Visual detection method for multiple pathogens based on top-speed PCR (Polymerase Chain Reaction)

Abstract

The invention discloses a multi-pathogen visual detection method based on top-speed PCR (Polymerase Chain Reaction), and belongs to the technical field of molecular biological detection. The invention designs specific primers and molecular beacons of salmonella Sa, vibrio parahaemolyticus VP and infectious subcutaneous and hematopoietic necrosis virus IHHNV for multiple PCR (Polymerase Chain Reaction), and three pathogens can be simultaneously amplified in a PCR system. In addition, the molecular beacon and the target are combined to expose the fluorophore, and the three pathogens can be distinguished without opening a cover by means of a self-made visual device. The multiple visual PCR detection method provided by the invention has the advantages of strong specificity, high sensitivity, short detection time, direct observation and the like, can realize simultaneous detection of multiple pathogens in a single sample, saves the cost, has higher economic value, and is suitable for on-site instant detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biological detection, and relates to the rapid visual detection of multiple pathogens. Specifically, it relates to an ultrafast visual detection system and method for Salmonella, Vibrio parahaemolyticus, and infectious hypodermal and hematopoietic necrosis virus, as well as an ultrafast visual detection system and method for Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus, which are pathogens related to mice. Background Art

[0002] Pathogens usually have high pathogenicity, the ability to spread rapidly, and cause widespread outbreaks. The presence of multiple strains further increases the pathogenic risk, exacerbates the severity of the disease, and complicates the detection. To address these challenges, there is an urgent need to develop a rapid, informative, sensitive, and specific method for simultaneously screening multiple pathogens.

[0003] Pathogen detection mainly relies on bacterial culture, immunoassay, and nucleic acid detection. Traditional bacterial culture-based methods are time-consuming and operationally intensive and complex. Immunoassays are specific and convenient, but the development of new antibodies to address emerging pathogens poses the challenge of a long process, and batch-to-batch differences in antibody performance further exacerbate this challenge. One major advantage of nucleic acid detection is the ability to directly target the pathogen's genome, enabling precise identification and facilitating adaptation to different environments. Polymerase chain reaction (PCR) is the gold standard for nucleic acid detection, and it benefits from the use of a single enzyme and relatively simple primer design, making it more promising for developing multiplex amplification. However, standard PCR typically takes up to two hours, and if sample transportation to a centralized laboratory is considered, the total turnaround time may exceed 24 hours. Therefore, establishing rapid nucleic acid amplification methods is crucial for the immediate detection of multiple pathogens.

[0004] Due to the simplicity, convenience, and low cost of visual detection methods, they show great potential for point-of-care testing. However, most of these methods rely on detecting amplification by-products generated, such as H + , Mg 2+ , and pyrophosphate, or use fluorescent dyes to identify the double-stranded structure of amplicons, rather than directly targeting the amplicon sequence. This limits their specificity and poses a challenge for achieving multiplex visual detection in a single reaction. In addition, many visual detection techniques involve open-lid and liquid handling steps, which greatly increase the risk of amplicon contamination. Therefore, an informative, specific, and sensitive one-pot visual detection method is crucial for simultaneously detecting multiple pathogens.

[0005] Herein, the present invention develops a rapid and highly specific multiplex visualization detection system by integrating rapid PCR and molecular beacons into a one-pot reaction. This method has the characteristics of visual observation, large amount of information, high sensitivity, good specificity, etc., and has good application prospects for on-site rapid detection. Summary of the Invention

[0006] The present invention aims to utilize rapid PCR technology to establish a multiplex pathogen visualization detection method to achieve rapid identification of multiple pathogens, so as to solve the problems of long time consumption, weak specificity, and easy contamination of amplicons existing in the prior art, and provide a new method for on-site detection.

[0007] To achieve the above object, the specific technical solutions adopted by the present invention are as follows: The present invention provides a multiplex pathogen visualization detection method based on rapid PCR, which is used for simultaneously detecting Salmonella Sa, Vibrio parahaemolyticus VP, and Infectious Hypodermal and Hematopoietic Necrosis Virus IHHNV, and includes the following steps: S1. DNA extraction of three pathogens, namely Salmonella, Vibrio parahaemolyticus, and Infectious Hypodermal and Hematopoietic Necrosis Virus; S2. Design three groups of upstream and downstream primer pairs and three probes for the pathogens to be detected in step S1 respectively; S3. Using the extracted genomic DNA as a template, perform a rapid multiplex PCR amplification reaction with the primer set and probes; S4. After the reaction ends, put the PCR tube into a self-made visualization device for fluorescence detection.

