Primer group, kit and detection method for detecting meriones unguiculatus hepatitis E virus

Abstract

The invention discloses a primer group, a kit and a detection method for detecting a meriones unguiculatus hepatitis E virus, and belongs to the technical field of virus detection. And the primer group comprises a primer Cl-HEV-F, a primer Cl-HEV-R, a primer Cl-HEV-qF, a primer Cl-HEV-qR and a primer Cl-HEV-qP. The detection method comprises the following steps: extracting virus RNA and reversely transcribing into cDNA; the method comprises the following steps: amplifying a capsid protein gene by using a Cl-HEV-F / R primer PCR (Polymerase Chain Reaction), connecting with a pMD19-T vector, and transforming DH5alpha cells for screening. The optimal conditions of RT-qPCR are as follows: the primer / probe concentration is 0.4 mu mol / L, and the annealing temperature is 60 DEG C. The method is high in specificity, the sensitivity reaches 4.33 * 10 copy number / mu L, and the repeatability is good. The detection system has high sensitivity, high specificity and good repeatability, and can be used for detection and quantitative analysis of the meriones unguiculatus hepatitis E virus.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and particularly relates to a primer set, a kit and a detection method for detecting hepatitis E virus in Mongolian gerbils. Background Art

[0002] Hepatitis E virus (HEV), as the pathogen of hepatitis E, belongs to the genus Hepatitis E virus of the family Hepatitis E virus, and is an unenveloped positive-sense single-stranded RNA virus with a genome of about 7.2 kb, containing three open reading frames (ORFs). Among them, the viral capsid protein encoded by ORF2 is the main target of neutralizing antibodies. HEV is divided into types I-IV. Types I and II only infect humans and are transmitted through fecal contamination of water sources; types III and IV mainly exist in animals such as pigs and wild boars and are transmitted to humans through animal-derived foods. HEV infection is mostly self-limiting hepatitis, but the condition is more severe in pregnant women, the elderly and immunocompromised individuals, and the fatality rate is significantly increased.

[0003] The study first discovered a novel hepatitis E virus (Cl-HEV) in Mongolian gerbils, which can effectively replicate in a cell culture system, has the potential for cross-species transmission, provides evidence for potential zoonotic pathogens, and also enriches the HEV host spectrum, highlighting the necessity of strengthening virus monitoring in wild animals.

[0004] Nucleic acid detection can directly screen for hepatitis E virus in Mongolian gerbils by targeting viral RNA, mainly including traditional reverse transcription PCR and real-time fluorescence quantitative PCR (qPCR) and other methods. These methods have high sensitivity and good specificity and can detect the virus before it spreads widely. Traditional RT-PCR needs to identify the amplification products through gel electrophoresis and other methods, and the operation is cumbersome; real-time fluorescence PCR can monitor the fluorescence signal in real time during amplification, and has the advantages of rapidity, high sensitivity and quantification. It is divided into a general dye method and a specific probe method (such as TaqMan fluorescence quantitative PCR). The latter is widely used in HEV detection due to its higher sensitivity and specificity.

[0005] However, the qPCR probe method faces challenges in practical applications: HEV samples often contain PCR inhibitors, which can reduce the amplification efficiency and lead to false negatives. It is necessary to strictly purify the samples and add internal standards to evaluate the degree of inhibition; the sequences of hepatitis E virus in Mongolian gerbils are highly diverse, and the differences may cause primer or probe mismatches, and there is a risk of missed detection of variant strains that are not covered; the detection requires RNA extraction and reverse transcription, and has high requirements for experimental conditions; the fluorescence probe reagents and real-time PCR equipment are expensive, resulting in increased costs. Although the qPCR probe method has high sensitivity, strong specificity and can be quantified, its problems such as complex sample pretreatment, high cost and potential missed detection risk still need to be solved to improve the reliability and applicability of HEV detection.

[0006] Based on this, the present invention designed a set of detection primers according to the capsid protein of Meriones unguiculatus hepatitis E virus, constructed a recombinant plasmid as a reaction template, and established a standard curve of the capsid protein for detection and quantification. SUMMARY OF THE INVENTION

[0007] One object of the present invention is to provide a primer set for detecting Meriones unguiculatus hepatitis E virus, and the primer set includes: Primer Cl-HEV-F, whose nucleotide sequence is shown in SEQ ID NO.1; Primer Cl-HEV-R, whose nucleotide sequence is shown in SEQ ID NO.2; Primer Cl-HEV-qF, whose nucleotide sequence is shown in SEQ ID NO.3; Primer Cl-HEV-qR, whose nucleotide sequence is shown in SEQ ID NO.4; Primer Cl-HEV-qP, whose nucleotide sequence is shown in SEQ ID NO.5.

