CN102140540B - Kit for detecting swine fever viruses - Google Patents

Abstract

A kit for detecting classical swine fever virus, comprising RNase inhibitor, AMV (avian myeloblastosis virus) reverse transcriptase, Taq enzyme (heat-resistant deoxyribonucleic acid polymerase), RNase-free water, One Step RNA PCR (one-step RNA polymerase chain reaction) mixture, negative and positive controls, and a mixture of primers from SEQ ID NO: 1 to SEQ ID NO: 6. In the detection process, the invention completes the reverse transcription and PCR amplification processes in one step reaction, so that the detection time is shortened from the original 4-5 hours to the current 2-3 hours, thereby facilitating rapid detection. At the same time, the present invention uses three pairs of primers, so that the method has higher sensitivity compared with the common PCR method, which is helpful for the prevention and control of CSFV and the elimination of latent virus-carrying animals, and avoids large-scale infection. The method is applicable to any laboratory and grassroots prevention and control units at all levels, veterinary stations, large, medium and small farms, etc.

Description

A kind of test kit for detecting swine fever virus

technical field

The present invention relates to a detection method of classical swine fever virus, in particular to a test kit for detection of classical swine fever virus, and the present invention also includes the detection method of the test kit. the

Background technique

Classical swine fever (CSF), referred to as classical swine fever, is an acute, severe, and contagious infectious disease of pigs caused by classical swine fever virus (CSFV). my country is one of the most endemic countries for swine fever. Every year, the direct economic loss caused by swine fever has billions of yuan, which seriously threatens the development of my country's pig industry and its international trade. my country is the country with the largest number of pigs in the world, and the pig industry has become a major pillar industry of my country's animal husbandry industry. However, the number of pigs that die each year due to swine fever infection accounts for 2% to 5% of the total amount of breeding, and the economic losses caused by the About 2 billion to 5 billion. my country adopts the method of inoculating the attenuated swine fever rabbit vaccine to prevent and control the epidemic of swine fever, and has successfully controlled the outbreak of swine fever. However, the epidemiological characteristics of swine fever have changed from a pandemic to endemic and sporadic, and occur at any time regardless of season. The clinical characteristics have also changed from typical swine fever to atypical swine fever, and diagnosis and prevention are becoming more and more difficult. Therefore, swine fever is still the number one infectious disease threatening pig farmers. the

RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method has been established at home and abroad. The application of PCR to diagnose CSFV abroad has made great progress. Many scholars have designed different primers based on the CSFV sequence and established a nested RT-PCR method for detecting CSFV nucleic acid. This method directly amplifies the expected fragments from the spleen, serum, lymph nodes and meat products of pigs, etc., but only two pairs of primers are used in this method, and a nucleotide fragment is generated by two PCR amplifications. Therefore, it takes 7 hours to detect a sample, which is time-consuming and labor-intensive. In addition, RNA (ribonucleic acid) virus has a high degree of variability, and one fragment is not enough to solve this problem. At the same time, CSFV is a highly contagious virus, so the existing detection methods do not have the characteristics of fast, sensitive and accurate, and need to be developed And develop fast, sensitive, accurate and cheap CSFV detection kits and detection methods to make up for the weak link in CSFV detection in my country, and at the same time help to eliminate recessive virus-carrying animals. the

Contents of the invention

The technical problem to be solved in the present invention is to overcome the inability of existing detection methods to meet the fast, sensitive and accurate detection requirements, thereby providing a kind of test kit for detecting swine fever virus that can detect viruses quickly, sensitively and accurately, The invention also provides a detection method of the kit. the

In order to solve the above problems, the present invention adopts the following technical solutions:

A kit for detecting classical swine fever virus, comprising RNase inhibitor, AMV (avian myeloblastosis virus) reverse transcriptase, Taq enzyme (heat-resistant deoxyribonucleic acid polymerase), RNase-free water, One Step RNA PCR (one-step RNA polymerase chain reaction) mixture, negative and positive controls, and a mixture of primers from SEQ ID NO: 1 to SEQ ID NO: 6. the