[0008] Preferably, in step S1, the method for extracting genomic DNA of the three pathogens is as follows: Take 1 mL of bacterial liquid for plate coating, then pick a single colony for secondary culture to obtain bacterial liquid, and use a commercial bacterial DNA extraction kit to extract genomic DNA, and store it in a refrigerator at -20 °C for standby.

[0009] Preferably, in step S2, the specific sequences of the three groups of primer pairs and probes are as follows: Specific primers and probes for detecting Salmonella Sa: Sa-FP: 5’-GCCCGGTAAACAGATGAGTA-3’ Sa-RP: 5’-TACCGCCAATAAAGTTCACA-3’ Sa-MB: 5’FAM-CCGAGGCGGCAATAGCGTCACCTTTCCTCGG-3’Dabcyl Specific primers and probes for detecting Vibrio parahaemolyticus VP: VP-FP: 5’-GAAAGTGCTTGAGATGAACG-3’ VP-RP: 5’-TCGAACAAGGCGTGAGTATC-3’ VP-MB: 5’HEX-CCGCGGGCTTTGTAGTACATCGCTTGTGCCTCCGCGG-3’BHQ1 Specific primers and probes for detecting Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV): IHHNV-FP: 5’-CGAAATCGACTGGAACCTACC-3’ IHHNV-RP: 5’-CCCAAATAGGCGAGGATTGTAG-3’ IHHNV-MB: 5’Texas Red-CCGAGGGAAGTTTGGCGTTCGTCCGGAGAGTCCTCGG-3’BHQ2 Preferably, in step S2, the amplification fragment length of the primer Sa-FP and the primer Sa-RP is 191 bp, the probe Sa-MB is modified with FAM, does not emit light when free, and emits green light after binding to the target.

[0010] Preferably, in step S2, the amplification fragment length of the primer VP-FP and the primer VP-RP is 99 bp, the probe VP-MB is modified with HEX, does not emit light when free, and emits yellow light after binding to the target; Preferably, in step S2, the amplification fragment length of the primer IHHNV-FP and the primer IHHNV-RP is 311 bp, the probe IHHNV-MB is modified with Texas Red, does not emit light when free, and emits red light after binding to the target.

[0011] Preferably, in step S3, the multiplex PCR amplification system is as follows: 5 U / μL Taq HS DNA Polymerase 1.25 μL, 10x PCR Buffer 2.5 μL, dNTP Mixture 2 μL, 1 μL each of the upstream and downstream primers of three groups of specific primers with a concentration of 10 μM each, 1.5 μL each of three probes with a concentration of 10 μM each, 1 μL of template each, and made up to 25 μL with enzyme-free water for PCR amplification.

[0012] Preferably, in step S3, the multiplex PCR reaction conditions are: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 2 s, annealing and extension at 55°C for 5 s, and amplification for 40 cycles.

[0013] Preferably, in step S4, the self-made visualization device mainly consists of an LED lamp, an excitation filter, an emission filter, a sample hole, and an observation hole.

[0014] Preferably, in step S4, during the visualization detection, if one or more of green fluorescence, yellow fluorescence, and red fluorescence are observed, it indicates that the detected pathogens are one or more of Salmonella, Vibrio parahaemolyticus, and Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV).

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention significantly shortens the thermal cycling time from several minutes to several seconds and reduces the total PCR reaction time from several hours to several minutes. The entire detection process can be completed within just 10 minutes.

[0016] (2) The primers provided by the present invention have strong specificity and can simultaneously amplify these three target genes from a mixed template of Salmonella, Vibrio parahaemolyticus, and IHHNV. The three molecular beacons provided directly target the three corresponding amplicons and are labeled with different fluorescent groups FAM, HEX, and Texas Red, thereby achieving one-pot multiplex detection.

