CN114107556A - Triple PCR detection kit for duck circular ring, duck adenovirus and duck tembusu virus - Google Patents

Abstract

The present disclosure relates to a triple PCR detection primer set for duck circovirus, duck adenovirus and duck Tembusu virus, wherein the triple PCR detection primer set comprises a first primer pair, a second primer pair and a third primer pair: the first primer pair has a nucleotide sequence pair shown in SEQ ID NO.1 and SEQ ID NO. 2; the second primer pair has a nucleotide sequence pair shown in SEQ ID NO.3 and SEQ ID NO. 4; the third primer pair has a nucleotide sequence pair shown in SEQ ID NO.5 and SEQ ID NO. 6. The invention also provides a triple PCR detection kit for duck circovirus, duck adenovirus and duck tembusu virus, and the detection kit disclosed by the invention can be used for simultaneously detecting the mixed infection or single infection condition of duck circovirus, duck adenovirus and duck tembusu virus, so that the workload is reduced, the detection time is greatly shortened, and the purpose of quickly detecting pathogeny is achieved.

Description

Triple PCR detection kit for duck circular ring, duck adenovirus and duck tembusu virus

Technical Field

The disclosure relates to the technical field of molecular biology, in particular to a triple PCR detection kit for duck circular ring, duck adenovirus and duck tembusu virus, and particularly relates to a triple PCR detection primer group for duck circular ring, duck adenovirus and duck tembusu virus and a detection kit obtained by the triple PCR detection primer group.

Background

Duck circovirus (DuCV) is currently responsible for chronic infectious wasting and immunosuppressive diseases in ducks, and is infectious for ducks of different breeds and growing stages. In recent years, DuCV has a marked upward trend in duck group infections and mixed infection cases. DuCV infection alone can cause disorder of feather, growth and development retardation and weight loss of infected ducks, and is easy to ignore because the disease symptoms are not obvious. However, DuCV infection mainly attacks the immune system of a host, so that immunosuppression is caused, the immune failure is caused, and secondary infection and mixed infection of bacteria and viruses are caused, so that the difficulty of controlling epidemic diseases is increased, the death rate of ducks is increased, and serious economic loss is caused to the duck breeding industry.

The duck adenovirus (DAdV) mainly comprises duck adenovirus type 1 (DAdV-1), duck adenovirus type 2 (DAdV-2), duck adenovirus type 3 (DAdV-3) and duck adenovirus type 4 (DAdV-4). Except for DAdV-1 (belonging to the genus FuAT adenovirus of the family adenoviridae), the other subtypes belong to the genus fowl adenovirus of the family adenoviridae, and are characterized mainly by swelling and bleeding of parenchymal organs, especially liver and spleen. Along with the rapid development of duck breeding industry in China, the infection rate of adenovirus in duck groups is higher and higher, and the production is seriously influenced.

Duck Tembusu Virus (DTMUV) is a novel flavivirus that begins to prevail in 2010 and can infect various breeds of Duck groups at each day of age. DTMUV infection can cause egg drop and even stop production of laying ducks; meat duck growth retardation, diarrhea, nervous symptoms and even death; the ducklings have nervous symptoms of unstable standing, incapability of falling down and the like, the death and culling rate is high, and huge economic loss is brought to the duck breeding industry.

DuCV, DAdV and DTMUV are common viruses seriously harming the duck breeding industry, and are frequently present in the form of mixed infection or secondary infection clinically, so that the growth and development of ducks are seriously hindered, even the ducks die and are eliminated, and the economic benefit of the duck breeding industry is seriously damaged. The diagnosis difficulty of duck diseases is increased due to mixed infection, misdiagnosis or missed diagnosis is easy to occur, and a method for rapidly detecting the three viruses is urgently needed to be established in actual production so as to guide clinical epidemic disease diagnosis and treatment scheme formulation, so that economic loss is reduced.

The traditional methods of virus separation, electron microscope observation, agar diffusion test, enzyme-linked immunosorbent assay and the like are time-consuming, have certain limitation in practical application, and are not beneficial to prevention and treatment of duck diseases. As a molecular biology technology, PCR has the advantages of simple and rapid operation, high sensitivity, good repeatability, strong specificity and the like, particularly, multiple PCR can simultaneously detect and identify multiple pathogens, and the PCR has unique advantages and high practical value in the aspect of differential diagnosis of clinical multiple pathogen mixed infection and has good application prospect.