The sequence SEQ ID NO: 1 to SEQ ID NO: 6 primer mixture includes:

The primer sequence of the 619bp band is:

SEQ ID NO: 1: Upstream primer TCAGGGGGATGTGCAGAGATGTGT

SEQ ID NO: 2: downstream primer GCTTTTTCCGCGCCTGATTGATA 395

The primer sequence of the bp band is:

SEQ ID NO: 3: Upstream primer CCGCCGGTGATTTCGTG

SEQ ID NO: 4: downstream primer ACTGCCTCTTTGCCCTTTCCTTAT 274

The primer sequence of the bp band is:

SEQ ID NO: 5: Upstream primer CTGGCCAAGAGGGGTGAGC

SEQ ID NO: 6: downstream primer ACAGGCCGTCTTGGGTATTC

The band sequence amplified by the primer mixture is:

Sequence SEQ ID NO: 1 to SEQ ID NO: 6 extended to the 5' end and/or 3' end;

619bp (NO: 7)

tcagggggatgtgcagagatgtgtggaagccatgaccaattatgcaagagagggtatccaatttatgaag tctcaagcactgaaggtgaaggaaacccctacttacaaggagacaatggacactgtgacggactatgtaaagaaattcatggaggcgctggcagacagtaaagaagacatcataaaatatgggctgtgggggacgcacacagccttatataagagcatcagtgccaggcttgggggtgagactgcgttcgctaccctggtagtgaagtggctggcatttgggggtgaatcaatagcagaccatgtcaaacaagcggccacagacttggtcgtttactatatcatcaacagacctcagttcccaggagacacggagacacaacaagacggaaggaaatttgtggccagcctactggcctcagctctagctacttacacatacaaaagctggaattacaataacctgtccaagatagttgaaccggctttggccactctgccctatgccgccacagctctcaaattatttgcccccacccgattagagagcgttgtcatattaagtaccgcaatctacaagacctacctatcaatcaggcgcggaaaaagc 

395bp: (NO: 8)

ccgccggtgatttcgtggacgagaagaaacctagagtcatacaataccctgaagcaaaaacaagactggccatcaccaaggtgatgtataagtgggtgaagcagaagccagtagttatacccgggtatgaagggaagacacctctattccaaatttttgacaaagtgaagaaggaatgggatcaatttcaaaatccagtggcagtgagcttcgacactaaggcgtgggacacccaggtaaccacaaaagatttggagctgataagggacatacaaaagtattatttcaagaagaaatggcacaaatttattgacaccctgaccacgcatatgtcagaagtacccgtgattagtgctgatggggaagtatacataaggaaagggcaaagaggcagt 

274bp: (NO: 9)

ctggccaagaggggtgagccaagaaccctgaagtggattagaaatttcaccgactgtccattgtgggttaccagttgctccgatgatggcgcgagtgggagtaaagagaagaagccagataggatcaacagaggcaaattaaaaatagccccaaaagagcatgagaaggacagcagaactaggccacctgacgctacgatcgtggtggaaggagtaaaataccaggtcaaaaagaaaggtaaagttaaaggaaagaatacccaagacggcctgt 

The One Step RNA PCR mixture contains PCR buffer, dNTP and Mg ²⁺ . The concentration of the PCR buffer is 10×, the concentration of dNTP is 10 mM, the concentration of Mg ²⁺ is 25 mM, and the ratio of the PCR buffer, dNTP and Mg ²⁺ is 5:4:3.

