A detection primer and kit for Vibrio parahaemolyticus and its application

Abstract

The present invention discloses a detection primer, a kit and its application for Vibrio parahaemolyticus, comprising an upstream primer F and a downstream primer R; the upstream primer R is selected from one or more of SEQ ID NO.10, SEQ ID NO.26 and SEQ ID NO.40, and the downstream primer R is selected from one or more of SEQ ID NO.11, SEQ ID NO.27 and SEQ ID NO.41. The primer set can be used in the detection of Vibrio parahaemolyticus and the preparation of Vibrio parahaemolyticus detection preparations and kits for non-diagnostic purposes. The present application establishes a standardized VpTPD pathogen detection method and develops a rapid, convenient, highly sensitive, highly specific, economical and practical detection primer and kit that can be widely promoted for pathogen detection, thereby solving the effective prevention and efficient monitoring of Vibrio parahaemolyticus.

Description

Technical Field

[0001] The present invention relates to the technical field of Vibrio detection, and in particular to a detection primer and a kit for Vibrio parahaemolyticus and applications thereof. Background Art

[0002] Transparent postlarva disease (TPD), also known as highly lethal vibriosis, is a disease that occurred in the coastal areas of my country in the spring of 2020. Penaeus vannamei A new disease has emerged in numerous shrimp hatcheries. Starting in March, the disease rapidly spread from southern shrimp hatcheries to northward, resulting in a sharp decline in the spring shrimp seed supply and posing a significant threat to my country's shrimp aquaculture industry. Shrimp seedlings infected with TPD exhibit decreased vitality and a dark, turbid body coloration, along with characteristic pathological changes such as hyaline lesions (blurred outlines and a pale yellow hue) in the hepatopancreatic tissue structure and empty stomachs in the jejunum. The disease progresses to acute death. Epidemiological surveys show that TPD primarily infects shrimp larvae 4 to 12 days of age (PL4-PL12), with a morbidity rate of up to 90% to 100% in severe cases. TPD is highly contagious through contact.

[0003] The causative agent of TPD is Vibrio parahaemolyticus ( Vibrio parahaemolyticus , VpTPD). Previously, the most common disease caused by Vibrio parahaemolyticus was acute hepatopancreatic necrosis disease (AHPND). Although TPD and AHPND are both caused by Vibrio parahaemolyticus and have similar clinical signs, their pathogenic factors are different. TPD does not carry the virulence factors (pirAvp and pirBvp) of AHPND. The two novel Vibrio highly virulence proteins VHVP-1 and VHVP-2 of VpTPD are the key virulence factors that cause TPD. They are composed of two tandem virulence genes located on an 187,791 bp plasmid. vhvp-1 and vhvp-2 Encoded, and vhvp-2 The gene is the key pathogenic factor that causes the death of whiteleg shrimp. Further analysis found that there is also a potential virulence gene on the 187791 bp plasmid. vhvp-3 Encoded VHVP-3 virulence protein.

[0004] However, effective prevention and control measures for vitreous seedling disease remain elusive, leaving farms to reduce production to mitigate losses. Due to the short onset of vitreous seedling disease in shrimp, clinical diagnosis is difficult to quickly and accurately detect the pathogen, and histopathological diagnosis is complex, hindering large-scale use. Real-time PCR (RT-PCR), a highly sensitive and specific temperature-dependent amplification method, has been widely used for molecular diagnosis of shrimp pathogens. This method uses fluorescently labeled probes or SYBR Green I fluorescent dye to double-strand target gene DNA, rapidly quantifying the sample by measuring fluorescence levels.

[0005] Patent document CN116024361B provides primers and a kit for detecting pathogenic Vibrio in shrimp seedlings. Real-time PCR is performed using TaqMan probes to detect Vibrio in shrimp seedlings. This method requires the design of specific probes. Compared with the SYBR Green I fluorescent dye method, the TaqMan probe method is complex to operate and expensive, making it unsuitable for large-scale promotion.

[0006] Therefore, it is necessary to establish a standardized VpTPD pathogen detection method and further develop fast, convenient, highly sensitive, highly specific, economical and practical detection primers and kits that can be widely promoted for pathogen detection, so as to solve the effective prevention and efficient monitoring of Vibrio parahaemolyticus in whiteleg shrimp farming and production. Summary of the Invention

[0007] The purpose of the present invention is to establish a standardized VpTPD pathogen detection method and further develop a rapid, convenient, highly sensitive, highly specific, economical and practical detection primer and kit that can be widely promoted for pathogen detection, thereby solving the effective prevention and efficient monitoring of Vibrio parahaemolyticus in the production of white shrimp farming. In order to solve this technical problem:

[0008] The first aspect of the present invention provides a primer set, which can be used in the detection of Vibrio parahaemolyticus, the primer set comprising an upstream primer F and a downstream primer R; the upstream primer F is selected from one or more of SEQ ID NO.10, SEQ ID NO.26 and SEQ ID NO.40, and the downstream primer R is selected from one or more of SEQ ID NO.11, SEQ ID NO.27 and SEQ ID NO.41.

[0009] SEQ ID NO.10 is vhvp-1 -F1:5'-CATAGATTCACCACCGAACCA-3';

[0010] SEQ ID NO.11 sequence is vhvp-1 -R1:5'GTTGAAGTATCCCACCGCA-3';

[0011] SEQ ID NO.26 is vhvp-2 -F3:5'GCTCTGGCACCACCGACCTAAT-3';

[0012] SEQ ID NO.27 is vhvp-2 -R3: 5'CCTTCAGGCAACGATACACGGAATG-3';

[0013] SEQ ID NO.40 is vhvp-3 -F4:5'GGCAACGAACAATAACTATACCACC-3';

[0014] SEQ ID NO.41 sequence is vhvp-3 -R4: 5'TGACCGCCTACAGTAAAGAACC-3'.

[0015] Further, vhvp-1 -F1 and vhvp-1 -R1 sequence for targeted detection vhvp-1 Gene; vhvp-2 -F3 and vhvp-2 -R3 sequence for targeted detection vhvp-2 Gene; vhvp-3 -F4 and vhvp-3 -R4 sequence for targeted detection vhvp- 3 Gene.

[0016] Further, vhvp-1 -The sequence of F1 is shown in SEQ ID NO.10, vhvp-1 - The sequence of R1 is shown in SEQ ID NO.11; vhvp-2 -The sequence of F3 is shown in SEQ ID NO.26, vhvp-2 - The sequence of R3 is shown in SEQ ID NO. 27; vhvp-3 -The sequence of F4 is shown in SEQ ID NO.40, vhvp-3 The sequence of -R4 is shown in SEQ ID NO.41.

[0017] Another aspect of the present invention provides a use of a primer set in preparing a Vibrio parahaemolyticus detection preparation for non-diagnostic purposes.

[0018] Another aspect of the present invention provides a detection kit comprising the above primer set.

[0019] Furthermore, the detection kit also includes a fluorescent dye, a negative control and a positive control; the negative control includes TE buffer; and the positive control includes Vibrio parahaemolyticus positive nucleic acid.