Currently, studies for detection and diagnosis of DuCV, DAdV and DTMUV using triple PCR techniques have not been seen.

Disclosure of Invention

In order to solve the technical problems or at least partially solve the technical problems, the present disclosure provides a triple PCR detection kit for duck circovirus, duck adenovirus and duck tembusu virus, which can simultaneously detect the mixed infection condition or single infection condition of duck circovirus, duck adenovirus and duck tembusu virus, reduce workload, greatly shorten detection time, and achieve the purpose of rapidly detecting pathogen.

In a first aspect, the present disclosure provides a triple PCR detection primer set for duck circovirus, duck adenovirus and duck tembusu virus, wherein the triple PCR detection primer set comprises a first primer pair, a second primer pair and a third primer pair:

the first primer pair has a nucleotide sequence pair shown in SEQ ID NO.1 and SEQ ID NO. 2; the second primer pair has a nucleotide sequence pair shown in SEQ ID NO.3 and SEQ ID NO. 4; the third primer pair has a nucleotide sequence pair shown in SEQ ID NO.5 and SEQ ID NO. 6.

The triple PCR detection primer group provided by the disclosure can be used for detecting mixed infection or single infection of duck circovirus, duck adenovirus or duck tembusu virus.

As a preferred technical scheme of the present disclosure, the molar ratio of the first primer pair, the second primer pair and the third primer pair is 1 (0.5-2) to (0.5-4).

As a preferred embodiment of the present disclosure, the molar ratio of the first primer pair, the second primer pair and the third primer pair is 1:1: 2.

In the present disclosure, a primer pair includes two primers, and when applied, each primer pair includes the same number of added moles of the two primers.

In a second aspect, the present disclosure provides a triple PCR detection kit for duck circovirus, duck adenovirus and duck tembusu virus, comprising the triple PCR detection primer set of the first aspect.

As a preferred technical scheme of the present disclosure, the triple PCR detection kit further comprises 2 XTAQ Master Mix (Dye Plus).

In a third aspect, the present disclosure provides the use of the triple PCR detection kit of the second aspect in triple PCR amplification.

As a preferred embodiment of the present disclosure, the annealing temperature of the triple PCR amplification is 52-59 ℃, preferably 57.4 ℃.

In a fourth aspect, the present disclosure provides a triple PCR detection method for simultaneously detecting duck circovirus, duck adenovirus and duck tembusu virus for non-diagnostic and therapeutic purposes, the triple PCR detection method comprising using the triple PCR detection primer set of the first aspect or the triple PCR detection kit of the second aspect.

As a preferred technical solution of the present disclosure, the triple PCR detection method includes the steps of:

(1) extracting RNA/DNA of a sample to be detected, and performing reverse transcription to obtain cDNA/DNA;

(2) carrying out PCR reaction by taking cDNA/DNA of a sample to be detected as a template, wherein a reaction system comprises the triple PCR detection primer group of the first aspect;

(3) and (3) carrying out agar gel electrophoresis detection on the PCR product obtained in the step (2), and judging whether the sample to be detected has duck circovirus, duck adenovirus or duck tembusu virus according to the detection result.

As a specific embodiment of the present disclosure, in the triple PCR detection method, the reaction system in step (2) is: 2 XTaq Master Mix (Dye Plus) 12.5. mu.L, sample cDNA/DNA mixture 2. mu.L, first primer pair and second primer pair each 0.5. mu.L, third primer pair 1. mu.L, and ddH₂O is complemented to 25 mu L, and the concentrations of the first primer pair, the second primer pair and the third primer pair are all 10 mu mol/L.

The PCR reaction conditions are as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 57.4 ℃ for 30s, and 72 ℃ for 30 s; preserving at 72 deg.C for 10min and 4 deg.C.

