Multiplex PCR (Polymerase Chain Reaction) detection primer combination, kit and method for simultaneously detecting multiple marine pathogenic bacteria

Abstract

The invention relates to the technical field of biology, and discloses a multiplex PCR (Polymerase Chain Reaction) detection primer combination, a kit and a method for simultaneously detecting multiple marine pathogenic bacteria. Specific primer pairs are respectively designed for marine pathogenic bacteria such as vibrio alginolyticus, vibrio vulnificus, photobacterium dama subspecies, vibrio harveyi and vibrio candersonii, a freeze-drying technology is combined, and the amplification efficiency and specificity are improved through primer design and optimization of an amplification system and detection program parameters. In addition, the method has relatively high accuracy, and shows an efficiency advantage, a relatively short detection period and a convenient result judgment characteristic when being used for detecting various marine pathogenic bacteria. Aiming at the field of detection of diseases caused by various marine pathogenic bacteria, the invention provides a reliable detection way, which is of great significance in practical application scenes such as aquaculture disease monitoring, prevention and control, marine organism health assessment and the like, and is helpful for promoting development and perfection of related disease detection technologies.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a multiplex PCR detection primer combination, a kit and a method for simultaneously detecting multiple marine pathogens. Background Art

[0002] Diseases play a key role in the development of the aquaculture industry and are one of the main factors restricting its development. Epidemiological studies in recent years have shown that Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii, Vibrio harveyi and Vibrio cannelli belong to the common pathogenic bacteria in the marine environment. These pathogens have significant characteristics such as strong pathogenicity and wide prevalence, which can cause extremely serious economic losses to the aquaculture industry. In the aquaculture system, once a marine bacterial disease breaks out, due to its rapid spread and difficulty in effective control, it often causes a large number of farmed organisms to become sick or even die, thus affecting the economic benefits and ecological balance of the entire aquaculture industry.

[0003] In the disease prevention and control strategy of the aquaculture industry, given the difficulty of preventing and controlling marine bacterial diseases, the current main approach is to focus on prevention. Among them, long-term routine monitoring of aquaculture fish, shrimp and aquaculture water is particularly important. As a commonly used method for pathogen detection, the PCR detection method has many inconveniences in practical applications. In order to ensure the effectiveness of reagents such as amplification tool enzymes and primers, they are usually stored in a low-temperature environment and need to be configured on site when used. This operating mode not only takes a lot of time in the reagent preparation link, but also places high requirements on the cleanliness of the detection environment. In the actual operation process, if there are mistakes in the operation of personnel or the reagents are exposed for too long, it is very easy to cause the risk of aerosol contamination of the reaction mother liquor, which in turn affects the accuracy and reliability of the test results. In addition, the cold chain transportation and storage of reagents further increase the detection cost, making it difficult to effectively promote and apply this detection method in remote areas or grassroots laboratories. At the same time, most current laboratories use a single primer pair for PCR testing when conducting pathogen detection. This detection method can only detect and analyze a single pathogen, has obvious singleness, and has relatively low detection efficiency, which is difficult to meet the actual needs of the aquaculture industry for rapid and efficient detection of multiple pathogens.

[0004] Therefore, there is an urgent need to develop a portable multi-pathogen rapid detection method, which should be efficient, accurate, convenient and low-cost, and can be widely used in different breeding environments and conditions, providing strong technical support and guarantee for disease prevention and control in the aquaculture industry. Summary of the invention

[0005] In view of this, the present invention proposes a multiplex PCR detection primer combination, a kit and a method for simultaneously detecting multiple marine pathogens.

[0006] The technical solution of the present invention is achieved in this way:

[0007] In the first aspect, the present invention provides a multiplex PCR detection primer combination for simultaneously detecting multiple marine pathogens, comprising the following components: a specific primer pair for Vibrio alginolyticus SEQ ID NO: 1-2; a specific primer pair for Vibrio vulnificus SEQ ID NO: 3-4; a specific primer pair for Photorhabditis elegans subspecies SEQ ID NO: 5-6; a specific primer pair for Vibrio harveyi SEQ ID NO: 7-8; and a specific primer pair for Vibrio cannelligi SEQ ID NO: 9-10.

[0008] In a second aspect, the present invention provides a multiplex PCR detection freeze-dried powder, which can simultaneously detect multiple marine pathogens, and its components include the multiplex PCR detection primer combination, 2×Taq enzyme Mix, 5×trehalose and dH2O; the preparation steps of the freeze-dried powder include the following:

[0009] A1 respectively dissolves SEQ ID NO: 1-2, SEQ ID NO: 3-4, SEQ ID NO: 5-6, SEQ ID NO: 7-8, SEQ ID NO: 9-10 with ddH2O to prepare primer working solutions of Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subsp. mermaidii, Vibrio harveyi, and Vibrio cannelli;

[0010] A2 adds the primer working solution to the PCR reaction master solution to obtain a mixed solution, and adds ddH2O; the PCR reaction master solution includes 2×Taq enzyme Mix and 5×trehalose;

[0011] A3 freeze-drying the mixed solution to obtain freeze-dried powder.

