CN106755572A - I types DHV and duck plague virus double fluorescent quantitative PCR method - Google Patents

Abstract

The invention provides a dual fluorescent quantitative PCR method for type I duck hepatitis virus and duck plague virus, and respectively designs a pair of specific detection primers DHV‑I‑F, DHV‑I‑R and a fluorescent primer for type I duck hepatitis virus Probe DHV‑I‑P, a pair of specific detection primers DPV‑F, DPV‑R and a fluorescent probe DPV‑P for duck plague virus, and confirmed the double detection of duck hepatitis virus type I and duck plague virus Concentrations of specific primers and fluorescent probes for fluorescent quantitative PCR method. The dual fluorescent quantitative PCR method provided by the invention can simultaneously detect and differentiate two types of duck hepatitis virus type I and duck plague virus, has the advantages of simple operation, high sensitivity, strong specificity, good repeatability, etc., and can not only save costs, It can also buy valuable time for epidemic monitoring and epidemic control, and provide effective means for the early rapid diagnosis, monitoring and epidemiological investigation of infectious duck diseases caused by these two viruses.

Description

Double Fluorescent Quantitative PCR Method for Type I Duck Hepatitis Virus and Duck Plague Virus

technical field

The invention belongs to the technical field of biological detection, and in particular relates to a double fluorescence quantitative PCR method for type I duck hepatitis virus and duck plague virus.

Background technique

Duck viral hepatitis is an acute, severe, highly contagious and highly lethal viral infectious disease characterized by hepatitis. The pathogen is duck hepatitis virus (DHV), which mainly causes ducklings under 3 weeks of age to become ill. There are mainly three serotypes of duck hepatitis virus, namely type I, type II, and type III. These three serotypes are independent of each other. DHV-I is mainly prevalent in my country. Type Ⅰ duck hepatitis virus is most likely to be popular in spring from March to April. The mortality rate of ducklings under 3 weeks old is extremely high after infection. The mortality rate of ducklings under one week old can reach more than 80%. Reaching about 50%, has seriously jeopardized the healthy development of duck industry all over the country.

Duck plague is an acute and highly fatal infectious disease caused by duck plague virus (DPV), also known as duck viral enteritis, commonly known as "big head plague". Duck plague virus is a herpes virus, and its virus particles are spherical. It can infect ducks of all ages and varieties. It can occur in all seasons throughout the year, but it is most popular at the turn of spring and summer and autumn. The disease is mainly transmitted through the digestive tract, and can also be transmitted through mating, eye conjunctiva and respiratory tract. Sick ducks, utensils and means of transportation contaminated by their excrement, and even contacts with epidemic areas or personnel in epidemic areas may cause the spread of duck plague. When duck plague is prevalent, the morbidity and death of adult ducks are more serious, and the incidence of ducklings under 1 month is less, which often brings huge economic losses to the duck industry.

At present, the detection methods of type Ⅰ duck hepatitis virus and duck plague virus mainly include isolation culture, serological detection, conventional PCR and real-time fluorescent quantitative PCR detection. Isolation culture has high requirements on culture environment, nutritional conditions, and separation operation, and it takes a long time; there are subjective factors in the interpretation of serological test results, the sensitivity and specificity are not high, and it is easy to cross with other microorganisms such as chlamydia Reaction and other problems; routine PCR detection has problems such as lack of specificity of primer binding, laboratory contamination, and presence of PCR inhibitors in clinical samples causing false negatives. After gene amplification, electrophoresis sequencing is required to verify, which is relatively time-consuming and costly. High; the real-time fluorescent probe PCR method distinguishes species based on sequence-specific probes, which improves the specificity and sensitivity of detection. For example, CN200810198528.9 discloses a molecular biology identification method for type I duck hepatitis virus, using fluorescent quantitative PCR The method is used to identify type Ⅰ duck hepatitis virus, but this method can only detect type Ⅰ duck hepatitis virus, and cannot detect type Ⅰ duck hepatitis virus and duck plague virus at the same time to make timely and accurate distinctions, which will easily delay epidemic monitoring and epidemic control.

