Salmonella enterica ultra-fast detection kit based on whole-process nucleic acid detection chip and detection method of salmonella enterica ultra-fast detection kit

Abstract

The invention discloses a salmonella enterica ultra-rapid detection kit based on a whole-process nucleic acid detection chip and a detection method thereof. The kit comprises a rapid hot start DNA polymerase, a PCR reaction mixed solution, a positive reference substance and a negative reference substance, the kit also comprises the following primers which take the salmonella enterica V3-V4 region as a target sequence and are designed by a PCR fluorescent probe method based on a whole-process nucleic acid detection chip technical platform: an upstream primer F, a downstream primer R and a detection probe P, and the nucleotide sequences of the three primers are shown as SEQ ID NO. 1-3. According to the detection kit disclosed by the invention, a V3-V4 target sequence can be rapidly detected on the micro-fluidic chip within more than ten minutes, so that the accuracy and the specificity of detecting the salmonella enterica gene are ensured; the invention provides the salmonella enterica gene detection kit which is more comprehensive in detection effect, high in specificity, good in sensitivity, low in omission ratio, convenient, simple and easy to operate.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent technology, specifically to an ultra-rapid detection kit for Salmonella enterica based on a full-process nucleic acid detection chip and its detection method. Background Art

[0002] Salmonella is a common foodborne pathogen. Contaminated raw eggs, egg products, and undercooked meat are the main routes of Salmonella infection in humans. In my country, 70%–80% of foodborne bacterial diseases are caused by Salmonella. Salmonella is an important member of the Enterobacteriaceae family, with 2 species, 42 serogroups, and over 2500 serotypes, of which more than 240 serotypes have been reported in my country. Different serotypes of Salmonella exhibit significantly different invasiveness and pathogenicity; therefore, exploring methods for Salmonella typing is crucial for identifying and controlling diseases caused by it. Even today, enteric Salmonella remains one of the more serious infectious diseases. Therefore, early, rapid, and accurate diagnosis is of great significance for the treatment and prevention of its spread.

[0003] There are three commonly used methods for the etiological diagnosis of enteric Salmonella: culture, serological methods, and PCR. The latter two are the main methods used in clinical practice.

[0004] Traditional methods for detecting Salmonella enterica are no longer sufficient to meet modern testing requirements due to their long testing cycles, complex procedures, and numerous required reagents. Pathogen nucleic acid detection technologies, such as polymerase chain reaction (PCR), also have practical problems. For example, conventional PCR requires specialized equipment and is prone to cross-contamination and cumbersome procedures. While real-time quantitative PCR (qPCR) effectively addresses cross-contamination and simplifies the process, its long testing time makes it unsuitable for rapid on-site testing. Immunological detection technologies are rapid, simple, and inexpensive, but require high-quality, highly stable monoclonal antibodies; otherwise, accuracy is insufficient, limiting them to auxiliary detection methods. Therefore, timely application of the latest advancements in biotechnology is crucial to meeting the ever-increasing demands for pathogen detection. Among these, rapid nucleic acid detection technology based on end-to-end nucleic acid detection chips represents a significant advancement in pathogen nucleic acid detection, and the established end-to-end nucleic acid detection chip technology platform offers numerous advantages.

[0005] Existing patents have reported a method for dual detection of the virulence island genes mgtC and sseL in Salmonella. However, its drawback is that the identification of bacteria should be designed in the variable region, and it does not have the advantage of specificity for designing specific genes. Moreover, the dual targets occupy more fluorescence channels.

[0006] Therefore, there is an urgent need to develop a gene detection kit and method for Salmonella enterica that has more comprehensive detection effects, higher specificity, and lower false negative rate. Summary of the Invention

[0007] The purpose of this invention is to provide an ultra-rapid detection kit for Salmonella enterica based on a full-process nucleic acid detection chip and its detection method, thereby solving the problems of long detection time, low specificity, low sensitivity and high false negative rate of existing Salmonella enterica gene detection kits.