[0012] In a third aspect, the present invention provides a multiplex PCR detection kit for simultaneously detecting multiple marine pathogens, comprising any of the multiplex PCR detection freeze-dried powders described above.

[0013] In a fourth aspect, the present invention provides a multiplex PCR detection method for simultaneously detecting multiple marine pathogens, which is non-disease diagnosis or treatment, and comprises the following steps:

[0014] B1 is configured with the multiplex PCR test lyophilized powder;

[0015] B2: adding freeze-dried powder and ddH2O to the genomic DNA of the sample to be tested, and then adding genomic DNA templates of Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subspecies mermaid, Vibrio harveyi, and Vibrio cannelli, and mixing to obtain a PCR reaction system; at the same time, setting a negative blank control of the sample;

[0016] B3 performs a multiplex PCR amplification reaction. After the reaction is completed, the amplification products are subjected to agarose gel electrophoresis or sequencing detection and analysis, and the type of pathogens contained in the sample to be tested is determined based on the detection results.

[0017] In a fifth aspect, the present invention provides the use of the above-mentioned primer combination, lyophilized powder, kit or detection method for non-disease diagnosis or treatment.

[0018] The beneficial effects of the present invention include at least the following:

[0019] The multiplex PCR detection method for simultaneously detecting multiple marine pathogens provided by the present invention uses five sets of primer combinations to cover multiple types of marine pathogen samples. By rigorously optimizing primer design, amplification system and detection program parameters, the amplification efficiency and specificity are effectively improved. The present invention has a high degree of accuracy and exhibits the characteristics of high efficiency, short detection cycle and convenient result determination in the process of detecting multiple marine pathogens. Its relatively high efficiency is reflected in the simultaneous amplification detection of multiple pathogens within a reasonable time range, which reduces the detection time to a certain extent; the shortening of the detection cycle is conducive to timely acquisition of test results, so as to take timely response measures in related scenarios such as aquaculture; the convenience of result determination makes the interpretation of the test results clearer and reduces the possibility of misjudgment.

[0020] The present invention adopts a test kit prepared by combining multiple primers with freeze-drying technology, which can be stably stored at room temperature, and gets rid of the dependence on cold chain transportation and special storage equipment. This feature significantly reduces the detection cost and breaks through the limitations of traditional detection reagents in transportation and storage. Due to the use of pre-formulated form, the time spent on on-site configuration of reagents and the risk of aerosol contamination that may be caused are effectively avoided. In the field of animal disease detection, especially for diseases caused by various marine pathogens, the test kit provides a practical, stable and reliable detection method. In practical application scenarios, such as aquaculture disease monitoring and prevention, marine biological health assessment, etc., it is of great significance and helps to promote the development and improvement of disease detection technology in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1The SEQ ID NOs: 1-10 provided in the present invention are respectively amplification patterns of single genomic DNA of pathogenic bacteria of Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subspecies mermaid, Vibrio harveyi and Vibrio cannelli; lanes 1-5 are respectively Vibrio cannelli, Vibrio harveyi, Photobacterium mermanii subspecies mermaid, Vibrio vulnificus and Vibrio alginolyticus; lanes 6-10 are corresponding negative controls;

[0023] Figure 2 The SEQ ID NOs: 1-10 provided in the present invention respectively show DNA PCR amplification patterns of five marine pathogens, namely, Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subspecies, Vibrio harveyi and Vibrio cannelligi; specifically, they are as follows:

[0024] (1) Lanes 1-5 are PCR amplifications of DNA from the five marine pathogens mentioned above using SEQ ID NOs: 9-10, and only the DNA of Vibrio cannelligi was amplified; Lane 6 was not spotted and no operation was performed;

[0025] (2) Lanes 7-11 are PCR amplifications of DNA from the five marine pathogens mentioned above using SEQ ID NOs: 7-8, and only the DNA of Vibrio harveyi was amplified;

[0026] (3) Lanes 12-16 are PCR amplifications of DNA from the five marine pathogens mentioned above using SEQ ID NOs: 5-6, and only the DNA of Photobacterium mermaniformis subspecies mermaidii was amplified;

[0027] (4) Lanes 17-21 are PCR amplifications of DNA from the five marine pathogens mentioned above using SEQ ID NOs: 3-4, and only the DNA of Vibrio vulnificus was amplified;

[0028] (5) Lanes 22-26 are PCR amplifications of DNA from the five marine pathogens mentioned above using SEQ ID NO: 1-2, and only the DNA of Vibrio alginolyticus was amplified;

[0029] Figure 3 The results of the sensitivity test of the multiplex PCR detection kit for marine pathogens provided by the present invention are shown in FIG. 1 , wherein the DNA sample is a mixed DNA sample of five marine pathogens; wherein:

[0030] (1) Lane 1 is the PCR amplification pattern of DNA extracted from 20 CFU / mL of five marine pathogens, and no target band was amplified;