Contents of the invention

In order to solve the above technical problems, the invention provides a double fluorescent quantitative PCR method for type I duck hepatitis virus and duck plague virus, in the method:

The sequences of the specific primers and probes designed to detect type I duck hepatitis virus are respectively:

Upstream primer DHV-I-F: 5'-TGGGATACCCAGGAGTACACGTA-3' (as shown in SEQ ID No.1);

Downstream primer DHV-I-R: 5'-TGTTAAGCTGGGAGGTGTCTTGT-3' (as shown in SEQ ID No.2);

Fluorescent probe DHV-I-P:

5'-VIC-CACCCACTGGCTTTGGAGCTGTGC-TAMRA-3' (as shown in SEQ ID No.3);

The sequences of specific primers and probes designed to detect duck plague virus are:

Upstream primer DPV-F: 5'-CGGTTTTGGGAAGGCTTTCG-3' (as shown in SEQ ID No.4);

Downstream primer DPV-R: 5'-ACCCTAACGGCTCCTGTAG-3' (as shown in SEQ ID No.5);

Fluorescent probe DPV-P:

5'-FAM-TCCACTGGCATTTGCTTGATTTCCGC-TAMRA-3' (as shown in SEQ ID No. 6).

Further, the method includes:

(1) Design and synthesize primers and probes;

(2) prepare positive plasmid standard substance;

(3) Optimizing the reaction conditions of double fluorescent quantitative PCR, and establishing a double fluorescent quantitative PCR detection system;

(4) The virus was detected by a dual fluorescent quantitative PCR detection system.

Further, the specific process of preparing positive plasmid standard items includes:

A. Synthesize primers for preparing positive plasmid standards;

DHV primers:

Upstream primer P1: 5'-GTTTTGGAAGGGAAGATACAGTGGT-3';

Downstream primer P2: 5'-GTAGGGTAGGGAATAGTAAAGTAAA-3';

The amplified target fragment is 516bp;

DPV primers:

Upstream primer P3: 5'-GCAGGGCATTTGTTAGACGATA-3';

Downstream primer P4: 5'-TGCCGCCAATTTGTAAAG-3';

The amplified target fragment is 337bp;

B. Extract type I duck hepatitis virus RNA and duck plague virus DNA with commercial kits, and store them at -70°C for future use;

C. carry out reverse transcription reaction to the RNA of type I duck hepatitis virus and obtain cDNA, the reaction system is to contain 0.2-1000ng/μL template RNA, 0.5μM downstream primer P2, 1mM dNTP, 1U/μL Rnase Inhibition (reverse transcriptase inhibition agent), 5×Primescript Buffer (5 times of reverse transcription buffer) of 200U Prime Script reverse transcriptase and the mixed solution of DEPC water, wherein 5×Primescript Buffer means that its volume fraction in the mixed solution is 20%;

The specific steps are: put the RNA template and downstream primer P2 in a water bath at 65°C for 10 minutes, then ice bath for 5 minutes, add dNTP, Rnase Inhibition, Prime Script reverse transcriptase, 5×Primescript Buffer and DEPC water after brief centrifugation, and keep at 42°C Water bath for 1 hour, water bath at 70°C for 10 minutes, ice bath for 5 minutes, and store at -20°C for later use;

D. carry out PCR amplification reaction with the cDNA of type I duck hepatitis virus and the DNA of duck plague virus respectively as template, then reclaim and purify the amplified product;

Among them, the reaction system of the PCR amplification reaction of the cDNA of duck hepatitis virus type I contains 0.5-1000ng/μL template cDNA, 0.4μM upstream primer P1, 0.4μM downstream primer P2, 0.8mM dNTP, 1.6mM MgCl ₂ , 0.1U /μL Ex Taq 10×Buffer (10 times buffer solution) and ddH ₂ O mixed solution, where 10× Buffer means its volume fraction in the mixed solution is 10%;

The amplification reaction conditions are: 95°C pre-denaturation for 5 minutes, 94°C for 60s, 51.8°C for 40s, 72°C for 60s, 30 cycles, and 72°C for 10 minutes;

