Detection kit for simultaneously detecting staphylococcus aureus and salmonella based on cdLAMP and detection method thereof

Abstract

The invention discloses a cdLAMP-based detection kit for simultaneously detecting staphylococcus aureus and salmonella and a detection method thereof, and relates to the field of biological detection.The detection kit comprises a silicon-based nano porous chip, a primer probe combination, 20 mM Tris-HCl, 10 mM KCl, 8 mM MgSO4, 10 mM (NH4) 2SO4, 0.05% Tween-20, a 2.5 mu L DNA template, 1.4 mM dNTP, 0.16 U / mu L Bst DNA polymerase and DEPC; the primer probe combination comprises a primer probe and a primer probe, wherein the primer probe is designed according to a nuc gene of staphylococcus aureus: GenBank registration number EF529607.1, and the primer probe is designed according to a fimY gene of salmonella: GenBank registration number JQ665438.1; according to the invention, dual detection is realized through a chip and a two-color fluorescence channel carried by an imaging analyzer, and compared with the existing detection method, the method has higher sensitivity and accuracy.

Description

Technical Field

[0001] The invention relates to the field of biological detection, in particular to a detection kit and a detection method thereof for simultaneously detecting Staphylococcus aureus and Salmonella based on cdLAMP. Background Art

[0002] Foodborne diseases are an increasingly serious public health problem, greatly increasing the global burden of disease and mortality and causing considerable socioeconomic impacts. According to the World Health Organization, nearly one in ten people worldwide become ill after consuming contaminated food each year, resulting in more than 420,000 deaths. Foodborne pathogens are one of the main factors causing foodborne diseases. Usually, the foodborne pathogens that cause diseases mainly include Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus, Staphylococcus aureus, and pathogenic Escherichia coli. It is reported that there are about 90 million cases of Salmonella infection worldwide each year. Among them, Salmonella enteritidis accounts for the largest proportion. Staphylococcus aureus (STA) is a Gram-positive bacterium that can cause foodborne diseases. Ingestion of food contaminated with STA may cause symptoms such as febrile illness, enteritis, and food poisoning. Therefore, the detection of STA and Salmonella (SAL) is crucial for food safety.

[0003] Culture methods are considered the gold standard for bacterial detection. By using selective media for enrichment, bacterial populations can be concentrated and target pathogens can be selectively proliferated to facilitate their isolation and detection. Culture methods have high accuracy, but the detection process is long. In recent years, molecular detection technologies have shown great potential in pathogen detection, characterized by extremely high sensitivity and rapid turnaround time. Detection methods based on polymerase chain reaction (PCR) have achieved three significant technological advances. The first generation of PCR was limited to qualitative analysis. The second generation of quantitative PCR (qPCR) uses relative quantification to determine the amount of target nucleic acid using a standard curve or reference gene. This method has become the gold standard for laboratory diagnosis. At the end of the 20th century, Vogelstein proposed the concept of digital PCR (dPCR), marking the arrival of the third generation of PCR in the field of molecular diagnosis. Based on the principle of Poisson distribution, dPCR divides nucleic acid samples into many tiny reaction units to achieve single molecule template amplification, fluorescence detection and subsequent statistical analysis. This methodology can achieve absolute quantification of target genes, thereby simplifying the analysis process.

[0004] In recent years, dPCR has played an important role in pathogen detection, nucleic acid quantification, copy number variation (CNV) detection, and chromosome abnormality detection. However, thermal cycling has hindered the application of this technology in resource-limited settings. Digital nucleic acid amplification technology includes digital thermal cycling amplification technology and digital isothermal amplification technology. Digital isothermal amplification technology combines the concept of digitization with the isothermal nucleic acid amplification method, overcoming the inherent temperature regulation limitations of dPCR and the challenges of traditional isothermal technology in quantification. Loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA) are outstanding representatives of isothermal amplification technologies that are becoming increasingly popular in molecular diagnosis and pathogen detection because they can operate at a constant temperature, have high efficiency, fast processing, and low equipment requirements. Among them, loop-mediated isothermal amplification (LAMP) technology has a high degree of specificity even in the presence of non-target DNA because it uses 4 to 6 primers to react simultaneously. In 2011, Gansen et al. first developed digital LAMP (dLAMP) technology on a microfluidic chip. Compared with dPCR, dLAMP technology not only provides higher sensitivity and accuracy, but also shows stronger anti-interference ability and higher specificity in the face of inhibitory substances. In addition, dLAMP reduces the complexity of operation and equipment requirements.

[0005] The current dLAMP technology is mainly divided into three categories: droplet-based, i.e., ddLAMP, chip-based, i.e., cdLAMP, and other types of dLAMP. Currently, most of the dLAMPs on the market are ddLAMPs, which usually use microfluidics to produce monodisperse droplets of uniform volume. During operation, droplets are prone to evaporation, rupture, and fusion, resulting in aerosol contamination and relatively large errors. In terms of droplet reading, one method is to read the fluorescence image of stationary droplets, which is labor-intensive and difficult to scale up. Another method is to detect the fluorescence image of droplets in a flow-based manner, which has high throughput, but it requires the use of complex equipment such as lasers and photomultiplier tubes to excite and detect signals, which makes the cost of the platform relatively high. With the development of microfabrication technology, high-throughput nanoliter or even picoliter cdLAMP reactions have been achieved on planar substrates. cdLAMP automatically and evenly distributes the configured reaction solution to the microreaction chambers on the chip for amplification. After the amplification is completed, the fluorescence signal of each microreaction chamber is scanned to perform quantitative analysis of the sample. Currently, digital systems are implemented using pressure, gel, valve, vacuum, or sliding chips. Although many improvements have been made, complex fluid control is still required. In addition, in order to avoid potential contamination caused by cumbersome cleaning and reuse, there is an urgent need for a method that can sensitively and accurately detect Staphylococcus aureus and Salmonella in food, providing a powerful analytical platform for food safety testing. The present invention solves such a problem. Summary of the invention