[0008] To solve the above problems, the present invention adopts the following technical solution:

[0009] According to a first aspect of the present invention, an ultra-rapid detection kit for Salmonella enterica based on a full-process nucleic acid detection chip is provided, comprising a rapid hot-start DNA polymerase, a PCR reaction mixture, a positive control, and a negative control, and further comprising the following primers designed using a PCR fluorescent probe method with the V3-V4 region of Salmonella enterica as the target sequence, based on a microfluidic chip technology platform: upstream primer F, downstream primer R, and a detection probe P, wherein the three primers are as follows:

[0010] Upstream primer F: 5'-AAGAAGCACCGGCTAACTCC-3' (SEQ ID NO. 1);

[0011] Downstream primer R: 5'-ACATCCGACTTGACAGACCG-3' (SEQ ID NO. 2);

[0012] Detection probe P: 5'-CTGCGTGCGCTTTAC-3' (SEQ ID NO. 3).

[0013] Preferably, the concentrations of the upstream primer F and the downstream primer R are each 0.4~0.8 μM, and the concentration of the detection probe P is 0.4~0.8 μM; most preferably, the concentrations of the upstream primer F and the downstream primer R are 0.8 μM, and the concentration of the detection probe P is 0.8 μM.

[0014] Preferably, the rapid hot-start DNA polymerase has an enzyme concentration of 0.25 U / μL to 0.5 U / μL, and more preferably an enzyme concentration of 0.25 U / μL.

[0015] Preferably, the PCR reaction mixture contains: 1×PCR Buffer, 0.3125~0.625mM dNTPs, 0.4~0.8μM upstream primer F / downstream primer R, and 0.4~0.8μM detection probe P;

[0016] Preferably, the PCR reaction mixture contains: 1×PCR Buffer, 0.3125mM dNTPs, 0.8μM upstream primer F / downstream primer R, and 0.8μM detection probe P.

[0017] Preferably, the positive control is an enteric Salmonella plasmid.

[0018] Preferably, the negative control is water.

[0019] According to a second aspect of the present invention, a method for detecting Salmonella enterica genes is provided, comprising the following steps:

[0020] (1) DNA was extracted using a pathogen DNA / RNA extraction kit as a sample template;

[0021] (2) A full-process nucleic acid detection chip based on EWOD is provided. The full-process nucleic acid detection chip includes, from bottom to top, a SiO2 support plate, a SiO2 glass, a dielectric layer, a first Teflon hydrophobic layer, a support layer, a second Teflon hydrophobic layer, and an ITO glass layer. A magnetic bead nucleic acid extraction area and a dual-temperature nucleic acid amplification area are formed between the first Teflon hydrophobic layer and the second Teflon hydrophobic layer. The magnetic bead nucleic acid extraction area includes, in sequence, a lysis chamber, a first washing chamber, a second washing chamber, and an elution chamber. The dual-temperature nucleic acid amplification area includes a low-temperature isothermal area and a high-temperature isothermal area. Interdigitated electrodes and EWOD driving electrodes are provided on the SiO2 glass. The interdigitated electrodes are aligned with the lysis chamber to realize the electrolysis of bacterial samples. The EWOD driving electrodes are aligned with the elution chamber and the dual-temperature nucleic acid amplification area.

[0022] (3) Provide the Salmonella enterica gene detection kit as described above, and add 0.2 volumes of rapid hot-start DNA polymerase, 3.8 volumes of PCR reaction mixture, and 8 volumes of sample template DNA to the full-process nucleic acid detection chip; by controlling the EWOD driving electrode to be energized so that its surface is hydrophilic, the droplets circulate in the low temperature isothermal zone and the high temperature isothermal zone to achieve amplification;

[0023] (4) After the reaction is complete, if there is an S-type amplification curve and the Ct value is within the reference range, it is positive; otherwise, it is negative.

[0024] The reaction conditions in step (3) are 95℃ for 50s; 95℃ for 2s, 60℃ for 2s, 45 cycles.

[0025] The rapid nucleic acid detection technology based on a full-process nucleic acid detection chip utilizes a fast-start DNA polymerase and three primers designed according to the target gene sequence (i.e., upstream primer F, downstream primer R, and detection probe P) to specifically identify a specific region on the target sequence and complete the detection rapidly within a few minutes. In the ultrafast reagent system, the concentrations of components in the PCR reaction mixture and the concentration of the fast-start DNA polymerase are both greater than those in traditional PCR reagents, which greatly increases the probability of molecular and ion collisions in the microfluidic chip, and allows amplification and detection to be completed in a short time under the action of the ultrafast amplification enzyme.