[0017] (3) The portable visualization device designed by the present invention allows direct visual identification of fluorescence, with a cost of only 10 yuan, showing high economic benefits.

[0018] Therefore, the detection system of the present invention uses specially designed molecular beacons to target pathogen sequences, facilitating specific multiplex visualization detection, and shortening the entire detection time to 10 minutes. In terms of sensitivity, the detection method shows high sensitivity, and the detection limit of the multiplex detection reaction is as low as 23 copies. In terms of specificity, the detection system of the present invention can only detect Salmonella, Vibrio parahaemolyticus, and Infectious Hypodermal and Hematopoietic Necrosis Virus, and does not react with other common pathogens such as Klebsiella pneumoniae, Helicobacter pylori, Escherichia coli, Pseudomonas aeruginosa, and Severe Acute Respiratory Syndrome Coronavirus 2. In addition, the detection system of the present invention achieves a 100% positive detection rate and 100% specificity in practical applications, and its performance is comparable to that of standard PCR. The detection system of the present invention uses a one-pot reaction, without the need for opening the lid and liquid handling steps, greatly simplifying the operation and avoiding the risk of amplicon contamination. It is easy to implement and can quickly adapt to various fields, including food safety detection and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a graph showing the optimized results of ultra-fast PCR time; Figure 1 a~ Figure 1 c is Figure 1Optimization of denaturation and annealing times for ultrafast PCR as shown in the ultrafast PCR time optimization results graph, denaturation at 95 °C for 1 - 3 s, annealing at 55 °C for 1 - 7 s; Figure 1 d is Figure 1 Feasibility of multiplex ultrafast PCR pathogen detection as shown in the ultrafast PCR time optimization results graph, lane M: 50 bp DNA Marker, lanes 1 - 6: single - ultrafast PCR amplification of Sa, VP, and IHHNV, lanes 7 - 8: triple - ultrafast PCR amplification of the three pathogens; Figure 2 is the multiplex visualization detection result graph for pathogen detection; Figure 2 a is Figure 2 Molecular beacon design and performance evaluation as shown in the multiplex visualization detection result graph for pathogen detection. Specific molecular beacons designed for Sa, VP, and IHHNV are labeled with FAM, HEX, and Texas Red respectively. The fluorescence change △F = F - F0, where F represents the real - time fluorescence value and F0 represents the initial fluorescence value; Figure 2 b is Figure 2 Detailed schematic diagram of the self - made portable visualization device as shown in the multiplex visualization detection result graph for pathogen detection. It includes an LED lamp, a sample well, and excitation and emission filters. After PCR amplification, the reaction tube is placed in the sample well, and under the excitation of the LED lamp, the fluorescence generated in each tube can be directly observed by the naked eye through the observation hole; Figure 2 c is Figure 2 Visualization graph observed using the self - made portable device as shown in the multiplex visualization detection result graph for pathogen detection. Green fluorescence corresponds to the FAM - labeled molecular probe, representing the presence of Salmonella, yellow fluorescence corresponds to the HEX - labeled molecular beacon, representing the presence of Vibrio parahaemolyticus, and red fluorescence corresponds to the Texas Red - labeled molecular beacon, representing the presence of infectious hypodermal and hematopoietic necrosis virus; Figure 3 is the molecular beacon concentration optimization result graph. The fluorescence intensity at different beacon concentrations (0.1 - 1 μM) is observed using a portable cartridge. Here, "-" indicates no template added, while "+" indicates template added; Figure 4 is the sensitivity and specificity detection result graph; Figure 4a is Figure 4 Sensitivity and specificity detection result graph shows the use of 10 - fold serial dilutions of genomic DNA templates (10 7 -10 0Visual detection results of MVPCR for Sa, VP, and IHHNV with different copy numbers. For each concentration, three replicates were analyzed, and only one representative fluorescence image is shown; Figure 4b Yes Figure 4 Results of standard PCR real-time fluorescence detection of Sa, VP, and IHHNV using 10-fold serial dilutions of genomic DNA templates (10 7 -10 0 copy numbers). △F = F - F0, where F represents the real-time fluorescence value and F0 represents the initial fluorescence value (n = 3); Figure 4c Yes Figure 4 3% agarose gel electrophoresis shown in the sensitivity and specificity detection results was used to evaluate the specificity of MVPCR. Lane M: 50 bp DNA Marker, Lanes 1 - 8: Sa, VP, IHHNV, Kleb, HP, E.Coli, SARS-CoV-2, PA, Lane 9: NTC, NTC: no-template control; Figure 4d Yes Figure 4 Specificity detection of Sa, VP, IHHNV, Kleb, HP, E.Coli, SARA-CoV2, and PA using standard PCR as shown in the sensitivity and specificity detection results (n = 3), ***P < 0.001, n = 3, ns: no significant difference; Figure 4e Yes Figure 4 Visualization images observed using a self-made portable visualization device as shown in the sensitivity and specificity detection results; Figure 5 Is the detection result map of spiked samples; Figure 5a Yes Figure 5 Flow chart of MVPCR for detecting spiked samples as shown in the detection result map of spiked samples; Figure 5b Yes Figure 5 Standard PCR real-time fluorescence curves and MVPCR visualization results of 20 spiked samples as shown in the detection result map of spiked samples. Among them, "-" indicates no template added, and "+" indicates template added; Figure 6 Is the detection result map of triple detection of mouse pathogens; Figure 6 a is Figure 6 Schematic diagram of MVPCR detection of triple mouse pathogens as shown in the detection result map of triple detection of mouse pathogens; Figure 6 b is Figure 6Triplex test results for mouse pathogens Standard PCR triplex test results shown in the figure; Figure 6 c is Figure 6 MVPCR visualization results shown in the figure for triplex detection of mouse pathogens. DETAILED DESCRIPTION