The reaction system of PCR amplification reaction with duck plague virus DNA as template is containing 0.2-1000ng/μL template DNA, 0.4μM upstream primer P3, 0.4μM downstream primer P4, 0.8mM dNTP, 1.6mM MgCl ₂ , 0.1U/μL Ex A mixture of Taq 10×Buffer and ddH ₂ O, where 10×Buffer means that its volume fraction in the mixture is 10%;

The amplification reaction conditions are: 95°C pre-denaturation for 5 minutes, 94°C for 60s, 51.8°C for 40s, 72°C for 60s, 30 cycles, and 72°C for 10 minutes;

E. Ligate the purified PCR products of type I duck hepatitis virus and duck plague virus to the carrier pMD18-T; then introduce the ligated vector into E. coli competent cells for transformation and plating; finally extract to obtain positive Standard grains.

Further, the dual fluorescent quantitative PCR detection system specifically includes: extracting RNA and DNA of the sample to be tested by conventional methods, and performing reverse transcription reaction on the RNA to obtain cDNA; then performing double fluorescent quantitative PCR reaction using cDNA and DNA as templates.

Further, the reaction system of the reverse transcription reaction is 5×Primescript Buffer containing 0.2-1000ng/μL template RNA, 0.5μM random primer, 1mM dNTP, 1U/μL Rnase Inhibition, 200U Prime Script reverse transcriptase and DEPC Water mixture, where 5×Primescript Buffer means its volume fraction in the mixture is 20%.

Further, the reaction conditions of the reverse transcription reaction are: water bath at 42° C. for 1 hour, water bath at 70° C. for 10 minutes, and ice bath for 5 minutes to obtain viral cDNA.

Further, the reaction system of the double fluorescent quantitative PCR reaction contains 0.5-1000ng/μL template cDNA, 0.2-1000ng/μL template DNA, 0.28μM upstream primer DHV-I-F, 0.28μM downstream primer DHV-I-R, 0.36μM fluorescent A mixture of probe DHV-I-P, 0.28 μM upstream primer DPV-F, 0.28 μM downstream primer DPV-R, 0.36 μM fluorescent probe DPV-P, 2×Premix Ex Taq and DEPC water, where 2×Premix Ex Taq means Its volume fraction in the mixed solution is 50%.

Further, the reaction conditions of the double fluorescent quantitative PCR reaction are: 95°C for 30s; 95°C for 5s, 60°C for 50s, 40 cycles.

Further, the optimization of the double fluorescent quantitative PCR reaction conditions includes:

a. Optimization of annealing temperature: using the plasmid as a template, perform gradient quantitative PCR at 53°C-61°C, and select the optimal annealing temperature according to the amplification reaction Ct, the fluorescence signal intensity of the amplification curve, and the melting curve;

b. Optimizing the concentration of primers and probes: using the plasmid as a template, the concentration of primers and probes is 0.04-0.4 μM, and performing fluorescent quantitative PCR to determine the optimal concentration of primers and probes.

The double fluorescence quantitative PCR method of type I duck hepatitis virus and duck plague virus established by the present invention can simultaneously detect and distinguish the two viruses of type I duck hepatitis virus and duck plague virus, and has the advantages of simple operation, high sensitivity, strong specificity and repeatability Good and other advantages, not only can save costs, but also can gain valuable time for epidemic monitoring and epidemic control, and provide research on early and rapid diagnosis, monitoring and epidemiological investigations of infectious duck diseases caused by these two viruses. effective means.

Description of drawings

Fig. 1 is the typical curve of the double fluorescent quantitative PCR method of the I type duck hepatitis virus of the specific embodiment of the present invention;

Fig. 2 is the standard curve of the double fluorescence quantitative PCR method of the duck plague virus of the specific embodiment of the present invention;

Fig. 3 is the amplification curve of the sensitivity analysis of the double fluorescence quantitative PCR method of the I type duck hepatitis virus of the specific embodiment of the present invention;

Fig. 4 is the amplification curve of the sensitivity analysis of the double fluorescent quantitative PCR method of the duck plague virus of the specific embodiment of the present invention;

Fig. 5 is the amplification curve of the specificity analysis of the double fluorescent quantitative PCR method of the I duck hepatitis virus of the specific embodiment of the present invention;

Fig. 6 is the amplification curve of the specificity analysis of the dual fluorescent quantitative PCR method of the duck plague virus according to the specific embodiment of the present invention.