[0008] The length of the target product amplified by the amplification primers is 397 bp, and the length of the target product of the detection primer set is 145 bp.

[0009] The nucleotide sequence of the primer Cl-HEV-F is shown in SEQ ID NO.1: 5’-GCTRACTGTGTCYGTCCCAA -3’ The nucleotide sequence of the primer Cl-HEV-R is shown in SEQ ID NO.2: 5’-TGAGCCACRCCATTRGTCTC -3’; The nucleotide sequence of the primer Cl-HEV-qF is shown in SEQ ID NO.3: �- GCTRACTGTGTCYGTCCCAA -3’; The nucleotide sequence of the primer Cl-HEV-qR is shown in SEQ ID NO.4: 5’-GCCTCAGTAGCCATGRTRTGTGT -3’; The nucleotide sequence and modification are as follows: 5’- FAM-CTGCTACTAATGCTGTTTTGTATGCTGC-BHQ-1 -3’, and the nucleotide sequence when making the sequence list is shown in SEQ ID NO.5: 5’-CTGCTACTAATGCTGTTTTGTATGCTGC-3’.

[0010] Another object of the present invention is to provide a detection kit, and the kit contains the detection primer set described in claim 1.

[0011] Furthermore, the kit further includes: a premixed probe-based qPCR reagent, and enzyme-free water.

[0012] The premixed probe-based qPCR reagent is purchased from Hunan Aikery Bioengineering Co., Ltd.

[0013] The second object of the present invention is to provide a method for constructing a recombinant plasmid, comprising the following steps: (1) Design amplification primers for the HEV capsid protein gene of Meriones unguiculatus to construct a recombinant plasmid as a standard plasmid; (2) Perform PCR on the HEV positive sample with the amplification primers to amplify the complete target gene; (3) Determine the fragment length by agarose gel electrophoresis to be consistent with the target gene length; (4) Recover and purify the PCR product in (2) that is determined to be consistent with the target fragment length, and use a common agarose gel DNA recovery kit for PCR product recovery and purification; (5) Use the T-vector ligation kit pMD19-T Vector Cloning Kit from Takara Biotechnology (Beijing) Co., Ltd. to ligate the purified and recovered product in (4) with the pMD19-T vector. Gently mix and place it in a 16°C constant temperature metal bath for ligation for 2 - 3.5 h to obtain a recombinant plasmid; (6) Transform the recombinant plasmid into DH5α competent cells; (7) Inoculate the colonies into a shaking tube containing the 1 mL LB medium with ampicillin, place it in a 37°C shaker for 6 - 8 h, and take it out until the bacterial solution in the shaking tube becomes turbid; (8) Perform PCR amplification and agarose gel electrophoresis on the transformed bacterial solution using the pMD19-T vector universal primers, and send the bacterial solution with the expected band size to Beijing Tsingke Biotechnology Co., Ltd. for Sanger sequencing; (9) Extract the recombinant plasmid. Use a pipette to aspirate 200 μL of the successfully prepared bacterial solution and add it to 5 mL of LB liquid medium (containing ampicillin). Culture it in a 37°C constant temperature shaker at 180 rpm for 6 - 8 h until the bacterial solution becomes turbid, then extract the plasmid. Use the nested PCR method to identify the recombinant plasmid, and after preliminary identification by agarose gel electrophoresis, send it to Beijing Tsingke Biotechnology Co., Ltd. for first-generation sequencing. Use a NanoDrop 2000 ultraviolet spectrophotometer to measure the concentration of the constructed recombinant plasmid, and store the plasmid at -20°C in the refrigerator for standby.

[0014] Furthermore, in step (1), the recombinant plasmid primers are primer Cl-HEV-F and primer Cl-HEV-R.

[0015] The nucleotide sequence of primer Cl-HEV-F is shown in SEQ ID NO. 1: 5'-GCTRACTGTGTCYGTCCCAA-3', and the nucleotide sequence of primer Cl-HEV-R is shown in SEQ ID NO. 2: 5'-TGAGCCACRCCATTRGTCTC-3'. The target length of the recombinant plasmid is 397 bp; Furthermore, the PCR reagent in step (2) used LA TaqVersion 2.0 plus dye produced by Bio-Rad Biotechnology (Beijing) Co., Ltd.; Furthermore, the conventional agarose gel DNA recovery kit in step (4) was from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Furthermore, the DH5α competent cells in step (6) were purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; Furthermore, in the bacterial solution PCR identification results in step (8), lanes 1-6 are recombinant plasmid bacterial solutions, and the sequencing results are consistent with the original sequence alignment; Furthermore, the plasmid extraction in step (9) was performed using a plasmid extraction kit from Tiangen Biotechnology Co., Ltd.