[0026] The aforementioned end-to-end nucleic acid detection chip technology platform is described in Chinese Invention Patent CN118956584A, entitled "An End-to-End Nucleic Acid Detection Chip Based on EWOD and Its Application." It should be understood that the relevant content disclosed in Chinese Invention Patent CN118956584A is incorporated herein by reference.

[0027] The end-to-end nucleic acid detection chip comprises, from bottom to top, a SiO2 support plate, SiO2 glass, a dielectric layer, a first Teflon hydrophobic layer, a support layer, a second Teflon hydrophobic layer, and an ITO glass layer. A magnetic bead nucleic acid extraction region and a dual-temperature nucleic acid amplification region are formed between the first and second Teflon hydrophobic layers. The magnetic bead nucleic acid extraction region includes, in sequence, a lysis chamber, a first washing chamber, a second washing chamber, and an elution chamber. The dual-temperature nucleic acid amplification region includes a low-temperature isothermal region and a high-temperature isothermal region. The SiO2 glass is equipped with interdigitated electrodes and an EWOD driving electrode. The interdigitated electrodes are aligned with the lysis chamber to achieve electrolysis of bacterial samples. The EWOD driving electrode is aligned with the elution chamber and the dual-temperature nucleic acid amplification region. By controlling the EWOD driving electrode to be energized, its surface becomes hydrophilic, and droplets circulate within the low-temperature and high-temperature isothermal regions, achieving amplification.

[0028] According to the present invention, the rapid nucleic acid detection technology based on a full-process nucleic acid detection chip utilizes real-time quantitative PCR to detect fluorescence signals on a microfluidic chip, completing the detection within a few minutes under the action of an ultrafast amplification enzyme. This rapid nucleic acid detection technology based on a full-process nucleic acid detection chip overcomes the inherent disadvantages of traditional PCR, such as long detection time, susceptibility to contamination, and high detection cost. The original real-time quantitative PCR detection method suffers from an excessively long detection time, typically around 2 hours. Furthermore, this detection method requires lower technical skills from the testing personnel, is extremely simple to operate, and does not require special reagents or instruments, which is beneficial for establishing a low-cost rapid screening system. The rapid nucleic acid detection technology based on a full-process nucleic acid detection chip is a simple, rapid, and highly specific gene amplification method. Comparing silicon-based microfluidic technology with PCR technology (including real-time quantitative PCR technology), it can be found that this technology is equivalent to or superior to PCR technology in terms of methodological indicators such as sensitivity, specificity, and detection range, significantly reducing detection time, and with lower instrument and detection costs.

[0029] The key inventive points of this invention are as follows: First, a detection system is designed using the V3-V4 variable nucleic acid sequence region of *Salmonella enterica* as the target sequence. The V3-V4 region is a specific variable region of *Salmonella enterica*. Compared to existing designs targeting virulence island genes (such as mgtC and sseL), this approach more accurately ensures the specificity and accuracy of detection, reducing false negatives or cross-reactions caused by insufficient conservation of the target sequence. Second, based on the PCR fluorescent probe method, three specific sequences (SEQ ID NO. 1-3) are designed. These three primers specifically bind to the V3-V4 target sequence, achieving accurate detection through fluorescence signal amplification. Furthermore, concentration optimization (optimal 0.8 μM) further improves reaction efficiency, ensuring a low false negative rate. Thirdly, relying on the full-process nucleic acid detection chip technology platform (based on EWOD microfluidic technology), combined with rapid hot-start DNA polymerase (concentration 0.25~0.5 U / μL) and optimized PCR reaction system (such as dNTPs concentration 0.3125~0.625mM), the detection process is made ultra-fast, with the entire amplification process taking only a dozen minutes (traditional PCR takes more than 2 hours); the reaction conditions are optimized to: 95℃ for 50s pre-denaturation, followed by 45 cycles of 95℃ for 2s / 60℃ for 2s. By shortening the reaction time of each step and the dual-temperature zone design of the microfluidic chip (high temperature isothermal zone and low temperature isothermal zone), the rapid circulation and movement of droplets are achieved, greatly improving efficiency. Fourth, it adopts an EWOD-based full-process nucleic acid detection chip, which integrates a magnetic bead nucleic acid extraction area (lysis chamber, washing chamber, elution chamber) and a dual-temperature nucleic acid amplification area. It achieves electrolysis through interdigital electrodes and controls droplet movement through EWOD-driven electrodes, realizing integrated automated operation of "sample lysis-nucleic acid extraction-amplification detection" without manual intervention, reducing operational complexity and contamination risk.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1) The detection kit of the present invention can perform rapid detection of V3-V4 target sequences on a microfluidic chip within ten minutes, while the detection time on other technology platforms is at least 2 hours;