detailed description

The commercialization reagent adopted in the specific embodiment of the present invention mainly comprises:

RNA in vitro extraction kit Total RNA kit (R6934) was purchased from OMEGA;

DNA virus kit (D3809-01) was purchased from OMEGA;

Ex taq enzyme was purchased from TAKARA company;

RNA reverse transcription kit Prime script RT Kit (RR047) was purchased from TAKARA company;

DNA Fragment Recovery Kit (08214RE1) was purchased from AXY Company;

Plasmid mini-extraction kit (06313KA1) was purchased from AXY Company;

Fluorescence quantitative Premix Ex Taq enzyme (RR390A) was purchased from TAKARA Company.

Unless otherwise specified, the following specific embodiments are all in accordance with conventional experimental conditions.

A double fluorescent quantitative PCR method for type I duck hepatitis virus and duck plague virus, comprising:

(1) Design and synthesize primers and probes;

Referring to the gene sequence of duck hepatitis virus type I in NCBI, using Primer 5 software, a pair of primers for amplifying the target fragment of 516bp was designed and synthesized, and the amplified product was used to prepare a standard product, and a conservative sequence was selected in the standard product sequence , using Beacon designer 7.5 software to design and synthesize a pair of fluorescent quantitative PCR primers and TaqMan fluorescent probes capable of amplifying 78bp, the sequences of the designed specific primers and probes for detecting type I duck hepatitis virus are respectively:

Upstream primer DHV-I-F: 5'-TGGGATACCCAGGAGTACACGTA-3';

Downstream primer DHV-I-R: 5'-TGTTAAGCTGGGAGGTGTCTTGT-3';

Fluorescent probe DHV-I-P:

5'-VIC-CACCCACTGGCTTTGGAGCTGTGC-TAMRA-3';

Referring to the conserved sequences in UL6 and UL7 of duck plague virus, using Primer 5 software, a pair of primers for amplifying the target fragment of 337bp were designed and synthesized, and the amplified products were used to prepare standard products. Using the standard products as templates, Beacondesigner was used to 7.5 software designed and synthesized a pair of fluorescent quantitative PCR primers and TaqMan fluorescent probes capable of amplifying 78bp, the sequences of the designed specific primers and probes for detecting duck plague virus are respectively:

Upstream primer DPV-F: 5'-CGGTTTTGGGAAGGCTTTCG-3';

Downstream primer DPV-R: 5'-ACCCTAACGGCTCCTGTAG-3';

Fluorescent probe DPV-P:

5'-FAM-TCCACTGGCATTTGCTTGATTTCCGC-TAMRA-3';

(2) prepare positive plasmid standard substance;

A. Synthesize primers for preparing positive plasmid standards;

DHV primers:

Upstream primer P1: 5'-GTTTTGGAAGGGAAGATACAGTGGT-3';

Downstream primer P2: 5'-GTAGGGTAGGGAATAGTAAAGTAAA-3';

The amplified target fragment is 516bp;

DPV primers:

Upstream primer P3: 5'-GCAGGGCATTTGTTAGACGATA-3';

Downstream primer P4: 5'-TGCCGCCAATTTGTAAAG-3';

The amplified target fragment is 337bp;

B. Extract type I duck hepatitis virus RNA and duck plague virus DNA with commercial kits, and store them at -70°C for future use;

C. Perform reverse transcription reaction on RNA of type I duck hepatitis virus to obtain cDNA, the reaction system is containing 20ng/μL template RNA, 0.5μM downstream primer P2, 1mM dNTP, 1U/μL Rnase Inhibition, 200U Prime Script reverse transcriptase The mixture of 5×Primescript Buffer and DEPC water;

The specific steps are: put the RNA template and downstream primer P2 in a water bath at 65°C for 10 minutes, then ice bath for 5 minutes, add dNTP, Rnase Inhibition, Prime Script reverse transcriptase, 5×Primescript Buffer and DEPC water after brief centrifugation, and keep at 42°C Water bath for 1 hour, water bath at 70°C for 10 minutes, ice bath for 5 minutes, and store at -20°C for later use;