The concentration of the primers is 50 pmol/μL, and the ratio of the three pairs of primers for amplifying bands of 274bp, 395bp and 619bp is 13:12:5. the

The negative control is normal cell culture supernatant, and the blank control is RNase-free water. the

The positive control is CSFV C strain cytotoxicity. the

The present invention also provides the method for detecting swine fever virus with described kit, comprises the following steps:

a. Take 400 μL tissue sample grinding supernatant (cytotoxic culture supernatant or serum), blank control and negative and positive controls to extract total RNA;

b. Use the detection kit of the present invention to perform RT-PCR amplification, wherein the conditions are as follows: reverse transcription at 50°C for 30 minutes, pre-denaturation at 94°C for 2 minutes, 94°C for 40 seconds, annealing temperature at 57°C for 40 seconds, 72°C ℃ for 50 seconds, after 30 cycles, 72℃ for 8 minutes;

c. Electrophoresis voltage 80V-100V, or current 40mA-50mA. Electrophoresis 15min-25min;

d. Results analysis, compared with the standard molecular weight of DL2000, if the positive control has three bands of 619bp, 395bp and 274bp or any of the three bands, the sample amplification product has any band of 274bp, 395bp and 619bp, while the negative control If there is no amplification band, the sample can be judged as positive, otherwise it is negative. the

The sample may be the spleen, blood, lymph nodes and meat products of the pig to be tested. the

In the detection process, the invention completes the reverse transcription and PCR amplification processes in one step reaction, so that the detection time is shortened from the original 4-5 hours to the current 2-3 hours, thereby facilitating rapid detection. At the same time, the present invention uses three pairs of primers, so that the method has higher sensitivity compared with the common PCR method, which is helpful for the prevention and control of CSFV and the elimination of latent virus-carrying animals, and avoids large-scale infection. The method is applicable to any laboratory and grassroots prevention and control units at all levels, veterinary stations, large, medium and small farms, etc. the

Description of drawings

Fig. 1 is the detection electrophoresis diagram of the sample of the present invention. the

In the figure: Lane 1 is the molecular weight standard DL2000; Lane 2 is the positive control; Lanes 3-6 are pig spleen, whole blood, serum and lymph nodes respectively, among which lanes 3 and 4 are strong positive samples; lanes 5 and 6 are positive samples ; Swimming lane 7 is a negative control; Swimming lane 8 is a blank control. the

Detailed ways

Below in conjunction with embodiment the present invention is described in further detail

Example 1

1. Design and preparation of primers

Refer to GenBank (gene bank) to search for multiple strains, search for regions that are conserved on multiple sequences for sequence comparison, and design three pairs of relatively conservative specific primers according to the comparison results. The primer sequences are as follows:

The primer sequence of the 619bp band is:

Upstream primer TCAGGGGGATGTGCAGAGATGTGT (SEQ ID NO: 1)

Downstream primer GCTTTTTCCGCGCCTGATTGATA (SEQ ID NO: 2)

The primer sequence of the 395bp band is:

The upstream primer is CCGCCGGTGATTTCGTG (SEQ ID NO: 3)

The downstream primer is ACTGCCTCTTTGCCCTTTTCCTTAT (SEQ ID NO: 4)

The primer sequence of the 274bp band is:

The upstream primer is CTGGCCAAGAGGGGTGAGC (SEQ ID NO: 5)

The downstream primer is ACAGGCCGTCTTGGGTATTC (SEQ ID NO: 6)

The above primers were synthesized by Dalian Bao Biological Engineering Co., Ltd. the

Positive control: The positive control of the kit of the present invention is the inactivated CSFV C strain cytotoxicity, which has been subcultured and mass-cultured by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. the

2. Sample preparation

(1) Take positive control, porcine spleen tissue grinding supernatant, negative control, RNase-free water (i.e. blank control) 400μl respectively, put in 1.5mL eppendorf tube, add 1000μl Trizol (nucleic acid extraction reagent), mix well, 4 ℃ for 5 minutes. the

(2) Add 200 μl of chloroform, cover and shake for 15 seconds, and place at room temperature for 3 minutes. the

(3) Centrifuge at 12,000 rpm and 4°C for 15 minutes, and separate into three layers. the

(4) Transfer the upper aqueous phase (about 700 μl) to another 1.5ml tube, add an equal amount (700 μl) of isopropanol, mix well, and stand at room temperature for 10 minutes. the

(5) Centrifuge at 10,000 rpm and 4°C for 10 minutes, discard the supernatant. the