[0020] Another aspect of the present invention provides an application of a detection kit in detecting Vibrio parahaemolyticus for non-diagnostic and therapeutic purposes.

[0021] Another aspect of the present invention provides a method for detecting Vibrio parahaemolyticus for non-diagnostic and therapeutic purposes using a primer set, comprising the following steps: Step A: collecting a sample to be tested and / or extracting DNA from the sample to be tested; Step B: using the sample to be tested or the DNA of the sample to be tested as a template, using the primer set to perform RT-PCR detection on the sample to be tested or the DNA of the sample to be tested; Step C: determining whether Vibrio parahaemolyticus is present based on a threshold cycle number of the RT-PCR; when the threshold cycle number is greater than 0, Vibrio parahaemolyticus is present, and the detection rate of Vibrio parahaemolyticus is 100%.

[0022] Furthermore, the sample to be tested in step A includes a recombinant plasmid, bacterial liquid, aquaculture water or feed.

[0023] Furthermore, the reaction system for RT-PCR detection in step B is as follows: 1 μL of the sample to be tested or the DNA of the sample to be tested, 0.4 μL of both the upstream primer F and the downstream primer R in the primer set, the concentrations of the upstream primer F and the downstream primer R being 10 μM, 10 μL of SYBR qPCR fast premix, and DEPC water to make up the RT-PCR reaction system to 20 μL; the reaction procedure for RT-PCR detection is as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, annealing at 55°C~60°C for 30 s, and 40 cycles from denaturation to annealing.

[0024] The three SYBR Green I RT-PCR detection methods developed in this application not only provide high sensitivity for laboratories Vp TPD detection tool. Through precise targeting Vp A novel virulence gene of TPD ( vhvp-1 、 vhvp-2 、 vhvp-3 ), three sets of primer pairs with high specificity and high sensitivity were screened out ( vhvp-1 -F1 / vhvp-1 -R1 (SEQ ID NO.10~11), vhvp-2 -F3 / vhvp-2 -R3 (SEQ ID NO. 26~27), vhvp-3 -F4 / vhvp-3-R4 (SEQ ID NO. 40-41)), successfully constructed three efficient, economical and stable real-time fluorescence quantitative detection methods. The high sensitivity and anti-interference ability of the three sets of primers provide key technical support for the detection of Vibrio parahaemolyticus. By culturing tissue samples of white shrimp, it is possible to quickly detect whether the tissue culture fluid of aquaculture animals such as white shrimp is infected. Vp TPD, whether the aquaculture water contains Vp TPD, or whether the feed and live bait are Vp TPD pollution, etc., thus solving the effective prevention and efficient monitoring of diseases in the production of white shrimp, and achieving Vp This method lays the foundation for rapid TPD detection. In the future, combined with portable RT-PCR equipment and automated nucleic acid extraction technology, this method is expected to enable on-site instant detection, facilitating early warning and precise disease prevention and control, and promoting the sustainable development of the shrimp farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above content and the following specific embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be noted that the accompanying drawings are only examples of the technical solutions claimed.

[0026] Figure 1 For Example 1 vhvp-1 、 vhvp-2 and vhvp-3 Agarose gel electrophoresis of gene excision and recovery (M is marker; A is vhvp-1 Gene, B vhvp-2 Gene, C is vhvp-3 Gene);

[0027] Figure 2 The agarose gel electrophoresis diagram of the second amplification products of vHVP-1, vHVP-2 and vHVP-3 in Example 1 (M is a marker; A is the second amplification product of vHVP-1, B is the second amplification product of vHVP-2, and C is the second amplification product of vHVP-3);

[0028] Figure 3 The agarose gel electrophoresis diagram of the third amplification products of vHVP-1, vHVP-2 and vHVP-3 in Example 2 (M is a marker; 1 to 6 in A are the electrophoresis results of the third amplification product of vHVP-1 under the primer sequences of SEQ ID NOs. 10 to 21; 1 to 6 in B are the electrophoresis results of the third amplification product of vHVP-2 under the primer sequences of SEQ ID NOs. 22 to 33; 1 to 6 in C are the electrophoresis results of the third amplification product of vHVP-3 under the primer sequences of SEQ ID NOs. 34 to 45);

[0029] Figure 4 The SYBR Green I RT-PCR primer screening amplification curve in Example 2 (where A, B, and C correspond to vhvp-1 , vhvp-2 , vhvp-3 Gene primer screening amplification curve, the horizontal axis is the cycle number, and the vertical axis is the fluorescence signal intensity);

[0030] Figure 5 The melting curve diagram of SYBR Green I RT-PCR primer screening in Example 2 (where a, b, and c are respectively Figure 4 Melting curves of primer screening of vhvp-1, vhvp-2, and vhvp-3 genes corresponding to A, B, and C in the figure. The horizontal axis is temperature in °C, and the vertical axis is the negative derivative of the fluorescence signal with temperature, that is, the rate of decrease of fluorescence intensity with temperature);

[0031] Figure 6 The standard curve of the recombinant plasmid in Test Example 1 (wherein the horizontal axis is the logarithm of the starting template copy number, the vertical axis is the threshold cycle number; A is the logarithm of the vhvp-1 recombinant plasmid at vhvp-1 -F1 / vhvp-1 -R1 primer under the standard curve, B is the vhvp-2 recombinant plasmid in vhvp-2 -F3 / vhvp-2 -R3 primers under the standard curve, C is the vhvp-3 recombinant plasmid vhvp- 3- F4 / vhvp-3 -R4 primer standard curve);

[0032] Figure 7 The three primers in test example 1 detected vhvp-1, vhvp-2 and vhvp-3 Gene sensitivity amplification curve (where A, B and C are the target vhvp-1 genetic vhvp-1 -F1 / vhvp-1 -R1 primer, targeting vhvp-2 genetic vhvp-2 -F3 / vhvp-2 -R3 primer and targeting vhvp-3 genetic vhvp-3- F4 / vhvp-³ -R4 primer sensitivity amplification curve, the horizontal axis is the cycle number, the vertical axis is the fluorescence signal intensity);

[0033] Figure 8 The three primers in test example 1 detected vhvp-1, vhvp-2 and vhvp-3 Sensitivity melting curve of the gene (where a, b and c are respectively Figure 7In the sensitivity melting curves corresponding to A, B, and C, the horizontal axis is temperature in °C, and the vertical axis is the negative derivative of the fluorescence signal with temperature, that is, the rate of decrease of fluorescence intensity with temperature);

[0034] Figure 9 The three primers in test example 2 detected Vp TPD specificity result graph (where A is the specific amplification curve, the horizontal axis is the cycle number, and the vertical axis is the fluorescence signal intensity; B is the melting curve graph corresponding to A, the horizontal axis is the temperature in °C, and the vertical axis is the negative derivative of the fluorescence signal with temperature, that is, the rate of decrease of fluorescence intensity with temperature);