[0032] 2) This invention selects the variable nucleic acid sequence region of Salmonella V3-V4 for primer design and uses three primers to ensure the accuracy and specificity of the detection kit for Salmonella intestinal genes.

[0033] 3) The gene rapid diagnostic kit of the present invention can determine the presence or absence of target substances based on whether there is an S-type amplification curve and whether the Ct value is within the reference range, thus having high specificity;

[0034] 4) The gene rapid diagnostic kit of the present invention is easy to identify and the results can be analyzed by software.

[0035] In summary, this invention provides an enteric Salmonella gene detection kit that offers more comprehensive detection results, higher specificity, better sensitivity, lower false negative rate, and is convenient, simple, and easy to operate. Attached Figure Description

[0036] Figure 1 This is an exploded view of a full-process nucleic acid detection chip according to a preferred embodiment of the present invention;

[0037] Figure 2 Is it like this? Figure 1 The diagram shows a plan view of the entire nucleic acid detection chip process.

[0038] Figure 3 Is it like this? Figure 1 A cross-sectional schematic diagram of the entire nucleic acid detection chip process is shown.

[0039] Figure 4 The results of the fluorescence quantitative PCR sensitivity experiment using a microfluidic chip for Salmonella enterica in Example 2 are shown. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0041] Example 1: Preparation of a Salmonella Intestinal Gene Detection Kit

[0042] Based on publicly available primers for the universal V3 and V4 variable regions of fungi, sequences of the V3-V4 regions were downloaded from NCBI. Primer Blast and Nucleotide Blast were used to design screening and identification primers. The design is as follows:

[0043] (1) Synthesize oligodeoxynucleotide primers according to the following sequence using a DNA synthesizer:

[0044] Upstream primer F: 5'-AAGAAGCACCGGCTAACTCC-3' (SEQ ID NO. 1);

[0045] Downstream primer R: 5'-ACATCCGACTTGACAGACCG-3' (SEQ ID NO. 2);

[0046] Detection probe P: 5'-CTGCGTGCGCTTTAC-3' (SEQ ID NO. 3).

[0047] (2) Prepare DNA polymerase and reverse transcriptase: Mix the fast hot-start DNA polymerase (purchased from Roche) and reverse transcriptase (purchased from TaKaRa) in a container;

[0048] (3) Preparation of PCR reaction mixture: The reaction mixture contains 1×PCR Buffer, 0.3125mM dNTPs, 0.8μM upstream and downstream primers F / R, and 0.8μM detection probe P, and is placed in a container;

[0049] (4) Prepare positive control: Salmonella enterica plasmid (purchased from Sangon Biotech), placed in a container;

[0050] (5) Prepare negative control: deionized water.

[0051] (6) Pack the above 4 containers into a reagent kit and seal it.

[0052] The preparation process is briefly described below:

[0053] 1. After synthesizing and purifying the upstream primer F, downstream primer R, and detection probe P, dilute with 1×TE, detect the concentration, and sample for quality inspection.

[0054] 2. Aseptically dispense the liquid prepared in steps (2) to (3) above, and sample for quality inspection;

[0055] 3. Prepare the positive control by dilution, dispense, and sample for quality inspection;

[0056] 4. Prepare, package, and sample the negative control standard for quality inspection;

[0057] 5. Assemble the reagent kit.