(6) Add 1000 μl of 75% ethanol prepared with ribonuclease-free water, rinse the precipitate once, centrifuge at 8000 rpm and 4° C. for 5 minutes, and discard the supernatant. the

(7) Centrifuge at 8000rpm at 4°C for 2min, discard the supernatant, and fully dry the RNA pellet at room temperature. the

(8) Add 20 μl of RNase-free water to dissolve the RNA precipitate, which can be used for PCR amplification, and the rest of the RNA precipitate is stored at -20°C for later use. the

3. Prepare the kit

This kit consists of the following components:

a. One Step RNA PCR mixture 10μl, concentration 10x

b. RNase inhibitor 0.5μl, the concentration is 40U/μl

c.AMV reverse transcriptase 0.5μl, the concentration is 5U/μl

d.Taq enzyme 0.5μl, the concentration is 5U/μl

e. 1.5 μl of three pairs of primer mixtures with primer concentrations of 50 pmol/μl, 0.65 μl of 274bp primers, 0.6 μl of 395bp primers, and 0.5 μl of 619bp primers. the

f. RNase-free water 7μl

g. Positive control 5μl

h. Negative control 5μl

4, detect swine fever virus with kit of the present invention

(1) The total PCR system is 25 μl. In the kit of the present invention, a.One StepRNA PCR mixed solution 10 μl; b.RNase inhibitor 0.5 μl; c.AMV reverse transcriptase 0.5 μl; d.Taq enzyme 0.5 μl; .7 μl of RNase-free water was added to four 0.2ml amplification tubes;

(2) Add 5 μl of positive control, 5 μl of RNA template extracted from pig spleen, 5 μl of negative control, 5 μl of RNase-free water (blank control) to the above-mentioned amplification tube, centrifuge at 12000 rpm for 5-30 seconds, and put the amplification tube into In the amplification instrument, amplify under the following setting program: reverse transcription at 50°C for 30 minutes, pre-denaturation at 94°C for 2 minutes, 94°C for 40s, annealing temperature at 57°C for 40s, 72°C for 50s, and extension at 72°C for 8 minutes after 30 cycles , to obtain the amplified product. the

Example 2

1. Design and preparation of primers: same as Example 1

2. Sample preparation

(1) Take 400 μl of pig whole blood to be tested, put it into a 1.5mL eppendorf tube, add 1000 μl Trizol (nucleic acid extraction reagent), mix well, and place at 4°C for 5 minutes. the

Step (2)-step (8) is the same as 2 in embodiment 1

3, the preparation kit is the same as in Example 1 3

4, detect swine fever virus with kit of the present invention

(1) The total PCR system is 25 μl. 10 μl of a.One Step RNA PCR mixed solution in the kit of the present invention; b.RNase inhibitor 0.5 μl; c.AMV reverse transcriptase 0.5 μl; d.Taq enzyme 0.5 μl; . Add 7 μl of RNase-free water to the 0.2ml amplification tube;

(2) Add 5 μl of RNA template extracted from pig whole blood to the above-mentioned amplification tube, centrifuge at 12,000 rpm for 5-30 seconds, put the amplification tube into the amplification instrument, and amplify under the following setting program: 50°C Reverse transcription for 25 minutes, pre-denaturation at 94°C for 2 minutes, 94°C for 30s, annealing temperature at 57°C for 35s, 72°C for 40s, 25 cycles and extension at 72°C for 8 minutes to obtain the amplified product. the

Example 3

1. Design and preparation of primers: same as Example 1

2. Sample preparation

(1) Take 400 μl of porcine serum to be tested, put it in a 1.5mL eppendorf tube, add 1000 μl Trizol (nucleic acid extraction reagent), mix well, and place at 4°C for 5 minutes. the

Step (2)-step (8) is the same as 2 in embodiment 1

3, the preparation kit is the same as in Example 1 3

4, detect swine fever virus with kit of the present invention

(1) The total PCR system is 25 μl. In the kit of the present invention, a.One StepRNA PCR mixed solution 10 μl; b.RNase inhibitor 0.5 μl; c.AMV reverse transcriptase 0.5 μl; d.Taq enzyme 0.5 μl; . Add 7 μl of RNase-free water to the 0.2ml amplification tube;