[0016] A third object of the present invention is to optimize the specific reaction conditions of TaqMan RT-qPCR of the RT-qPCR detection primer set containing capsid protein fragments, including primer concentration, probe concentration and annealing temperature.

[0017] The fourth purpose of the present invention is to conduct sensitivity, specificity and repeatability tests on capsid protein RT-qPCR detection primers.

[0018] The fifth object of the present invention is to provide a hepatitis E probe method RT-qPCR standard curve.

[0019] The standard curve was further established based on the test results of 10-fold dilution of the positive plasmid. The copy number of the plasmid standard was used as the horizontal axis and the cycle number was used as the vertical axis to draw the standard curve and calculate the correlation coefficient R 2 The RT-qPCR standard curve of the capsid protein fragment of the gerbil hepatitis E virus is y=-2.748x+34.89, R 2 =0.9953. R 2 The value of the standard curve for HEV capsid protein RT-qPCR was greater than 0.99, and the standard curve had a good linear relationship.

[0020] The sixth object of the present invention is to provide a method for detecting and quantifying hepatitis E virus in gerbils based on hepatitis E capsid protein primers and a standard curve, comprising the following steps: (1) Extract viral RNA from samples; (2) Reverse transcribe RNA into cDNA; (3) Perform TaqMan RT-qPCR using cDNA as a template to obtain Ct value; (4) Substitute the Ct value into the standard curve equation of the hepatitis E virus capsid protein y = -2.748x + 34.89 to calculate the virus copy number.

[0021] Further, for the extraction of viral RNA in step (1), the RNeasy Mini Kit Qiagen from Qiagen, Germany is used.

[0022] Further, for the reverse transcription reagent in step (2), the PrimeScript IV 1st strand cDNA Synthesis Mix from Takara Biotechnology (Beijing) Co., Ltd. is used. The reverse transcription system includes: 2 μL of 5×PrimeScript IV cDNA Synthesis Mix, 1 μL of Random 6 mers, 3 μL of enzyme-free water, and 4 μL of RNA; the reverse transcription reaction conditions are: 42°C for 30 min, 85°C for 5 min, and 4°C for 10 min.

[0023] Further, for the qPCR premix reagent in step (3), the ProTaq HS Premix Probe Method qPCR Kit from Hunan Aikery Biotechnology Co., Ltd. is used. The reaction components include: 10 μL of 2×Pro Taq HS Probe Premix, 0.8 μL of primer Cl-HEV-qF, 0.8 μL of primer Cl-HEV-qR, 0.8 μL of probe Cl-HEV-qP, 2 μL of Cl-HEV plasmid, and 5.6 μL of enzyme-free water; the qPCR reaction conditions are: pre-denaturation at 95°C for 10 min; PCR reaction at 95°C for 15 s, 60°C for 30 s, for 45 cycles; the standard curve is plotted using GraphPad Prism 8.0; Further, when performing qPCR detection in step (3), 3 parallel replicate wells are set up for each sample; and a negative control is set, and the negative control is enzyme-free water; Further, in step (3), the Ct value of the sample takes the average of 3 replicates as the final result.

[0024] The hepatitis E virus detection system for Mongolian gerbils provided by the present invention has the following technical effects: Strong pertinence in primer design: Designed for the conserved region of the virus capsid protein, including ordinary PCR and fluorescence quantitative primers, which can accurately identify the virus sequence.

[0025] The recombinant plasmid is standard and reliable: verified by sequencing to be consistent with the original sequence, accurately measured in concentration, and stably preserved.

[0026] The reaction conditions are optimized: the optimal primer / probe concentration is determined to be 0.4 μmol / L and the annealing temperature is 60 °C, with high detection efficiency.

[0027] The detection performance has significant advantages: High sensitivity: The lowest detection limit reaches 4.33×10 0 copies / μL.

[0028] Strong specificity: Only specific amplification occurs for the target virus.

[0029] Good repeatability: The coefficient of variation of Ct values at each concentration is small, and the results are reliable.

[0030] Accurate quantitative analysis: The standard curve has a good linear relationship (R² = 0.9953), and the virus copy number can be accurately calculated.