D. carry out PCR amplification reaction with the cDNA of type I duck hepatitis virus and the DNA of duck plague virus respectively as template, then reclaim and purify the amplified product;

Among them, the reaction system of the PCR amplification reaction of the cDNA of duck hepatitis virus type I contains 20ng/μL template cDNA, 0.4μM upstream primer P1, 0.4μM downstream primer P2, 0.8mMdNTP, 1.6mM MgCl ₂ , 0.1U/μL Ex Mixture of Taq 10×Buffer and ddH ₂ O;

The amplification reaction conditions are: 95°C pre-denaturation for 5 minutes, 94°C for 60s, 51.8°C for 40s, 72°C for 60s, 30 cycles, and 72°C for 10 minutes;

The reaction system of the PCR amplification reaction with duck plague virus DNA as template is containing 20ng/μL template DNA, 0.4μM upstream primer P3, 0.4μM downstream primer P4, 0.8mM dNTP, 1.6mM MgCl ₂ , 0.1U/μL Ex Taq Mixture of 10×Buffer and ddH ₂ O;

The amplification reaction conditions are: 95°C pre-denaturation for 5 minutes, 94°C for 60s, 51.8°C for 40s, 72°C for 60s, 30 cycles, and 72°C for 10 minutes;

Recover and purify the amplified product using Axygen's DNA Column Gel Recovery Kit for gel recovery;

E. Preparation of positive standard plasmids, specifically including: connecting the PCR products of the purified type I duck hepatitis virus and duck plague virus to the vector pMD18-T respectively; and then introducing the ligated vector into E. coli competent cells for transformation and plate; finally extract to obtain the positive standard plasmid;

(3) Optimizing the reaction conditions of double fluorescent quantitative PCR, and establishing a double fluorescent quantitative PCR detection system;

S1. Preparation of standard curve for dual fluorescent quantitative PCR detection system

The plasmids of type I duck hepatitis virus and duck plague virus of 10 ⁸ -10 ² copies/μL were used as templates to carry out PCR amplification reaction respectively, and the standard curve (Fig. 1) and The standard curve of the double fluorescent quantitative PCR method for duck plague virus (Fig. 2), it can be seen from the figure that the template copy number in each reaction system has a good linear relationship in the range of 10 ⁸ -10 ² copies/μL, and the correlation coefficient is 0.999, the amplification efficiency of type I duck hepatitis virus is E=100.8%, the amplification efficiency of duck plague virus is E=95.7%;

S2. Optimization of reaction conditions for dual fluorescent quantitative PCR

a. Annealing temperature optimization: use the standard plasmid as a template, carry out gradient quantitative PCR at 53°C-61°C, and select the optimal annealing temperature according to the amplification reaction Ct, the fluorescence signal intensity of the amplification curve, and the melting curve;

b. Optimization of the concentration of primers and probes: use the standard plasmid as a template, the concentration of primers and probes is 0.04-0.4 μM, perform fluorescent quantitative PCR, and determine the optimal concentration of primers and probes;

Groping through experiments shows that the final concentration of the primers of type I duck hepatitis virus and duck plague virus is 0.28 μM, the final concentration of probes of type I duck hepatitis virus and duck plague virus is 0.36 μM, and the optimal annealing temperature is 60 ℃;

S3. Establish a dual fluorescent quantitative PCR detection system, which specifically includes:

①A commercial kit is used to extract the RNA and DNA of the sample to be tested using conventional methods, and the RNA is subjected to reverse transcription reaction to obtain cDNA. The reaction system of the reverse transcription reaction is 0.2-1000ng/μL template RNA, 0.5μM Mixture of random primers, 1mM dNTP, 1U/μL Rnase Inhibition, 200U PrimeScript reverse transcriptase in 5×PrimescriptBuffer and DEPC water;

The random primers, dNTP, Rnase Inhibition, Prime Script reverse transcriptase, 5×Primescript Buffer, and DEPC water in the reaction system of the reverse transcription reaction are all from the RNA reverse transcription kit Prime script RT Kit (RR047);

Reaction conditions: 42°C water bath for 1 hour, 70°C water bath for 10 minutes, and ice bath for 5 minutes to obtain viral cDNA;

