplify the DAdV-2598bp and DuCV297bp specific bands, which are consistent with the single PCR result, as shown in FIG. 4.
Example 2
Dual PCR primer set specificity assay:
the established double PCR method is utilized to carry out the DNA template mixing DAdV-2 and DuCV, the single cDNA template of duck virus hepatitis I, duck reovirus and duck Tembusu virus, the single DNA template of duck adenovirus type 2, duck circovirus, Muscovy duck parvovirus, duck plague virus, duck colibacillus, Riemerella anatipestifer and duck source fowl pasteurella multocida and ddH2O, and the amplification result shows that only the mixed DNA template of DAdV-2 and DuCV and the single DNA template of DAdV-2 and DuCV amplify the band corresponding to the size of the target fragment as shown in FIG. 5. And (3) carrying out gene sequencing on the PCR amplification product of the amplified DuCV virus, carrying out bioinformatics software analysis on the sequence of the amplification product and the sequence of a reference primer, wherein the nucleotide homology of the amplification product and the sequence of the reference primer is more than 96%, and the analysis result proves that the PCR amplification product is the DuCV virus.
Example 3
Double PCR primer set sensitivity test:
DNA concentrations of DAdV-2 and DuCV were determined to be 67ng and 52ng, respectively, using a spectrophotometer, and sterilizedThe double distilled water is used for 10 times of gradient dilution. Then take 10-1～10-5The dilution was detected, and PCR amplification was performed using each diluted DNA as a template, and the result of detection of the minimum amount detected is shown in FIG. 6.
As shown in FIG. 6, the amplification results of the two virus DNA mixtures showed that the minimum amounts of DNA detected by this method were 0.67pg and 5.2pg for DAdV-2 and DuCV, respectively.
Example 4
Clinical application
The double PCR primer set established by the invention is used for carrying out PCR detection on a sample which comprises 83 suspected dead duck viscera collected in 2019-2020 by Shangqin Meilan bioengineering Co., Ltd as a clinical detection sample, and the result is shown in Table 4.
And comparing and analyzing the detection result with the single PCR detection result, wherein the detection result shows that the coincidence rate of the clinical pathological material detection result and the single PCR detection result of the dual PCR detection kit disclosed by the invention reaches 100%. And (3) carrying out gene sequencing on the amplified positive product of the DAdV-2, carrying out bioinformatics software analysis on the positive product and a reference primer sequence, wherein the nucleotide homology of the positive product and the reference primer sequence is more than 98%, and the analysis result proves that the PCR amplified product is the DAdV-2 virus.
TABLE 4 clinical sample test results
SEQUENCE LISTING
<110> Shanqiu Meilan bioengineering Co., Ltd
<120> double PCR primer group for rapidly detecting duck type 2 adenovirus and duck circovirus and detection method thereof
And applications thereof
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> for duck virus detection
<400> 1
cgatggaacg acagtaaa 18
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> for duck virus detection
<400> 2
aaacgaaaaa gcaagagc 18
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> for duck virus detection
<400> 3
gtggtgggac ggttactcgg 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> for duck virus detection
<400> 4
tttattggga acgggagggt 20
Claims (6)
1. A dual PCR primer set for rapidly detecting DAdV-2 and DuCV is characterized in that the dual PCR primer set consists of a primer for detecting DAdV-2 and a primer for detecting DuCV,
the nucleotide sequence of the primer for detecting DAdV-2 is as follows:
the upstream primer F1: 5'-CGATGGAACGACAGTAAA-3'
The downstream primer R1: 5'-AAACGAAAAAGCAAGAGC-3'
The nucleotide sequence of the primer used for detecting DuCV is:
the upstream primer F2: 5'-GTGGTGGGACGGTTACTCGG-3'
The downstream primer R2: 5'-TTTATTGGGAACGGGAGGGT-3' are provided.
2. Use of the primer set according to claim 1 for preparing a reagent for detecting DAdV-2 and DuCV.
3. A kit comprising the dual PCR primer set of claim 1.
4. The method for simultaneously detecting DAdV-2 and DuCV using the dual PCR primer set of claim 1, comprising the steps of:
(1) synthesizing a dual PCR primer set for detecting DAdV-2 and DuCV;
(2) extracting total DNA of a sample to be detected, and performing PCR amplification reaction by using the extracted total DNA as a template and the double PCR primer group synthesized in the step (1);
(3) and analyzing the PCR amplification product, and judging the result.
5. The method of claim 4, wherein the reaction system of the PCR amplification in step (2) is: the upstream primer F1, the downstream primer R1, the upstream primer F2 and the downstream primer R2 are respectively 0.5 mu L, and the final concentration of each primer is 0.4 pmol/mu L; 2 × Taq Master Mix enzyme 12.5 μ L; 1. mu.L of each DNA template; sterilized ddH2O is complemented to 25 mu L; the conditions of the PCR amplification reaction are as follows: pre-denaturation at 95 ℃ for 5 min; 90s at 94 ℃, 20s at 94 ℃, 30s at 54 ℃, 20s at 72 ℃, 30 cycles, and a final cycle with an extension of 5min at 72 ℃.
6. The method according to claim 4, wherein the analyzing the PCR amplification product in step (3) is specifically to perform agarose gel electrophoresis analysis on the PCR amplification product, and determine the type of virus in the sample according to the electrophoresis result; when DAdV-2 exists in the sample, a 598bp band appears in an amplification product; when DuCV exists in the sample, a 297bp band appears in an amplification product; when the sample contains mixed infection of DAdV-2 and DuCV, two bands of 598bp and 297bp appear in the amplification product.
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| CN202011594770.5A CN112575122B (en) | 2020-12-29 | 2020-12-29 | Dual PCR primer set for rapidly detecting duck type 2 adenovirus and duck circovirus, and detection method and application thereof |
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| CN202011594770.5A CN112575122B (en) | 2020-12-29 | 2020-12-29 | Dual PCR primer set for rapidly detecting duck type 2 adenovirus and duck circovirus, and detection method and application thereof |
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| CN112575122B (en) | 2024-04-09 |
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