Nested PCR (Polymerase Chain Reaction) amplification primer and kit for internally-running sheep disease virus and application of nested PCR amplification primer and kit

Abstract

The invention discloses a nested PCR (Polymerase Chain Reaction) amplification primer for an internally-running sheep disease virus, which comprises a first-round upstream primer, a first-round downstream primer, a second-round upstream primer and a second-round downstream primer, the nucleotide sequence of the first-round upstream primer is as shown in SEQ ID NO: 1, the nucleotide sequence of the second-round upstream primer is as shown in SEQ ID NO: 3, and the nucleotide sequence of the second-round downstream primer is as shown in SEQ ID NO: 4. The nucleotide sequences of the first round of downstream primer and the second round of downstream primer are as shown in SEQ ID NO: 2. The kit has the advantages of excellent specificity and sensitivity, low cost and good stability.

Description

Technical Field

[0001] The invention belongs to the field of virus detection, and in particular relates to a Nairobi sheep disease virus nested PCR amplification primer, a kit and an application thereof. Background Art

[0002] Nairobi sheep disease (NSD) is a zoonosis caused by tick-borne Nairobi sheep disease virus (NSDV). The virus belongs to the family Nairoviridae of the order Bunyavirales. Nairobi sheep disease is an acute, febrile infectious disease. In the early stage of infection, it is often difficult to observe its typical pathological changes. As the course of the disease prolongs, typical symptoms of the gastrointestinal digestive system gradually become obvious. It mainly infects sheep and goats. In the early stage of the disease, animals often show fever and thin feces. Later, mucus or blood appears in the feces and accompanied by a decrease in body temperature, with a high morbidity and mortality rate. Nairobi virus is an enveloped single-stranded RNA virus. Its genome is divided into three segments, namely L (large) segment, M (middle) segment and S (small) segment, which encode RNA polymerase protein related to virus replication, at least two or more envelope glycoproteins and nucleocapsid-related proteins, respectively.

[0003] At present, relevant studies have shown that NSDV has been reported in sheep, goats and ticks abroad, and NSDV has been detected in ticks on sheep in China. The virus can not only infect goats and sheep, but also infect humans and cause symptoms such as fever and headache. It is a zoonotic virus with important public health significance. In previous studies, no molecular biological detection methods for NSDV have been found. The virus has a certain latent or pathogenic infection in Chinese sheep flocks, which not only poses a potential threat to the healthy development of the sheep industry, but also poses a huge threat to public health safety. Therefore, an epidemiological investigation of the virus is imminent. At present, the detection methods for Nairobi sheep disease virus are mainly agar gel immunodiffusion test and indirect fluorescent antibody test.

[0004] Polymerase Chain Reaction (PCR) and Quantitative Real-time PCR (qPCR) are common laboratory detection methods. PCR uses a pair of oligonucleotide fragments that are complementary to the 5' and 3' ends of the template as primers. Under the action of DNA polymerase, they extend along the template chain according to the mechanism of semi-conservative replication until new DNA synthesis is completed. By repeating this process, the target DNA fragment can be amplified in a short time to obtain a large number of target fragments. Commonly used qPCR methods are divided into dye method (SYBR Green I method) and probe method (Taqman probe method). Both add fluorescent groups during the PCR reaction and use the increase in fluorescent signals to reflect the amplification of the product. Compared with ordinary PCR, fluorescent quantitative PCR has better sensitivity and specificity, but the cost of equipment and consumables is much higher than that of ordinary PCR.

[0005] Ordinary PCR has the advantages of simple operation and low cost, but its specificity and sensitivity are poor; fluorescent quantitative PCR has better specificity and sensitivity than ordinary PCR, but the instrument is more expensive, the cost of a single test is higher than ordinary PCR, and the operator needs to have relevant experience, which places high demands on the operator. Summary of the invention

[0006] An object of the present invention is to provide a nested PCR primer for detecting Nairobi sheep disease virus with excellent specificity and good sensitivity in view of the above technical problems to be solved.

[0007] Another object of the present invention is to provide a kit comprising the nested PCR primers.

[0008] Another object of the present invention is to provide application of the nested PCR primers.