[0031] (2) Lanes 2-7 are 2×10 of five marine pathogens 2 CFU / mL, 2×10 3 CFU / mL, 2×10 4 CFU / mL, 2×105 CFU / mL, 2×10 6 CFU / mL and 2×10 7 CFU / mL bacterial quantity extracted DNA PCR amplification pattern, amplification was normal;

[0032] (3) Lane 8 is the negative control;

[0033] Figure 4 The figure shows the effect of different primer concentrations on the amplification of genomic DNA of marine pathogenic bacteria in the present invention; wherein: the primer concentrations of lanes 1-6 are 200nM, 300nM, 400nM, 500nM, 600nM, and 1000nM, respectively; lane 7 is a negative control of a primer concentration of 200nM, and lane 8 is a negative control of a primer concentration of 1000nM;

[0034] Figure 5 The results of amplification of different combinations of marine pathogenic bacteria genomic DNA by the multiple primer combination PCR detection kit of the present invention; wherein:

[0035] (1) Lanes 1-5 are single sample genomic DNA amplifications, which are Vibrio cannelli, Vibrio harveyi, Photorhabdus mermanii subsp. mermaidii, Vibrio vulnificus, and Vibrio alginolyticus, respectively;

[0036] (2) Lanes 6-8 show the simultaneous amplification of genomic DNA from two samples, namely, Vibrio harveyi and Photorhabdus mermanii subsp. mermaidii, Vibrio harveyi and Vibrio vulnificus, and Vibrio vulnificus and Vibrio alginolyticus;

[0037] (3) Lanes 10-12 show the simultaneous amplification of genomic DNA from three samples, namely, Vibrio harveyi, Photorhabdus mermani subsp. mermaidus, and Vibrio vulnificus; Photorhabdus mermani subsp. mermaidus, Vibrio vulnificus, and Vibrio alginolyticus; and Vibrio harveyi, Photorhabdus mermani subsp. mermaidus, and Vibrio alginolyticus;

[0038] (4) Lanes 13-14 show the simultaneous amplification of genomic DNA from four samples, namely, Vibrio cannelli, Vibrio harveyi, Vibrio vulnificus, and Vibrio alginolyticus; Vibrio cannelli, Vibrio harveyi, Photobacterium mermanii subspecies mermaidii, and Vibrio vulnificus;

[0039] (5) Lanes 15-18 are negative controls corresponding to the above combinations;

[0040] Figure 6 It is the result of simultaneous amplification of genomic DNA of five marine pathogens, namely, Vibrio alginolyticus, Vibrio vulnificus, Photorhabdus mermanii subspecies, Vibrio harveyi and Vibrio cannelligi, by the multiple primer combination PCR detection kit of the present invention; wherein: lane 1 is a negative blank, and lane 2 is the effect of simultaneous amplification of genomic DNA of five marine pathogens. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out under conventional conditions or conditions recommended by the manufacturer. The methods used in the following embodiments are all conventional methods unless otherwise specified. The various ways of obtaining biological materials described in the embodiments only provide an experimental way to obtain them in order to achieve the specific disclosed purpose, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the embodiments.

[0042] Example 1

[0043] This example uses the multiplex PCR detection primer combination, kit and method for marine pathogens provided by the present invention to simultaneously perform multiplex PCR detection on five marine pathogens, namely, Vibrio alginolyticus, Vibrio vulnificus, Photobacterium damselae subsp. damselae, Vibrio harveyi and Vibrio campbellii. The specific method is as follows:

[0044] (I) PCR detection of various marine pathogens based on the specific primer pairs provided by the present invention

[0045] 1. Primer design and synthesis

[0046] The primer combination and target fragment sequence information provided by the present invention are shown in Table 1 and Table 2 respectively:

[0047] Regarding the selection of target fragments and the design of primers: The target fragments of Vibrio alginolyticus were selected based on the specific sequences of the ToxR / ToxS genes of Vibrio alginolyticus published on NCBI, including LR860673.1, LR860774.1, LR860912.1, AB372523.1, AB372527.1, KJ579443.1, AB372525.1 and AB372524.1. The target fragments of Vibrio vulnificus were selected based on the specific sequences of the mdh gene of Vibrio vulnificus published on NCBI, including KP224100.1, KP224104.1, EF642732.1, KP224105.1, KP224115.1, KP224111.1, KP224090.1 and EF642717.1. The target fragments of Photorhabdus mermaniformis were selected by citing the specific sequences of toxR gene of Photorhabdus mermaniformis subspecies published on NCBI, including MW362860.1, MW362862.1, MW362857.1, MT176619.1, KU760754.1, KU760747.1, AF170887.1, and AF170886.1. The target fragments of Vibrio harveyi were selected by citing the specific sequences of toxR gene of Vibrio harveyi 101 published on NCBI, including DQ640259.1, JF930600.1, DQ640258.1, LR860968.1, AY247418.1, JF930598.1, JF930595.1, and FM202682.1. The target fragment of Vibrio cannelliger was selected by referring to the TLH gene-specific sequences of Vibrio cannelliger published on NCBI, including DQ663484.1, DQ434996.1, DQ434995.1, AB271109.1 and DQ356918.1. The software MEG11 was used to align the sequences of Vibrio alginolyticus, Vibrio vulnificus, Photorhabdus mermanii subspecies, Vibrio harveyi and Vibrio cannelliger, and the conserved sequences were found. In the conserved sequence region, Primer Premier 6 software was used to design heterosexual primers for Vibrio alginolyticus, Vibrio vulnificus, Photorhabdus mermanii subspecies, Vibrio harveyi and Vibrio cannelliger.