[0035] Figure 10 The stability results of the VpTPD SYBR Green I RT-PCR detection method in Test Example 3 (where A, B and C are the target vhvp-1 genetic vhvp-1 -F1 / vhvp-1 -R1 primer, targeting vhvp-2 genetic vhvp-2 -F3 / vhvp-2 -R3 primer and targeting vhvp-3 genetic vhvp-3- F4 / vhvp-3 -R4 primer detection cycle number difference analysis results; in A, ns represents no significant difference; in B, ** represents significant difference, *** represents extremely significant difference; in C, ns represents no significant difference, ** represents significant difference; D, E, and F represent the endpoint fluorescence value difference analysis results corresponding to A, B, and C, respectively, the horizontal axis is the concentration of total DNA in the hepatopancreas of white shrimp (ng / μL), and the vertical axis is the threshold cycle number; in D, **** represents extremely significant difference; in E, * represents significant difference, ** represents extremely significant difference; in F, ns represents no significant difference);

[0036] Figure 11 Infections numbered 1 to 50 in test case 4 Vp Statistics of TPD detection cycles of white shrimp hepatopancreas tissue samples;

[0037] Figure 12 In case 4, the number of infections is 51 to 100. Vp Statistics of TPD detection cycles in hepatopancreatic tissue samples of whiteleg shrimp. DETAILED DESCRIPTION

[0038] The detailed features and advantages of the present invention are described in detail below in the specific embodiments, and the content is sufficient to enable any person skilled in the art to understand the technical content of the present invention and implement it accordingly. Based on the description, claims and drawings disclosed in this specification, those skilled in the art can easily understand the relevant purposes and advantages of the present invention.

[0039] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0040] To make the objectives, technical solutions, and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0041] (1) Source of sample materials

[0042] The samples used in this experiment were whiteleg shrimp purchased from Nanbin Market in Yazhou District, Sanya City, Hainan Province, and whiteleg shrimp provided by Hainan Lanyin Aquatic Seed Technology Co., Ltd.

[0043] Sequence and primer synthesis: performed by Sangon Biotech (Shanghai) Co., Ltd., hereinafter referred to as Sangon.

[0044] (2) Sources of reagents and consumables

[0045] Phusion™ high-fidelity DNA polymerase was purchased from Thermo Fisher, Trans1-T1 cloning competent cells and pEASY®-Blunt Zero Cloning Kit were purchased from Quanshijin, ChamQ Universal SYBR qPCR MasterMix, 4S Red Plus nucleic acid dye, DL2000 DNA Marker, DL5000 DNA Marker, and 10× DNA loading buffer were purchased from Novezan, Premix Taq™ enzyme, DL1000 DNA Marker, and DL10000 DNA Marker were purchased from Takara, agarose DNA recovery kit, plasmid miniprep kit, and marine animal tissue genomic DNA extraction kit were purchased from Tiangen, agarose was purchased from Qingke Biotechnology, agar powder and 20× PBS were purchased from Solebo, tryptone and yeast powder were purchased from Oxoid, 2216E liquid medium and 2216E agar were purchased from Haibo Biotechnology, sodium chloride and ampicillin were purchased from Shanghai Shenggong, and white eight-strip quantitative tubes and non-fluorescent quantitative eight-strip tubes were purchased from Biosharp.

[0046] (3) Source of instruments and equipment

[0047] The snowflake ice maker was purchased from Xinzhi, the constant temperature metal bath was purchased from Youning, the centrifuge and pipette were purchased from Eppendorf, the ultrapure water preparation system was purchased from Merk Millipore, the gel imaging system, PCR instrument and qPCR instrument were all purchased from Analytik Jena, the vertical pressure steam sterilizer was purchased from Yamato, the electronic balance was purchased from Ohaus, the constant temperature shaking incubator and the electric constant temperature incubator were all purchased from Zhicheng, the micro-spectrophotometer was purchased from Thermo Fisher, the ultra-low temperature refrigerator, medical refrigerator-freezer, clean bench and liquid nitrogen tank were all purchased from Haier, and the UV gel cutting instrument was purchased from PORABIO.

[0048] (4) Solutions and preparation methods required for the experiment

[0049] LB liquid medium: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl. Autoclave at 121°C for 20 min.

[0050] 2) LB solid medium: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) NaCl, 1.5% (w / v) agar powder, sterilized at 121°C for 20 min.

[0051] 3) Ampicillin (stock solution): Accurately weigh 2.5 g of ampicillin into a 50 mL centrifuge tube. Add 40 mL of sterile water. Mix thoroughly to dissolve, then dilute to 50 mL. Sterilize by filtering through a 0.22 μm filter. Aliquot (1 mL / tube) and store at -20°C.

[0052] 4) Kanamycin (stock solution): Accurately weigh 2.5 g of kanamycin into a 50 mL centrifuge tube. Add 40 mL of sterile water. Mix thoroughly to dissolve, then dilute to 50 mL. Sterilize by filtering through a 0.22 μm filter. Aliquot (1 mL / tube) and store at -20°C.

[0053] 5) 2216E solid medium: 5.24% (w / v) 2216E solid medium, sterilized at 121°C for 20 min.

[0054] 6) 2216E liquid medium: 3.74% (w / v) 2216E liquid medium, sterilized at 121°C for 20 min.

[0055] Example 1

[0056] vhvp-1 、 vhvp-2and vhvp-3 The construction and extraction of gene recombinant plasmids include the following steps:

[0057] S1, vhvp-1 、 vhvp-2 and vhvp-3 The specific steps for constructing gene recombination plasmids are as follows:

[0058] (1) According to the previous research of this laboratory Vp The results of sequencing the TPD strain revealed three pathogenic genes of Penaeus vannamei TPD. vhvp-1 (SEQ ID NO. 1), vhvp-2 (SEQ ID NO. 2) and vhvp-3 (SEQ ID NO.3) nucleotide sequence was synthesized by direct synthesis by Bioengineering vhvp-1 、 vhvp-² and vhvp-3 Synthesize and design gene-specific primers using Primer Premier 6.0. The primer sequences are as follows:

[0059] vhvp-1 -cF: 5'-ATGTCTAATAGATCACCGTCG-3' (SEQ ID NO.4);

[0060] vhvp-1 -cR: 5'-TTAGTCAGTAAGGATGGTGTAGC-3' (SEQ ID NO.5);

[0061] vhvp-2 -cF: 5'-ATGCAAAATATAAATAATCTGAAACTGGAAACCCCTAC-3' (SEQ ID NO. 6);

[0062] vhvp-2 -cR: 5'-TCATGCGGTATCGTTTTC-3' (SEQ ID NO. 7);

[0063] vhvp-3 -cF: 5'-ATGAGTACATCATTATTCAGCAATACCC-3' (SEQ ID NO.8);

[0064] vhvp-3 -cR: 5'-CTATGAGCGGGGTAACATTCG-3' (SEQ ID NO. 9).

