CN111705165A - Primer set, kit and detection method for double PCR detection of fish rhabdovirus - Google Patents

Abstract

The invention discloses a primer group, a kit and a detection method for double PCR detection of fish rhabdovirus, wherein the detection method comprises the following steps: extracting RNA from the kidney tissue of the fish to be detected, synthesizing cDNA by taking the RNA as a template, and performing double PCR amplification by taking the synthesized cDNA as the template and adopting a synthesized primer group; and (4) carrying out agarose gel electrophoresis on the product obtained by the double PCR amplification, and placing the gel in an imaging system for observing the result. The invention designs related specific primers aiming at the conserved gene sequence of the snakehead vesiculovirus and appropriate PCR reaction conditions, establishes a double PCR detection method of the snakehead vesiculovirus, has the characteristics of strong specificity, high sensitivity and the like, can quickly and accurately detect the snakehead vesiculovirus, provides an important technical means for clinical diagnosis and prevention of the snakehead vesiculovirus, and provides a technical scheme for carrying out researches on identification, separation, epidemiological investigation and the like of the snakehead vesiculovirus.

Description

Primer set, kit and detection method for double PCR detection of fish rhabdovirus

technical field

The invention relates to a primer set, a kit and a detection method for double PCR detection of fish rhabdovirus.

Background technique

PCR stands for polymerase chain reaction technology, a process similar to DNA replication in a reference cell. The technology uses DNA as a template, adds PCR enzyme and corresponding specific primers to carry out a programmed reaction in a PCR instrument, and replicates and amplifies to generate new complementary DNA fragments. The process consists of three steps of denaturation-annealing-extension. After multiple cycles, DNA amplification can be amplified millions of times within 2-3 hours. Because of its high amplification efficiency, strong specificity and high sensitivity, it has been widely used in the field of molecular biology research.

Double PCR is an improved solution based on the common PCR method. The difference from the general PCR technology is that double PCR needs to add two pairs of primers at one time, and simultaneously amplify in the same reaction system to obtain Compared with general PCR, the target band with higher specificity has the advantages of high efficiency, speed, economy, simplicity and so on.

Snakehead fish vesicular virus (SHVV) belongs to Rhabdoviridae family, Perhabdovirus genus. In 2014, it was isolated from the diseased hybrid snakehead in the breeding pond. The virus can infect snakehead fish, mandarin fish, and eel. In recent years, the virus has caused great harm to some aquaculture areas. In some artificially cultured ponds, the diseased fish had no obvious symptoms in the early stage of infection, their food intake was weakened, and their physique decreased; Protruding, floating on the edge of the pool, sometimes tumbling and dying soon after. At present, there is no effective treatment method, and there is no special literature and technology for the identification of this virus.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the defects of the prior art, and to provide a primer set, a kit and a detection method for double PCR detection of fish rhabdovirus, which can quickly and accurately detect fish rhabdovirus, which is the best solution for the fish rhabdovirus. The clinical diagnosis and prevention of the virus provides an important technical means, and provides a technical method for the identification, isolation, epidemiological investigation and other studies of the virus.

In order to solve the above-mentioned technical problems, the present invention provides a primer set for double PCR detection of fish rhabdovirus, the primer set includes primer SHVV-1 and primer SHVV-2, and the nucleotide sequence of the primer SHVV-1 is as follows: The nucleotide sequences of the primer SHVV-2 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequences of the primer SHVV-2 are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

The present invention also provides a kit for double PCR detection of fish rhabdovirus, the kit comprising the above-mentioned primer set.

The present invention also provides a fish rhabdovirus double PCR detection method, including:

Extract RNA from the kidney tissue of the fish to be tested, and use RNA as a template to synthesize cDNA;

Take the synthetic cDNA as a template, adopt the primer set described in claim 1, carry out double PCR amplification;

The products obtained from the double PCR amplification were subjected to agarose gel electrophoresis, and the gel was placed in an imaging system to observe the results.

Preferably, the RNA extraction adopts Trizol method.

Preferably, the reaction system of the double PCR amplification is: cDNA template × 1 μL, primer SHVV-1F × 1 μL, primer SHVV-1R × 1 μL, primer SHVV-2F × 1 μL, primer SHVV-2R × 1 μL, Premix Taq × 10 μL, ddH ₂ O×5 μL, total amount 20 μL.

Preferably, the reaction procedure of the double PCR amplification is as follows: 95°C for 5 min, 95°C for 30s, 56°C for 30s, 72°C for 1 min, 30 cycles; 72°C, 5min extension, and 4°C storage.

Preferably, the agarose gel electrophoresis is 1.0% agarose gel electrophoresis at 120V for 30min.