[0020] The implementation of the present invention is described in detail below in conjunction with the embodiments of the present invention. The following embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operating processes, but the protection scope of the present invention is not limited to the following embodiments.

[0021] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. Among them: The bacterial DNA extraction kit was purchased from Tiangen Biotechnology Co., Ltd., and Taq HS DNA Polymerase, 10xPCR Buffer, dNTP Mixture, and 50bp DNA Marker were purchased from TaKara Company. Example

[0022] Extraction of pathogen genomic DNA in this example: Take 6 μL of glycerol bacteria and add 6 mL of LB liquid culture medium. The culture was incubated at 37 °C with shaking at 180 rpm for 16 hours. Then 1 mL of the bacterial suspension was streaked onto an LB agar plate and incubated upside down at 37 °C for 12-16 hours. Pick a single colony and culture it in liquid culture medium for another 12-16 hours to obtain a bacterial suspension. Use a bacterial DNA extraction kit to extract the genomic DNA of Sa, VP and IHHNV and store it in a refrigerator at -20 °C for later use. Example

[0023] In this example, primers and probes were designed based on the gene sequence information included in GenBank. The sequences of three sets of specific primers and probes and the sizes of amplified fragments are shown in Table 1.

[0024] Table 1 Multiplex PCR primer and probe sequences

[0025] Example 3 This example is an extremely fast PCR time detection of three pathogens.

[0026] Using the above-extracted and preserved genomic DNA (Sa, VP, IHHNV) as a template, add 1.25 μL of 5 U / μL Taq HS DNA Polymerase, 2.5 μL of 10x PCR Buffer, 2 μL of dNTP Mixture, 1 μL each of the upstream and downstream primers of three groups of specific primers with a concentration of 10 μM, and make up to 25 μL reaction system with water. Perform a hot start at 95°C for 30 s, then denature at 95°C (1 s or 2 s or 3 s) and anneal and extend at 55°C (1 s or 3 s or 5 s or 7 s) for a total of 40 cycles. The amplified products were analyzed by 3% agarose gel and run at 120 V for 40 min. Although longer thermal cycling times produce more amplified products, the shortest and most effective extreme PCR conditions are denaturation at 95°C for 2 s and annealing and extension at 55°C for 5 s, see Figure 1 a. Next, the feasibility of triple amplification under rapid cycling conditions was tested by combining three primer sets in a one-pot reaction. Lane 8 successfully showed three distinct bands, and each band corresponded to the target amplicon in a single reaction, confirming the effectiveness of extreme PCR amplification, see Figure 1 b. Example

[0027] This example is a study on the detection performance of a portable cartridge.