[0065] (2) Through the first round of PCR reaction vhvp-1 、 vhvp-2 and vhvp-3Gene amplification, wherein the first round of PCR reaction system is: synthetic vhvp-1 、 vhvp-2 or vhvp-3 1 μL of gene template DNA corresponding to the gene vhvp-1 、 vhvp-2 or vhvp-3 1 μL each of upstream primer F and downstream primer R (10 μM), 5 μL HF buffer, 0.5 μL dNTP, 0.2 μL Phanta Max Master Mix (high-fidelity DNA polymerase), and DEPC water were added to make the total volume of the system 25 μL. The mixture was vortexed and centrifuged briefly before the first round of PCR reaction.

[0066] The first round of PCR reaction program was: pre-denaturation at 98℃ for 30 s, denaturation at 98℃ for 10 s, annealing at 55℃ for 30 s, and extension at 72℃ for 1.5 to 3.0 min (wherein, vhvp-1 and vhvp-2 gene, 72°C for 1.5 min; vhvp-3 The gene was amplified at 72°C for 3.0 min), and a total of 30 cycles of denaturation to extension were performed, followed by full extension at 72°C for 10 min and storage at 4°C to obtain the first amplification product of vHVP-1, vHVP-2 or vHVP-3.

[0067] (3) After the first round of PCR reaction, the first amplified products of vhvp-1, vhvp-2 or vhvp-3 were separated by 1.5% agarose gel electrophoresis, and the bands of about 8000 bp, 4500 bp and 3000 bp were cut out respectively using a UV gel cutting instrument, and then recovered using an agarose DNA recovery kit to obtain the target gene. vhvp-1 、 vhvp-2 and vhvp-3 Gene, specific electrophoresis results see Figure 1 .

[0068] (4) Follow the instructions of the pEASY®-Blunt Zero Cloning Kit to clone the target gene. vhvp-1 、 vhvp-2 and vhvp-3 The enzyme ligation reaction was performed with the Blunt Zero cloning vector respectively. The enzyme ligation reaction system was: 1 μL of target gene, 1 μL of Blunt Zero cloning vector, and 3 μL of DPEC water. After gently pipetting to mix, the PCR instrument was controlled at 37°C for 30 minutes. After the reaction was completed, the first ligation products of vhvp-1, vhvp-2, and vhvp-3 were obtained and placed on ice for later use.

[0069] (5) Trans-T1 competent cells (pEASY ® Thaw the competent cells included with the 5-Blunt Zero Cloning Kit on ice. Add the primary ligation products of vhvp-1, vhvp-2, and vhvp-3 to 50 μL of Trans-T1 competent cells, mix thoroughly by flicking, and quickly place on ice for 30 minutes. Heat shock in a 42°C metal bath for 30 seconds, then immediately place on ice for 2 minutes. Under sterile conditions, add the transformed bacterial solution to 250 μL of resistance-free LB liquid medium and incubate on a shaker at 37°C, 220 rpm, for 1 hour. This is the primary transformation solution for vhvp-1, vhvp-2, and vhvp-3. Subsequently, 200 μL of the first transformed bacterial liquid of vhvp-1, vhvp-2 and vhvp-3 were spread on ampicillin (Amp, Amipicilline)-resistant LB solid culture medium, and cultured upside down in an incubator at 37°C overnight to form the first plates of vhvp-1, vhvp-2 and vhvp-3.

[0070] (6) Use a sterile pipette tip to pick up 8 monoclonal plaques from the first plate of vhvp-1, vhvp-2, and vhvp-3 and place them in 500 μL LB liquid medium (containing ampicillin antibiotics at a final concentration of 100 μg / mL) and culture at 200 rpm and 37°C for 6 hours to obtain vhvp-1, vhvp-2, and vhvp-3 monoclonal bacterial liquids. Using the bacterial liquid as a template, use the universal primers M13F and M13R or M13F and the R primer corresponding to the gene clone (i.e., the downstream primers for PCR amplification of the vhvp-1, vhvp-2, and vhvp-3 genes) to perform bacterial liquid PCR identification, which is the second round of PCR reaction.

[0071] The second-round PCR reaction system was as follows: 1 μL of vhvp-1, vhvp-2, or vhvp-3 monoclonal bacterial solution, 1 μL of M13F (10 μM), 1 μL of M13R or R primer (10 μM), 12.5 μL of Premix Taq™, and DEPC water to adjust the reaction system to 25 μL. The second-round PCR reaction program was as follows: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 3.0 min, for a total of 30 cycles of denaturation and extension, followed by full extension at 72°C for 10 min and storage at 4°C to obtain the second amplification products of vhvp-1, vhvp-2, and vhvp-3.

[0072] The results were verified by 1.5% agarose gel electrophoresis. Figure 2As shown, the second amplification products of vhvp-1, vhvp-2 and vhvp-3 have brighter bands at around 8000 bp, 4500 bp and 3000 bp, respectively.

[0073] S2, vhvp-1 、 vhvp-2 and vhvp-3 The specific steps for extracting gene recombinant plasmid are as follows:

[0074] The vhvp-1, vhvp-2, and vhvp-3 monoclonal bacterial suspensions in S1 were used to extract plasmids using a plasmid mini-extraction kit. The plasmid extraction steps were performed according to the instructions. The following reagents were all provided with the plasmid mini-extraction kit.

[0075] 1. Collect the cells: Take 1-4 mL of overnight cultured bacterial solution, centrifuge at 12,000 rpm for 1 min, discard the supernatant, and collect the bacterial pellet. If there is too much bacterial solution, centrifuge it several times to merge the pellets; 2. Resuspend the cells: Add 150 μL of P1 solution premixed with RNase A and TIANRed, and vortex or pipette to thoroughly suspend the cells; 3. Lysis: Add 150 μL of solution P2 and gently invert 6 to 8 times until the solution turns clear purple (TIANRed indicates that lysis is complete); 4. Neutralization and precipitation: Immediately add 350 μL of solution P5, quickly invert and mix 12 to 20 times until the solution turns clear yellow (indicating complete neutralization), centrifuge at 12,000 rpm for 2 min, and take the supernatant; 5. Adsorption column treatment: Transfer the supernatant to adsorption column CP3, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid; 6. Wash and dry: Add 300 μL of rinse solution PWT, centrifuge for 30 s, and repeat once; centrifuge for 1 minute 7. Elution: Move the adsorption column to a new centrifuge tube, add 50-100 μL of buffer TB, and centrifuge for 30 seconds to collect the plasmid DNA.

[0076] After plasmid extraction, the plasmid concentration was determined using a NanoDrop Lite ultramicro spectrophotometer. The concentration of the vhvp-1 recombinant plasmid was determined to be 112.3 ng / μL, the concentration of the vhvp-2 recombinant plasmid was determined to be 402.2 ng / μL, and the concentration of the vhvp-3 recombinant plasmid was determined to be 132 ng / μL.

[0077] The copy number of recombinant plasmid per microliter was calculated according to the following formula: copy number (copies / μL) = [M (ng / μL) × 6.02 × 10 23 (copies / mol) × 10 -9)] / [N (bp) × 660 (g / mol / bp)], where M is the concentration of the recombinant plasmid, N is the base pair copy number of the prepared recombinant plasmid, and the average weight of one base pair is 660 Da.