②Using cDNA and DNA as templates for double fluorescence quantitative PCR reaction, the reaction system contains 0.5-1000ng/μL template cDNA, 0.2-1000ng/μL template DNA, 0.28μM upstream primer DHV-I-F, 0.28μM downstream primer DHV-I-R, Mixture of 0.36 μM fluorescent probe DHV-I-P, 0.28 μM upstream primer DPV-F, 0.28 μM downstream primer DPV-R, 0.36 μM fluorescent probe DPV-P, 2×Premix Ex Taq and DEPC water;

The reaction conditions are: 95°C for 30s; 95°C for 5s, 60°C for 50s (collecting fluorescent signals), 40 cycles;

S4. Check the established dual fluorescent quantitative PCR detection system

① Sensitivity analysis of dual fluorescent quantitative PCR detection method

Dilute the plasmid standard 10 times, take 1×10 ⁸ -1×10 ² copies/μL of each 2 μL of the standard plasmid as a mixed template for double fluorescent quantitative PCR detection, determine the minimum detection amount and set up a negative control;

7 groups of experiments were set up, and the plasmids of type I duck hepatitis virus and duck plague virus (1×10 ⁸ -1×10 ² copies/μL) diluted by 10 times were used as templates for PCR amplification, and the test group numbers and plasmids The corresponding relationship of concentration is shown in Table 1:

Table 1 Correspondence between test group number and plasmid concentration

Test group number Plasmid concentration (copies/μL) 1 1×10 ⁸ 2 1×10 ⁷ 3 1×10 ⁶ 4 1×10 ⁵ 5 1×10 ⁴ 6 1×10 ³ 7 1×10 ²

Sensitivity results are shown in Figure 3 and Figure 4, respectively. It can be seen from the figure that the PCR detection of type I duck hepatitis virus and duck plague virus still has fluorescence curves using 1×10 ² copies/μL plasmid as a template, indicating that the detection method The sensitivity is 100copies/μL;

② Specificity analysis of dual fluorescent quantitative PCR detection method

Apply the dual fluorescent quantitative PCR detection system provided by the present invention to detect various virus samples including type I duck hepatitis virus and duck plague virus respectively, and verify the effectiveness of the double fluorescent quantitative PCR method for type I duck hepatitis virus and duck plague virus specificity;

Apply the double fluorescent quantitative PCR detection method provided by the present invention to detect 12 groups of virus samples, and the virus types contained in each group of virus samples are as shown in Table 2:

Table 2 The types of viruses contained in each group of virus samples

The results of specificity are shown in Figure 5 and Figure 6. It can be seen from the figures that only type I duck hepatitis virus and duck plague virus have amplification fluorescence curves, and other pathogenic nucleic acid detections have no amplification fluorescence curves. Therefore, the detection method has relatively high specificity. it is good;

③Repeatability analysis of double fluorescent quantitative PCR detection method

Repetitive tests are divided into repeated tests within groups and repeated tests between groups.

Repeated experiments within the group: In the same experiment, use 10 ⁶ copies/μL of the plasmid standard mixture template of type I duck hepatitis virus and duck plague virus to do 5 parallel reactions, and calculate the standard deviation and variation coefficient of the fluorescence Ct values of each concentration , to verify the stability of the dual fluorescent quantitative PCR method for type I duck hepatitis virus and duck plague virus;

Repeated test results in the group are as shown in table 3, 5 parallel reactions are done to the same sample, and the repeatability in the group of the double fluorescent quantitative PCR method of type I duck hepatitis virus and duck plague virus is good;

Table 3 Repeatability test within the double fluorescent quantitative PCR group

Repeated test between groups: Repeat the test on the same mixed template above after 3 days and 7 days under the same reaction conditions, calculate the standard deviation and coefficient of variation of the Ct value, and verify the double fluorescent quantitative PCR method of type I duck hepatitis virus and duck plague virus stability;

Repeated test results between groups are shown in table 4, using the same nucleic acid template, with the same detection conditions, repeat the test after 3d and 7d, the average coefficient of variation of the Ct value of type 1 duck hepatitis virus and duck plague virus is 0.30% respectively and 1.2%, the repeatability between groups of the double fluorescent quantitative PCR method of type I duck hepatitis virus and duck plague virus is good;

Table 4 Repeatability test between groups of double fluorescent quantitative PCR

(4) The virus is detected by a dual fluorescent quantitative PCR detection system;

Using the established dual fluorescent quantitative PCR detection system to detect 166 anal swabs collected from ducks in Xuzhou, Jiangsu, the results detected 13 copies of type I duck hepatitis virus (positive rate 7.8%) and 75 copies of duck plague virus (positive rate 45%).