[0028] Using the above-extracted and preserved genomic DNA (Sa, VP, IHHNV) as a template, add 1.25 μL of 5 U / μL Taq HS DNA Polymerase, 2.5 μL of 10x PCR Buffer, 2 μL of dNTP Mixture, 1 μL each of the upstream and downstream primers of three groups of specific primers with a concentration of 10 μM, and 1.5 μL each of three probes with a concentration of 10 μM, and make up to 25 μL reaction system with water. For real-time fluorescence monitoring, use standard PCR reaction conditions (denaturation at 95°C for 20 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, for a total of 40 cycles), and a real-time increase in fluorescence was observed, confirming the successful hybridization of the beacon with the target, and the fluorescence channels of the 3 beacons work independently without cross-interference, see Figure 2 a. In addition, to reduce the dependence on dedicated equipment, a portable cartridge was designed, equipped with an LED and a filter, a sample well and an observation well, suitable for different excitation and emission wavelengths. The specific information is shown in Table 2, allowing direct visual detection with the naked eye.

[0029] Table 2 Different excitation and emission wavelengths

[0030] During PCR amplification, a large number of beacon MBs hybridize with their targets, resulting in the disruption of the stem-loop structure. The exposed fluorophores are excited by the LED light passing through the excitation filter, emitting bright fluorescence. The emitted fluorescence passes through the emission filter again to produce monochromatic light, making it visible to the naked eye. In the portable cartridge, the FAM-labeled probe targeting Salmonella emits green fluorescence, the HEX-labeled probe targeting Vibrio parahaemolyticus emits yellow fluorescence, and the TEX-labeled probe targeting IHHNV emits red fluorescence. See Figure 2 b. Example

[0031] This example is for optimizing the beacon concentration.

[0032] Beacon concentrations of 0.1 - 1 μM were selected for rapid PCR, and the corresponding visual detection effects were evaluated using a portable cartridge. As the beacon concentration increased successively, the observed fluorescence intensity also gradually increased, and the discrimination between positive and negative samples became clearer. However, at a beacon concentration of 1 μM, weak fluorescence was also observed in negative samples. The optimal beacon concentration was 0.6 μM. See Figure 3 for details. Example

[0033] This example is for detecting the sensitivity and specificity of the MVPCR method.

[0034] The sensitivity of the MVPCR method was further evaluated using 10-fold serial dilutions of genomic DNA templates from three pathogens. The detection limits of Salmonella, Vibrio parahaemolyticus, and infectious hypodermal and hematopoietic necrosis virus were visually observed to be 23, 1.6×10², and 2.1×10² copies per reaction, respectively. See Figure 4a for details. In addition, by standard PCR detection, the detection limits of Salmonella, Vibrio parahaemolyticus, and IHHNV were 23, 1.6×10², and 2.1×10² copies per reaction, respectively. The MVPCR method showed comparable sensitivity to the standard PCR method. See Figure 4b for details. This finding confirmed that the MVPCR method does not affect the amplification sensitivity. Even under rapid thermal cycling conditions, the beacon can successfully hybridize with the target, causing the stem to open and producing fluorescence visible to the naked eye. Therefore, the proposed MVPCR method is extremely fast (<10 min), highly sensitive (detection limit as low as 23 copies), and can distinguish results by the naked eye, making it suitable for point-of-care testing.

[0035] The specificity of the MVPCR method was evaluated using three target pathogens, Salmonella Sa, Vibrio parahaemolyticus, and infectious hypodermal and hematopoietic necrosis virus IHHNV, and five other non-target pathogens (Klebsiella Kleb, Helicobacter pylori HP, severe acute respiratory syndrome coronavirus 2, Escherichia coli E.Coli, Pseudomonas aeruginosa PA). Gel electrophoresis showed that only the MVPCR amplification products of Salmonella, Vibrio parahaemolyticus, and infectious hypodermal and hematopoietic necrosis virus were detected, see Figure 4c . Similarly, the standard PCR results showed that only the fluorescence signals of Sa, VP, and IHHNV were monitored, which was consistent with the MVPCR results, see Figure 4d . In addition, using a portable cartridge for visual observation, only Salmonella Sa was detected in the FAM channel, only Vibrio parahaemolyticus VP was detected in the HEX channel, and only infectious hypodermal and hematopoietic necrosis virus IHHNV was detected in the TEX channel, see Figure 4e . These results verified the high specificity of the MVPCR method and demonstrated the absence of fluorescence crosstalk. Therefore, the MVPCR method showed high specificity for visualizing multiplex pathogen detection. Example

[0036] This example is the detection of spiked shrimp samples.

