Application of LYZ lysozyme mRNA expression quantity as swine transmissible gastroenteritis diagnostic marker

Abstract

The invention discloses application of LYZ lysozyme mRNA expression quantity as a swine transmissible gastroenteritis diagnostic marker, and relates to the technical field of molecular biology. The invention also discloses a primer for detecting the lysozyme mRNA (messenger Ribonucleic Acid) of the diagnostic marker LYZ of the transmissible gastroenteritis of the swine, wherein the primer comprises an upstream primer sus-LYZ-F: 5 '-ACTAGGCGTTAAAGCATGGGTG-3' and a downstream primer sus-LYZ-R: 5 '-AGAGACAAGGTGAGCTGAAGA-3'. The invention also discloses a kit for detecting the lysozyme mRNA of the diagnostic marker LYZ of the transmissible gastroenteritis of the swine. According to the invention, a porcine transmissible gastroenteritis virus TGEV is used for infecting a porcine ileum epithelial cell line IPI-2I, transcriptome sequencing is carried out, differential expression mRNA is screened, LYZ lysozyme mRNA in a TGEV infection group is obviously higher than that in a Mock group, so that the marker gene for detecting porcine transmissible gastroenteritis infection can be used. The detection method is time-saving, labor-saving, easy to detect and accurate in result.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to the application of LYZ lysozyme mRNA as a diagnostic marker for porcine transmissible gastroenteritis. Background Art

[0002] Porcine transmissible gastroenteritis (TGE) is a highly contagious disease caused by porcine transmissible gastroenteritis virus (TGEV) characterized by vomiting, diarrhea, and dehydration in pigs, causing huge losses to the pig industry. The disease can infect pigs of different ages, but the mortality rate for newborn piglets is as high as 100%. Adult pigs are mildly infected after infection, and may experience growth disorders and agalactia. In recent years, the disease has occurred frequently around the world. It has also been prevalent in many regions of my country, such as Sichuan, Hubei, Jilin, Beijing, and Guangzhou, and is on the rise. Since TGEV is often mixed with other viruses or often accompanied by secondary bacterial infections, the clinical symptoms are complex and diverse. Mixed infections and secondary infections greatly increase the difficulty of TGEV diagnosis. Therefore, establishing a method for rapid and accurate detection of TGEV is of great significance for the prevention and control of porcine transmissible gastroenteritis and the cultivation of new varieties resistant to transmissible gastroenteritis.

[0003] LYZ lysozyme, also known as muramidase or N-acetylmuramide glycanohydrlase, is an alkaline enzyme that can hydrolyze mucopolysaccharides in pathogenic bacteria. It mainly breaks down the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, decomposing the insoluble mucopolysaccharides in the cell wall into soluble glycopeptides, causing the cell wall to rupture and the contents to escape, thereby dissolving the bacteria. Lysozyme can also directly bind to negatively charged viral proteins, forming complex salts with DNA, RNA, and apoproteins to inactivate the virus. Therefore, the enzyme has antibacterial, anti-inflammatory, and antiviral effects.

[0004] However, there are no reports on the relationship between LYZ lysozyme and porcine transmissible gastroenteritis. Summary of the invention

[0005] The application of the LYZ lysozyme mRNA expression level as a diagnostic marker for porcine transmissible gastroenteritis provided by the present invention is intended to solve the problems existing in the above-mentioned background technology.

[0006] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0007] In a first aspect, the present invention discloses the use of LYZ lysozyme mRNA expression as a diagnostic marker for porcine transmissible gastroenteritis.

[0008] In a second aspect, the present invention discloses the use of a reagent for detecting the expression amount of LYZ lysozyme mRNA in the preparation of a porcine transmissible gastroenteritis detection or diagnosis product.

[0009] In a preferred embodiment of the present invention, the reagent for detecting LYZ lysozyme mRNA includes an upstream primer sus-LYZ-F: 5'-ACTAGGCGTTAAAGCATGGGTG-3' and a downstream primer sus-LYZ-R: 5'-AGAGACAAGGTGAGCTGAAGAA-3'.

