CN102618668B - Duplex PCR (polymerase chain reaction) detection kit for duck Newcastle disease virus and duck circovirus - Google Patents

Abstract

The invention discloses a double PCR detection kit for duck Newcastle disease virus and duck circovirus. The present invention provides a primer set for detecting Newcastle disease virus and duck circovirus, consisting of primer 1, primer 2, primer 3 and primer 4; said primer 1, said primer 2, said primer 3 and said primer The nucleotide sequences of primer 4 are respectively sequence 1, sequence 2, sequence 3 and sequence 4 in the sequence list. The experiment of the present invention proves, this research has designed 2 pairs of primers, has established the detection method of double PCR of Newcastle disease virus and duck circovirus, can detect and distinguish Newcastle disease virus and duck circovirus two kinds of pathogens simultaneously. Heavy PCR technology has the advantages of simple operation, high sensitivity, strong specificity and good repeatability.

Description

Duck Newcastle Disease Virus and Duck Circovirus Duplex PCR Detection Kit

technical field

The invention relates to the field of biotechnology, in particular to a double PCR detection kit for duck Newcastle disease virus and duck circovirus.

Background technique

Duck Newcastle disease is an acute, highly contagious and fatal infectious disease caused by duck-derived Newcastle disease virus (Duck Newcastle Diseases duck NDV). Early studies have found that ducks have strong resistance to pathogenic APMV-1, and only behave as healthy carriers, even if they are infected with strong viruses, they will not cause disease. However, in recent years, it has been found that the natural prevalence of duck-derived Newcastle disease virus, which causes high pathogenicity and mortality, is seriously harmful to the duck industry. Hebei, Fuzhou, Zhejiang, Shanxi, Shandong, Henan and other places have successively reported the occurrence and prevalence of the disease. The above studies show that the infection of duck-derived Newcastle disease virus is on the rise, which will cause great harm to the duck industry in my country.

Duck circovirus (Duck circovirus, DuCV) is a member of the genus Circovirus in the family Circoviridae and was first reported in Germany. In recent years, there have been reports of duck infection or disease caused by duck circovirus in Hungary, the United States, Taiwan, China, Shandong, Liaoning, Fujian, Guangdong and Guangxi. The main clinical manifestations of duck disease caused by DuCV are dysplasia, weight loss and messy feathers. Histiocytosis. The gradual atrophy of lymphoid tissue in infected animals leads to decreased immune function or immune response suppression, which increases the probability of double or multiple infections. The characteristics of circovirus infection and mixed infection with other pathogenic bacteria or viruses are very complicated. Chai Tongjie's investigation of duck circovirus and mixed infection in Cherry Valley ducks in Shandong showed that the mixed infection rate of duck circovirus, duck type I hepatitis, Riemerella anatipestifer and Escherichia coli was relatively high. Qu Sujie and others investigated the infection of duck circovirus in some areas of Guangxi, and the results showed that there was a phenomenon of mixed infection with pathogens such as R. anatipestifer, Newcastle disease virus, avian influenza virus and duck hepatitis virus.

Waterfowl circovirus cannot grow on cells and poultry embryos, and the virus mostly appears in the form of subclinical infection, so it cannot be diagnosed by traditional virus isolation and animal test methods. The initial diagnosis of circovirus was mainly based on pathological tissue and electron microscope observation, but both methods required professional skills and were not very sensitive.

Mixed infection has brought many difficulties to the diagnosis of duck diseases, which cannot be diagnosed only by clinical manifestations, and molecular diagnostic techniques are needed. These methods such as virus isolation, agar diffusion test and enzyme-linked immunosorbent assay are time-consuming and are not conducive to virus control. The PCR method has the advantages of simple operation, high sensitivity, strong specificity and good repeatability, and has become an important method for animal pathogen detection. In particular, multiplex PCR has the outstanding feature of simultaneous detection and identification of multiple pathogens, and has unique advantages and high practical value in the differential diagnosis of clinical mixed infection of multiple pathogens. However, due to the existence of multiple templates and primers in a PCR system, it is necessary to prevent non-specific binding between primers and templates, to avoid the appearance of non-specific bands, and to avoid the generation of primer-dimers as much as possible. At the same time, it is very important to have a suitable gradient for the size of the target fragment and the annealing conditions of each primer to be as identical as possible to ensure that the amounts of the two amplification products are relatively balanced. Condition exploration is very important.