(2) Add 5 μl of RNA template extracted from pig whole blood to the above-mentioned amplification tube, centrifuge at 12,000 rpm for 5-30 seconds, put the amplification tube into the amplification instrument, and amplify under the following setting program: 50°C Reverse transcription for 40 minutes, pre-denaturation at 94°C for 5 minutes, 94°C for 60s, annealing temperature at 57°C for 60s, 72°C for 90s, and 40 cycles of extension at 72°C for 15 minutes to obtain amplified products. the

Example 4

1. Design and preparation of primers: same as Example 1

2. Preparation of sample 4

(1) Take 400 μl of the porcine lymph node grinding supernatant to be tested, put it in a 1.5ml eppendorf tube, add 1000 μl Trizol (nucleic acid extraction reagent), mix well, and place at 4°C for 5 minutes. the

Step (2)-step (8) is the same as 2 in embodiment 1

3, the preparation kit is the same as in Example 1 3

4, detect swine fever virus with kit of the present invention

(1) The total PCR system is 25 μl. In the kit of the present invention, a. One Step RNAPCR mixed solution 10 μl; b. RNase inhibitor 0.5 μl; c. AMV reverse transcriptase 0.5 μl; d. Taq enzyme 0.5 μl; e. three pairs of primers 1.5 μl; . Add 7 μl of RNase-free water to the 0.2ml amplification tube;

(2) Add 5 μl of RNA template extracted from pig whole blood to the above-mentioned amplification tube, centrifuge at 12,000 rpm for 5-30 seconds, put the amplification tube into the amplification instrument, and amplify under the following setting program: 50°C Reverse transcription for 30 minutes, pre-denaturation at 94°C for 2 minutes, 94°C for 60s, annealing temperature at 57°C for 50s, 72°C for 50s, and 35 cycles of extension at 72°C for 10 minutes to obtain amplified products. the

Result judgment

1. Weigh 1.0g agarose, add it to 100ml 1×TAE buffer, heat to melt, add 5μL (100mg/ml) ethidium bromide, mix well, and pour it into a horizontal gel plate with an 8-hole comb , the thickness of the gel plate is 5mm, pull out the comb after the gel is cooled, put the gel into the electrophoresis tank, add 1×TAE buffer to submerge the gel surface. the

2. Take 5 μl of the PCR amplification product obtained in the above-mentioned Examples 1-4 and 2 μL of 6xloading buffer (loading buffer), mix well, add to the sample well, and add 5 μl of DL2000 molecular weight standard at the same time. the

3. 80V-100V voltage or 40mA-50mA current electrophoresis for 15min-25min. Take out the gel plate and put it on the UV transilluminator, turn on the UV lamp to observe and take pictures, the results are shown in Figure 1. the

As shown in the figure, compared with the standard molecular weight of DL 2000, it can be seen that the electrophoresis results of pig spleen in Example 1 and pig whole blood in Example 2 showed three different bands, namely 619bp, 395bp and 274bp; There were two bands in the lymph nodes in the serum and the examples, which were consistent with the positive control, while the negative control and the blank control had no amplification bands, so it was determined that samples 1-sample 4 were positive in Examples 1-Example 4, that is, the The swine spleen, whole blood, serum and lymph nodes to be tested contained classical swine fever virus. the

Claims (7)