[0010] In a third aspect, the present invention discloses a primer for detecting LYZ lysozyme, a diagnostic marker of porcine transmissible gastroenteritis, comprising an upstream primer sus-LYZ-F: 5'-ACTAGGCGTTAAAGCATGGGTG-3' and a downstream primer sus-LYZ-R: 5'-AGAGACAAGGTGAGCTGAAGAA-3'.

[0011] In a fourth aspect, the present invention discloses a use of the primer as described in the third aspect in the preparation of a product for detecting or diagnosing infectious gastroenteritis.

[0012] In a fifth aspect, the present invention discloses a kit for detecting porcine transmissible gastroenteritis virus, the kit comprising the primers described in the third aspect.

[0013] In a sixth aspect, the present invention discloses the use of the kit as described in the fifth aspect in the preparation of a product for detecting or diagnosing porcine transmissible gastroenteritis.

[0014] In a seventh aspect, the present invention discloses a method for quantitatively detecting the expression of LYZ lysozyme mRNA in pig gastrointestinal cells, comprising the following steps:

[0015] Step 1, primer design and synthesis: design and synthesize primers and internal reference primers for amplifying LYZ lysozyme;

[0016] Step 2, extracting total RNA from the pig ileum epithelial cells to be tested and performing reverse transcription to obtain cDNA;

[0017] Step 3, real-time fluorescence quantitative PCR: using the primers described in step (1) respectively, and using the cDNA of the gastrointestinal cells of the pig to be tested as a template to perform fluorescence quantitative PCR;

[0018] Step 4: Quantitatively detect the expression level of LYZ lysozyme mRNA in pig gastrointestinal cells: Statistically analyze the Ct value of the real-time fluorescence quantitative PCR in step (3) to determine the expression level of LYZ lysozyme mRNA in pig gastrointestinal cells.

[0019] In a preferred embodiment of the present invention, the primers used to amplify LYZ lysozyme include an upstream primer sus-LYZ-F: 5'-ACTAGGCGTTAAAGCATGGGTG-3' and a downstream primer sus-LYZ-R: 5'-AGAGACAAGGTGAGCTGAAGAA-3'; the internal reference primers include an upstream primer βactin-sus-F: 5'-CTGGCACCACACCTTCTACAA-3' and a downstream primer βactin-sus-R: 5'-GTGTTGAAGGTCTCGAACATGAT-3'.

[0020] In a preferred embodiment of the present invention, in step 4, when the relative expression level of LYZ lysozyme mRNA in porcine gastrointestinal cells in the tested cells is lower than 1.41, it can be used to indicate that the cells are not infected by TGEV, otherwise, they are infected by TGEV.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention infects porcine ileal epithelial cell line IPI-2I with porcine transmissible gastroenteritis virus TGEV, performs transcriptome sequencing, and screens differentially expressed mRNAs, wherein LYZ lysozyme mRNA in the TGEV infection group is significantly higher than that in the Mock group, and thus a marker gene for detecting porcine transmissible gastroenteritis infection can be used.

[0023] The detection method of the present invention is time-saving, labor-saving, easy to detect and produces accurate results. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention.

[0025] Figure 1 The statistical bar graph of differentially expressed genes;

[0026] Figure 2 This is a comparison of LYZ mRNA expression levels between the Mock group and the TGEV infection group after infection. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0028] Example 1

[0029] 1. Construction of transcriptome sequencing library

[0030] The porcine transmissible gastroenteritis virus TGEV toxic beads used in this experiment were P9 generation TGEV stored in the veterinary department of our unit, 2 ml.

[0031] In this experiment, cells from the TGEV group and the MOCK group were collected, with 4 samples in each group. The TGEV group represented porcine ileal epithelial cells infected with TGEV, and the MOCK group represented porcine ileal epithelial cells not infected with TGEV, serving as a blank control.

[0032] TGEV was diluted 10-fold and then inoculated, and samples were collected after 12 hours.