[0058] Example 2: Application of the Salmonella Intestinal Gene Detection Kit

[0059] 1 Materials and Methods

[0060] 1.1 Materials

[0061] 1.1.1 Pathogens

[0062] This invention uses 14 pathogen strains, mainly from the Chinese Center for Disease Control and Prevention, the Shanghai Municipal Center for Disease Control and Prevention, and the Pudong New Area Center for Disease Control and Prevention. See Table 1 for details.

[0063] Table 1. Strains and their sources

[0064]

[0065] 1.2 Sample Preparation

[0066] 1.2.1 Take anal swab samples and add them to a solution containing lysis buffer, proteinase K, and magnetic beads for lysis and nucleic acid adsorption;

[0067] 1.2.2 After the magnetic beads adsorbed with nucleic acid were washed three times and eluted once, sample template DNA was obtained.

[0068] 1.3 This paper provides a complete nucleic acid detection chip based on EWOD. Combined with... Figures 1-3 As shown, the specific structure is explained in detail below:

[0069] For details, please refer to the following: Figure 1 The entire process of nucleic acid detection chip includes, from bottom to top, the following layers: SiO2 support plate 1, SiO2 glass 2, PET dielectric layer 3, first Teflon hydrophobic layer 4, support layer 5, second Teflon hydrophobic layer 6, and ITO glass layer 7.

[0070] See Figure 2 A magnetic bead nucleic acid extraction region 10 and a dual-temperature nucleic acid amplification region 20 are formed between the first Teflon hydrophobic layer 4 and the second Teflon hydrophobic layer 6. The magnetic bead nucleic acid extraction region 10 includes a lysis chamber 11, a first washing chamber 12, a second washing chamber 13, and an elution chamber 14. The dual-temperature nucleic acid amplification region 20 includes a low-temperature isothermal region 21 and a high-temperature isothermal region 22.

[0071] Combination Figure 2 , Figure 3 As shown, the SiO2 glass 2 is provided with interdigitated electrodes 30 and EWOD driving electrodes 40. The interdigitated electrodes 30 are aligned with the lysis chamber 11 to realize the electrolysis of bacterial samples. The EWOD driving electrodes 40 are aligned with the elution chamber 14 and the dual-temperature nucleic acid amplification region 20. By controlling the EWOD driving electrodes 40 to make its surface hydrophilic, the droplets circulate in the low temperature isothermal region 21 and the high temperature isothermal region 22 to realize amplification.

[0072] 1.4 The reaction process of the full-process nucleic acid detection chip rapid nucleic acid detection technology using the kit of Example 1.

[0073] 1.5 Reagent preparation and reaction

[0074] 1.5.1 The reaction was carried out on a 12 μL microfluidic chip. The reaction system was first prepared in a 200 μL PCR reaction tube: 0.2 μL of rapid hot-start DNA polymerase, 3.8 μL of PCR reaction mixture, and 8 μL of sample template DNA.

[0075] 1.5.2 The prepared reaction system reagents were reacted on a microfluidic chip under the following conditions: 95℃ for 50s; 95℃ for 2s, 60℃ for 2s, for 45 cycles. Specifically: First, the test sample was added to the lysis chamber 11, and the test sample was lysed by applying a voltage (AC voltage 30V, frequency 100,000Hz) at a certain frequency to the interdigitated electrode 30. After electrolysis, the magnetic rod 50 controlled the magnetic bead 60 to enter the lysis chamber 11, and the mixture was stirred by moving the magnetic rod 50. Then, the magnetic rod 50 was moved to the elution chamber 14, allowing the magnetic bead to pass through the silicone oil and enter the first washing chamber 12. The above process was repeated to allow the magnetic bead to enter the second washing chamber 13. The EWOD driving electrode 40 below the elution chamber 14 was powered on, and the extraction solution was added to the elution chamber 14. The magnetic rod 50 was controlled to move the magnetic bead 60 to the elution chamber 14 for elution, and then the magnetic rod 50 was controlled again to move the magnetic bead 60 back to the second washing chamber 13. The EWOD driving electrodes were energized to move the reaction reagents in the elution chamber to a 95°C temperature zone for 50 seconds of pre-denaturation. After this, the droplets were cyclically moved between the high-temperature isothermal zone 22 (95°C) and the low-temperature isothermal zone 21 (60°C) by controlling the energization and de-energization of the EWOD driving electrodes. In each cycle, the droplets spent 2 seconds in the high-temperature isothermal zone 22 and 2 seconds in the low-temperature isothermal zone 21. Under these conditions, the droplets moved between the high-temperature isothermal zone 22 and the low-temperature isothermal zone 21 for 45 cycles, with the entire amplification time not exceeding 7 minutes. The movement time of the droplets between adjacent electrodes was set to 500 ms to 1000 ms to ensure stable droplet movement. The fastest droplet movement between electrodes in this invention can reach 100 ms.