[0048] Primers were synthesized by Shanghai Bioengineering Co., Ltd. ddH2O was purchased from Nanjing Novozyme Biotechnology Co., Ltd. with the catalog number P071-P1. 2×Taq enzyme Mix was purchased from Nanjing Novozyme Biotechnology Co., Ltd. with the catalog number P222-04. Trehalose was purchased from Best One Lab Supplies Mall, Sinopharm Reagent, analytical grade, content 99%. Pathogenic strains of Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermaidii, Vibrio harveyi and Vibrio cannelli were isolated and purified from the lesion tissues of grouper and golden pomfret cultured in a certain aquatic product company base and fish raft in Hainan using culture medium plates, and then obtained through 16S first-generation sequencing.

[0049] Table 1

[0050]

[0051]

[0052] Table 2

[0053]

[0054]

[0055] 2. Sample source and genomic DNA extraction

[0056] (1) Sample source: Take the diseased tissue, skin, liver, spleen, kidney, etc. of aquatic animals such as grouper, golden pomfret, shrimp, etc. infected with the corresponding marine pathogens and inoculate them on solid plates, separate them by streaking, and place them in a constant temperature incubator and culture them at 30℃ for 24-36 hours to obtain single colonies of marine pathogens for use.

[0057] (2) Pick a single colony from the plate and place it in a sterile EP tube pre-filled with 100 μL of sterile ddH2O. Mix by repeatedly blowing or shaking to disperse the bacteria in the ddH2O water.

[0058] (3) Move the EP tube to a 100℃ water bath for 5 minutes, then place it directly in a 4℃ refrigerator to cool for 5 minutes.

[0059] (4) The cooled EP tube is placed in a centrifuge and centrifuged at 12,000 rpm for 10 min. The system is precipitated into cell debris and other substances, while the DNA is dispersed in the supernatant, and the DNA of the sample can be obtained.

[0060] 3. Use the multiplex PCR primer combination provided by the present invention to detect the DNA of the sample

[0061] 3.1、PCR reaction system configuration:

[0062] (1) Amplification of negative control and sample genomic DNA was performed using the Vibrio alginolyticus specific primer pair (SEQ ID NO: 1-2) shown in Table 2. The specific reaction system is shown in Table 3:

[0063] Table 3

[0064]

[0065] (2) Negative control and sample genomic DNA were amplified using the Vibrio vulnificus specific primer pairs (SEQ ID NO: 3-4) shown in Table 2. The total volume of the reaction system was 25 μL, which was the same as the reaction system shown in Table 3, except that the primers SEQ ID NO: 1-2 were replaced by SEQ ID NO: 3-4.

[0066] (3) Amplification of negative control and sample genomic DNA was performed using the Photorhabditis mermaniformis subspecies-specific primer pairs (SEQ ID NOs: 5-6) shown in Table 2. The total volume of the reaction system was 25 μL, which was the same as the reaction system shown in Table 3, except that primers SEQ ID NOs: 1-2 were replaced with SEQ ID NOs: 5-6.

[0067] (4) Amplification of negative control and sample genomic DNA was performed using the Vibrio harveyi specific primer pair (SEQ ID NO: 7-8) shown in Table 2. The total volume of the reaction system was 25 μL, which was the same as the reaction system shown in Table 3, except that the primers SEQ ID NO: 1-2 were replaced by SEQ ID NO: 7-8.

[0068] (5) Amplification of negative control and sample genomic DNA was performed using the Vibrio cannelligensis-specific primer pairs (SEQ ID NOs: 9-10) shown in Table 2. The total volume of the reaction system was 25 μL, which was the same as the reaction system shown in Table 3, except that primers SEQ ID NOs: 1-2 were replaced with SEQ ID NOs: 9-10.

[0069] 3.2. PCR amplification reaction conditions are as follows:

[0070]

[0071] 4. Perform agarose gel electrophoresis on the amplified product

[0072] 5. Results

[0073] Figure 1 The amplification patterns of single genomic DNA of the five marine pathogenic bacteria (Vibrio alginolyticus, Vibrio vulnificus, Photorhabditis mermanni subsp. mermaidica, Vibrio harveyi and Vibrio cannelli) using the above primer pairs of SEQ ID NO: 1-10 are shown.

[0074] The results showed that these primer pairs can produce specific amplification products when amplifying the genomic DNA of their respective marine pathogens, which can be used for subsequent pathogen detection and identification.