[0033] The specific operation process is as follows:

[0034] Day 1: Plating cells

[0035] 1. Digest the cells full of porcine transmissible gastroenteritis virus with trypsin and spread them on a 12-well plate (1 mL / well);

[0036] For cell passage, resuspend 6 T25 cells in 50 mL, of which 40 mL is used for plate attachment and the remaining 10 mL is used for further cell passage;

[0037] 2. Culture overnight in a 37°C, CO2 incubator;

[0038] Day 2: TGEV infection of cells

[0039] 1. Take out the 12-well plate, discard the supernatant, and wash twice with 5 μg / mL pancreatic enzyme maintenance solution, 500 μL / well;

[0040] 2. TGEV was diluted 10-fold with pancreatic enzyme maintenance solution containing 5 μg / mL, and then infected into porcine ileal epithelial cell line IPI-2I, with 400 μL / well of 12-well plate, and incubated in a 37°C incubator for 2 h;

[0041] 3. Discard the supernatant and wash twice with 5 μg / mL pancreatic enzyme maintenance solution, 500 μL / well;

[0042] 4. Add 5 μg / mL pancreatic enzyme maintenance solution, 1 mL / well of 12-well plate; continue to culture in 37°C incubator for 9 hours;

[0043] Day 3: Sample collection

[0044] 1. Wash the cells three times with PBS, 500 μL / well, and discard the supernatant;

[0045] 2. Add AIDzol lysis buffer, add 400μLL / well to 12-well plate, let stand at room temperature for 5 minutes, and collect the samples into 1.5mL EP tubes (enzyme-free EP tubes for RNA extraction) after repeated blow-ups with a blue pipette tip. Mark them: ZL-Mock-1, ZL-Mock-2, ZL-Mock-3, ZL-Mock-4, ZL-TGEV-1, ZL-TGEV-2, ZL-TGEV-3, and ZL-TGEV-4.

[0046] 3. Send to Meiji Company for RNA extraction and transcriptome sequencing.

[0047] 2. Analysis of transcriptome sequencing results

[0048] 1. Screening of differentially expressed genes

[0049] The software edgeR was used to analyze the difference in gene expression, and the screening threshold was: |log2FC|>=1.000, pvalue<0.05. The results are as follows Figure 1 As shown, a total of 379 differentially expressed genes were obtained, including 209 up-regulated genes and 170 down-regulated genes. The top 5 genes with the most significant expression differences are shown in Table 1, among which LYZ gene ranks first.

[0050] Table 1 Top 5 genes with different expression levels

[0051]

[0052] 3. Verification of transcriptome sequencing results

[0053] 1 The total RNA extracted from the porcine ileal epithelial cells of the TGEV group and the MOCK group was reverse transcribed using the Ibotek RNA reverse transcription kit (RK20429). The reaction system and reaction conditions were as follows:

[0054] Reverse transcription reaction system:

[0055] On ice, add the following components to an RNase-free PCR tube, mix well, and centrifuge briefly:

[0056] Components Addition amount 5×ABScript III RT Mix 4μL 20×gDNA Remover Mix 1μL Total RNA 10pg-1 μg* <![CDATA[Nuclease-free H2O]]> Make up to 20 μL

[0057] *Note: Add the appropriate amount of RNA according to the experimental requirements. When the RNA template volume is large, make sure that the RNA is dissolved in water instead of TE Buffer, because TE will inhibit the reverse transcription reaction.

[0058] Reverse transcription reaction conditions

[0059] Perform reverse transcription reaction on a PCR instrument according to the following reaction program:

[0060] temperature time 37℃ 2min 55℃ 15min 85℃ 5min 4℃ Hold

[0061] The reverse transcription product (RT reaction solution) can be used for subsequent qPCR reaction immediately, or stored at -20℃ and used within half a year. For long-term storage, it is recommended to store at -80℃ after aliquoting. Repeated freezing and thawing of cDNA should be avoided.

[0062] 2. Fluorescence quantitative PCR detection

[0063] The reverse transcription product cDNA was used as a template for fluorescence quantitative PCR reaction. The reaction conditions were as follows: reaction system 10ul: cDNA 0.5ul, upstream / downstream primers 0.25ul each, mix 5ul, ddH2O 4ul.

