CN103320540B - Duck tembusu virus, egg-decreasing syndrome virus and the Triplex RT-PCR kit of Avian pneumo-encephalitis virus - Google Patents

Abstract

The invention discloses a triple RT-PCR kit for duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus. The kit contains three pairs of specific primers. Experiments have proved that the application of the present invention requires only one RT-PCR reaction to simultaneously detect and identify three pathogens of duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus, and has strong specificity, high sensitivity, low cost, and high efficiency. Efficiency and other advantages; and the present invention can directly determine the amplification result by using the difference in the length of the amplified fragment in primer design, which is more convenient, intuitive and practical. The test sample of the present invention can be from healthy animal or dead animal, can be DNA and/or RNA, minimum can detect 100pg duck Tembusu virus RNA, 10pg egg drop syndrome virus DNA and 1ng Newcastle disease virus RNA simultaneously, this It provides accurate diagnostic results for the early onset of the three viruses, is of great significance to cutting off their transmission routes, has broad application prospects, and has important practical significance for the sustainable development of the duck breeding industry.

Description

Triple RT-PCR of Duck Tembusu Virus, Egg Drop Syndrome Virus and Newcastle Disease Virus Reagent test kit

technical field

The invention belongs to the technical field of PCR kits, and in particular relates to a triple RT-PCR kit for duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus, and a pair of primers for identifying or assisting in identifying the three viruses.

Background technique

Duck Tanbusu Virus (DTBSV) is the pathogen of a viral infectious disease that causes a sharp drop in laying duck egg production, death, and neurological symptoms and death in ducklings. The disease mainly affects adult ducks in the egg-laying period, and can reduce the egg-laying rate of female ducks in the peak egg-laying period to less than 20% or even 0 within a few days, and can also cause neurological symptoms and death in ducklings. , is one of the most serious infectious diseases that endanger the duck industry. Egg Drop Syndrome Virus (EDSV) is an adenovirus that mainly causes duck egg production decline. Laying ducks infected with Tambusu virus or egg drop syndrome virus will also cause a drop in egg production, and lesions such as ovarian hemorrhage and congestion will also be seen in necropsy. Newcastle disease Virus (NDV) is a virus that causes serious lesions in the respiratory tract, digestive tract and nervous system of chickens and other poultry and birds. It was previously considered to be less pathogenic to ducks and other waterfowl. Reports of waterfowl becoming ill after being infected with Newcastle disease virus indicate that the virus has gradually adapted to the body of waterfowl and has become pathogenic to waterfowl. Laying ducks infected with virulent Newcastle disease will also cause a decline in egg production, and lesions such as ovarian hemorrhage and congestion will also be seen in necropsy. At present, the detection methods of duck Tembusu virus include indirect ELISA and PCR, the detection methods of egg drop syndrome virus include HI, PCR, etc., and the detection methods of Newcastle disease virus include HI, PCR, etc.

Due to its high sensitivity, good specificity, rapidity and simplicity, PCR technology has entered clinical diagnostic laboratories and is widely used in the detection of various poultry disease pathogens, including the detection of DTBSV, EDSV and NDV. Triple RT-PCR is a special form of PCR. Its outstanding feature is that one RT-PCR reaction can detect and identify three pathogens at the same time, which has high clinical application value.

Contents of the invention

The technical problem to be solved in the present invention is to provide a triple RT-PCR reagent for duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus with good sensitivity, high specificity, rapid method, low cost and high efficiency. cassettes, and sets of primer pairs that identify or aid in the identification of the three viruses.

In order to solve the above-mentioned technical problems, the present invention adopts the following technical scheme: the triple RT-PCR primer set of duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus, including three pairs of specific primers, which are respectively primers 1 and 2 ( Identification of Newcastle disease virus), primers 3 and 4 (identification of egg drop syndrome virus), primers 5 and 6 (identification of duck Tembusu virus), which have sequence listings SEQ.ID.No.1 to SEQ.ID.No .6 base sequence.

The molar ratio of primer 1, primer 2, primer 3, primer 4, primer 5, and primer 6 is 5:5:5:5:3:3.