[0009] In order to achieve the above invention objectives, the present invention provides a Nairobi sheep disease virus nested PCR amplification primer, the nested PCR amplification primer comprises a first-round upstream primer, a first-round downstream primer, a second-round upstream primer and a second-round downstream primer, the nucleotide sequence of the first-round upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the second-round upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequences of the first-round downstream primer and the second-round downstream primer are shown in SEQ ID NO: 2.

[0010] On the other hand, the present invention also provides a kit, which comprises the Nairobi sheep disease virus nested PCR amplification primers.

[0011] On the other hand, the present invention also provides the use of the Nairobi sheep disease virus nested PCR amplification primers in preparing a kit for detecting Nairobi sheep disease virus.

[0012] As a preferred embodiment, in the application, the steps of performing the detection are as follows:

[0013] (1) Extracting viral nucleic acid from the sample to be tested;

[0014] (2) performing a nested PCR reaction using the Nairobi sheep disease virus nested PCR amplification primers;

[0015] (3) Gel electrophoresis was performed to observe whether the final PCR product contained a band of 906 bp in length. If so, it indicated the presence of Nairobi sheep disease virus.

[0016] As a preferred embodiment, the first-round PCR amplification reaction system is: PrimeScript 1StepEnzyme Mix 2.5μL, 2×1Step Buffer 25μL, first-round upstream primer 1.5μL with a concentration of 10μM, first-round downstream primer 1.5μL with a concentration of 10μM, RNA template 5.0μL, and ddH2O is added to a total volume of 50.0μL.

[0017] As a preferred embodiment, the first round of PCR amplification program is as follows: reverse transcription at 50°C for 30 min; pre-denaturation at 94°C for 3 min; denaturation at 94°C for 40 s, annealing at 53°C for 40 s, extension at 72°C for 60 s, 35 cycles; extension at 72°C for 5 min.

[0018] As a preferred embodiment, the second-round PCR amplification reaction system is: PrimeScript 1StepEnzyme Mix 2.5μL, 2×1Step Buffer 25μL, second-round upstream primer 1.5μL with a concentration of 10μM, second-round downstream primer 1.5μL with a concentration of 10μM, first-round PCR product template 5.0μL, and ddH2O is added to a total volume of 50.0μL.

[0019] As a preferred embodiment, the second round of PCR amplification program is as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 40 s, annealing at 53°C for 40 s, extension at 72°C for 45 s, 35 cycles; and extension at 72°C for another 5 min.

[0020] As a preferred embodiment, the sample is from sheep, including but not limited to goats and sheep.

[0021] As a preferred embodiment, the sample is sheep spleen.

[0022] Nested PCR is a variant of polymerase chain reaction (PCR) that uses two pairs of PCR primers to amplify complete fragments. The first pair of PCR primers is called outer primers and is used in the first round of amplification to enrich the target gene fragment. The second pair of primers is called inner primers or nested primers, which bind to the inside of the first round of PCR products and are used in the second round of amplification. The length of the second round of PCR amplification products is usually shorter than that of the first round of amplification. Nested PCR is low in cost, high in specificity, and has a sensitivity comparable to that of qPCR. It can be used for the detection of NSDV clinical samples and large-scale screening. In nested PCR, the ratio of the two rounds of amplification primers needs to be precisely controlled to ensure specific amplification. The appropriate primer sequence should also be reasonably designed to ensure its specificity and avoid the formation of primer dimers, which leads to non-specific amplification, consumption of primers and enzymes, and reduced yield of specific amplification products.

[0023] The present invention is based on nested PCR technology, takes conserved sequence nucleotides as target sequences, designs inner primers in the target amplification region of the L fragment, and greatly improves the specificity and sensitivity of the detection method through two PCR amplifications, while the detection cost is much lower than that of fluorescent quantitative PCR, which is more conducive to popularization and use in large-scale screening in the grassroots market. The nested PCR detection method of Nairobi sheep disease virus of the present invention has good specificity, stability and high sensitivity, only needs to directly use the nucleic acid sequence in the sample as a template, and can multiply the viral gene sequence in vitro, which greatly reduces the detection cost and detection time, and can be used for the detection of clinical samples and large-scale epidemiological surveys. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of PCR amplification using NDSV-F1 and NDSV-R primers with NSDV positive nucleic acid as a template are shown.

[0025] Figure 2 The results of the second round of PCR amplification are shown.