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be Modifications or equivalent replacements without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the claims of the present invention.

sequence listing

<110> Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences

<120> Double Fluorescent Quantitative PCR Method for Type I Duck Hepatitis Virus and Duck Plague Virus

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<170> PatentIn version 3.3

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Claims (7)

1. a kind of I types DHV and duck plague virus double fluorescent quantitative PCR method, it is characterised in that in methods described：

The specific primer of design detection I type DHVs and the sequence of probe are respectively：

Sense primer DHV-I-F：5’-TGGGATACCCAGGAGTACACGTA-3’；

Anti-sense primer DHV-I-R：5’-TGTTAAGCTGGGAGGTGTCTTGT-3’；

Fluorescence probe DHV-I-P：

5’-VIC-CACCCACTGGCTTTGGAGCTGTGC-TAMRA-3’；

The specific primer of design detection duck plague virus and the sequence of probe are respectively：

Sense primer DPV-F：5’-CGGTTTTGGGAAGGCTTTCG-3’；

Anti-sense primer DPV-R：5’-ACCCTAACGGCTCCTGTAG-3’；

Fluorescence probe DPV-P：

5’-FAM-TCCACTGGCATTTGCTTGATTTCCGC-TAMRA-3’。

2. I types DHV according to claim 1 and duck plague virus double fluorescent quantitative PCR method, its feature exist In methods described includes：

(1) design and synthetic primer and probe；

(2) positive plasmid standard items are prepared；

(3) optimization of double fluorescent quantitative PCR reaction condition is carried out, double fluorescent quantitative PCR detection architecture is set up；

(4) virus is detected using double fluorescent quantitative PCR detection architecture.

3. I types DHV according to claim 2 and duck plague virus double fluorescent quantitative PCR method, its feature exist In the double fluorescent quantitative PCR detection architecture is specifically included：Conventional method extracts the RNA and DNA of testing sample, and to RNA Carry out reverse transcription reaction and obtain cDNA；Again double fluorescent quantitative PCR reaction is carried out by template of cDNA and DNA.

4. I types DHV according to claim 3 and duck plague virus double fluorescent quantitative PCR method, its feature exist In the reaction system of, the reverse transcription reaction be containing 0.2-1000ng/ μ L template ribonucleic acids, 0.5 μM of random primer, 1mM dNTP, 5 × Primescript Buffer and DEPC of 1U/ μ L Rnase Inhibition, 200U Prime Script reverse transcriptase The mixed liquor of water.

5. I types DHV according to claim 4 and duck plague virus double fluorescent quantitative PCR method, its feature exist In the reaction condition of the reverse transcription reaction is：42 DEG C of water-baths 1h, 70 DEG C of water-bath 10min, ice bath 5min, obtain virus cDNA.

6. I types DHV according to claim 3 and duck plague virus double fluorescent quantitative PCR method, its feature exist In the reaction system of the double fluorescent quantitative PCR reaction is to contain 0.5-1000ng/ μ L template cDNA, 0.2-1000ng/ μ L Template DNA, 0.28 μM of sense primer DHV-I-F, 0.28 μM of anti-sense primer DHV-I-R, 0.36 μM of fluorescence probe DHV-I-P, 0.28 μM of sense primer DPV-F, 0.28 μM of anti-sense primer DPV-R, 2 × Premix Ex Taq of 0.36 μM of fluorescence probe DPV-P With the mixed liquor of DEPC water.

7. I types DHV according to claim 6 and duck plague virus double fluorescent quantitative PCR method, its feature exist In the reaction condition of the double fluorescent quantitative PCR reaction is：95℃30s；95 DEG C of 5s, 60 DEG C of 50s, 40 circulations.