[0078] The copy number of the recombinant plasmid per microliter was calculated as follows: 8.85× 10 9 The copy number of vhvp-2 recombinant plasmid is 4.46 × 10 10 The copy number of vhvp-3 recombinant plasmid is 1.76 × 10 10 The number of copies.

[0079] Then, an appropriate amount of plasmid was sent to Bioengineering for the first sequencing. The first sequencing results were compared and analyzed, and the similarity was 100%. Therefore, the recombinant plasmid vectors were successfully constructed, namely the vhvp-1 recombinant plasmid, vhvp-2 recombinant plasmid and vhvp-3 recombinant plasmid, which were labeled and stored in a -20℃ refrigerator.

[0080] Example 2

[0081] The primer screening test includes the following steps:

[0082] S1. Use PCR reaction to perform primer screening. The specific steps are as follows:

[0083] (1) respectively vhvp-1 (SEQ ID NO. 1) , vhvp-2 (SEQ ID NO. 2) and vhvp-3 (SEQ ID NO.3) gene sequence was used as the target gene. Gene-specific primers were designed using Primer Premier 6.0. Six pairs of primers were set for each gene (i.e., six upstream primers (F stands for Forward, indicating upstream primers) and six downstream primers (R stands for Reverse, indicating downstream primers) were set for each gene. The upstream and downstream primers of the unified primer pair were paired for use. For example, vhvp -1-F1 is vhvp -1 upstream primer 1, vhvp -1-R1 is vhvp -1 downstream primer 1, the two are used in pairs), a total of 18 pairs of primer sequences are as follows:

[0084] vhvp-1 -F1: 5'-CATAGATTCACCACCGAACCA-3' (SEQ ID NO. 10);

[0085] ​<h2 style=";text-align:left;direction:ltr">-R1:5'-GTTGAAGTATCCCACCGCA-3'(SEQ ID NO.11);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0086] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F2:5'-GCAAATCACAACAACCCGTC-3'(SEQ ID NO.12);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0087] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R2:5'-TCCATTGGAGTAAAGAAAGCCTCC-3'(SEQ ID NO.13);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0088] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F3:5'-CATTACAGCCGCACACCT-3'(SEQ ID NO.14);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0089] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R3:5'-ACGAACCGAACTGCCAC-3'(SEQ ID NO.15);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0090] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F4:5'-TCATTACAGCCGCACACCTT-3'(SEQ ID NO.16);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0091] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R4:5'-CCCTAATAGTGCGGGTAGCG-3'(SEQ ID NO.17);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0092] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F5:5'-GTTTTTAACGGTGGGGACGC-3'(SEQ ID NO.18);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0093] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R5:5'-GTGCCAAGTCGTCCGTATCT-3'(SEQ ID NO.19);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0094] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F6:5'-GCGTCGATGATGCAGGACTA-3'(SEQ ID NO.20);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0095] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R6:5'-CGGTACAGTGTGCTGAGGTT-3'(SEQ ID NO.21);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0096] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F1:5'-GCATTCCGTGTATCGTTGCC-3'(SEQ ID NO.22);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0097] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R1:5'-AATGGCACACCCAATGTTGC-3'(SEQ ID NO.23);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0098] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F2:5'-TCCGTGTATCGTTGCCTGAAGG-3'(SEQ ID NO.24);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0099] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R2:5'-GTTGCACTGTGAGATGAGGAATGG-3'(SEQ ID NO.25);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0100] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F3:5'-GCTCTGGCACCACCGACCTAAT-3'(SEQ ID NO.26);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0101] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R3:5'-CCTTCAGGCAACGATACACGGAATG-3'(SEQ ID NO.27);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0102] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F4:5'-GCCTGAAGGTACTCAGTATGATCGTA-3'(SEQ ID NO.28);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0103] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R4:5'-ATGTTGCACTGTGAGATGAGGAATG-3'(SEQ ID NO.29);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0104] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F5:5'-CTCATCTCCTCTGCCATCCA-3'(SEQ ID NO.30);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0105] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R5:5'-ACGACCCATTCAAGATACCCA-3'(SEQ ID NO.31);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0106] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F6:5'-ACGACCCATTCAAGATACCCA-3'(SEQ ID NO.32);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0107] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R6:5'-TCACGACCCATTCAAGATACC-3'(SEQ ID NO.33);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0108] <h2 style=";text-align:left;direction:ltr"> ​<h2 style=";text-align:left;direction:ltr">-F1:5'-GGGCAACCCCTTATCTGGAC-3'(SEQ ID NO.34);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0109] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R1:5'-CTCGCTGCCTTTTGTTTGCT-3'(SEQ ID NO.35);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0110] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F2:5'-TGCAATTAGGGCAACCCCTT-3'(SEQ ID NO.36);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0111] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R2:5'-TGCCTGCATCATAGCGGTAG-3'(SEQ ID NO.37);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0112] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F3:5'-CAACCCCTTATCTGGACGCC-3'(SEQ ID NO.38);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0113] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R3:5'-CGCTGCCTTTTGTTTGCTGA-3'(SEQ ID NO.39);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0114] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -F4:5'-GGCAACGAACAATAACTATACCACC-3'(SEQ ID NO.40);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0115] <h2 style=";text-align:left;direction:ltr"> ​ <h2 style=";text-align:left;direction:ltr"> -R4:5'-TGACCGCCTACAGTAAAGAACC-3'(SEQ ID NO.41);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0116] <h2 style=";text-align:left;direction:ltr"> vhvp-3 <h2 style=";text-align:left;direction:ltr"> -F5:5'-CAACGAACAATAACTATACCACCAC-3'(SEQ ID NO.42);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0117] <h2 style=";text-align:left;direction:ltr"> vhvp-3 <h2 style=";text-align:left;direction:ltr"> -R5:5'-CCGCCTACAGTAAAGAACCC-3'(SEQ ID NO.43);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0118] <h2 style=";text-align:left;direction:ltr"> vhvp-3 <h2 style=";text-align:left;direction:ltr"> -F6:5'-TCTTCATTCACGATAGCCTTACAG-3'(SEQ ID NO.44);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0119] <h2 style=";text-align:left;direction:ltr"> vhvp-3 <h2 style=";text-align:left;direction:ltr"> -R6:5'-CCACTCTTCTATAAAGTTGCCCA-3'(SEQ ID NO.45)。<h2 style=";text-align:left;direction:ltr">

[0120] (2) The above 18 pairs of primers were used to perform conventional PCR reaction on vhvp-1 plasmid, vhvp-2 plasmid and vhvp-3 plasmid, i.e. the third round of PCR reaction. The third round of PCR reaction system was: vhvp-1 recombinant plasmid, vhvp-2 recombinant plasmid or vhvp-3 recombinant plasmid (concentration was 10 per microliter) 5 The reaction mixture was diluted with 1 μL of 5% paraformaldehyde (100 copies), 1 μL of upstream primer F (10 μM), 1 μL of downstream primer (10 μM), 12.5 μL of Premix Taq™, and DEPC water to 25 μL. The second-round PCR reaction procedure was as follows: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 10 s, annealing at 55°C-60°C for 30 s, and extension at 72°C for 15 s, for a total of 30 cycles of denaturation and extension, followed by full extension at 72°C for 10 min. The cells were stored at 4°C to obtain the tertiary amplification products of vhvp-1, vhvp-2, and vhvp-3.