Compared with the prior art, the technical scheme provided by the embodiment of the disclosure has the following advantages:

(1) the three pairs of specific primer pairs provided by the disclosure can be used for detecting mixed infection or single infection of duck circovirus, duck adenovirus or duck tembusu virus;

(2) the triple PCR detection primer group and the triple PCR detection method provided by the disclosure can simultaneously determine the mixed infection and single infection conditions of duck circovirus, duck adenovirus and duck tembusu virus in a sample through one-time PCR reaction, reduce the workload, save the detection time and achieve the purpose of quickly detecting the pathogen;

(3) the triple PCR detection primer group and the triple PCR detection method provided by the disclosure have the advantages of strong specificity, rapidness, simplicity, convenience, easiness in operation and the like, and provide technical support for monitoring, prevention and control of duck circovirus, duck adenovirus and duck Tembusu virus;

(4) the triple PCR detection primer group and the triple PCR detection method provided by the disclosure have high sensitivity and can detect 10 at the lowest²The copied/mu L DuCVCap gene, the DAdVHexon gene and the DTMUV E gene plasmid provide an accurate detection method for the early onset of the three viruses and have important significance for timely cutting off the transmission of the three viruses.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and together with the description, serve to explain the principles of the disclosure.

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present disclosure, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive exercise.

FIG. 1 is an agarose gel electrophoresis image of the PCR products provided in examples 1-9 of the present disclosure.

FIG. 2 is an agarose gel electrophoresis image of the PCR products provided in examples 10-14 of the present disclosure.

Fig. 3 is a diagram of the result of the specificity verification of the triple PCR detection method provided by the present disclosure.

FIG. 4 is a graph of the sensitivity test results of the triple PCR test method provided by the present disclosure.

FIG. 5 is an agarose gel electrophoresis image of a portion of the PCR product in 50 samples in a clinical trial.

Detailed Description

In order that the above objects, features and advantages of the present disclosure may be more clearly understood, aspects of the present disclosure will be further described below. It should be noted that the embodiments and features of the embodiments of the present disclosure may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure, but the present disclosure may be practiced in other ways than those described herein; it is to be understood that the embodiments disclosed in the specification are only a few embodiments of the present disclosure, and not all embodiments.

The methods or reagents described in this disclosure, unless otherwise indicated, are all methods commonly used in the art and are commercially available; the specific materials and reagents used were as follows:

DuCV (DuCV), duck adenovirus (DAdV), duck Tembusu virus (DTMUV), H9 subtype avian influenza virus (H9AIV), Duck Hepatitis Virus (DHV), duck plague virus (DEV), Duck Paramyxovirus (DPMV), duck astrovirus (DAstV), Riemerella anatipestifer, Escherichia coli and salmonella are provided by the research institute of epidemic disease prevention and control technology of livestock and poultry in chat and city university.

The virus RNA/DNA rapid purification kit is purchased from Omega company, the reverse transcription kit is purchased from Thermo company, pMD-18T is purchased from doctor-Biotech Co., Ltd, the gel recovery kit is purchased from Shanghai biological engineering Co., Ltd, and the bacterial genome DNA extraction kit is purchased from Tiangen Biochemical technology Co., Ltd; the PCR instrument is manufactured by BIO-RAD company in the United states, and the low-temperature high-speed centrifuge is manufactured by Thermo company.

Example 1

The embodiment provides a method for establishing a triple PCR reaction system of duck circovirus, duck adenovirus and duck tembusu virus.

(1) And designing a primer group.

(a) Comparing duck circovirus sequences published on a reference GenBank, selecting a duck circovirus Cap gene, and designing and screening a specific primer pair (a first primer pair) aiming at the Cap gene segment, wherein the primer pair has nucleotide sequences shown in SEQ ID No.1 and SEQ ID No. 2;

(b) comparing duck adenovirus sequences published on a reference GenBank, selecting duck adenovirus Hexon genes, and designing and screening a specific primer pair (a second primer pair) aiming at a Hexon gene fragment, wherein the primer pair has nucleotide sequences shown in SEQ ID No.3 and SEQ ID No. 4;

(c) the duck tembusu virus E gene is selected by comparing duck tembusu virus sequences published on a reference GenBank, and a specific primer pair (a third primer pair) aiming at the E gene fragment is designed and screened, wherein the primer pair has nucleotide sequences shown in SEQ ID No.5 and SEQ ID No. 6.