So far, there have been no reports on the detection and diagnosis of Newcastle disease virus and duck circovirus by duplex PCR.

Contents of the invention

One object of the present invention is to provide a primer set for detecting Newcastle disease virus and duck circovirus.

The primer set provided by the present invention is composed of primer 1, primer 2, primer 3 and primer 4;

The nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are respectively sequence 1, sequence 2, sequence 3 and sequence 4 in the sequence list.

In the above primer set, the molar ratio of the primer 1, the primer 2, the primer 3 and the primer 4 is 0.1-0.5:0.1-0.5:1:1;

In the above primer set, the molar ratio of the primer 1, the primer 2, the primer 3 and the primer 4 is specifically 0.2:0.2:1:1.

Another object of the present invention is to provide a PCR reagent for detecting Newcastle disease virus and duck circovirus.

The PCR reagent provided by the present invention is composed of the above-mentioned primer set, PCR buffer and water;

The final concentration of primer 1 and primer 2 in the PCR reagent in the primer set is specifically 0.1 μmol/mL-0.5 μmol/mL; the final concentration of the primer 1 and primer 2 in the PCR reagent The concentration is further specifically 0.2 μmol/mL;

The final concentration of primer 3 and primer 4 in the PCR reagent in the primer set is specifically 0.8 μmol/mL-1.2 μmol/mL; the final concentration of the primer 3 and primer 4 in the PCR reagent The concentrations are all further specifically 1 μmol/mL.

The above PCR buffer is 2×Taq PCR Mix (purchased from Tiangen. KT201).

The third object of the present invention is to provide a PCR kit for detecting Newcastle disease virus and duck circovirus.

The PCR kit provided by the present invention includes the above-mentioned primer set or the above-mentioned PCR reagent.

The application of the above-mentioned primer set or the above-mentioned PCR reagent or the above-mentioned kit in the preparation and/or auxiliary detection of whether the product to be tested contains Newcastle disease virus and duck circovirus is also within the protection scope of the present invention.

The above-mentioned detection and/or auxiliary detection of whether the test sample contains Newcastle disease virus and duck circovirus is to use the above-mentioned primer set or the above-mentioned PCR reagent or the above-mentioned kit to perform double PCR amplification on the test sample.

In the above application, the annealing temperature of the duplex PCR amplification is 50°C-65°C, and the annealing temperature of the duplex PCR amplification is specifically 55°C.

The fourth object of the present invention is to provide a primer pair A for detecting Newcastle disease virus.

The primer pair A provided by the present invention is composed of the primer 1 and the primer 2 in the above primer set;

The fifth object of the present invention is to provide a primer pair B for detecting duck circovirus.

The primer pair B provided by the present invention consists of the primer 3 and the primer 4 in the above primer set.

The application of the above-mentioned primer pair A in the preparation of detection and/or auxiliary detection of whether the sample to be tested contains Newcastle disease virus products is also within the protection scope of the present invention;

The application of the above-mentioned primer pair B in preparing and/or assisting in detecting whether duck circovirus products are contained in the sample to be tested is also within the protection scope of the present invention.

The above-mentioned Newcastle disease virus can be of duck origin or non-duck origin.

The experiment of the present invention proves, this research has designed 2 pairs of primers, has established the detection method of double PCR of Newcastle disease virus and duck circovirus, can detect and distinguish Newcastle disease virus and duck circovirus two kinds of pathogens simultaneously. Heavy PCR technology has the advantages of simple operation, high sensitivity, strong specificity and good repeatability. Therefore, the double-PCR detection method of duck circovirus and duck Newcastle disease virus established in this study can be used for mixed infection of duck-derived Newcastle disease virus caused by decreased immunity caused by duck circovirus infection, which can save time. cost and reduce pollution.