The application of the triple RT-PCR primer set of duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus in PCR amplification, the annealing temperature of RT-PCR amplification is 50°C-60°C.

The annealing temperature for PCR amplification was 52°C.

Triple RT-PCR Kit for Duck Tembusu Virus, Egg Drop Syndrome Virus and Newcastle Disease Virus, the kit contains the following reagents: Liquid A: Primer Set and One-Step RT-PCR Kit; Primer Set includes three pairs Specific primers, respectively, primers 1 and 2, primers 3 and 4, and primers 5 and 6, the concentration of primers 1 to 4 in the primer set is 25 μM, and the concentration of primers 5 to 6 is 15 μM, each 1 μL, One Step RT-PCR The kit includes 2×one-step reaction mix (containing 4 kinds of dNTP, RT reaction buffer composition and PCR reaction buffer composition), TransScript one-step emzyme mix (containing reverse transcriptase and PCRTaq enzyme) and deionized water; Solution B: duck Tembusu virus cDNA, egg drop syndrome virus DNA and Newcastle disease virus cDNA, as a positive control; solution C: deionized water, as a negative control.

The application of the above-mentioned triple RT-PCR kits of duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus in RT-PCR amplification, the annealing temperature of RT-PCR amplification is 50°C-60°C.

The annealing temperature for PCR amplification was 52°C.

Aiming at the current lack of effective and reliable technologies for the simultaneous detection and diagnosis of duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus, the inventor studied and designed three pairs of specific primers, and established duck Tembusu virus , egg drop syndrome virus and Newcastle disease virus triple RT-PCR detection method, and prepared the corresponding detection kits. Experiments have proved that the application of the present invention requires only one RT-PCR reaction to simultaneously detect and identify the three pathogens of duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus, and has strong specificity, high sensitivity, low cost, and high efficiency. Efficiency and other advantages; and the present invention can directly determine the amplification result by using the difference in the length of the amplified fragment in primer design, which is more convenient, intuitive and practical. The test sample of the present invention can be from healthy animal or dead animal, can be DNA and/or RNA, minimum can detect 100pg duck Tembusu virus RNA, 10pg egg drop syndrome virus DNA and 1ng Newcastle disease virus RNA simultaneously, this It provides accurate diagnostic results for the early onset of the three viruses, is of great significance to cutting off their transmission routes, has broad application prospects, and has important practical significance for the sustainable development of the duck breeding industry.

Description of drawings

Figure 1 is the electropherogram of the sensitivity test results of triple RT-PCR, in the figure: M is the molecular weight standard 100bp DNAladder; 1 is 10ng NDV, 10ng EDSV and 10ng DTBSV; 2 is 1ng NDV, 1ng EDSV and 1ng DTBSV; 3 is 100pg NDV, 100pg EDSV and 100pg DTBSV; 4 is 10pg NDV, 10pg EDSV and 10pg DTBSV; 5 is 1pgNDV, 1pg EDSV and 1pg DTBSV; 6 is 100fg NDV, 100fg EDSV and 100fg DTBSV; 7 is negative control (water).

Figure 2 is the electrophoresis diagram of the specificity test results of triple RT-PCR, in the figure: M is the molecular weight standard 100bp DNAladder; 1 is DTBSV RNA; 2 is EDSV DNA; 3 is NDV RNA; 4 is the mixed sample of DTBSV RNA and EDSV RNA (concentration ratio 1:1); 5 is the mixed sample of NDV RNA and DTBSV RNA (concentration ratio 1:1); 6 is the mixed sample of EDSV RNA and NDV RNA (concentration ratio 1:1); 7 is NDV RNA, EDSV Mixed sample of RNA and DTBSV RNA (concentration ratio 1:1:1); 8 is H5 subtype avian influenza RNA; 9 is Muscovy duck parvovirus RNA; 10 is duck plague virus DNA; 11 is H9 subtype avian influenza RNA; 12 is the negative control (water).

detailed description

Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the materials and reagents used can be obtained from commercial sources. The specific materials and reagents used are as follows:

Newcastle disease virus (NDV) is recorded in "Isolation and Identification of Newcastle Disease Virus in Guangxi", Guangxi Animal Husbandry and Veterinary Medicine, 2002,18(6):5-7, the public can obtain it from the Veterinary Research Institute of Guangxi Zhuang Autonomous Region;

Egg Drop Syndrome Virus is recorded in "Survey of Egg Drop Syndrome Virus 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;

Newcastle disease virus is recorded in "Research on Newcastle Disease Vegetable Oil Emulsion Vaccine", Chinese Journal of Preventive Veterinary Medicine, 2000, (04): 23-27, and the public can obtain it from the Veterinary Research Institute of Guangxi Zhuang Autonomous Region;

Duck plague virus AV1221 strain was purchased from China Veterinary Drug Administration;

H9 subtype avian influenza is recorded in "Establishment of a method for 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, available to the public Obtained from Veterinary Research Institute of Guangxi Zhuang Autonomous Region;

H5 subtype avian influenza is recorded in "Establishment of multiple reverse transcription polymerase chain reaction detection method for H5 subtype avian influenza virus", Chinese Journal of Zoonotic Diseases, 2005, 21(9): 762-764, the public can download from Obtained by Veterinary Research Institute of Guangxi Zhuang Autonomous Region;

Duck Tambusu virus was recorded in "Isolation and preliminary identification of 4 strains of duck-derived Tambusu virus in Guangxi", China Animal Quarantine, which has been included and is to be published.

Viral RNA/DNA rapid purification kit and One-Step RT-PCR kit were purchased from Beijing Quanshijin Biotechnology Co., Ltd., PMD-18T was purchased from Dalian Bao Bioengineering Co., Ltd.; DNA Fragment Gel Recovery Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd. Shijin Biotechnology Co., Ltd. The PCR instrument is the PE9600 instrument produced by Perkin Elmer Cetus Company in the United States.

Embodiment 1, design and synthesis of primers

Three pairs of specific primers were designed and synthesized (Table 1) based on the conserved gene sequences of duck Tembusu virus (DTBSV), egg drop syndrome virus (EDSV) and Newcastle disease virus (NDV) that were published and verified by Blast. ).

Table 1 Primer sequences for identification of NDV, EDSV and DTBSV

Example 2, Triple RT-PCR Identification of Duck Tembusu Virus, Egg Drop Syndrome Virus and Newcastle Disease Virus

1. Establishment of triple RT-PCR system

1. Preparation of samples to be tested

According to the instructions of the rapid virus RNA/DNA purification kit, duck Tembusu virus RNA, Newcastle disease virus RNA and egg drop syndrome virus DNA were extracted. The concentration and purity of the nucleic acid were determined according to the Sambrook method, and stored at -70°C for later use.

2. Optimization of triple RT-PCR reaction conditions

RT-PCR was performed in one step using the One Step RT-PCR kit. Optimize each cycle parameter and each primer concentration of RT-PCR to determine the best RT-PCR mode.

By optimizing the concentration of RT-PCR primers, each reaction temperature, time and number of cycles, etc., it is finally determined that the best working final concentrations of NDV283-1 and NDV283-2 primers in RT-PCR are both 05 μM, DTBSV422-1 and DTBSV422 The optimal working final concentration of -2 primers is 0.5 μM, and the optimal working final concentration of EDSV741-1 and EDSV741-2 primers is 0.3 μM.

Reaction system (50μl): PrimeScript1Step Enzyme Mix2μl, 2×1Step Buffer25μl, NDV283-1 and NDV283-2 primers with a final concentration of 0.5μM, EDSV741-1 and EDSV741-2 primers with a final concentration of 0.5μM, and a final concentration of 0.3 μM DTBSV422-1 and DTBSV422-2 primers, 3 μL of NDV DNA, DTBSV RNA and EDSV DNA (volume ratio 1:1:1) were used as mixed templates, and 50 μl was supplemented with RNase-free dH ₂ O.

The best reaction mode for RT-PCR is reverse transcription at 50°C for 30 minutes, denaturation at 94°C for 2 minutes, then denaturation at 94°C for 1 minute, annealing at 52°C for 1 minute, extension at 72°C for 1 minute, and a total of 35 cycles , and finally extended at 72°C for 10 minutes, and then ended the reaction at 4°C.