[0075] (II) Detection of samples to be tested based on the multiplex PCR detection kit provided by the present invention

[0076] The present invention provides a marine pathogenic bacteria multiplex PCR detection kit, which comprises the above five pairs of primers SEQ ID NO: 1-10 and is combined with freeze-drying technology to synthesize PCR detection freeze-dried powder.

[0077] This PCR test freeze-dried powder is used to detect Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subspecies, Vibrio harveyi and Vibrio cannelli (hereinafter referred to as: five marine pathogens), and the kit also contains freeze-dried PCR reaction master solution, DNAMaker and ddH2O. Among them, DNAMaker contains 5 bands of specific amplification lengths, namely 580bp, 464bp, 307bp, 223bp, and 133bp. It is initially in a dry powder state and needs to be dissolved with 25μL ddH2O and stored at -20℃ when used. The specific steps are as follows:

[0078] 1. Composition of multiplex PCR reaction master mix

[0079] The multiplex PCR reaction master solution comprises: preparing 2×Taq enzyme Mix and 5×trehalose freeze-drying protective agent.

[0080] The 2×Taq enzyme Mix contains Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, and stabilizer; the preparation method of trehalose is to use 100 mL of deionized water to dissolve 70 g of analytical pure trehalose.

[0081] Reagents composition 2×Taq Enzyme Mix TaqDNA polymerase, dNTPs, MgCl2, reaction buffer, stabilizer 5× Trehalose Freeze Drying Protectant 100mL deionized water, 70g analytical grade trehalose

[0082] 2. The method for preparing the lyophilized powder for PCR detection of five marine pathogens by the bacterial multiple PCR detection kit comprises the following steps:

[0083] (1) Prepare primer working solution: Add ddH2O to dissolve the upstream and downstream primers of five marine pathogens and prepare primer working solutions with a concentration of 100 μM each.

[0084] (2) Preparation of PCR reaction master solution: 12.5 μL 2× Taq enzyme mix and 5 μL 5× trehalose as freeze-drying protective agent were prepared into PCR reaction master solution.

[0085] (3) Preparation of freeze-dried powder: 0.05 μL of each of the five sets of primer working solutions prepared was added to the above PCR reaction master solution to obtain a mixed solution, and ddH2O was added to make up to 25 μL. The mixed solution was then placed in a freeze drying instrument, the cold trap temperature was set to -65°C, the sample temperature was set to -45°C, pre-cooled for 1 hour to form ice cubes, and then vacuum dried for 2 hours, and finally frozen into dry powder, thereby obtaining the freeze-dried powder.

[0086] Reagents composition Primer working solution <![CDATA[ddH2O, upstream and downstream primers of five marine pathogenic bacteria (100 μM)]]> PCR reaction master mix 12.5 μL 2× Taq enzyme Mix, 5 μL 5× trehalose Freeze-dried powder Mix five sets of primer working solutions (0.05 μL each) and PCR reaction master solution, and add ddH2O to make up to 25 μL

[0087] 3. Preparation of multiplex PCR reaction system

[0088] The samples were tested using the multiplex PCR detection kit in this application, and the steps were as follows:

[0089] (1) Take the freeze-dried powder and determine the amount of ddH2O to be added according to the amount of genomic DNA contained in the amplified sample: when the amplified sample is 1 genomic DNA, add 24 μL ddH2O to dissolve it;

[0090] (2) If the sample to be amplified is 2 genomic DNAs, add 23 μL ddH2O to dissolve; if the sample to be amplified is 3 genomic DNAs, add 22 μL ddH2O to dissolve; if the sample to be amplified is 4 genomic DNAs, add 21 μL ddH2O to dissolve; if the sample to be amplified is 5 genomic DNAs, add 20 μL ddH2O to dissolve.

[0091] (3) After the dry powder is dissolved, 1 μL of DNA template of each sample is added thereto and mixed thoroughly to construct a PCR reaction system. During this process, a negative blank control of the sample is also set up.

[0092] 4. The reaction conditions for multiplex PCR amplification are as follows:

[0093]

[0094]

[0095] 5. Product testing and determination

[0096] The prepared PCR reaction system was amplified according to the above amplification procedure to obtain the PCR amplification products of the negative blank and sample. The amplification products were separated and detected using agarose gel electrophoresis technology, and compared and analyzed based on the amplification length bands of DNAMaker:

[0097] If no amplified bands appear in the PCR product, it is determined that Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subspecies, Vibrio harveyi and Vibrio cannelligi in the sample to be tested are all negative;

[0098] If a band with the same length as DNAMaker 580bp appears, the Vibrio alginolyticus in the sample to be tested is determined to be positive;

[0099] If a band with the same length as DNAMaker 464bp appears, Vibrio vulnificus is determined to be positive;

[0100] If a band with the same length as DNAMaker 307bp appears, the Photobacterium mermaniformis subspecies is determined to be positive;

[0101] If a band with the same length as DNAMaker 223 bp appears, Vibrio harveyi is determined to be positive;

[0102] If a band with the same length as DNAMaker 133bp appears, Vibrio cannelligii is determined to be positive.