[0121] The results were verified by 1.5% agarose gel electrophoresis. Figure 3 As shown, a total of 12 pairs of primers were screened. Among them, A is the agarose gel electrophoresis result of the third amplification product of vhvp-1 under the primer sequence of SEQ ID NO.10~21. Four groups of primer pairs with no non-specific amplification and high amplification efficiency were preliminarily screened, targeting vhvp-1 Initial gene screening vhvp- 1 -F1 / vhvp-1 -R1, vhvp-1 -F4 / vhvp-1 -R4, vhvp-1 -F5 / vhv p-1-R5, vhvp-1 -F6 / vhvp - 1 -R6; B is the agarose gel electrophoresis result of the third amplification product of vhvp-2 under the primer sequence of SEQ ID NO.22~33, targeting vhvp- 2 Initial gene screening vhvp-2 -F1 / vhvp-2 -R1, vhvp-2 -F3 / vhvp-2 -R3, vhvp-2 -F4 / vhvp-2- R4, vhvp-2 -F5 / vhvp-2 -R5; C is vhvp-3 The agarose gel electrophoresis results of the third amplification product under the primer sequence of SEQ ID NO.34~45, targeting vhvp-3 Initial gene screening vhvp-3- F1 / vhvp-3-R1, vhvp-3 -F2 / vhvp-3 -R2, vhvp-3 -F3 / vhvp-3- R3, vhvp-3- F4 / vhvp-3 -R4.

[0122] (3) RT-PCR was further performed using SYBR Green I to screen the 12 pairs of primers selected above. The reaction system for the first round of RT-PCR was: vhvp-1 recombinant plasmid, vhvp-2 recombinant plasmid or vhvp-3 recombinant plasmid (concentration of 10 per microliter) 5 The reaction mixture was diluted with 1 μL of primer (100 copies), 0.4 μL of upstream primer F (10 μM), 0.4 μL of downstream primer (10 μM), 10 μL of SYBR qPCR Master Mix, and DEPC water to a volume of 20 μL. The first-round RT-PCR reaction procedure was as follows: initial denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, and annealing at 55°C–60°C for 30 s, for a total of 40 cycles. The first amplification curves for vhvp-1, vhvp-2, and vhvp-3, as well as the first melting curves for vhvp-1, vhvp-2, and vhvp-3, were obtained, respectively.

[0123] The results are as follows Figure 4 and Figure 5 As shown, three sets of primers ( vhvp-1 -F1 / vhvp-1 -R1, vhvp-2 -F3 / vhvp-2 -R3 and vhvp-3- F4 / vhvp-3 -R4) had the smallest detection Ct value and the fluorescence intensity was higher than that of other primers in the same group (for example, vhvp-1 -F1 / vhvp-1 -R1 primer's minimum detection Ct value and fluorescence intensity were higher than vhvp-1 -F4 / vhvp-1 -R4, vhvp-1 -F5 / vhv p-1-R5 and vhvp-1 -F6 / vhvp - 1 -R6 three sets of primers, these three sets of primers are vhvp-1 -F1 / vhvp-1 -R1 and other primers in the same group), the melting curve showed a single peak between 75 and 85 ° C and the melting temperature was higher than that of other primers in the same group, showing the best amplification efficiency. vhvp-1 -F1 / vhvp-1 -R1, vhvp-2 -F3 / vhvp-2 -R3 and vhvp-3- F4 / vhvp-3 -R4 These three sets of primers can be used for the preliminary detection of VpTPD infection.

[0124] Test Example 1 Sensitivity Test

[0125] (1) To verify the sensitivity of SYBR Green I RT-PCR detection method (i.e., detection method using SYBR Green I for RT-PCR reaction) for detecting TPD, 10-fold serial dilutions of standard plasmids (i.e., the copy numbers of vhvp-1 recombinant plasmid, vhvp-2 recombinant plasmid, and vhvp-3 recombinant plasmid were 10, ... 5 -10 0 The second round of RT-PCR reaction was performed using the template of 10-fold serial dilution.

[0126] The reaction system of the second round of RT-PCR was vhvp-1 recombinant plasmid, vhvp-2 recombinant plasmid or vhvp-3 recombinant plasmid (concentration of 10 per microliter 5 1 μL of primer F (10 μM), 0.4 μL of primer R (10 μM), 10 μL of SYBR qPCR Master Mix, and DEPC water were used to adjust the reaction volume to 20 μL. The reaction procedure for the second round of RT-PCR was as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, and annealing at 55°C-60°C for 30 s (wherein, vhvp-1 -F1 / vhvp-1 -The annealing temperature of R1 is 55℃; vhvp-2 -F3 / vhvp-2 -R3 annealing temperature is 55℃; vhvp-3 -F4 / vhvp-3 -R4 annealing temperature was 56°C), and the denaturation to annealing process was performed for 40 cycles.

[0127] According to the three standard plasmids and their corresponding optimal primers ( vhvp-1 -F1 / vhvp-1 -R1, vhvp-2 -F3 / vhvp- 2 -R3 and vhvp-3- F4 / vhvp-3 -R4) Generate a standard curve and obtain the standard curve equation. The result is as follows Figure 6As shown, the standard curve equation of the vhvp-1 plasmid is Y=-3.39875x+35.518 (R²= 0.9467); the standard curve equation of the vhvp-2 plasmid is Y=-2.976x+34.517 (R²= 0.992); and the standard curve equation of the vhvp-3 plasmid is Y=-3.705x+33.945 (R²=0.9956v), where x represents the logarithm of the copy number and Y represents the threshold cycle number.

[0128] (2) Reuse the vhvp-1 -F1 / vhvp-1 -R1, vhvp-2 -F3 / vhvp-2 -R3 and vhvp-3- F4 / vhvp-3 The three primer sets of -R4 were used to perform the third round of RT-PCR reaction on the vhvp-1 recombinant plasmid, vhvp-2 recombinant plasmid and vhvp-3 recombinant plasmid respectively. The system and procedure of the third round of RT-PCR reaction were the same as those of the second round of RT-PCR reaction in step (1) of test example 1. vhvp-1 -F1 / vhvp-1 -R1, vhvp-2 -F3 / vhvp-2 -R3 and vhvp-3 -F4 / vhvp-3 -R4 sensitivity amplification curve and vhvp-1 -F1 / vhvp-1 -R1, vhvp-2 -F3 / vhvp-2 -R3 and vhvp-3 -F4 / vhvp-3 -R4 sensitivity melting curve.