Wherein, the specific primer pair aiming at the duck circovirus Cap:

SEQ ID No.1：5′-TCTCTCGTGCCCGGGGATCT-3′；

SEQ ID No.2：5′-GTCATTTCCCGAGTAACCGTCCC-3′；

wherein, the specific primer pair aiming at duck adenovirus Hexon is as follows:

SEQ ID No.3：5′-TGCGACAACTACCTGTGGAC-3′

SEQ ID No.4：5′-GCGTACGGAAGTAAGCCAT-3′

wherein, the specific primer pair aiming at the duck tembusu virus E gene comprises the following components:

SEQ ID No.5：5′-CATGGACAGGGTCATCAGC-3′

SEQ ID No.6：5′-AGAGTACTTCACTGGAATAGCTCC-3′

(d) amplifying a sample to be detected by using a conventional PCR amplification method, wherein the sizes of amplified fragments are 371bp (duck circovirus), 235bp (duck adenovirus) and 155bp (duck tembusu virus), and all primers are sterile ddH₂O (RNasefree) was prepared at a concentration of 10. mu. mol/L for use.

(2) Extraction of viral RNA/DNA and cDNA Synthesis

DuCV and DAdV DNA and DTMUV RNA were extracted according to the instructions of the viral RNA/DNA rapid purification kit, cDNA synthesis was performed with the RNA template according to the instructions of the reverse transcription kit, and all cDNA and DNA templates were stored at-80 ℃ for future use.

(3) Establishment of 25 mu L triple PCR reaction system

12.5. mu.L of 2 XTaq Master Mix (Dye Plus), 1. mu.L of each of DuCVDNA, DAdV DNA and DTMUV cDNA templates, 1. mu.L of each of the first primer pair, the second primer pair and the third primer pair obtained in step (1) (each primer added in an amount of 0.5. mu.L), and ddH was added to the mixture₂O is complemented to 25 mu L;

the reaction procedure is as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 57.4 deg.C, and annealing at 72 deg.C for 30s, and performing 30 cycles; finally, extension is carried out for 10min at 72 ℃ to complete PCR reaction.

Examples 2 to 9

The embodiment provides a method for establishing a triple PCR reaction system of duck circovirus, duck adenovirus and duck tembusu virus.

The difference from example 1 is that, in this example, the amounts of addition of the first primer pair, the second primer pair and the third primer pair are shown in table 1:

TABLE 1

Examples 10 to 14

The embodiment provides a method for establishing a triple PCR reaction system of duck circovirus, duck adenovirus and duck tembusu virus.

The difference from example 1 is that in this example, the annealing temperatures were 52.5 ℃ (example 10), 54.1 ℃ (example 11), 55.9 ℃ (example 12), 57.4 ℃ (example 13), and 58.4 ℃ (example 14) respectively during the PCR reaction.

Performance test 1

(1) The PCR reactions obtained in examples 1 to 9 were verified by 1.2% agarose gel electrophoresis, and the results were stored by photographing.

FIG. 1 is an agarose gel electrophoresis of PCR products provided in examples 1-9, where M in FIG. 1 is DNA Marker DL2000, and 1-9 correspond to examples 1-9, respectively.

As is clear from FIG. 1, in the present disclosure, the effects are most excellent when the amounts of the first primer set, the second primer set and the third primer set are 1. mu.L, 1. mu.L and 2. mu.L, respectively, and the molar ratios are 1:1:2, respectively.

(2) The PCR reactions obtained in examples 10 to 14 were verified by 1.2% agarose gel electrophoresis, and the results were stored by photographing.

FIG. 2 is an agarose gel electrophoresis of PCR products provided in examples 10-14, wherein M in FIG. 2 is DNA Marker DL2000, and 1-5 correspond to examples 10-14, respectively.

As can be seen from fig. 2, in the present disclosure, the optimum annealing temperature is 57.4 ℃.

Example 15

The embodiment provides a triple PCR detection method, which comprises the following steps:

(1) extracting RNA/DNA of a sample to be detected, and performing reverse transcription to obtain cDNA/DNA;

(2) carrying out PCR reaction by taking cDNA/DNA of a sample to be detected as a template:

the reaction system is as follows: 2 XTaq Master Mix (Dye Plus) 12.5. mu.L, sample cDNA/DNA mixture 2. mu.L, first primer pair and second primer pair each 1. mu.L, third primer pair 2. mu.L, and ddH₂O is complemented to 25 mu L, and the concentrations of the first primer pair, the second primer pair and the third primer pair are all 10 mu mol/L;

the PCR reaction conditions are as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 57.4 ℃, 30s at 72 ℃, 30 cycles, 10min at 72 ℃ and storage at 4 ℃;

(3) and (3) carrying out agar gel electrophoresis detection on the PCR product obtained in the step (2).