1. a test kit for detecting classical swine fever virus, comprising RNase inhibitor, AMV reverse transcriptase, Taq enzyme, RNase-free water, One Step RNA PCR mixed solution, negative and positive controls, characterized in that The kit also includes a mixture of primers with sequences SEQ ID NO: 1 to SEQ ID NO: 6. 2. a kind of test kit for detecting classical swine fever virus according to claim 1, is characterized in that described sequence SEQ ID NO: 1 to SEQ ID NO: 6 primer mixture comprises: The primer sequence of the 619bp band is: SEQ ID NO: 1: Upstream primer TCAGGGGGATGTGCAGAGATGTGT SEQ ID NO: 2: Downstream primer GCTTTTTCCGCGCCTGATTGATA The primer sequence of the 395bp band is: SEQ ID NO: 3: Upstream primer CCGCCGGTGATTTCGTG SEQ ID NO: 4: Downstream primer ACTGCCTCTTTGCCCTTTCCTTAT The primer sequence of the 274bp band is: SEQ ID NO: 5: Upstream primer CTGGCCAAGAGGGGTGAGC SEQ ID NO: 6: Downstream primer ACAGGCCGTCTTGGGTATTC. 3. a kind of test kit for detecting classical swine fever virus according to claim 1, is characterized in that the band sequence that described primer mixture amplifies is: Sequences of SEQ ID NO: 1 to SEQ ID NO: 6 extended to the 5' end and/or 3' end; 619bp (NO: 7) tcagggggatgtgcagagatgtgtggaagccatgaccaattatgcaagagagggtatccaatttatgaagtctcaagcactgaaggtgaaggaaacccctacttacaaggagacaatggacactgtgacggactatgtaaagaaattcatggaggcgctggcagacagtaaagaagacatcataaaatatgggctgtgggggacgcacacagccttatataagagcatcagtgccaggcttgggggtgagactgcgttcgctaccctggtagtgaagtggctggcatttgggggtgaatcaatagcagaccatgtcaaacaagcggccacagacttggtcgtttactatatcatcaacagacctcagttcccaggagacacggagacacaacaagacggaaggaaatttgtggccagcctactggcctcagctctagctacttacacatacaaaagctggaattacaataacctgtccaagatagttgaaccggctttggccactctgccctatgccgccacagctctcaaattatttgcccccacccgattagagagcgttgtcatattaagtaccgcaatctacaagacctacctatcaatcaggcgcggaaaaagc 395bp: (NO: 8) ccgccggtgatttcgtggacgagaagaaacctagagtcatacaataccctgaagcaaaaacaagactggccatcaccaaggtgatgtataagtgggtgaagcagaagccagtagttatacccgggtatgaagggaagacacctctattccaaatttttgacaaagtgaagaaggaatgggatcaatttcaaaatccagtggcagtgagcttcgacactaaggcgtgggacacccaggtaaccacaaaagatttggagctgataagggacatacaaaagtattatttcaagaagaaatggcacaaatttattgacaccctgaccacgcatatgtcagaagtacccgtgattagtgctgatggggaagtatacataaggaaagggcaaagaggcagt 274bp: (NO: 9) ctggccaagaggggtgagccaagaaccctgaagtggattagaaatttcaccgactgtccattgtgggttaccagttgctccgatgatggcgcgagtgggagtaaagagaagaagccagataggatcaacagaggcaaattaaaaatagccccaaaagagcatgagaaggacagcagaactaggccacctgacgctacgatcgtggtggaaggagtaaaataccaggtcaaaaagaaaggtaaagttaaaggaaagaatacccaagacggcctgt。 4. a kind of test kit for detecting classical swine fever virus according to claim 1, it is characterized in that described One Step RNA PCR mixture comprises PCR damping fluid, dNTP and Mg ²⁺ , the concentration of described PCR damping fluid The concentration of dNTP is 10 mM, the concentration of Mg ²⁺ is 25 mM, and the ratio of PCR buffer, dNTP and Mg ²⁺ is 5:4:3. 5. a kind of test kit for detecting classical swine fever virus according to claim 1, is characterized in that described primer concentration is 50pmol/μ L, the volume ratio of three pairs of primers of amplification 274bp, 395bp, 619bp band It is 13:12:5. 6. A kind of test kit for detecting classical swine fever virus according to claim 1, is characterized in that described negative control is normal cell culture supernatant, and blank control is RNase-free water. 7. a kind of test kit for detecting classical swine fever virus according to claim 1, is characterized in that described positive control is CSFV C strain cytotoxicity.

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