[0031] High practical application value: Easy to operate, controllable cost, suitable for epidemiological investigations, scientific research, clinical screening, etc. Description of the Drawings

[0032] Figure 1 It is the electrophoresis result diagram of the PCR amplification of the target gene provided by the present invention; Figure 2 It is the electrophoresis result diagram of the PCR identification of the recombinant plasmid bacterial solution provided by the present invention; Figure 3 It is the standard curve diagram of the probe-based RT-qPCR detection system for the hepatitis E virus of Mongolian gerbils provided by the present invention; Figure 4 It is the result diagram of the optimization of the primer concentration of the probe-based RT-qPCR for the hepatitis E virus of Mongolian gerbils provided by the present invention; Figure 5A It is the result diagram of the optimization of the annealing temperature at 54 °C of the probe-based RT-qPCR for the hepatitis E virus of Mongolian gerbils provided by the present invention; Figure 5B It is the result diagram of the optimization of the annealing temperature at 56 °C of the probe-based RT-qPCR for the hepatitis E virus of Mongolian gerbils provided by the present invention; Figure 5C It is the result diagram of the optimization of the annealing temperature at 58 °C of the probe-based RT-qPCR for the hepatitis E virus of Mongolian gerbils provided by the present invention; Figure 5D It is the result diagram of the optimization of the annealing temperature at 60 °C of the probe-based RT-qPCR for the hepatitis E virus of Mongolian gerbils provided by the present invention; Figure 6 It is the result diagram of the optimization of the probe concentration of the probe-based RT-qPCR for the hepatitis E virus of Mongolian gerbils provided by the present invention; Figure 7 This is the result diagram of the specificity test of the probe-based RT-qPCR detection system for hepatitis E virus in Meriones unguiculatus provided by the present invention; Figure 8 This is the result diagram of the sensitivity test of the probe-based RT-qPCR detection system for hepatitis E virus in Meriones unguiculatus provided by the present invention. Specific implementation manners

[0033] The present invention is illustrated by the following examples.

[0034] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0035] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0036] Example 1 Design of primers for amplifying the capsid protein of hepatitis E virus in Meriones unguiculatus and design of primers for RT-qPCR detection Download the existing full-length sequence of hepatitis E virus in Meriones unguiculatus (accession number: OR232328.1) from NCBI, select a conserved region in the capsid protein, design primers based on it as the subsequent RT-qPCR detection primers; and design ordinary PCR amplification to amplify the target gene template. The primer sequences are shown in Table 1 below: Table 1: Synthesis of Recombinant Plasmid in Example 2 Select a positive sample of gerbil hepatitis E virus to extract viral RNA. Reverse transcribe the RNA into cDNA. Using the cDNA as a template, perform PCR with common PCR virus detection primers to amplify the target gene.

[0037] Further, the components of the PCR reaction are as follows: For the outer reaction, 10 μL of 2×Taq PCR Mix, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of cDNA, and 7 μL of enzyme-free water. The amplification conditions are: pre-denaturation at 94°C for 3 min; PCR reaction at 94°C for 30 s, 52°C for 30 s, 72°C for 40 s, for 35 cycles; final extension at 72°C for 7 min. For the inner reaction, 15 μL of 2×Taq PCR Mix, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of the outer PCR product, and 12 μL of enzyme-free water. The amplification conditions are: pre-denaturation at 94°C for 3 min; PCR reaction at 94°C for 30 s, 53.5°C for 30 s, 72°C for 30 s, for 35 cycles; final extension at 72°C for 7 min.

[0038] Perform agarose gel electrophoresis to determine that the size of the PCR product conforms to the length of the target gene. As Figure 1 shown, M represents the 5000bp DNA molecular weight standard.

[0039] Use a common agarose gel DNA recovery kit to purify the PCR product. The specific steps are as follows: (1) Use an electronic balance to weigh the empty enzyme-free 1.5 mL centrifuge tube. After taking 50 μL of the PCR amplification product for gel electrophoresis, use a sterilized blade under a UV gel cutting instrument to cut the agarose gel block containing the target gene fragment as small and accurately as possible and place it in the centrifuge tube. (2) Weigh the centrifuge tube again with an electronic balance to calculate the weight of the gel block. (3) According to the standard of adding 100 μL of solubilization solution PN to every 0.1 g of gel block, add the required volume of PN solution to the gel block and dissolve the gel block in a 50°C metal bath. To ensure complete dissolution of the gel, gently rotate the centrifuge tube up and down every 2 - 3 min until the gel block is completely dissolved. (4) Place the CA2 adsorption column in the collection tube. Since the adsorption column has a stronger ability to bind DNA at room temperature, take out the centrifuge tube and place it at room temperature. After the solution temperature drops to room temperature, add all the solution to the adsorption column, let it stand at room temperature for 2 min, and centrifuge at 13400*g for 1 min. (5) Discard the lower-layer solution, add 600 μL of PW washing solution to the adsorption column. A certain volume of absolute ethanol needs to be added to the PW washing solution before use. Let it stand at room temperature for 5 min, and centrifuge at 13400*g for 1 min; (6) Repeat step (5); (7) Discard the lower-layer solution, centrifuge the empty column at 13400*g for 2 min to empty it, try to discard the washing solution in the collection tube as much as possible, and place the adsorption column at room temperature and let it stand until it is completely dry; (8) Place the adsorption column in a new enzyme-free centrifuge tube, add 30 μL of preheated DEPC water to the center of the adsorption column, centrifuge at 13400*g for 2 min, and store the collected DNA solution at -20°C for later use.