[0026] Figure 3 The results show that the nested PCR detection method of the present invention is specific to Nairobi sheep disease virus.

[0027] Figure 4 The results show the sensitivity of the nested PCR detection method of the present invention to Nairobi sheep disease virus.

[0028] Figure 5 Nested PCR results of sheep spleen samples are shown. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention, rather than to limit the scope of the present invention.

[0030] 1. Reagents

[0031] TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0, pMD TM 18-T VectorCloning Kit, PrimeScript TM One Step RT-PCR Kit Ver.2 and DL2000 Marker were purchased from Bao Biotechnology (Dalian) Co., Ltd.; Trans5α Chemically Competent Cell was purchased from Quanshijin Biotechnology Co., Ltd.; Gel Extraction Kit was purchased from Guangzhou Feiyang Biotechnology Co., Ltd.; SteadyPure Plasmid DNA Extraction Kit was purchased from Hunan Aikerui Biotechnology Co., Ltd.

[0032] 2. Instruments and Equipment

[0033] The clean bench was purchased from Suzhou Purification Equipment Co., Ltd.; the biological tissue grinder was purchased from Bertin Technologies; the ultra-micro-volume fluorescence spectrophotometer was purchased from DeNovix; the electric constant temperature water bath was purchased from Shanghai Yiheng Technology Co., Ltd.; the gradient PCR instrument was purchased from SENSO; and the gel imaging system was purchased from Azure Biosystems.

[0034] Example 1. Primer design and synthesis

[0035] According to the L segment genomic sequence of NSDV obtained by high-throughput sequencing by the inventor (accession number: OP558304.1) and the L segment genomic sequence of NSDV published in GenBank (accession number: NC_034387.1), MEGA5.1 software was used for comparison, the target gene sequence was selected, and the following specific nested PCR primers were designed using Primer Primer 5.0 (see Table 1). The primers were synthesized by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd.

[0036] Table 1. Nested PCR primer sequences

[0037]

[0038] Example 2. Construction of standard plasmid

[0039] According to TaKaRa PrimeScript TMOne Step RT-PCR Kit Ver.2 instruction manual, PCR amplification of NSDV positive nucleic acid (extracted from NSDV virus liquid). The reaction system is shown in Table 2.

[0040] Table 2: PCR amplification reaction system

[0041] Sample name volume PrimeScript 1Step Enzyme Mix 2.5μL 2×1Step Buffer 25μL First round upstream primer (10 μM) 1.5μL First round downstream primer (10 μM) 1.5μL RNA template 5.0μL <![CDATA[ddH2O]]> 14.5μL Total volume 50.0μL

[0042] The amplification program was as follows: reverse transcription at 50°C for 30 min; pre-denaturation at 94°C for 3 min; denaturation at 94°C for 40 s, annealing at 53°C for 40 s, extension at 72°C for 60 s, 35 cycles; and extension at 72°C for 5 min.

[0043] The amplified product was identified by 1% (w / v) agarose gel electrophoresis. Negative control: ddH2O was used instead of RNA template in the PCR reaction system.

[0044] Figure 1 The electrophoresis results of the products of PCR amplification using the NDSV-F1 and NDSV-R primers in Table 1 with NSDV positive nucleic acid as a template are shown, and a 994 bp target fragment (SEQ ID NO: 4) was successfully amplified. Figure 1 In the figure, M indicates DL 2000 DNA marker, lane 1 is NSDV positive amplification, and lane 2 is negative control.

[0045] The amplified target fragment was purified and recovered, and then purified according to pMD TM According to the instructions of the 18-T Vector Cloning Kit, the above DNA fragments were connected to the pMD18-T vector to construct a standard plasmid, and then the plasmid was transformed into Trans5α Chemically Competent Cell, positive clones were screened by PCR identification, and the positive strains were sent to the Guangzhou Branch of Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. The plasmid was extracted from the bacterial solution with the correct sequence according to the instructions of the SteadyPurePlasmid DNA Extraction Kit. The standard plasmid was named NSDV-18T-L and stored at -20°C for future use.

[0046] Example 3. Establishment of nested PCR detection method

[0047] Nested PCR is divided into two rounds of amplification.