[0076] 1.6 Post-reaction treatment

[0077] After the reaction is complete, if there is an S-shaped amplification curve and the Ct value is within the reference range, it is considered positive; otherwise, it is considered negative.

[0078] 1.7 Specificity Test

[0079] 1.7.1 The pathogen microfluidic chip technology platform was used for detection. Fourteen pathogens were amplified. Those with S-shaped amplification curves and Ct values ​​within the reference range were considered positive, otherwise they were considered negative, thus verifying the specificity of the method.

[0080] 1.7.2 Mixed nucleic acid detection of several pathogens: 8 μL of an equal volume mixture of DNA from Salmonella enterica, Escherichia coli, Staphylococcus aureus, and Haemophilus influenzae was used for detection.

[0081] 1.8 Sensitivity Test

[0082] DNA extracted from Salmonella intestinalis was diluted to E3, and the gradient concentrations of E3 were detected. The results are shown in [Figure number missing]. Figure 4 .

[0083] 1.9 Repeatability Test

[0084] The specificity test and sensitivity test were each repeated twice.

[0085] 2 Results

[0086] 2.1 Establishment of a whole-process nucleic acid detection chip method for Salmonella enterica genes

[0087] 2.2 Specificity test: The test result for enteric Salmonella was positive, while the results for 13 non-enteric Salmonella strains were negative. The mixed DNA of enteric Salmonella and four pathogens (Escherichia coli, Staphylococcus aureus, and Haemophilus influenzae) was positive, as shown in Table 2. The results indicate that the kit has high specificity.

[0088] Table 2. Specificity test results using the method of the present invention

[0089]

[0090] 2.3 Sensitivity Test

[0091] The microfluidic chip method for Salmonella enterica DNA can detect copy numbers up to 1000 copies / mL. Microfluidic chip PCR results are shown below. Figure 4 .

[0092] 2.4 Repeatability test: The specificity test was repeated twice, and the results were consistent. The sensitivity test was repeated twice, and the results were of the same order of magnitude.

[0093] Example 3: Screening of primer and probe concentrations for the Salmonella Intestinal Gene Detection Kit

[0094] 1 Materials and Methods

[0095] 1.1 Materials

[0096] Plasmid.

[0097] 1.2 The reaction process of the full-process nucleic acid detection chip rapid nucleic acid detection technology using the kit of Example 1.

[0098] 1.3 Screening of primer and probe concentrations

[0099] Two sets of final concentrations were designed: the first set of primers had a concentration of 0.4 μM and the second set of probes had a concentration of 0.8 μM and the third set of probes had a concentration of 0.8 μM.

[0100] 1.4 Reagent preparation and reaction

[0101] 1.4.1 The reaction was carried out on a 12 μL microfluidic chip. The reaction system was first prepared in a 200 μL PCR reaction tube: 0.2 μL of rapid hot-start DNA polymerase, 3.8 μL of PCR reaction mixture, and 8 μL of sample template DNA.

[0102] 1.4.2 The prepared reaction system reagents were reacted on a microfluidic chip under the following conditions: 95℃ for 50s; 95℃ for 2s, 60℃ for 2s, for 45 cycles.