[0037] Fresh shrimp were purchased from a local supermarket, decapitated, and washed with deionized water. Then the shrimp were placed in a biosafety cabinet and exposed to ultraviolet light for 30 min. After that, the shrimp were immersed in a 6 mL bacterial suspension (Sa, VP, and IHHNV) of a series of concentrations in a 15 mL test tube and incubated at room temperature for 30 minutes. For single pathogen infection, the shrimp were immersed in the corresponding bacterial suspension, while for infections involving two or three pathogens, they were immersed in a mixed bacterial suspension containing the same proportion of each pathogen. After incubation, the shrimp samples were transferred to a clean plate and placed in the biosafety cabinet for another 30 minutes to promote bacterial attachment. Then the spiked shrimp samples were put into a mortar and ground, and then 200 μL of deionized water was added to elute the pathogens.

[0038] DNA extraction was performed using a NaOH-based method. Specifically, 50 μL of the eluted pathogen suspension was mixed with 200 μL of 0.5 M NaOH, incubated at room temperature for 3 min, and 1 μL of the cell lysate was directly used as a template for MVPCR detection. At the same time, standard PCR was performed as a reference. The presence of Salmonella was indicated by observing green fluorescence with the aid of a portable cartridge, the presence of Vibrio parahaemolyticus was indicated by observing yellow fluorescence, and the presence of infectious hypodermal and hematopoietic necrosis virus was confirmed by observing red fluorescence, see Figure 5aMVPCR was used to analyze 20 pathogen-spiked shrimp samples, with standard PCR as a reference. The visual detection results were consistent with the real-time fluorescence curves of standard PCR, demonstrating the reliability, stability and high specificity of the MVPCR method, see Figure 5b Therefore, by using a portable cartridge (4 × 5 × 2 cm³, 25 g, 10 RMB), the MVPCR method has shown high applicability for multiple pathogen detection in point-of-care testing. Example

[0039] This example is the detection of mouse feces samples.

[0040] Further expansion, the MVPCR method was applied to multiple practical tests in mice. Mice are widely regarded as an indispensable model organism in laboratory research. In order to maintain biosafety and ensure uninterrupted research, regular screening of infected mice is essential. Mice are susceptible to a variety of pathogens, including Pseudomonas aeruginosa PA, Klebsiella pneumoniae Kleb, and Staphylococcus aureus S.au.

[0041] In this example, based on the gene sequence information included in GenBank, three sets of specific primers and probe sequences and amplified fragment sizes designed for mouse pathogens are shown in Table 3.

[0042] Table 3 Primer and probe sequences for mouse multiplex PCR

[0043] The proposed method was applied to detect all three pathogens simultaneously. Standard PCR was performed in parallel as a reference. A series of stool samples were spiked with one or more pathogens, and the concentration of bacterial suspensions ranged from 10 to 10³ CFU / mL. To further challenge the proposed method, we selected low-concentration samples with Ct values ​​exceeding 35 in the standard PCR, see Figure 6 b. Based on the visual inspection results, we can easily identify the infected samples and determine the specific pathogens present, with only Pseudomonas aeruginosa PA detected in the FAM channel, Klebsiella pneumoniae Kleb detected in the HEX channel, and Staphylococcus aureus S.au detected in the TEX channel. Infected samples exhibited fluorescence visible to the naked eye, while uninfected samples showed no fluorescence. See Figure 6 c. Therefore, the established MVPCR method has strong feasibility and can be easily applied to multiplex nucleic acid detection in various fields.

[0044] The embodiments of the present invention have been specifically described above. However, the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A multiple pathogen visualization detection method based on ultra-fast PCR, characterized in that: The following steps are involved: S1, DNA extraction of three pathogens Salmonella, Vibrio parahaemolyticus, and IHHNV; S2, designing three sets of upstream and downstream primer pairs and three probes for the pathogen to be detected in step S1; S3, using the extracted genomic DNA as a template, a rapid multiplex PCR amplification reaction was performed using primer sets and probes; S4. After the reaction is completed, the PCR tube is placed in a homemade visualization device for fluorescence detection.