2. Sensitivity test of triple RT-PCR

Use the samples prepared in step 1 and 1 as RNA templates of Duck Tembusu virus, Egg Drop Syndrome Virus and Newcastle Disease Virus, add an appropriate amount to the same test tube and adjust the final concentration with water to make the final concentration consistent, and then carry out 10 times According to the gradient dilution, amplify according to the optimized PCR reaction conditions in steps 1 and 2, and test its sensitivity; at the same time, set a negative control that replaces the same amount of template with water.

After the reaction, mix 50 μl RT-PCR product with 5 μl bromophenol blue, electrophoresis in 10 g/L agarose gel, stain with ethidium bromide, observe and take pictures under ultraviolet light, and compare with DNA standard molecular weight, Analyze and record the results. Cut the desired fragments with a blade under ultraviolet light, and then purify and recover them with a DNA Fragment Gel Recovery Kit. Take an appropriate amount of purified and recovered PCR product, connect it with PMD-18T (from the PME-18T kit) at 16°C for 4 hours, and transform DH5α Escherichia coli. The white colony grown on the ampicillin-containing selective medium was picked and cultured at 37°C, and the PCR method was used for rapid identification. The positive clones were sent to Dalian Baosheng Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared and analyzed by Blast.

The results of agarose gel electrophoresis are shown in Figure 1. The triple RT-PCR can detect at least 100pg duck Tembusu virus RNA, 10pg egg drop syndrome virus RNA and 1ng Newcastle disease virus RNA. The Newcastle disease virus was amplified to a band of about 300bp in the templates of various concentrations above the minimum detection line, the egg drop syndrome virus was amplified to a band of about 750bp, and the duck Tembusu virus was amplified The size of the band is about 400bp, and the sequencing results further confirmed the RT-PCR amplification products of Duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus. The sizes are 283bp, 741bp and 422bp respectively, which are consistent with the experimental design , and the nucleic acid sequence of the PCR product is consistent with the homology of the gene corresponding fragment of the primer design template. The above results indicated that the simultaneous detection of Duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus by triple RT-PCR had high sensitivity.

3. Specificity test of triple RT-PCR

1. Preparation of samples to be tested

EDSV RNA, DTBSV RNA, AIV H5RNA, AIV H9RNA, AIV H9RNA, duck plague virus DNA, Muscovy duck parvovirus RNA and NDV RNA were extracted according to the instructions of the viral RNA/DNA rapid purification kit. The concentration and purity of the nucleic acid were determined according to the Sambrook method, and stored at -70°C for later use.

2. Triple RT-PCR amplification

Amplify according to the optimized PCR reaction conditions in steps 1 and 2, except that the templates are as follows: DTBSV RNA; EDSV DNA; NDV RNA; a mixed sample of DTBSV RNA and EDSV RNA (concentration ratio 1:1); NDV RNA Mixed sample with DTBSV RNA (concentration ratio 1:1); Mixed sample of EDSV RNA and NDV RNA (concentration ratio 1:1); Mixed sample of NDV RNA, EDSV RNA and DTBSV RNA (concentration ratio 1:1:1); H5 Subtype avian influenza RNA; Muscovy duck parvovirus RNA; duck plague virus DNA; H9 subtype avian influenza RNA; negative control water.

After the reaction, mix 50 μl RT-PCR product with 5 μl bromophenol blue, electrophoresis in 10 g/L agarose gel, stain with ethidium bromide, observe and take pictures under ultraviolet light, and compare with DNA standard molecular weight, Analyze and record the results. Cut the desired fragments with a blade under ultraviolet light, and then purify and recover them with a DNA Fragment Gel Recovery Kit. Take an appropriate amount of purified and recovered PCR product, connect it with PMD-18T (from the PME-18T kit) at 16°C for 4 hours, and transform DH5α Escherichia coli. The white colony grown on the ampicillin-containing selective medium was picked and cultured at 37°C, and the PCR method was used for rapid identification. The positive clones were sent to Dalian Baosheng Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared and analyzed by Blast.