[0129] The results of test case 1 are as follows Figure 7 and Figure 8 As shown, vhvp-1、vhvp-2 and vhvp-3 The genes can be stably amplified in gradients, with a single melting curve peak, no primer dimers or non-specific peaks, and no negative amplification was observed. vhvp-1 The sensitivity of SYBR Green I RT-PCR detection was 8.85×10 1 The copy number (i.e. the lowest concentration gradient is 8.85×10 1 copies / µL); vhvp-2 The sensitivity of SYBR Green I RT-PCR detection was 8.92×10 0 The copy number (i.e. 8.92×10 0 copies / µL); vhvp-3The sensitivity of SYBR Green I RT-PCR detection was 1.76×10 1 The copy number (i.e. 1.76× 10 1 The comparative analysis showed that the detection method targeting the vhvp-2 gene had the highest sensitivity, which was 8.92×10 0 copies / µL.

[0130] Test Example 2 Specificity Test

[0131] The recombinant plasmids of white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VpAHPND), infectious myonecrosis virus (IMNV), and yellow head virus (YHV, GAV) available in the laboratory were used as templates. SYBR Green I real-time PCR was used to detect the three sets of primers (targeting vhvp-1 、 vhvp-2 and vhvp-3 The fourth round of RT-PCR reaction was performed using vhvp-1, vhvp-2, and vhvp-3 recombinant plasmid standards as positive controls to verify the specificity of the gene.

[0132] The reaction system of the fourth round of RT-PCR is: DNA template (10 5 The reaction mixture was prepared by adding 1 μL of primer F (10 μM), 0.4 μL of upstream primer F, 0.4 μL of downstream primer R (10 μM), 10 μL of SYBR qPCR Master Mix, and DEPC water to a volume of 20 μL. The reaction procedure for the fourth round of RT-PCR was the same as that for the second round of RT-PCR in Test Example 1.

[0133] The results of test case 2 are as follows Figure 9 As shown, the above three primer sets are only Vp Positive products were amplified from TPD, but no specific amplification was observed in the recombinant plasmids infected with WSSV, IHHNV, VpAHPND, IMNV, YHV, and GAV. vhvp-1 genetic vhvp-1 -F1 / vhvp-1 -R1 primer, targeting vhvp-2 genetic vhvp-2 -F3 / vhvp-2 -R3 primer and targeting vhvp-3 genetic vhvp-3- F4 / vhvp-3 The -R4 primer has high specificity and does not cross-react with other common shrimp pathogens.

[0134] Test Example 3 Stability Test

[0135] In order to further evaluate the interference of Penaeus vannamei DNA on the SYBR Green I RT-PCR detection method, the stability test was performed using the total DNA of the hepatopancreas of healthy Penaeus vannamei extracted in Test Example 2.

[0136] In the presence of 0-2000 ng gradient diluted total DNA of the hepatopancreas of whiteleg shrimp (i.e., in the presence of background DNA of shrimp), the vhvp-1 recombinant plasmid, vhvp-2 recombinant plasmid and vhvp-3 recombinant plasmid (concentration of 10 5 The fifth round of RT-PCR reaction was performed using the three sets of primers in Test Example 1.

[0137] Among them, targeted vhvp-1 genetic vhvp-1 -F1 / vhvp-1 -R1 primers were used for the detection of total DNA from the hepatopancreas of healthy white shrimp at a concentration gradient of 0, 200 ng / μL, 400 ng / μL, 600 ng / μL, 800 ng / μL, 1000 ng / μL and 1200 ng / μL; vhvp-2 genetic vhvp-2 -F3 / vhvp-2 -R3 primers were used for the detection of total DNA from the hepatopancreas of healthy white shrimp at a concentration gradient of 0, 400 ng / μL, 800 ng / μL, 1000 ng / μL, 1500 ng / μL and 2000 ng / μL; vhvp-3 genetic vhvp-3- F4 / vhvp-3 -R4 primer was used for detection, and the concentration gradient of total DNA from the hepatopancreas of healthy white shrimp was 0, 400 ng / μL, 600 ng / μL, 800 ng / μL and 1000 ng / μL.

[0138] The fifth round of RT-PCR reaction system is: vhvp-1 recombinant plasmid, vhvp-2 recombinant plasmid or vhvp-3 recombinant plasmid (concentration of 10 5 1 μL of primer (10 copies / μL), 0.4 μL of upstream primer F (10 μM), 0.4 μL of downstream primer R (10 μM), 0-2000 ng of total hepatopancreas DNA from whiteleg shrimp, 10 μL of SYBR qPCR Master Mix, and DEPC water to a total volume of 20 μL. The fifth-round RT-PCR reaction procedure was the same as the second-round RT-PCR reaction procedure in Test Example 1.

[0139] The results of test case 3 are as follows Figure 10 As shown, vhvp-1 Target gene detection method ( vhvp-1 -F1 / vhvp-1 -R1) is not affected when the background DNA content of shrimp is less than 1200 ng ( Figure 9 A and D); vhvp-2 Target gene detection method ( vhvp-2 -F3 / vhvp-2 -R3) is not affected when the background DNA content of shrimp is less than 1500ng ( Figure 9 B and E); vhvp- 3 Target gene detection method ( vhvp-3 -F4 / vhvp-3 -R4) is not affected when the background DNA content of shrimp is less than 1000 ng ( Figure 9 C and F). All three detection methods only interfere when the background concentration of shrimp DNA is very high (greater than 1000 ng), and this interference has a very small impact on sensitivity. This means that in actual testing, even if the sample contains a large amount of shrimp DNA, there is no need to specifically reduce the shrimp "background DNA" content. The SYBR Green I RT-PCR detection method can be used directly to quickly complete pathogen detection, saving time in the purification step and ensuring the reliability of the test results.

[0140] Test Example 4: Whiteleg shrimp sample test

[0141] The three sets of primers in Test Example 1 were used to test the virus infection experiment in this laboratory (infection Vp SYBR Green I RT-PCR was performed on 100 hepatopancreas tissue samples of Penaeus vannamei obtained by TPD.

[0142] The sixth-round RT-PCR reaction system consisted of 1 μL of test sample DNA (100 samples of hepatopancreatic tissue from Litopenaeus vannamei), 0.4 μL of upstream primer F (10 μM), 0.4 μL of downstream primer R (10 μM), 10 μL of SYBR qPCR Master Mix, and DEPC water to a total of 20 μL. The sixth-round RT-PCR reaction procedure was the same as the second-round RT-PCR procedure in Test Example 1.

[0143] The results are as follows Figure 11 and Figure 12 As shown, the above three sets of primers were used to detect by SYBR Green I RT-PCR detection method. vhvp-1 、 vhvp-2 and vhvp-3The detection rates of all genes were 100%, and the cycle numbers (Ct) ranged from 20.26 to 33.47.