[0048] The first round of PCR amplification reaction system: PrimeScript 1Step Enzyme Mix 2.5μL, 2×1StepBuffer 25μL, NDSV-F1 primer with a concentration of 10μM 1.5μL, NDSV-R primer with a concentration of 10μM 1.5μL, RNA template 5.0μL, ddH2O to make up to a total volume of 50.0μL.

[0049] The first round of PCR amplification program was as follows: reverse transcription at 50°C for 30 min; pre-denaturation at 94°C for 3 min; denaturation at 94°C for 40 s, annealing at 53°C for 40 s, extension at 72°C for 60 s, 35 cycles; extension at 72°C for 5 min.

[0050] The second round of PCR amplification reaction system: PrimeScript 1Step Enzyme Mix 2.5μL, 2×1StepBuffer 25μL, NDSV-F2 primer with a concentration of 10μM 1.5μL, NDSV-R primer with a concentration of 10μM 1.5μL, first round PCR product template 5.0μL, ddH2O is added to the total volume of 50.0μL.

[0051] The second round of PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 40 s, annealing at 53°C for 40 s, extension at 72°C for 45 s, 35 cycles; and extension at 72°C for another 5 min.

[0052] After the amplification was completed, the amplified product was detected on a 1% (w / v) agarose gel. The results showed that a 906 bp target fragment (SEQ ID NO: 5) was successfully amplified. Figure 2 shown. Figure 2 In the figure, M indicates DL 2000 DNA marker, and lanes 1 and 2 indicate NSDV amplification products.

[0053] Example 4. Specificity test of nested PCR detection method

[0054] The nested PCR amplification conditions of Example 3 of the present invention were used to amplify the mixed positive nucleic acid of Clostridium putrefaciens and Clostridium perfringens (extracted from the quadruple dry powder inactivated vaccine (multi-link Bing) of Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. for sheep quick epidemic, sudden sniper, lamb dysentery, and enterotoxemia), the mixed positive nucleic acid of peste des petits ruminants virus and goat pox virus (extracted from the two-in-one live vaccine (Clone9 strain + AV41 strain) of Huapai Bioengineering Group Co., Ltd. for peste des petits ruminants and goat pox), the positive nucleic acid of Brucella (extracted from the Brucellosis live vaccine (S2 strain) of Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.), the positive nucleic acid of Escherichia coli (extracted from the inactivated vaccine (Ebbin) of sheep Escherichia coli disease of Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.), and the positive nucleic acid of Mycoplasma goat (extracted from the inactivated vaccine (C87-1 strain) of goat contagious pleuropneumonia of Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.) as templates, and ddH2O was used as a negative control and the positive nucleic acid of NSDV was used as a positive control to detect the specificity of the nested PCR method of the present invention.

[0055] The results are as follows Figure 3 shown. Figure 3 In the figure, M indicates DL 2000 DNA marker, lane 1 indicates mixed positive nucleic acid of Clostridium putrefaciens and Clostridium perfringens, lane 2 indicates mixed positive nucleic acid of Peste des Petits Ruminants virus and goat pox virus, lane 3 indicates positive nucleic acid of Brucella, lane 4 indicates positive nucleic acid of Escherichia coli, lane 5 indicates positive nucleic acid of Mycoplasma goat, lane 6 indicates negative control, and lane 7 indicates positive nucleic acid of NSDV. Figure 3 It can be seen that except for the positive nucleic acid of NSDV, no target bands were found in the negative control and other viruses, indicating that this method has good specificity.

[0056] Example 5. Sensitivity test of nested PCR detection method

[0057] The mass concentration of NSDV-18T-L standard plasmid was determined by ultra-micro UV spectrophotometer. 1 ~10 9 The template copy number after dilution was 3.7×10 8 ~3.7×10 0 2 μL of each was taken as a template, and nested PCR amplification was performed according to the amplification conditions of Example 3. The amplified products were detected by 1% (w / v) agarose gel electrophoresis, and the sensitivity of the detection method was determined.

[0058] Test results such as Figure 4 shown. Figure 4 In the figure, M represents the DL 2000 DNA marker, and lanes 1 to 9 represent the copy number of 3.7×10 8 , 3.7×107 , 3.7×10 6 , 3.7×10 5 , 3.7×10 4 , 3.7×10 3 , 3.7×10 2 , 3.7×10 1 , 3.7×10 0 copies / μL of the standard plasmid, and lane 10 represents the negative control. The results showed that the minimum detection limit was 3.7×10 1 copies / μL.