[0064] The designed primers are as follows:

[0065]

[0066] Fluorescence quantitative PCR reaction conditions: The fluorescence quantitative dye used was ABclonal Genious 2XSYBR Green Fast qPCRMix (No ROX) from Ibotek, pre-denaturation at 95°C for 3 min; 95°C for 10 s, 64.5°C for 30 s for 40 cycles; the melting curve program was as follows:

[0067]

[0068] 3. Gene expression analysis: Microsoft Excel was used to process the original data of gene expression, and 2-ΔΔCt was used to calculate the relative expression level of LYZ lysozyme mRNA in the TGEV infection group (using TGEV infected cells) and the Mock group (uninfected cells, as blank control). GraphPad Prism9 software was used for statistical analysis. The results showed that the expression level of LYZ lysozyme gene in the uninfected group (Mock group) was lower than 1.41 relative to the internal reference gene βactin, and the relative expression level of LYZ lysozyme gene was significantly different between the two groups (P < 0.001). The experimental results are as follows Figure 2 shown.

[0069] Depend on Figure 2It can be seen that when the relative expression level of LYZ lysozyme mRNA in the tested cells is lower than 1.41, it can be used to indicate that the cells are not infected by TGEV, indicating that LYZ lysozyme mRNA can be used as a marker gene for detecting porcine transmissible gastroenteritis infection.

[0070] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of LYZ lysozyme mRNA expression as a diagnostic marker for porcine transmissible gastroenteritis.

2. Application of reagents for detecting LYZ lysozyme mRNA in the preparation of porcine transmissible gastroenteritis detection or diagnosis products.

3. The use according to claim 2, characterized in that: The reagent for detecting LYZ lysozyme mRNA comprises an upstream primer sus-LYZ-F: 5'-ACTAGGCGTTAAAGCATGGGTG-3' and a downstream primer sus-LYZ-R: 5'-AGAGACAAGGTGAGCTGAAGAA-3'.

4. A primer for detecting LYZ lysozyme, a diagnostic marker for porcine transmissible gastroenteritis, characterized in that: The primers include an upstream primer sus-LYZ-F: 5'-ACTAGGCGTTAAAGCATGGGTG-3' and a downstream primer sus-LYZ-R: 5'-AGAGACAAGGTGAGCTGAAGAA-3'.

5. Use of the primer according to claim 4 in preparing a product for detecting or diagnosing infectious gastroenteritis.

6. A kit for detecting porcine transmissible gastroenteritis virus, characterized in that: The kit comprises the primers according to claim 4.

7. Use of the kit as claimed in claim 6 in the preparation of a product for detecting or diagnosing porcine transmissible gastroenteritis.

8. A method for quantitatively detecting the expression of LYZ lysozyme mRNA in pig gastrointestinal cells, characterized in that: The steps include: Step 1, primer design and synthesis: design and synthesize primers and internal reference primers for amplifying LYZ lysozyme; Step 2, extracting total RNA from the pig ileum epithelial cells to be tested and performing reverse transcription to obtain cDNA; Step 3, real-time fluorescence quantitative PCR: using the primers described in step (1) respectively, and using the cDNA of the gastrointestinal cells of the pig to be tested as a template to perform fluorescence quantitative PCR; Step 4: Quantitatively detect the expression level of LYZ lysozyme mRNA in pig gastrointestinal cells: Statistically analyze the Ct value of the real-time fluorescence quantitative PCR in step (3) to determine the expression level of LYZ lysozyme mRNA in pig gastrointestinal cells.

9. The method for quantitatively detecting the expression of LYZ lysozyme mRNA in pig gastrointestinal cells according to claim 8, characterized in that: The primers used to amplify LYZ lysozyme include upstream primer sus-LYZ-F: 5'-ACTAGGCGTTAAAGCATGGGTG-3' and downstream primer sus-LYZ-R: 5'-AGAGACAAGGTGAGCTGAAGAA-3'; the internal reference primers include upstream primer βactin-sus-F: 5'-CTGGCACCACACCTTCTACAA-3' and downstream primer βactin-sus-R: 5'-GTGTTGAAGGTCTCGAACATGAT-3'.

10. The method for quantitatively detecting the expression of LYZ lysozyme mRNA in pig gastrointestinal cells according to claim 8, characterized in that: In step 4, when the relative expression level of porcine gastrointestinal cell LYZ lysozyme mRNA in the tested cells is lower than 1.41, it can be used to indicate that the cells are not infected by TGEV, otherwise, they are infected by TGEV.

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