[0103] Example 2 Accuracy and specificity verification

[0104] 1. First-generation sequencing verifies the accuracy and specificity of primers

[0105] The DNA genome amplification products of the five marine pathogenic bacteria in Example 1 were subjected to first-generation sequencing, and the sequences obtained by first-generation sequencing (as shown in Table 4) were compared with the NCBI database.

[0106] The target fragment of Vibrio alginolyticus (SEQ ID NO: 11 in Table 1) and the amplified product (SEQ ID NO: 16 in Table 4) were analyzed as examples. From the consistency (98.5%) and similarity (98.5%) indicators, the two sequences are highly similar. This means that the sequencing result of the PCR amplification product is very close to the target sequence, reflecting that the primers have good specificity and can accurately amplify a sequence that is highly similar to the target.

[0107] Table 4

[0108]

[0109]

[0110] In the sequence alignment, there are only 5 gaps, accounting for 0.9% of the total length, which indicates that there are a small number of mismatches or insertions / deletions. These differences may be due to errors in the PCR process, such as base mismatches, slippage, etc., or slight variations in the target sequence itself. The high overall score (2602.0) indicates that the two sequences match well overall, further supporting the conclusion that the PCR amplification product sequence is highly correlated with the target sequence.

[0111] In summary, from the comparison results of the amplified product and its target fragment in the NCBI database, it can be seen that when SEQ ID NO: 1-2 is used, the amplified product is Vibrio alginolyticus; when SEQ ID NO: 3-4 is used, it is Vibrio vulnificus; when SEQ ID NO: 5-6 is used, it is Photobacterium mermanii subspecies; when SEQ ID NO: 7-8 is used, it is Vibrio harveyi; when SEQ ID NO: 9-10 is used, it is Vibrio cannelli. It can be seen that the primer combination provided by the present invention has high accuracy and specificity.

[0112] 2. Primer specificity verification

[0113] (1) According to the PCR reaction system and conditions of Example 1, PCR amplification was performed on the DNA templates of Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subspecies mermaid, Vibrio harveyi and Vibrio cannelli. After the amplification was completed, the products were detected by electrophoresis. The experimental results were repeated several times (such as Figure 2 ) showed that only the DNA template of Vibrio alginolyticus produced specific bands.

[0114] (2) Similarly, according to the above method, the DNA of the five marine pathogens was amplified by PCR using primers SEQ ID NO: 3-4. The results showed that only the DNA of Vibrio vulnificus produced a specific band; when SEQ ID NO: 5-6 was used for PCR amplification, only the DNA of Photorhabdus mermaidus subspecies mermaid produced a specific band; when SEQ ID NO: 7-8 was used for PCR amplification, only the DNA of Vibrio harveyi produced a specific band; when SEQ ID NO: 9-10 was used for PCR amplification, only the DNA of Vibrio canneri produced a specific band (such as Figure 2 shown).

[0115] (3) In summary, the multiplex PCR detection primer combination, kit and method for marine pathogenic bacteria provided by the present invention have high specificity.

[0116] Example 3 Sensitivity Verification of Multiplex PCR Detection Kit

[0117] According to the reaction system and conditions of Example 1, the sensitivity test was carried out using positive samples of five marine pathogens, namely, Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subspecies, Vibrio harveyi and Vibrio cannelli. The specific operation is as follows:

[0118] 1. Bacterial liquid dilution

[0119] The bacterial solutions of the five marine pathogens were diluted with PBS buffer solution. For each pathogen, seven concentration gradients were set, namely 2×107 CFU / mL, 2×10 6 CFU / mL, 2×10 5 CFU / mL, 2×10 4 CFU / mL, 2×10 3 CFU / mL, 2×10 2 CFU / mL and 20 CFU / mL were used for subsequent experiments.

[0120] 2. Nucleic acid extraction and amplification

[0121] Nucleic acid extraction was performed on the bacterial suspensions of these five marine pathogens at different concentrations, and the extracted nucleic acids were then dissolved in freeze-dried powder and amplified by PCR.

[0122] 3. Test and results

[0123] The PCR amplification products were verified by agarose gel electrophoresis. The results showed that all pathogenic microorganisms were 2 CFU / mL concentration, these five marine pathogens can be detected simultaneously, which shows that the multiplex PCR detection primer combination and the kit provided by the present invention have high sensitivity (results are shown in Figure 3 shown).

[0124] Example 4 Optimization of multiplex PCR detection kit

[0125] In this example, positive samples of five marine pathogens, namely, Vibrio alginolyticus, Vibrio vulnificus, Photorhabditis mermanii subspecies, Vibrio harveyi and Vibrio cannelli, were used as test objects, and the primer combination and the freeze-drying addition concentration of trehalose were optimized to determine the optimal PCR freeze-drying system.