Beneficial effects achieved by the present invention:

Compared with traditional virus morphological identification and virus serotype identification, molecular biology technology is applied to the detection of virus, which has the advantages of fast speed, low cost and simple operation. Compared with ordinary PCR, the specificity of double PCR is greatly improved, and the probability of false positives is reduced. At the same time, it can better distinguish viruses with higher homology and similar species, and it can be used in clinical detection with higher accuracy and reliability.

Aiming at the conserved gene sequence of snakehead vesicular virus, the invention designs relevant specific primers and suitable PCR reaction conditions to establish a double PCR detection method for snakehead vesicular virus, which has the characteristics of strong specificity and high sensitivity, and can detect quickly and accurately The discovery of snakehead vesicular virus provides an important technical means for clinical diagnosis and prevention of snakehead vesicular virus, and provides technical solutions for the identification, isolation and epidemiological investigation of snakehead vesicular virus.

Description of drawings

Figure 1 shows the double PCR detection results: M: Marker DL2000; 1: SHVV-1F, SHVV-1R amplification results; 2: SHVV-2F, SHVV-2R amplification results; 3: SHVV-1F, SHVV-1R, SHVV -2F, SHVV-2R two pairs of primers co-amplification results.

Figure 2 shows the specificity detection results of double PCR: M: Marker DL2000; 1: Snake snake vesicular virus; 2: Red sea bream iridovirus; 3: Infectious spleen and kidney necrosis virus; 4: Carp spring viremia virus; 5: ddH ₂ O negative control.

Figure 3 shows the detection results of double PCR sensitivity: M:Marker DL2000; 1-5: 10 ⁰ , 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , dilution factor of virus solution.

Figure 4 is the nested PCR clinical sample detection: M: Marker DL2000; 1-21: samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21; ₂₂ : ddH2O negative control.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings. The following examples are only used to illustrate the technical solutions of the present invention more clearly, and cannot be used to limit the protection scope of the present invention.

The establishment of embodiment 1 double PCR detection method

1. Experimental materials: The diseased fish samples were collected from the hybrid snakehead breeding farm in Shunde City, Guangdong Province, and were stored in the laboratory at -80°C.

Experimental reagents: TRIpure Reagent (Beijing Aidelai Biotechnology); isopropanol, chloroform, 75% ethanol (Sinopharm Chemical Reagent Factory); DEPC water (product of Biosharp); Premix Taq (TaKaRa Version 2.0plusdye), anti-reagent Transcriptase, DL2000 DNA Marker (product of TaKaRa Company); DNA gel recovery kit (manufactured by Quanzhou Gold Company); agarose (Biowest Agarose); 50×TAE buffer.

2. Primer design and synthesis

According to NCBI (National Center for Biotechnology) has published related gene sequences of snakehead vesicular virus, using Clone Manager software, according to the conserved sequence of snakehead vesicular virus nucleocapsid protein gene. Using primer design software (Primer primer), two pairs of PCR primers (SHVV-1F, SHVV-1R; SHVV-2F, SHVV-2R) of snakehead vesicular virus were designed. The primer sequences are shown in Table 1.

Table 1: SHVV duplex PCR primer sequences

3. RNA extraction and cDNA synthesis

RNA extraction: Take the kidney tissue of the fish to be tested, and extract the viral RNA of the sample to be tested according to the RNA extraction instructions (Trizol method). The extracted RNA is easily degraded and needs to be reverse transcribed immediately or stored at -20°C.

cDNA synthesis: According to the RNA reverse transcription instructions, take a 0.2ml non-enzyme centrifuge tube and prepare the following mixture (10μL): RNA template × 5μL, Oligo Dt Primer (50μM) × 1μL, dNTP Mixture (10Mm each) × 1μL, RNaseFree dH ₂ O × 3 μL, keep it in a PCR machine at a constant temperature of 65 °C for 5 min, and quickly insert it into ice to cool; the above steps can improve the efficiency of reverse transcription. The above reaction mixture × 10 μL, RNase Free dH ₂ O × 4.5 μL, Prime Script II RTase (200U/μL) × 1 μL, 5 × Prime Script II Buffer × 4 μL, RNase Inhibitor (40 U/μL) × 0.5 μL, gently pipetting and shaking. Put the centrifuge tube into the PCR machine, synthesize cDNA at 45°C for 50 minutes, inactivate the enzyme at 95°C for 5 minutes, and then immediately put it in an ice box to cool for 3 minutes, which is the cDNA template, and store it at -20°C for later use.

4. Establishment of double PCR system

Duplex PCR requires two pairs of primers to be completed in one amplification, and the total amount of reaction preparation is 20 μL.