[0040] Ligate the purified PCR product with the vector. The components of the ligation reaction are: 1 μL of pMD19-T Vector, 4 μL of the DNA target fragment, and 5 μL of Solution Ⅰ. The reaction conditions are: ligate at 16°C for 2 - 3.5 h.

[0041] Transform the recombinant plasmid into DH5α competent cells. The specific steps are as follows: (1) After the DH5α competent cells are completely melted on ice, transfer them to a new 1.5 mL enzyme-free centrifuge tube. At the same time, add 10 μL of the reaction system to it, gently mix, and place it on ice for 30 min; (2) After the reaction is completed, place the reaction solution in a 42°C metal bath for heat shock for 90 s, and then quickly place it in an ice-water bath for 2 min. The operation process should be gentle; (3) In the biosafety cabinet, add 900 μL of LB liquid medium without ampicillin to this centrifuge tube, and then place it in a 37°C constant-temperature shaker and shake it at 180 rpm for 1 h; (4) Use a high-speed centrifuge to centrifuge the shaken liquid at 3000 rpm for 5 min, discard the supernatant, and leave about 200 μL of liquid in the centrifuge tube. Gently pipette and mix it with a pipette tip to form a suspension; (5) In the biosafety cabinet, take out the pre-prepared LB agar solid medium containing Amp, and use a sterile triangular spreading rod to evenly spread the suspension up, down, left, right, and in the middle on the medium. Seal the edge with a sealing film, and place it upside down in a 37°C constant-temperature incubator for overnight culture for 11 - 16 h; (6) After overnight culture, pick 5 - 10 suspected single white colonies from the culture dish, and inoculate them into shaking tubes containing 1 mL of LB medium with ampicillin respectively. Place them in a constant-temperature shaker and culture at 37°C for 6 - 8 h until the bacterial liquid in the shaking tube becomes turbid, and then take them out.

[0042] Colony PCR amplification, the specific steps are as follows: (1) Use the universal primers of pMD19-T vector to perform PCR amplification on the transformed bacterial solution. The reaction system and reaction conditions refer to the general PCR experimental method.

[0043] (2) Take 5 μL of the bacterial solution PCR product for agarose gel electrophoresis. Aliquot 100 μL of the bacterial solution with the expected band size and send it to Beijing Tsingke Biotechnology Co., Ltd. for first-generation sequencing.

[0044] (3) Expand the culture of the bacterial solution with positive sequencing results.

[0045] For plasmid extraction, use the plasmid mini-prep kit from Tiangen Biotech. Pipette 200 μL of the successfully prepared bacterial solution into 5 mL of LB liquid medium (containing ampicillin), and culture it at 37°C in a constant temperature shaker at 180 rpm for 6 - 8 h until the bacterial solution becomes turbid, and then extract the plasmid. The specific steps are as follows: (1) Take 5 mL of the overnight cultured bacterial solution, add it to a 1.5 mL centrifuge tube in 5 portions, and centrifuge at 13400*g for 1 min using a high-speed centrifuge. Try to discard the supernatant as much as possible and collect the bacterial cell pellet. (2) Add RNase A and TIAN Red to the P1 solution in advance and mix it until it becomes a clear red. Add 150 μL of P1 to the centrifuge tube, and use a vortex oscillator to mix the bacterial solution. After mixing, it becomes a turbid red. (3) The P2 solution can be placed in a 37°C metal bath and heated for 5 min before use. Add 150 μL of the P2 solution to the centrifuge tube, and gently invert the bacterial solution up and down until it becomes clear and viscous. (4) Add 350 μL of the P5 solution to the centrifuge tube, quickly invert and mix until flocculent precipitates appear, then centrifuge at 13400 rpm for 2 min. The P5 solution can be placed in a 37°C metal bath and heated for 5 min before use. (5) Use a pipette to aspirate the supernatant and add it to the adsorption column CP3, and centrifuge at 13400 rpm for 30 s. (6) Pour out the lower-layer solution, add 300 μL of the washing solution PWT to the adsorption column. A certain volume of absolute ethanol should be added to the washing solution PWT before use, and centrifuge at 13400 rpm for 30 s. (7) Pour out the lower-layer solution, place the adsorption column back into the collection tube again, and centrifuge at 13400 rpm without load for 1 min to remove the residual washing solution in the adsorption column. (8) Place the adsorption column in a new 1.5 mL enzyme-free centrifuge tube, let it stand at room temperature for 5 - 10 min. Add 50 μL of pre-warmed elution buffer TB to the middle of the adsorption column, centrifuge at 13400 rpm for 30 s, repeat the step for secondary elution to obtain the recombinant plasmid.