2. The method for visualizing multiple pathogen detection based on ultra-fast PCR according to claim 1, characterized in that: In step S1, the method for extracting DNA of the three pathogens is: taking 1 mL of bacterial solution for plate coating, then picking a single colony for secondary culture to obtain bacterial solution, using a commercial bacterial DNA extraction kit to extract genomic DNA, and storing it in a refrigerator at -20°C for later use.

3. The method for visualizing multiple pathogen detection based on ultra-fast PCR according to claim 1, characterized in that: In step S2, the specific sequences of the primer set and probe are as follows: Specific primers and probes for detecting Salmonella Sa: Sa-FP:5'-GCCCGGTAAACAGATGAGTA-3' Sa-RP:5'-TACCGCCAATAAAGTTCACA-3' Sa-MB: 5'FAM-CCGAGGCGGCAATAGCGTCACCTTTCCTCGG-3'Dabcyl Specific primers and probes for detecting Vibrio parahaemolyticus VP: VP-FP:5'-GAAAGTGCTTGAGATGAACG-3' VP-RP:5'-TCGAACAAGGCGTGAGTATC-3' VP-MB: 5'HEX-CCGCGGGCTTTGTAGTACATCGCTTGTGCCTCCGCGG-3'BHQ1 Specific primers and probes for the detection of infectious hypodermal and hematopoietic necrosis virus IHHNV: IHHNV-FP: 5'-CGAAATCGACTGGAACCTACC-3' IHHNV-RP: 5'-CCCAAATAGGCGAGGATTGTAG-3' IHHNV-MB: 5'Texas Red-CCGAGGGAAGTTTGGCGTTCGTCCCGGAGAGTCCTCGG-3'BHQ2.

4. The method for visualizing multiple pathogen detection based on ultra-rapid PCR according to claim 3, characterized in that: The amplified fragment length of the primer Sa-FP and the primer Sa-RP is 191 bp, and the probe Sa-MB is modified with FAM, does not emit light when free, and emits green light after binding to the target; The amplified fragment length of the primer VP-FP and the primer VP-RP is 99 bp, and the probe VP-MB is modified with HEX, does not emit light when free, and emits yellow light after binding to the target; The amplified fragment length of the primer IHHNV-FP and the primer IHHNV-RP is 311 bp, and the probe IHHNV-MB is modified with Texas Red, does not emit light when free, and emits red light after binding to the target.

5. The method for visualizing multiple pathogen detection based on ultra-fast PCR according to claim 1, characterized in that: In step S3, the multiplex PCR amplification system is 25 μL, including 1.25 μL of 5U / μL Taq HS DNA Polymerase, 2.5 μL of 10x PCR Buffer, 2 μL of dNTP Mixture, 1 μL of each of the upstream and downstream primers of three sets of specific primers with a concentration of 10 μM, 1.5 μL of each of three probes with a concentration of 10 μM, and 1 μL of each template, which is filled to 25 μL with enzyme-free water for PCR amplification.

6. The method for visualizing multiple pathogen detection based on ultra-fast PCR according to claim 1, characterized in that: In step S3, the multiplex PCR reaction conditions are: pre-denaturation at 95°C for 30s, denaturation at 95°C for 1-3s, annealing and extension at 55°C for 1-7s, and amplification for 40 cycles.

7. The method for visualizing multiple pathogen detection based on ultra-fast PCR according to claim 1, characterized in that: In step S4, the homemade visualization device is mainly composed of an LED lamp, an excitation filter, an emission filter, a sample hole and an observation hole.

8. The method for visualizing multiple pathogen detection based on ultra-fast PCR according to claim 1, characterized in that: In step S4, when the visualization device performs detection, if one or more of green fluorescence, yellow fluorescence, and red fluorescence are observed, it indicates that the detected pathogens are one or more of Salmonella, Vibrio parahaemolyticus, infectious hypodermic and hematopoietic necrosis viruses, respectively.

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