Performance test 2

(1) Specificity verification of triple PCR detection method

According to the instructions of the virus RNA/DNA rapid purification kit, extracting the RNA/DNA of DuCV, DAdV, DTMUV, H9AIV, DHV, DEV, DPMV and DAstV; the RNA template refers to the specification of a reverse transcription kit to synthesize cDNA, the genome DNA of Riemerella anatipestifer, Escherichia coli and salmonella is extracted according to the specification of a bacterial genome DNA extraction kit, and all the cDNA and DNA templates are stored at the low temperature of-80 ℃ for later use.

Triple PCR reactions were carried out using DNA/cDNA as templates and the PCR detection method provided in example 15, respectively, and the triple PCR products were detected by agarose gel electrophoresis.

FIG. 3 is a diagram showing the result of the specificity verification of the triple PCR detection method provided by the present disclosure, in which M is DNA Marker DL2000, and 1 is DuCV and DAdMixed samples of V and DTMUV, 2-12 for DuCV, DAdV, DTMUV, H9AIV, DHV, DEV, DPMV, DAstV, Riemerella anatipestifer, E.coli and Salmonella, respectively, and 13 for negative controls (ddH)₂O)。

As can be seen from fig. 3, the triple PCR detection primer set provided by the present disclosure can only specifically target to DuCV, DAdV, and DTMUV, and can be used for detecting pathogens frequently encountered by other ducks: amplification of H9AIV, DHV, DEV, DPMV, DAstV, Riemerella anatipestifer, Escherichia coli and salmonella is negative, and no specific band exists, so that the detection primer group and the detection method provided by the disclosure have good specificity, and can specifically identify DuCV, DAdV and DTMUV.

(2) Sensitivity test

PCR amplification is carried out by using DuCVCap gene, DAdVHexon gene and DTMUV E gene primers and DuCV, DAdV and DTMUV DNA and cDNA as templates respectively to obtain full-length target fragments of Cap gene, Hexon gene and E gene, and the three gene fragments are linked to pMD18-T vector respectively.

The recombinant vectors containing the correct sequences of DuCVCap gene, DAdVHexon gene and DTMUV E gene fragments are named as DuCV-T, DAdV-T and DTMUV-T respectively, the plasmids of DuCV-T, DAdV-T and DTMUV-T are extracted by a plasmid extraction kit, the nucleic acid concentration of the plasmids is measured by a trace nucleic acid detector, the corresponding copy number is calculated according to the molecular mass and the nucleic acid concentration, the DuCV-T, DAdV-T and the DTMUV-T are mixed in equal volume and diluted by 10 times, the concentration obtained by each method is 10 times⁷-10¹The sensitivity of triple PCR was determined using DuCV-T, DAdV-T and DTMUV-T mixed plasmid DNA in copies/. mu.L as a template, wherein the conditions of the triple PCR reaction were as in example 15, the criteria were the same as in example 15, and a negative control (ddH) was provided₂O)。

FIG. 4 is a graph of the sensitivity test results of the triple PCR test method provided by the present disclosure, in which:

M：DNA Marker DL2000；

1：4.64×10⁷copy/. mu.LDuCV, 1.62X 10⁷Copy/. mu.LDAdV, 3.34X 10⁷Copy/. mu.LDTMUV;

2：4.64×10⁶copy/. mu.LDuCV, 1.62X 10⁶Copy/. mu.LDAdV, 3.34X 10⁶Copy/. mu.LDTMUV;

3：4.64×10⁵copy/. mu.LDuCV, 1.62X 10⁵Copy/. mu.LDAdV, 3.34X 10⁵Copy/. mu.LDTMUV;

4：4.64×10⁴copy/. mu.LDuCV, 1.62X 10⁴Copy/. mu.LDAdV, 3.34X 10⁴Copy/. mu.LDTMUV;

5：4.64×10³copy/. mu.LDuCV, 1.62X 10³Copy/. mu.LDAdV, 3.34X 10³Copy/. mu.LDTMUV;

6：4.64×10²copy/. mu.LDuCV, 1.62X 10²Copy/. mu.LDAdV, 3.34X 10²Copy/. mu.LDTMUV;

7：4.64×10¹copy/. mu.LDuCV, 1.62X 10¹Copy/. mu.LDAdV, 3.34X 10¹Copy/. mu.LDTMUV;

8: negative control (ddH)₂O)。

As can be seen from FIG. 4, the triple PCR method for detecting DuCV, DAdV and DTMUV was found to be at least 10 detectable²Copies/. mu.L of plasmid DNA (see FIG. 4), indicating that the simultaneous detection of DuCV, DAdV and DTMUV by this triple PCR has a higher sensitivity.