Description of drawings

Figure 1 is the double PCR temperature optimization experiment

Figure 2 is the optimization result of double PCR

Figure 3 is the double PCR specificity test

Fig. 4 is the sensitivity of detecting DuCV and DuNDV by double PCR method

Figure 5 is the detection of some clinical samples by double PCR

Figure 6 is the routine PCR detection of DuCV virus in clinical samples

Figure 7 is routine PCR detection of NDV virus in clinical samples

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Viruses, reagents used in the following examples are specifically as follows:

Duck plague virus AV1221 was purchased from China Veterinary Drug Control Institute;

Newcastle disease virus is duck Newcastle disease virus, NDV-F48E9, and NDV-Lasota, among which duck Newcastle disease virus is recorded in "Research on the preparation of duck-derived Newcastle disease oil emulsion inactivated vaccine", a master's thesis of Shandong Agricultural University, and the public can get it from Guangxi Zhuang Obtained from Veterinary Research Institute of Autonomous Region; NDV-F48E9 and NDV-Lasota were purchased from China Veterinary Drug Control Institute;

Duck type I hepatitis virus AV2111 strain (hereinafter referred to as DHV I) was purchased from China Veterinary Drug Control Institute;

Muscovy duck parvovirus (MDPV) is recorded in "Isolation and Identification of Muscovy Duck Parvovirus in Guangxi", Guangxi Animal Husbandry and Veterinary Medicine, 2002, 18(6): 5-7, and the public can obtain it from the Veterinary Research Institute of Guangxi Zhuang Autonomous Region;

Duck circovirus is recorded in "Survey of Duck Circovirus Infection in Some Areas of Guangxi", China Animal Husbandry and Veterinary Medicine, 2010, 37(11): 156-158, and the public can obtain it from the Veterinary Research Institute of Guangxi Zhuang Autonomous Region;

Goose plague virus is recorded in "Establishment of Fluorescent Quantitative PCR Detection Method for Goose Plague Virus", Shanghai Animal Husbandry and Veterinary Communication, 2008, 160(06): 30-31, the public can obtain it from the Veterinary Research Institute of Guangxi Zhuang Autonomous Region;

H9 subtype avian influenza virus is recorded in "Establishment of rapid detection and identification of H9 subtype avian influenza virus by multiple reverse transcription polymerase chain reaction", Chinese Journal of Zoonotic Diseases, 2006, (09): 858-860, public Available from the Veterinary Research Institute of Guangxi Zhuang Autonomous Region;

Avian Pasteurella multocida is recorded in "Research on Detection of Avian Pasteurella Multocida Using Polymerase Chain Reaction", Chinese Journal of Preventive Veterinary Medicine, 1999, (06) The public can obtain it from the Veterinary Research Institute of Guangxi Zhuang Autonomous Region;

Escherichia coli 0157:H7 is recorded in "Guangxi Livestock Escherichia coli 0157:H7 Epidemiological Investigation", Chinese Journal of Zoonoses, 2011, 27(12): 1151-1155, and the public can obtain it from the Institute of Veterinary Medicine of Guangxi Zhuang Autonomous Region get;

Riemerella anatipestifer is recorded in "Research on the Establishment and Application of Real-time Fluorescent Quantitative PCR Rapid Detection Method for Riemerella anatipestifer", Biotechnology, 2010, 37(10): 87-91, the public can obtain it from Veterinarian of Guangxi Zhuang Autonomous Region obtained by the Institute;

Reagents: 2×Taq PCR Mix was purchased from Beijing Tiangen Biotechnology Company; TIANamp Blood/Cell/Tissue Gene DNA Extraction Kit was purchased from Tiangen Company; RNA extraction reagent TRIzol LS Reagent was purchased from Invitrogen Company.

Embodiment 1, design and synthesis of primers

The F gene of Newcastle disease virus and the V1/rep gene of duck circovirus in GenBank were compared by using Lasergene software, and primers were designed in the conserved regions by using the online software Primer Premier 5.0. Primers were synthesized by Shanghai Invitrogen Company. The primer sequences are listed in Table 1.