[0103] 2 Results

[0104] The results of the first set of primer and probe concentrations showed an amplification curve with a relatively large Ct value and a relatively small fluorescence signal value; the results of the second set of primer and probe concentrations showed an amplification curve with a relatively small Ct value and a relatively large fluorescence signal value.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A rapid detection kit for Salmonella enterica based on a full-process nucleic acid detection chip, comprising a rapid hot-start DNA polymerase, a PCR reaction mixture, a positive control, and a negative control, characterized in that, It also includes the following primers designed using the PCR fluorescent probe method based on a microfluidic chip technology platform, targeting the V3-V4 region of Salmonella enterica: upstream primer F, downstream primer R, and a detection probe P. The three primers are as follows: Upstream primer F: 5'-AAGAAGCACCGGCTAACTCC-3'; Downstream primer R: 5'-ACATCCGACTTGACAGACCG-3'; Detection probe P: 5'-CTGCGTGCGCTTTAC -3'.

2. The ultra-rapid detection kit for Salmonella enterica according to claim 1, characterized in that, The concentrations of the upstream primer F and the downstream primer R are each 0.4~0.8 μM, and the concentration of the detection probe P is 0.4~0.8 μM.

3. The ultra-rapid detection kit for Salmonella enterica according to claim 2, characterized in that, The concentrations of the upstream primer F and the downstream primer R are both 0.8 μM, and the concentration of the detection probe P is 0.8 μM.

4. The ultra-rapid detection kit for Salmonella enterica according to claim 1, characterized in that, The concentration of the rapid hot-start DNA polymerase is 0.25 U / μL to 0.5 U / μL.

5. The ultra-rapid detection kit for Salmonella enterica according to claim 4, characterized in that, The concentration of the rapid hot-start DNA polymerase is 0.25 U / μL.

6. The ultra-rapid detection kit for Salmonella enterica according to claim 1, characterized in that, The PCR reaction mixture contains: 1×PCR Buffer, 0.3125~0.625mM dNTPs, 0.4~0.8μM upstream primer F / downstream primer R, and 0.4~0.8μM detection probe P.

7. The ultra-rapid detection kit for Salmonella enterica according to claim 6, characterized in that, The PCR reaction mixture contains: 1×PCR Buffer, 0.3125mM dNTPs, 0.8μM upstream primer F / downstream primer R, and 0.8μM detection probe P.

8. The ultra-rapid detection kit for Salmonella enterica according to claim 1, characterized in that, The positive control is a plasmid of Salmonella enterica; the negative control is water.

9. A method for detecting Salmonella enterica genes using the kit described in any one of claims 1 to 8, characterized in that, Includes the following steps: (1) DNA was extracted using a pathogen DNA / RNA extraction kit as a sample template; (2) A full-process nucleic acid detection chip based on EWOD is provided, the full-process nucleic acid detection chip comprising, from bottom to top: a SiO2 support plate, a SiO2 glass, a dielectric layer, a first Teflon hydrophobic layer, a support layer, a second Teflon hydrophobic layer and an ITO glass layer; a magnetic bead nucleic acid extraction area and a dual-temperature nucleic acid amplification area are formed between the first Teflon hydrophobic layer and the second Teflon hydrophobic layer; The magnetic bead nucleic acid extraction area includes, in sequence: a lysis chamber, a first washing chamber, a second washing chamber, and an elution chamber; The dual-temperature nucleic acid amplification region includes: a low-temperature isothermal region and a high-temperature isothermal region; The SiO2 glass is provided with interdigitated electrodes and EWOD driving electrodes. The interdigitated electrodes are aligned with the lysis chamber to realize the electrolysis of bacterial samples. The EWOD driving electrodes are aligned with the elution chamber and the dual-temperature nucleic acid amplification region. (3) Provides an ultra-rapid detection kit for Salmonella enterica according to any one of claims 1 to 8, wherein 0.2 volumes of rapid hot-start DNA polymerase, 3.8 volumes of PCR reaction mixture, and 8 volumes of sample template DNA are added to the full-process nucleic acid detection chip; by controlling the EWOD driving electrode to be energized so that its surface becomes hydrophilic, the droplets circulate and move in the low temperature isothermal zone and the high temperature isothermal zone to achieve amplification; (4) After the reaction is complete, if there is an S-type amplification curve and the Ct value is within the reference range, it is positive; otherwise, it is negative.

10. The method for detecting Salmonella enterica genes according to claim 9, characterized in that, The reaction conditions described in step (3) are 95℃ for 50s; 95℃ for 2s, 60℃ for 2s, for 45 cycles.

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