(3) Clinical trial

50 samples were randomly sampled for testing from the censorship in 2019-2021 chat, texas, chaze, Weifang, etc. using the triple PCR test method provided in example 15, FIG. 5 is an agarose gel electrophoresis image of a portion of the PCR products in 50 samples, in which:

m is DNA Marker DL 2000;

1 is a positive control (DuCVDNA, DAdV DNA and DTMUV cDNA);

2: negative control (ddH)₂O)；

4. 11: a DuCV positive sample;

7: DuCV and DTMUV mixed infection samples;

8: a DAdV positive sample;

10: a DTMUV positive sample;

13: DuCV and DAdV mixed infection samples;

3. 5, 6, 9, 12, 14: and (4) negative samples.

And then, detecting and verifying the 50 samples by using a conventional PCR method, wherein the result shows that the detection result is completely the same as the detection result of the triple PCR detection method provided by the disclosure, and the coincidence rate is 100%, which indicates that the triple PCR detection method provided by the disclosure is suitable for rapid detection of duck circovirus, duck adenovirus and duck tembusu virus.

It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The foregoing are merely exemplary embodiments of the present disclosure, which enable those skilled in the art to understand or practice the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the disclosure. Thus, the present disclosure is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> chat university

Triple PCR detection kit for <120> duck circular ring, duck adenovirus and duck tembusu virus

<130> KP2114664.3

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tctctcgtgc ccggggatct 20

<210> 2

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtcatttccc gagtaaccgt ccc 23

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgcgacaact acctgtggac 20

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcgtacggaa gtaagccat 19

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

catggacagg gtcatcagc 19

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agagtacttc actggaatag ctcc 24

Claims (9)

1. The triple PCR detection primer group for duck circovirus, duck adenovirus and duck tembusu virus is characterized by comprising a first primer pair, a second primer pair and a third primer pair:

the first primer pair has a nucleotide sequence pair shown in SEQ ID NO.1 and SEQ ID NO. 2; the second primer pair has a nucleotide sequence pair shown in SEQ ID NO.3 and SEQ ID NO. 4; the third primer pair has a nucleotide sequence pair shown in SEQ ID NO.5 and SEQ ID NO. 6.

2. The triple PCR detection primer set according to claim 1, wherein the molar ratio of the first primer pair, the second primer pair and the third primer pair is 1 (0.5-2) to (0.5-4).

3. The triple PCR detection primer set according to claim 2, wherein the molar ratio of the first primer pair, the second primer pair and the third primer pair is 1:1: 2.

4. Triple PCR detection kit for duck circovirus, duck adenovirus and duck Tembusu virus, characterized by comprising the triple PCR detection primer set of any one of claims 1 to 3.

5. The triple PCR assay kit of claim 4, wherein the triple PCR assay kit further comprises 2 XTAQA Master Mix (Dye Plus).

6. Use of the triple PCR assay kit of claim 4 or 5 for triple PCR amplification.

7. Use according to claim 6, wherein the annealing temperature of the triple PCR amplification is 52-59 ℃, preferably 57.4 ℃.

8. Triple PCR detection method for simultaneously detecting duck circovirus, duck adenovirus and duck tembusu virus for non-diagnostic and therapeutic purposes, characterized in that the triple PCR detection method comprises the use of the triple PCR detection primer set of any one of claims 1 to 3 or the triple PCR detection kit of claim 4 or 5.

9. The triple PCR detection method according to claim 8, wherein the triple PCR detection method comprises the steps of:

(1) extracting RNA/DNA of a sample to be detected, and performing reverse transcription to obtain cDNA/DNA;

(2) carrying out PCR reaction by taking cDNA/DNA of a sample to be detected as a template, wherein the reaction system comprises a triple PCR detection primer set according to any one of claims 1-3;

(3) and (3) carrying out agar gel electrophoresis detection on the PCR product obtained in the step (2), and judging whether the sample to be detected has duck circovirus, duck adenovirus or duck tembusu virus according to the detection result.

Images