Table 1 is the double PCR primer sequence

Embodiment 2, double PCR detection

1. Extraction of nucleic acid

Refer to the TIANamp blood/cell/tissue gene DNA extraction kit to extract duck circovirus, Muscovy duck parvovirus, duck plague virus AV1221, gosling plague virus, Riemerella anatipestifer, Escherichia coli, avian multocida Bacillus DNA;

According to the instruction manual of TRIzol LS Reagent, extract the RNA of duck Newcastle disease virus, Newcastle disease virus NDV-F48E9, Newcastle disease virus NDV-Lasota, DHV I, and H9 subtype avian influenza virus respectively, reverse transcribe into cDNA respectively, and store at -70℃ Save for later.

2. Establishment of double PCR amplification system

1. Reaction system optimization

The amount of duck Newcastle disease virus primers, duck circovirus primers, templates, etc. was optimized, and the optimal reaction amount was determined after repeated experiments. Finally, the double PCR reaction system was determined to be 25 μL: 2×Taq PCR Mix (purchased from Tiangen .KT201) 12.5 μL, 0.1ul each of DuNDV upstream and downstream primers (the primer concentration is 50 μmol/mL, and the final concentration in the reaction system is 0.2 μmol/mL), DuCV upstream and downstream primers (both primer concentrations are 50 μmol/mL, , the final concentration in the reaction system is 1 μmol/mL) each 0.5 μL, each template 2 μL, add water to 25 μL.

2. Optimization of reaction conditions

According to the above reaction system, the template is an equal-volume mixture of duck Newcastle disease virus cDNA and duck circovirus DNA, and the annealing temperature is increased sequentially from 50°C to 65°C, and the optimal annealing temperature is determined after repeated experiments.

The optimization results of reaction annealing temperature are shown in Figure 1, where, M: 100bp DNA ladder; 1: 50.0°C; 2: 50.4°C; 3: 51.5°C; 4: 53.0°C; 5: 54.8°C; 6: 56.6°C; 7 : 58.4℃; 8: 60.2℃; 9: 62.0℃; 10: 63.5℃; 11: 64.6℃; 12: 65.0℃. It can be seen that the optimum annealing temperature is 55℃.

Determine the best reaction conditions: 94°C for 5min; 94°C for 1min; 55°C for 1min; 72°C for 1min, 35 cycles; 72°C for 10min. According to the above reaction system, the templates are the cDNA of duck Newcastle disease virus and the cDNA of duck circovirus. An equal volume mixture of DNA, DNA of duck circovirus, cDNA of duck Newcastle disease virus, was reacted.

The results are shown in Figure 2, wherein, M: 100bp DNA ladder; 1: equal volume mixture of cDNA of Duck Newcastle Disease Virus and DNA of Duck Circovirus; 2: DNA of Duck Circovirus; 3: Duck Newcastle Disease Virus It can be seen that this condition can amplify the target fragment, and the amplification effect is good.

3. Specificity test of double PCR

Perform double PCR amplification according to the above-mentioned two optimized PCR reaction systems and optimized reaction conditions. The difference is that the templates are as follows:

Duck Newcastle Disease Virus cDNA + Duck Circovirus DNA (equal volume mix), NDV-F48E9 cDNA + Duck Circovirus DNA (equal volume mix), NDV-Lasota cDNA + Duck Circovirus DNA (equal volume mix) , DNA of Muscovy duck parvovirus, cDNA of DHV I, DNA of goose plague virus, DNA of duck plague virus AV1221, cDNA of H9 subtype influenza virus, DNA of Riemerella anatipestifer, DNA of Escherichia coli, poultry DNA from Pasteurella multocida. The results are shown in Figure 3, wherein, M: 100bp DNA ladder; 1: the cDNA of duck Newcastle disease virus+duck circovirus DNA (mixed in equal volume); 2: the cDNA+duck circovirus of NDV-F48E9 (mixed in equal volume ) DNA; 3: the cDNA of NDV-Lasota+duck circovirus DNA (equal volume mixing); 4: the DNA of Muscovy duck parvovirus; 5: the cDNA of DHV I; 6: the DNA of goose plague virus; 7: DNA of duck plague virus AV1221; 8: cDNA of H9 subtype influenza virus; 9: DNA of Riemerella anatipestifer; 10: DNA of Escherichia coli; 11: DNA of Pasteurella avium multocida; 12: negative As for the control (water), it can be seen that 1-3 have target fragments of 493bp and 218bp, and the rest have no target fragments.