[0144] Test Example 5 Bacterial Liquid Test

[0145] Referring to Test Example 4, prepare 10 TPD-infected and 10 healthy whiteleg shrimp. Use a toothpick to pick diseased tissue from each plate and streak it onto solid enrichment medium. After incubating overnight, pick a single colony from the plate and transfer it to liquid enrichment medium. Incubate on a constant-temperature shaker for 4-16 hours, then expand the culture at a 1:100 ratio. The liquid enrichment medium is prepared with 20 g of peptone, 40 g of sodium chloride, 5 mL of 0.01% crystal violet solution, and 1 L of distilled water. Adjust the pH to 9.0, autoclave at 121°C for 15 minutes, and cool before use.

[0146] Using the cultured bacterial solution as a template, or extracting the cultured bacterial solution DNA as a template, the three sets of primers in Test Example 1 were used to detect using the SYBR Green I RT-PCR detection method to obtain the same Figure 10 and Figure 11 Basically the same cycle number result graph, and vhvp-1 、 vhvp-2 and vhvp-3 The detection rate of genes was 100%, indicating that the culture solution contained Vp TPD.

[0147] Test Example 6: Test of feed solution for whiteleg shrimp farming

[0148] Referring to Test Example 4, 100 μL of 0.1 mg / mL TPD-infected whiteleg shrimp feed solution was tested, and the three sets of primers in Test Example 1 were used to detect the SYBR Green I RT-PCR detection method, and the results were the same as those in Test Example 4. Figure 10 and Figure 11 Basically the same cycle number result graph, and vhvp-1 、 vhvp-2 and vhvp-3 The detection rate of the gene was 100%, indicating that the feed solution of whiteleg shrimp culture contained Vp TPD.

[0149] Test Example 7: Water test for whiteleg shrimp aquaculture

[0150] Referring to Test Example 4, 100 μL of 0.1 mg / mL TPD-infected whiteleg shrimp aquaculture water was tested, and the three sets of primers in Test Example 1 were used to detect the SYBR Green I RT-PCR detection method, and the results were the same as those in Test Example 4. Figure 10 and Figure 11Basically the same cycle number result graph, and vhvp-1 、 vhvp-2 and vhvp-3 The detection rate of the gene was 100%, indicating that the water body of the whiteleg shrimp farming contained Vp TPD.

[0151] It can be concluded that the three SYBR Green I RT-PCR detection methods developed in this application not only provide the laboratory with high sensitivity Vp TPD detection tool. Through precise targeting Vp A novel virulence gene of TPD ( vhvp-1 、 vhvp-2 、 vhvp-3 ), three sets of primer pairs with high specificity and high sensitivity were screened out ( vhvp-1 -F1 / vhvp-1 -R1 (SEQ ID NO. 10~11), vhvp-2 -F3 / vhvp-2 -R3 (SEQ ID NO. 26~27), vhvp-3 -F4 / vhvp-3 -R4 (SEQ ID NO. 40-41)), successfully constructed three efficient, economical and stable real-time fluorescence quantitative detection methods (SYBR Green IRT-PCR detection methods). The high sensitivity and anti-interference ability of the three sets of primers provide key technical support for the detection of Vibrio parahaemolyticus. By culturing tissue samples of white shrimp, it is possible to quickly detect whether the tissue culture fluid of aquatic animals such as white shrimp is infected. Vp TPD, whether the aquaculture water contains Vp TPD, or whether the feed and live bait are Vp TPD pollution, etc., thus solving the effective prevention and efficient monitoring of diseases in the production of white shrimp, and achieving Vp This method lays the foundation for rapid TPD detection. In the future, combined with portable RT-PCR equipment and automated nucleic acid extraction technology, this method is expected to enable on-site instant detection, facilitating early warning and precise disease prevention and control, and promoting the sustainable development of the shrimp farming industry.

[0152] The terms and expressions used herein are for descriptive purposes only, and the present invention should not be limited to these terms and expressions. The use of these terms and expressions is not intended to exclude any equivalent features illustrated and described (or portions thereof), and it should be recognized that various modifications are also within the scope of the claims. Other modifications, variations, and substitutions are possible. Accordingly, the claims are intended to cover all such equivalents.

[0153] Similarly, it should be pointed out that although the present invention has been described with reference to the current specific embodiments, ordinary technicians in this technical field should realize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions can be made without departing from the spirit of the present invention. Therefore, as long as the changes and modifications to the above embodiments are within the scope of the essential spirit of the present invention, they will fall within the scope of the claims of the present invention.

Claims

1. Use of a primer set in preparing a SYBR Green I Real Time PCR detection preparation for Vibrio parahaemolyticus, characterized in that: The primer set consists of an upstream primer F and a downstream primer R; The sequence of the upstream primer F is shown in SEQ ID NO.10, and the sequence of the downstream primer R is shown in SEQ ID NO.

11.

2. Use of a primer set in the preparation of a SYBR Green I Real Time PCR detection kit for Vibrio parahaemolyticus, characterized in that: The primer set consists of an upstream primer F and a downstream primer R; The sequence of the upstream primer F is shown in SEQ ID NO.10, and the sequence of the downstream primer R is shown in SEQ ID NO.

11.

3. The use according to claim 2, characterized in that The detection kit also includes a fluorescent dye, a negative control and a positive control; The negative control includes TE buffer; The positive control includes a positive nucleic acid for Vibrio parahaemolyticus.

4. The use according to claim 2, characterized in that The application is for non-diagnostic treatment purposes.

5. A method for detecting Vibrio parahaemolyticus for non-diagnostic and therapeutic purposes using a primer set, characterized in that: The following steps are involved: Step A: Collect the sample to be tested and extract the DNA of the sample to be tested; Step B: Using the DNA of the sample to be tested as a template, the primer set is used to perform SYBR Green I Real Time PCR detection on the DNA of the sample to be tested; the primer set consists of an upstream primer F and a downstream primer R; the sequence of the upstream primer F is shown in SEQ ID NO.10, and the sequence of the downstream primer R is shown in SEQ ID NO.11; Step C: Determine whether the Vibrio parahaemolyticus exists in the DNA of the sample to be tested according to the cycle threshold value of SYBR Green I Real Time PCR detection. vhvp-1 Gene; When the cycle threshold value is greater than 0 and less than 35, the Vibrio parahaemolyticus is present. vhvp-1 Gene.

6. The method according to claim 5, characterized in that The sample to be tested in step A includes a recombinant plasmid, a bacterial liquid, aquaculture water or feed.

7. The method according to claim 5, characterized in that The reaction system for SYBR Green I Real Time PCR detection in step B is as follows: 1 μL of the sample DNA to be tested, 0.4 μL of both the upstream primer F and the downstream primer R in the primer set, with the concentrations of the upstream primer F and the downstream primer R being 10 μM, 10 μL of SYBR qPCR fast premix, and DEPC water to make up the volume of the SYBR Green I Real Time PCR detection reaction system to 20 μL; The reaction procedure for SYBR Green I Real-Time PCR detection was as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, and annealing at 55°C–60°C for 30 s, with a total of 40 cycles from denaturation to annealing.

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