[0046] (9) Identify the recombinant plasmid using the ordinary PCR method. The obtained PCR amplification products are preliminarily identified by agarose gel electrophoresis. As Figure 2 shown, M: 2000 bp DNA molecular weight standard. Send the recombinant plasmid to Beijing Tsingke Biotechnology Co., Ltd. for Sanger sequencing, and use Geneious software (version 2021.2.2) to perform sequence alignment and analysis on the obtained nucleotide sequence.

[0047] Example 3 Optimization of the primer concentration of the capsid protein in the TaqMan RT-qPCR reaction of gerbil hepatitis E virus In the TaqMan RT-qPCR reaction, the primer concentration was optimized according to four concentration gradients of 0.1, 0.2, 0.3, and 0.4 μL. Use a positive plasmid with a copy number of 4.33×10 7 copies / μL as the template, Cl-HEV-qF and Cl-HEV-qR as the detection primers, and Cl-HEV-qP as the probe. The primer concentration with the smallest Ct Ct value and the highest fluorescence intensity is the optimal primer concentration. The specific operation steps are as follows: Add 10 μL of 2×Pro Taq HS Probe Premix, 2 μL of Cl-HEV plasmid, 0.8 μL of Cl-HEV-qP to the reaction tube. Under the condition of a primer concentration of 0.1 μmol / L, add 0.2 μL of the upstream and downstream primers respectively; under the condition of a primer concentration of 0.2 μmol / L, add 0.4 μL of the upstream and downstream primers respectively; under the condition of a primer concentration of 0.3 μmol / L, add 0.6 μL of the upstream and downstream primers respectively; under the condition of a primer concentration of 0.4 μmol / L, add 0.8 μL of the upstream and downstream primers respectively. Finally, supplement with enzyme-free water to a total reaction system of 20 μL. Each sample well is subjected to 3 technical replicates. The reaction conditions are: qPCR reaction conditions: pre-denaturation at 95°C for 10 min; PCR reaction at 95°C for 15 s, 60°C for 30 s, for 45 cycles.

[0048] The amplification results of the optimized primer concentration of the gerbil hepatitis E virus capsid protein RT-qPCR are as Figure 4 shown. It can be seen that the optimal primer concentration for the capsid protein RT-qPCR is 0.4 μmol / L.

[0049] The optimization results of primer concentration in the TaqMan RT-qPCR reaction of the capsid protein of Meriones unguiculatus hepatitis E virus are shown in Table 2 below: Table 2: Example 4 Optimization of Probe Concentration in the TaqMan RT-qPCR Reaction of Meriones unguiculatus Hepatitis E Virus Based on the completion of primer concentration optimization, probe concentration optimization was carried out. In the TaqMan RT-qPCR reaction, four concentration gradients of 0.1, 0.2, 0.3, and 0.4 μL were used for probe concentration optimization. Using a positive plasmid with a copy number of 4.33×10 7 copies / μL as a template, Cl-HEV-qF and Cl-HEV-qR were used as detection primers, and Cl-HEV-qP was used as a probe. With Ct the probe concentration with the smallest value and the highest fluorescence intensity being the optimal probe concentration. The specific operation steps were as follows: Add 10 μL of 2×Pro Taq HS Probe Premix to the reaction tube, 2 μL of the Meriones unguiculatus hepatitis E virus plasmid, add 0.8 μL of each of the upstream and downstream primers in the RT-qPCR reaction of the capsid protein, add 0.2 μL of the probe under the condition of a probe concentration of 0.1 μmol / L, add 0.4 μL of the probe under the condition of a probe concentration of 0.2 μmol / L, add 0.6 μL of the probe under the condition of a probe concentration of 0.3 μmol / L, add 0.8 μL of the probe under the condition of a probe concentration of 0.4 μmol / L, and finally supplement with enzyme-free water to a total reaction system of 20 μL. Each sample well was subjected to 3 technical replicates. The reaction conditions were: The qPCR reaction conditions were: pre-denaturation at 95°C for 10 min; PCR reaction at 95°C for 15 s, 60°C for 30 s, for 45 cycles.