The results of agarose gel electrophoresis are shown in Figure 2. All samples containing Duck Tembusu virus, Egg Drop Syndrome Virus and Newcastle Disease Virus nucleic acid templates can amplify amplified bands consistent with the size of the experimental design, namely Newcastle disease virus amplifies the target band with a size of about 300bp, egg drop syndrome virus amplifies the target band with the size of about 750bp, duck Tembusu virus amplifies the target band with the size of about 400bp; and Other control pathogens did not have any amplified bands at the same position. The sequencing results further confirmed that the RT-PCR amplification products of Duck Tembusu virus, Egg Drop Syndrome Virus and Newcastle Disease Virus were 283bp, 741bp and 422bp respectively, which were consistent with the size of the experimental design, and the nucleic acid sequences of the PCR products were consistent with the primers The homology of the corresponding fragment of the gene of the design template is consistent. This result indicated that the detection of Duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus by this triple RT-PCR had strong specificity.

Embodiment 3, the assembly of detection kit

According to the research results of Examples 1 and 2, a detection kit was assembled for easy use.

Solution A: primer set and One-Step RT-PCR kit; the primer set includes three pairs of specific primers, namely primers 1 and 2, primers 3 and 4, primers 5 and 6, and the concentration of primers 1 to 4 is 25 μM. The concentration of primers 5 to 6 is 15 μM, 1 μL each, and the OneStep RT-PCR kit includes 2×one-step reaction mix (containing 4 kinds of dNTPs, RT reaction buffer composition and PCR reaction buffer composition), TransScript one-step emzyme mix (containing reverse transcriptase and PCR Taq enzyme) and deionized water;

Solution B: duck Tembusu virus cDNA, egg drop syndrome virus cDNA and Newcastle disease virus cDNA, as positive controls;

Liquid C: deionized water, as a negative control.

Therefore, the primer set of the present invention, the kit and the detection method established therefrom can be used to identify whether the sample to be tested is infected with duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus: if a 283bp fragment is obtained, the test sample The sample contains Newcastle disease virus, and vice versa; if a fragment of 741bp is obtained, the sample to be tested contains egg drop syndrome virus, otherwise there is no; if a fragment of 422bp is obtained, the sample to be tested contains duck Tembusu virus , and vice versa.

Claims (6)

1. a triple RT-PCR kit of duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus, is characterized in that the kit contains following reagents: Liquid A: primer set and One-Step RT-PCR kit; the primer set includes three pairs of specific primers, respectively primers 1 and 2, primers 3 and 4, primers 5 and 6, and primers 1 to 4 in the primer set The concentration of primers 5 to 6 is 25 μM, the concentration of primers 5 to 6 is 15 μM, 1 μL each, and the One Step RT-PCR kit includes 2×one-step reaction mix, TransScript one-stepemzyme mix and deionized water; Solution B: duck Tembusu virus cDNA, egg drop syndrome virus DNA and Newcastle disease virus cDNA, as positive controls; Solution C: deionized water, as a negative control; The primers 1 and 2, primers 3 and 4, and primers 5 and 6 are the base sequences of SEQ.ID.No.1 to SEQ.ID.No.6 in the sequence table, respectively. 2. the non-diagnostic application of the triple RT-PCR kit of duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus described in claim 1 in PCR amplification, it is characterized in that: the annealing of described PCR amplification The temperature is 50°C-60°C. 3. The non-diagnostic application according to claim 2, characterized in that: the annealing temperature of the PCR amplification is 52°C. 4. The triple RT-PCR primer set of duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus, is characterized in that comprising three pairs of specific primers, is respectively primer 1 and 2, primer 3 and 4, primer 5 and 6. They are the base sequences of SEQ.ID.No.1 to SEQ.ID.No.6 in the sequence table respectively, and the molar ratio of primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6 is 5:5:5:5:3:3. 5. the non-diagnostic application of the triple RT-PCR primer set of duck Tembusu virus, egg drop syndrome virus and Newcastle disease virus described in claim 4 in PCR amplification, it is characterized in that: the annealing of described PCR amplification The temperature is 50°C-60°C. 6. The non-diagnostic application according to claim 5, wherein the annealing temperature of the PCR amplification is 52°C.