Reaction system: cDNA template in step 2 × 1 μL, SHVV-1F × 1 μL, SHVV-1R × 1 μL, SHVV-1F × 1 μL, SHVV-1R × 1 μL, Premix Taq × 10 μL, ddH ₂ O × 5 μL. PCR reaction program: 95°C for 5 min, 95°C for 30s, 56°C for 30s, 72°C for 1 min, 30 cycles; 72°C for 5min extension, cooling at 12°C, and storage at 4°C.

5. Electrophoresis detection

Prepare 1×TAE buffer and 1.0% agarose gel; spot 5μL, DL2000Marker 6μL as reference, and use gel electrophoresis apparatus for 120V electrophoresis for 30min. The results showed that the positive bands were clear and bright (see Figure 1). The band was glued back and tested by related sequencing, and the amplified band was consistent with the target gene sequence.

Example 2 Evaluation of the double PCR method

1. Specific detection

The DNA or cDNA of snakehead vesicular virus (SHVV), infectious spleen and kidney necrosis virus (ISKNV), spring carp viremia virus (SVCV) and red sea bream iridovirus (RSIV) were taken as detection templates, and ddH ₂ O was set as negative. The control was detected according to the double PCR detection method established in the present invention. The results showed that SHVV was positive, and two target bands of 845bp and 313bp were amplified. The experimental control samples ISKNV, SVCV, RSIV and ddH ₂ O had no bands and were negative (see Figure 2).

2. Sensitivity detection

10-fold specific dilution (10 ⁰ , 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 ) was performed on the virus liquid with 1TCID ₅₀ /mL, RNA was extracted according to the detection method established in the present invention, and cDNA was synthesized. Using this as the PCR template, double PCR amplification was carried out with the corresponding system and procedure. At the same time, a set of ddH ₂ O was set as a negative control for the template. The results showed that bright double bands could still be observed when the dilution concentration of the tested sample virus liquid was within 1×10 ^-2 . When the dilution concentration of the sample virus solution reaches 1×10 ^-3 , there are faint bands, and when the dilution concentration of the sample virus solution reaches 1×10 ^-3 or more, there is no specific band (see Figure 3). It shows that the double PCR method established in this experiment can detect snakehead vesicular virus at a minimum concentration of 1×0.01TCID ₅₀ /mL.

Embodiment 3 Clinical sample detection

Select 21 clinical samples of diseased hybrid snakehead kidney tissue, set ddH ₂ O as a negative control, and detect according to the double PCR detection method established in the present invention. The results showed that 21 samples amplified specific bands and were positive; the negative control ddH ₂ O had no bands and was negative (see Figure 4 ). The positive band was recovered and sequenced, and the gene sequence of the positive sample was consistent with the gene sequence of snakehead vesicular virus (SHVV) published by NCBI.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the technical principle of the present invention, several improvements and modifications can also be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

sequence listing

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Claims (7)

1. the primer set of fish rhabdovirus double PCR detection, is characterized in that, described primer set comprises primer SHVV-1 and primer SHVV-2, and the nucleotide sequence of described primer SHVV-1 is as SEQ ID NO:1 and SEQ ID NO: 2, the nucleotide sequences of the primer SHVV-2 are shown in SEQ ID NO: 3 and SEQ ID NO: 4. 2. A kit for double PCR detection of fish rhabdovirus, wherein the kit comprises the primer set of claim 1. 3. Fish Rhabdovirus double PCR detection method is characterized in that, comprises: Extract RNA from the kidney tissue of the fish to be tested, and use RNA as a template to synthesize cDNA; Take the synthetic cDNA as a template, adopt the primer set described in claim 1, carry out double PCR amplification; The products obtained from the double PCR amplification were subjected to agarose gel electrophoresis, and the gel was placed in an imaging system to observe the results. 4. fish rhabdovirus double PCR detection method according to claim 3 is characterized in that, described RNA extraction adopts Trizol method. 5. fish rhabdovirus double PCR detection method according to claim 3 is characterized in that, the reaction system of described double PCR amplification is: cDNA template × 1 μL, primer SHVV-1F × 1 μL, primer SHVV-1R ×1 μL, primer SHVV-2F × 1 μL, primer SHVV-2R × 1 μL, Premix Taq × 10 μL, ddH ₂ O × 5 μL, total amount 20 μL. 6. The fish rhabdovirus double PCR detection method according to claim 3, wherein the reaction program of the double PCR amplification is: 95 ℃ for 5 min, 95 ℃ for 30 s, 56 ℃ for 30 s, 72 ℃ 1 min, 30 cycles; 72°C, 5 min extension, and 4°C storage. 7. fish Rhabdovirus double PCR detection method according to claim 3, is characterized in that, described agarose gel electrophoresis is 1.0% agarose gel electrophoresis, 120V electrophoresis 30min.

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