[0050] The amplification results of the optimization of the RT-qPCR probe concentration of the Meriones unguiculatus hepatitis E virus capsid protein are as Figure 6 shown, and it can be seen that the optimal probe concentration for the RT-qPCR of the capsid protein is 0.4 μmol / L.

[0051] The optimization results of the probe concentration in the TaqMan RT-qPCR reaction of the capsid protein of Meriones unguiculatus hepatitis E virus are shown in Table 3 below: Table 3: Example 5 Optimization of Annealing Temperature in the TaqMan RT-qPCR Reaction of Meriones unguiculatus Hepatitis E Virus Based on the optimization of the upstream and downstream primer concentrations and probe concentration, the annealing temperature was optimized. In the TaqMan RT-qPCR reaction, the annealing temperature was optimized using four temperature gradients of 54 °C, 56 °C, 58 °C, and 60 °C. A positive plasmid with a copy number of 4.33×10 7 copies / μL was used as the template, Cl-HEV-qF and Cl-HEV-qR were the detection primers, and Cl-HEV-qP was the probe. The annealing temperature with the smallest Ct value and the highest fluorescence intensity was taken as the optimal annealing temperature. The specific operation steps were as follows: Add 10 μL of 2×Pro Taq HS Probe Premix, 2 μL of Cl-HEV plasmid, 0.8 μL of Cl-HEV-qP, and 0.8 μL of each of the upstream and downstream primers in the capsid protein RT-qPCR reaction to the reaction tube, and finally add enzyme-free water to make up a total reaction system of 20 μL. Each sample well was subjected to 3 technical replicates. The reaction conditions were: The qPCR reaction conditions were: pre-denaturation at 95 °C for 10 min; PCR reaction at 95 °C for 15 s, 54 °C / 56 °C / 58 °C / 60 °C for 30 s, for 45 cycles.

[0052] The amplification results of the annealing temperature optimization of the RT-qPCR of the HEV capsid protein of Meriones unguiculatus are shown in Figure 5A -D. It can be seen that the optimal annealing temperature for RT-qPCR is 60 °C.

[0053] The optimization results of the annealing temperature in the TaqMan RT-qPCR reaction of the HEV capsid protein of Meriones unguiculatus are shown in Table 4 below: Table 4: Example 6 Specificity test of the RT-qPCR reaction of the HEV capsid protein of Meriones unguiculatus Using the real-time fluorescence quantitative RT-qPCR detection system for HEV of Meriones unguiculatus to detect the positive and negative plasmids of HEV of Meriones unguiculatus, as Figure 7 shown, the results showed that only the HEV of Meriones unguiculatus showed a specific amplification curve, indicating that this method can specifically detect the HEV of Meriones unguiculatus.

[0054] Example 7 Sensitivity test of the RT-qPCR reaction of the HEV capsid protein of Meriones unguiculatus Using the optimized detection system, the positive plasmid standard was serially diluted 10-fold (4.33×10 0 ~10 10 copies / μL) as the template, and 3 replicate wells were set for each gradient. As Figure 8, The results showed that the lowest detection limit of real-time fluorescence quantitative RT-qPCR for hepatitis E virus in Mongolian gerbils was 4.33×10 0 copies / μL. It indicated that the RT-qPCR detection of the capsid protein of hepatitis E virus in Mongolian gerbils had relatively high sensitivity.

[0055] Results of the RT-qPCR reaction repeatability test for the capsid protein of hepatitis E virus in Mongolian gerbils in Example 8 Consistent with the sensitivity test, the optimized detection system was used to detect the positive plasmid standards of different dilutions. Three replicate wells were set for each dilution to test the repeatability of this method and calculate the Ct mean value, standard deviation and coefficient of variation of the Ct values. The results showed that the coefficient of variation of the