[0126] 1. Primer concentration optimization

[0127] A bacterial multiple primer combination PCR detection kit was used, SEQ ID NO: 1-10 primers were added thereto, and the primer concentrations were set to 200nM, 300nM, 400nM, 500nM, 600nM and 1000nM, respectively, to amplify the genomic DNA templates of the above five marine pathogens. The results showed that when the concentration of SEQ ID NO: 1-10 primers was in the range of 200-1000nM, genomic DNA amplification was achieved. After comprehensive consideration, the optimal primer concentration was determined to be 200nM (such as Figure 4 shown).

[0128] 2. Optimization of trehalose freeze-drying protective agent concentration

[0129] Trehalose was added as a freeze-drying protective agent in the bacterial multiple primer combination PCR detection kit, and its added content was set to 5%, 10%, 15%, and 20%, respectively, to amplify the genomic DNA templates of the above five marine pathogens. The experiment found that when the trehalose added content was in the range of 5%-20%, the genomic DNA amplification could be achieved. The specific results are shown in the following table:

[0130] Sample No. Trehalose ratio in PCR reaction system Amplification effect 1 0％ No amplified band 2 5％ normal 3 10％ normal 4 15％ normal 5 20％ normal

[0131] Based on the optimization results of primer concentration and trehalose concentration, the optimal bacterial multiple primer combination freeze-dried powder system (25 μL) was determined. The system contains 12.5 μL of 2×Taq enzyme Mix, 0.05 μL of upstream and downstream primer working solutions (100 μM) of Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subsp. mermaidii, Vibrio harveyi and Vibrio cannelligi, 5 μL of trehalose and 7 μL of ddH2O.

[0132] 3. Determination of the optimal PCR freeze-dried powder system

[0133] Combined with the above optimization results, the preparation method of the optimal bacterial multiple primer combination freeze-dried powder system (25 μL) is shown in the following table:

[0134] Reagents Dosage 2×Taq Enzyme Mix 12.5μL Upstream and downstream primers working solution of five marine pathogens (100μM) 0.05μL each Trehalose 5μL <![CDATA[ddH2O]]> 7μL

[0135] The results showed that in this system, the freeze-dried system containing 20% ​​trehalose had the best storage effect at room temperature and could be stored at room temperature for up to 6 months, which is of great significance for storage and transportation convenience in practical applications.

[0136] Example 5

[0137] In this example, positive samples of five marine pathogens, namely, Vibrio alginolyticus, Vibrio vulnificus, Photorhabditis mermanii subspecies, Vibrio harveyi and Vibrio cannelligi, were used as test objects to verify the effectiveness of the kit in detecting single and multiple positive samples.

[0138] 1. Use freeze-dried powder to prepare the PCR reaction system. The specific steps are as follows:

[0139] (1) When the amplification sample is a genomic DNA, take the freeze-dried powder, add 24 μL ddH2O to dissolve it, then add 1 μL of the DNA template of the sample, and mix well to obtain the PCR reaction system.

[0140] (2) If the amplification sample is a mixture of two genomic DNAs, take the freeze-dried powder, add 23 μL ddH2O to dissolve it, then add 1 μL of the DNA template of the two samples respectively, and mix well to obtain a PCR reaction system.

[0141] (3) For the amplification sample, three genomic DNAs were mixed, freeze-dried powder was taken, 22 μL ddH2O was added to dissolve, and then 1 μL of DNA template of each sample was added and mixed to prepare a PCR reaction system.

[0142] (4) When the amplification sample is a mixture of four genomic DNAs, take the freeze-dried powder, add 21 μL of ddH2O to dissolve it, then add 1 μL of the DNA template of each sample, and mix well to obtain a PCR reaction system.

[0143] (5) If the amplification sample is a mixture of 5 genomic DNAs, take the freeze-dried powder, add 20 μL ddH2O to dissolve it, then add 1 μL of the DNA template of each sample and mix well to obtain a PCR reaction system.

[0144] 2. Amplification effect detection

[0145] Set the sample to be tested and the negative blank sample, and amplify them. The amplification effect is as follows Figure 5-6 shown.

[0146] Figure 5 In the figure, single sample genomic DNA amplification (lanes 1-5); two samples genomic DNA amplification (lanes 6-8); three samples genomic DNA amplification (lanes 10-12); four samples genomic DNA amplification (lanes 13-14); negative control (lanes 15-18). Figure 6 In the figure, lane 1 is a negative blank; lane 2 is the result of simultaneous amplification of 5 genomic DNAs of Vibrio alginolyticus, Vibrio vulnificus, Photorhabdus mermanii subsp. mermaidii, Vibrio harveyi and Vibrio cannelli.

[0147] From this we can see that Figure 5 Lanes 6-14 show the simultaneous amplification of genomic DNA of samples in 2, 3, and 4 different combinations, and corresponding amplification bands appeared in all of them, indicating that the kit can achieve the simultaneous amplification of multiple pathogen genomic DNA combinations in the same reaction system, has the ability to detect multiple different pathogens at the same time, and can meet the detection needs when multiple pathogens are mixed.