[0059] Example 6. Clinical sample testing

[0060] TaKaRa MiniBEST Viral RNA / DNAExtraction Kit Ver.5.0 was used to extract nucleic acids from 60 sheep spleen samples collected from a certain area in Henan Province, and the nested PCR detection method of the present invention (amplification conditions of Example 3) was used to detect spleen tissue nucleic acids. ddH2O was used as a negative control.

[0061] Figure 5 The results of detecting 60 clinical sheep spleen samples using the nested PCR detection method of the present invention are shown. Figure 5 In the figure, M represents DL 2000 DNA marker, lanes 1 to 48, 50 to 61 represent 60 clinical samples, and lane 49 represents the negative control. The results showed that a total of 3 positive samples were detected.

[0062] The positive samples detected were amplified using the following amplification system: PrimeScript 1Step Enzyme Mix 2.5μL, 2×1Step Buffer 25μL, NDSV-F2 primer 1.5μL at a concentration of 10μM, NDSV-R primer 1.5μL at a concentration of 10μM, first-round PCR product template 5.0μL, ddH2O to a total volume of 50.0μL. Amplification program: 95℃ pre-denaturation for 3min; 95℃ denaturation for 40s, 53℃ annealing for 40s, 72℃ extension for 45s, 35 cycles; 72℃ extension for another 5min. Then, the nucleic acid was purified using GelExtraction Kit, connected to the pMD18-T vector, and sent to Guangzhou Branch of Sangon Biotech (Shanghai) Co., Ltd. for sequencing to determine the detection effect of this method. Subsequent sequencing comparison results showed that the amplified sequences were all NSDV sequences, indicating that this method can be used for clinical detection.

Claims

1. A nested PCR amplification primer for Nairobi sheep disease virus, characterized in that It includes a first-round upstream primer, a first-round downstream primer, a second-round upstream primer and a second-round downstream primer. The nucleotide sequence of the first-round upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the second-round upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequences of the first-round downstream primer and the second-round downstream primer are shown in SEQ ID NO:

2.

2. A kit comprising the Nairobi sheep disease virus nested PCR amplification primers according to claim 1.

3. Use of the Nairobi sheep disease virus nested PCR amplification primers according to claim 1 in preparing a kit for detecting Nairobi sheep disease virus.

4. The use according to claim 1, characterized in that: The steps to perform the test are as follows: (1) Extracting viral nucleic acid from the sample to be tested; (2) performing a nested PCR reaction using the Nairobi sheep disease virus nested PCR amplification primers; (3) Gel electrophoresis was performed to observe whether the final PCR product contained a band of 906 bp in length. If so, it indicated the presence of Nairobi sheep disease virus.

5. The use according to claim 1, characterized in that: The first round of PCR amplification reaction system is: PrimeScript 1Step Enzyme Mix 2.5μL, 2×1Step Buffer 25μL, first-round upstream primer 1.5μL at a concentration of 10μM, first-round downstream primer 1.5μL at a concentration of 10μM, RNA template 5.0μL, and ddH2O to make up to a total volume of 50.0μL.

6. The use according to claim 1, characterized in that: The first round of PCR amplification program was as follows: reverse transcription at 50°C for 30 min; pre-denaturation at 94°C for 3 min; denaturation at 94°C for 40 s, annealing at 53°C for 40 s, extension at 72°C for 60 s, 35 cycles; extension at 72°C for 5 min.

7. The use according to claim 1, characterized in that: The second round of PCR amplification reaction system is: PrimeScript 1Step Enzyme Mix 2.5μL, 2×1Step Buffer 25μL, second-round upstream primer 1.5μL with a concentration of 10μM, second-round downstream primer 1.5μL with a concentration of 10μM, first-round PCR product template 5.0μL, ddH2O to make up to a total volume of 50.0μL.

8. The use according to claim 1, characterized in that: The second round of PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 40 s, annealing at 53°C for 40 s, extension at 72°C for 45 s, 35 cycles; and extension at 72°C for another 5 min.

9. The use according to claim 1, characterized in that: The samples were from sheep.

10. The use according to claim 1, characterized in that: The sample is sheep spleen.

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