[0056] values of each concentration of this detection system was less than [[ID=!16]]Table 5: Example 9 Establishment of the RT-qPCR standard curve for the capsid protein of hepatitis E virus in Mongolian gerbils The positive plasmid of hepatitis E virus in Mongolian gerbils was diluted by 10-fold serial dilution. The recombinant plasmid at a dilution of 4.33×10 0 ~10 10 copies / μL was used as the template. The reaction components were: 10 μL of 2×Pro Taq HS Probe Premix, 2 μL of Cl-HEV plasmid, 0.8 μL of the upstream primer Cl-HEV-qF, 0.8 μL of the downstream primer Cl-HEV-qR, 0.8 μL of the probe Cl-HEV-qP, and 5.6 μL of nuclease-free water. The reaction conditions were: pre-denaturation at 95°C for 10 min; PCR reaction at 95°C for 15 s, 60°C for 30 s, for 45 cycles. Three parallel replicate wells were set for each gradient. According to the detection results at each dilution, with the copy number of the plasmid standard as the abscissa and the Ct value as the ordinate, a standard curve was plotted as Figure 3 , y = 2.748x + 34.89, R 2 = 0.9953. This equation can be used for the quantitative detection of samples in later in vivo and in vitro experiments, that is, when the Ct value of the sample to be tested is known, the initial copy number of the sample to be tested can be calculated, so as to perform quantitative detection on the sample to be tested.

Claims

1. A primer set for detecting hepatitis E virus in Mongolian gerbils, characterized in that, The primer set includes: Primer Cl-HEV-F, whose nucleotide sequence is as shown in SEQ ID NO.1; Primer Cl-HEV-R, whose nucleotide sequence is as shown in SEQ ID NO.2; Primer Cl-HEV-qF, whose nucleotide sequence is as shown in SEQ ID NO.3; Primer Cl-HEV-qR, whose nucleotide sequence is as shown in SEQ ID NO.4; Primer Cl-HEV-qP, whose nucleotide sequence is as shown in SEQ ID NO.

5.

2. A kit, characterized in that, The kit contains the primer set for the detection described in claim 1.

3. The kit according to claim 2, wherein The kit further includes: a pre-mixed probe-based qPCR reagent and nuclease-free water.

4. A method for detecting hepatitis E virus of Mongolian gerbils by using the primer set according to claim 1, characterized in that, It includes the following steps: (1) Extract the RNA of the hepatitis E virus of Mongolian gerbils and perform reverse transcription to synthesize cDNA; (2) Obtain the target gene of the viral capsid protein by PCR amplification and construct a recombinant plasmid; (3) Optimize the RT-qPCR reaction conditions; (4) Evaluate the sensitivity, specificity, and repeatability of the detection method; (5) Perform 10-fold serial dilutions on the recombinant plasmid. Using the copy number of the plasmid standard as the abscissa and Ct the value as the ordinate, plot the standard curve for capsid protein detection; (6)Detection sample acquisition Ct Value, substitute it into the standard curve equation to calculate the virus copy number.

5. The detection method according to claim 4, characterized in that, The steps for constructing the recombinant plasmid in step (2) are as follows: (1) Use the viral detection primers to PCR amplify the target gene of the capsid protein; the primers are the upstream primer Cl-HEV-F and the downstream primer Cl-HEV-R; The nucleotide sequence of the primer Cl-HEV-F is as shown in SEQ ID NO.1; The nucleotide sequence of the primer Cl-HEV-R is as shown in SEQ ID NO.2; (2) Purify the PCR product; (3) Ligate the purified target gene with the vector to obtain a recombinant plasmid; (4) Transform the recombinant plasmid into DH5α competent cells for screening.

6. The detection method according to claim 4, characterized in that, The primers include the upstream primer Cl-HEV-qF, the downstream primer Cl-HEV-qR, and the fluorescent probe primer Cl-HEV-qP; The nucleotide sequence of the primer Cl-HEV-qF is as shown in SEQ ID NO.3; The nucleotide sequence of the primer Cl-HEV-qR is as shown in SEQ ID NO.4; The nucleotide sequence of the primer Cl-HEV-qP is as shown in SEQ ID NO.

5.

7. According to the detection method described in claim 4, wherein The primer concentration range is 0.1 - 0.4 μmol / L; The probe concentration range is 0.1 - 0.4 μmol / L; The annealing temperature range is 54 - 60 °C.

8. The detection method according to claim 4, characterized in that, The steps for establishing the RT-qPCR standard curve in step (5) are as follows: (1) Dilute the recombinant plasmid by 10-fold serial dilution to 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 , for a total of eleven dilution factors; (2)Perform RT-qPCR detection using the recombinant plasmids at each dilution as templates, and record the corresponding Ct value; (3)With the copy number of the recombinant plasmid standard as the abscissa, Ct and the value as the ordinate, plot the standard curve.

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