[0148] Figure 5 Lanes 15-18 and Figure 6 Lane 1 was used as a negative control, and no amplification bands appeared (or the results were consistent with those of a negative control). This indicates that there were no interfering factors such as nonspecific amplification during the entire detection process, which indirectly confirms the specificity of the kit during the detection process and the reliability of the results.

[0149] Figure 6Lane 2 presents the simultaneous amplification patterns of five genomic DNAs, including Vibrio alginolyticus, Vibrio vulnificus, Photorhabditis elegans subspecies, Vibrio harveyi and Vibrio cannelligi, further proving that the kit is capable of detecting multiple pathogens simultaneously with good results. Overall, it shows that the kit has high effectiveness and accuracy in detecting single or multiple positive samples of multiple marine pathogens, and can be used in actual related pathogen detection work.

[0150] In summary, the multiple primer combination PCR detection kit provided by the present invention shows good amplification effect in the detection of these five marine pathogens, whether it is a single sample or multiple samples, and has high specificity, accuracy and practicality, and can meet the corresponding detection needs.

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A multiplex PCR primer combination for simultaneously detecting multiple marine pathogens, characterized in that: The components include: Specific primer pair for Vibrio alginolyticus SEQ ID NO: 1-2; A pair of primers specific for Vibrio vulnificus SEQ ID NOs: 3-4; Specific primer pair for Photorhabditis mermaniformis subspecies mermaniformis SEQ ID NOs: 5-6; a primer pair specific for Vibrio harveyi SEQ ID NO: 7-8; and The specific primer pair for Vibrio cannellii is SEQ ID NO: 9-10.

2. A multiplex PCR detection freeze-dried powder, characterized in that: Simultaneously detecting multiple marine pathogens, the components include the multiplex PCR detection primer combination of claim 1, 2×Taq enzyme Mix, 5×trehalose and dH2O; the preparation steps of the freeze-dried powder include the following: A1 respectively dissolves SEQ ID NO: 1-2, SEQ ID NO: 3-4, SEQ ID NO: 5-6, SEQ ID NO: 7-8, SEQ ID NO: 9-10 with ddH2O to prepare primer working solutions of Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subsp. mermaidii, Vibrio harveyi, and Vibrio cannelli; A2 adds the primer working solution to the PCR reaction master solution to obtain a mixed solution, and adds ddH2O; the PCR reaction master solution includes 2×Taq enzyme Mix and 5×trehalose; A3 freeze-drying the mixed solution to obtain freeze-dried powder.

3. The multiplex PCR detection freeze-dried powder according to claim 2, characterized in that: Also includes the following: In the step A1, 100 μM primer working solutions of Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subspecies, Vibrio harveyi, and Vibrio cannelligi are prepared respectively; In step A2, each 25 μL of the mixed solution contains: 12.5 μL of 2×Taq enzyme Mix, 5 μL of 5×trehalose, and 0.05 μL of each of the five primer working solutions.

4. The multiplex PCR detection freeze-dried powder according to claim 2, characterized in that: Also includes: In step A3, the mixed solution is placed in a freeze drying apparatus, the cold trap temperature is set to -65°C, the sample temperature is set to -45°C, pre-cooled for 1 hour to form ice cubes, and then vacuum dried for 2 hours.

5. The multiplex PCR detection freeze-dried powder according to claim 2, characterized in that: Also includes: The 2×Taq enzyme Mix comprises: Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, and stabilizer; The 5× trehalose comprises: 70 g of analytical pure trehalose dissolved in every 100 mL of deionized water.

6. A multiplex PCR detection kit for simultaneously detecting multiple marine pathogens, characterized in that: The invention comprises the multiplex PCR detection freeze-dried powder according to any one of claims 2 to 5.

7. A multiplex PCR detection method for simultaneously detecting multiple marine pathogens, characterized in that: Non-disease diagnosis or treatment; including the following steps: B1 is configured with the multiplex PCR detection freeze-dried powder described in any one of claims 2 to 5; B2: adding freeze-dried powder and ddH2O to the genomic DNA of the sample to be tested, and then adding genomic DNA templates of Vibrio alginolyticus, Vibrio vulnificus, Photobacterium mermanii subspecies mermaid, Vibrio harveyi, and Vibrio cannelligi samples, and mixing them to obtain a PCR reaction system; At the same time, set up a negative blank control for the sample; B3 performs a multiplex PCR amplification reaction. After the reaction is completed, the amplification products are subjected to agarose gel electrophoresis or sequencing detection and analysis, and the type of pathogens contained in the sample to be tested is determined based on the detection results.

8. The multiplex PCR detection method according to claim 7, characterized in that: In step B3, the multiplex PCR amplification reaction conditions are as follows:

9. The multiplex PCR detection method according to claim 7, characterized in that: In step B2, the components of each 25 μL PCR reaction system include the following: 。 10. Use of the primer combination according to claim 1, the lyophilized powder according to any one of claims 2 to 5, the detection kit according to claim 6, or the detection method according to any one of claims 7 to 9, characterized in that: Not for diagnosis or treatment of disease.

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