It shows that the primer and method of the present invention have high specificity.

Therefore, the above-mentioned primers and methods can be applied to identify whether the unknown sample is infected with Newcastle disease virus (can be duck source or not) and duck circovirus:

If a 493bp fragment is obtained, the sample contains Newcastle disease virus, otherwise it does not;

If a 218bp fragment is obtained, the sample contains duck circovirus, otherwise it does not;

If the fragments of 493bp and 218bp are obtained, the sample contains Newcastle disease virus and duck circovirus, otherwise it does not.

4. Sensitivity test of double PCR

Use DU 800 ultraviolet spectrophotometer to measure the cDNA concentration of duck Newcastle disease virus and the concentration of duck circovirus DNA to be 40ng/ul and 20ng/ul, respectively. The two were diluted 10 times and then mixed in equal volumes as templates. Two optimized PCR reaction system and optimized reaction conditions for double PCR amplification,

The results are shown in Figure 4, M: 100bp DNA ladder; 1: 40ng duck Newcastle disease virus cDNA and 20ng duck circovirus DNA; 2: 4ng duck Newcastle disease virus cDNA and 2ng duck circovirus DNA; 3: 400pg duck Newcastle Epidemic virus cDNA and 200pg Duck Circovirus DNA; 4: 40pg Duck Newcastle Disease Virus cDNA and 20pg Duck Circovirus DNA; 5: 4pg Duck Newcastle Disease Virus cDNA and 2pg Duck Circovirus DNA; 6: 400rg Duck Newcastle Disease Virus cDNA and 200fg duck circovirus DNA; 7: 40fg duck newcastle disease virus cDNA and 20fg duck circovirus DNA; 8: 4fg duck newcastle disease virus cDNA and 2fg duck circovirus DNA; it can be seen that 1-7 all have The target fragment was amplified, so the established double PCR method had a minimum detection limit of 40 fg of nucleic acid for duck Newcastle disease virus and a minimum detection limit of 20 fg for duck circovirus.

Embodiment 3, double PCR detection sample to be tested

38 copies (numbered 1-38) of duck disease materials (liver collection) submitted for inspection in Yulin, Nanning, and Haikou were extracted from duck disease materials DNA and RNA respectively, and the RNA was reverse transcribed to obtain cDNA. The DNA and cDNA of each sample were Mix (volume ratio 1:1) to obtain mixed samples numbered 1-38.

The above-mentioned mixed samples numbered 1-38 were respectively used as templates, and double PCR amplification was performed according to the optimized PCR reaction system and optimized reaction conditions in Example 2.

If a 493bp fragment is obtained, the sample contains Newcastle disease virus, otherwise it does not;

If a 218bp fragment is obtained, the sample contains duck circovirus, otherwise it does not;

If the fragments of 493bp and 218bp are obtained, the sample contains Newcastle disease virus and duck circovirus, otherwise it does not.

The amplification result is shown in Figure 5, M is 100bp DNA ladder; Y is positive control (duck Newcastle disease virus cDNA and duck circovirus DNA are mixed in equal volume); 39 is negative control (water); 1-38 are numbers respectively It is the duck disease material of 1-38; it can be seen that 5, 13, 22, 24, and 32 have only 218bp target fragments, and the rest have no other target fragments, indicating that only duck circovirus is contained in the duck disease material numbered 1-38 , the infection rate of duck circovirus was 13.1% (5/38).

The results of conventional methods to detect 1-38 duck disease materials:

The duck disease material DNA numbered 1-38 was used as a template, and the specific primer of DuCV virus was used upstream: 5'-TATATTATTACCGGCGC(C/T)TGTA-3'; downstream: 5'-TCAGGAATCCCTG(A/C)AGGTGA-3 ′, (see the article "Development of a polymerase chain reaction procedure for detection and differentiation of duck and goose circovirus"; the length of the amplified fragment is 228bp. The amplification result is shown in Figure 6, M is 100bp DNA ladder; D is duck circle Virus positive control; 39 is negative control (water); 1-38 are the duck disease materials numbered 1-38 respectively; it can be seen that 5, 13, 22, 24, and 32 have 228bp target fragments, and the rest have no target fragments , indicating that the duck disease feeds numbered 1-38 have duck circovirus, and the infection rate containing duck circovirus is 13.1% (5/38).

The duck disease material cDNA numbered 1-38 was used as a template, and the specific primers of NDV virus P1: 5'-AGGCCTCTTGCRGCTGC-3'; P2: 5'-TTGTTCCCTAYACCTAAC-3'; The establishment of the virulent strain of epidemic virus and the attenuated vaccine strain method "); NDV strong strain and the universal fragment of 671bp of the attenuated vaccine strain (this fragment is the 103nt-774nt nucleotide of the F gene of Newcastle disease virus). The amplification results are shown in Figure 7, M is 100bp DNA ladder; N is the positive control for Newcastle disease; 1-38 are duck disease materials numbered 1-38 respectively; it can be seen that 1-38 has no target fragments, indicating that There was no Newcastle disease virus in the duck disease feeds numbered 1-38.

Claims (12)

1. detect the primer sets of Avian pneumo-encephalitis virus and duck circovirus, formed by primer 1, primer 2, primer 3 and primer 4;

The nucleotide sequence of described primer 1, described primer 2, described primer 3 and described primer 4 is followed successively by sequence 1, sequence 2, sequence 3 and the sequence 4 in the sequence table respectively.

2. primer sets according to claim 1 is characterized in that:

The mol ratio of described primer 1, described primer 2, described primer 3 and described primer 4 is 0.1-0.5:0.1-0.5:1:1.

3. primer sets according to claim 2 is characterized in that:

The mol ratio of described primer 1, described primer 2, described primer 3 and described primer 4 is 0.2:0.2:1:1.

4. detect the PCR reagent of Avian pneumo-encephalitis virus and duck circovirus, formed by arbitrary described primer sets, PCR damping fluid and water among the claim 1-3;

Primer 1 in the described primer sets and the primer 2 final concentration in described PCR reagent is 0.1 μ mol/mL-0.5 μ mol/mL;

Primer 3 in the described primer sets and primer 4 final concentration in described PCR reagent is 0.8 μ mol/mL-1.2 μ mol/mL.

5. PCR reagent according to claim 4 is characterized in that:

Primer 1 in the described primer sets and the primer 2 final concentration in described PCR reagent is 0.2 μ mol/mL;

Primer 3 in the described primer sets and primer 4 final concentration in described PCR reagent is 1 μ mol/mL.

6. detect the PCR test kit of Avian pneumo-encephalitis virus and duck circovirus, comprise arbitrary described primer sets or claim 4 or 5 described PCR reagent among the claim 1-3.

Among the claim 1-3 arbitrary described primer sets or claim 4 or 5 described PCR reagent or the described test kit of claim 6 preparation detect and/or the auxiliary detection testing sample in whether contain application in Avian pneumo-encephalitis virus and the duck circovirus product.

8. according to the described application of claim 7, it is characterized in that: whether contain Avian pneumo-encephalitis virus and duck circovirus in described detection and/or the auxiliary detection testing sample for arbitrary described primer sets or claim 4 among the claim 1-3 or 5 described PCR reagent or the described test kit of claim 6 described testing sample being carried out double pcr amplification.

9. according to claim 7 or 8 described application, it is characterized in that:

The annealing temperature of described double pcr amplification is 50 ℃-65 ℃.

10. according to the described application of claim 9, it is characterized in that:

The annealing temperature of described double pcr amplification is 55 ℃.

11. a primer that detects Avian pneumo-encephalitis virus to A, is made up of the described primer 1 in arbitrary described primer sets among the claim 1-3 and described primer 2;

Or a kind of primer that detects duck circovirus is made up of the described primer 3 in arbitrary described primer sets among the claim 1-3 and described primer 4 B.

12. the described primer of claim 11 to A preparation detect and/or the auxiliary detection testing sample in whether contain application in the Avian pneumo-encephalitis virus product;

Or the described primer of claim 11 to B preparation detect and/or the auxiliary detection testing sample in whether contain application in the duck circovirus product.

Images