[0006] To address the deficiencies of the prior art, the present invention aims to provide a detection kit and a detection method for simultaneously detecting Staphylococcus aureus and Salmonella based on cdLAMP, which can sensitively and accurately detect Staphylococcus aureus and Salmonella typhimurium in aquatic products.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A cdLAMP-based detection kit for simultaneous detection of Staphylococcus aureus and Salmonella, including: a silicon-based nanoporous chip, a primer-probe combination, 20 mM Tris-HCl, 10 mM KCl, 8 mM MgSO 4 , 10 mM (NH 4 ) 2 SO 4 , 0.05% Tween-20, 2.5 μL DNA template, 1.4 mM dNTP, 0.16 U / μL Bst DNA polymerase and DEPC; the primer probe combination includes: a primer probe designed according to the nuc gene of Staphylococcus aureus: GenBank accession number EF529607.1, and a primer probe designed according to the fimY gene of Salmonella: GenBank accession number JQ665438.1.

[0009] The aforementioned detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP, the primer probe combination includes: 0.2μM outer primer, 1.2μM FIP, 0.4μM FIP probe, 1.6μM BIP and 0.8μM loop primer; the outer primers include: STA-F3, STA-B3, SAL-F3 and SAL-B; FIP includes: STA-FIP and SAL-FIP; FIP probe includes: STA-FIP probe and SAL-FIP probe; BIP includes: STA-BIP and SAL-BIP; loop primers include: STA-LF, STA-LB and SAL-LF.

[0010] In the aforementioned detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP, the sequence of STA-F3 is SEQ ID No: 01, the sequence of STA-B3 is SEQ ID No: 02, the sequence of STA-FIP is SEQ ID No: 03, the sequence of STA-BIP is SEQ ID No: 04, the sequence of STA-LF is SEQ ID No: 05, the sequence of STA-LB is SEQ ID No: 06, the sequence of STA-FD is SEQ ID No: 07, the sequence of STA-Probe is SEQ ID No: 08, the sequence of SAL-F3 is SEQ ID No: 09, the sequence of SAL-B3 is SEQ ID No: 10, the sequence of SAL-FIP is SEQ ID No: 11, the sequence of SAL-BIP is SEQ ID No: 12, the sequence of SAL-LF is SEQ ID No: 13, the sequence of SAL-FD is SEQ ID No: 14, and the sequence of SAL-Probe is SEQ ID No: 15.

[0011] The aforementioned detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP, the silicon-based nanoporous chip contains 20,000 micropores, each micropore has a uniform volume and a pore size of 50μm, and the micropores include: micropore side walls, micropore surfaces, nano-hydrophilic layers, and nano-hydrophobic layers modified on the micropore surfaces.

[0012] In the aforementioned cdLAMP-based detection kit for simultaneous detection of Staphylococcus aureus and Salmonella, the nano-hydrophobic layer was modified with silane by microcontact printing. The specific method included: ultrasonically cleaning the chip with trichloroethylene, acetone and ethanol in sequence, and then immersing it in H 2 SO 4 :H 2 O 2 The chip was treated at 60°C for 1.5 hours in a solution with a volume ratio of 7:3, then ultrasonically cleaned twice with deionized water and ethanol, and blown dry with nitrogen; a mold with a thickness of about 0.2 mm was immersed in (1H,1H,2H,2H-perfluorodecyl)trimethoxysilane and dried under a nitrogen flow to remove excess reagents; the mold was placed on the chip, pressed for 20 seconds, and then removed; the chip was heated to 90°C for 30 minutes; the chip was rinsed with deionized water and purged with nitrogen to remove residual reagents.

[0013] The detection method of the detection kit for simultaneously detecting Staphylococcus aureus and Salmonella based on cdLAMP, such as the detection method of the aforementioned detection kit, comprises the following steps:

[0014] Step 1, DNA preparation;

[0015] Step 2: Design of LAMP primers and probes;

[0016] Step 3, preparing the cdLAMP reaction system;

[0017] Step 4, preparing the chip;

[0018] Step 5: Sample allocation:

[0019] Using a disposable chip with a size of 1×1 cm, a scraper with a size of 10×43 mm and a cover plate, 20 μL of sample is introduced into the scraper, and the sample is fixed at the bottom of the scraper; when the preparation instrument is started, the sample is scraped into the micropores of the chip; after the sample is loaded, the instrument automatically seals with oil, and the amount of sealing oil for each chip is 250 μL; finally, the cover plate is covered, and the chip preparation is completed; the chip is placed on the amplification instrument for reaction, and after the amplification is completed, the chip is placed in the imaging analyzer;

[0020] Step 6: Capture fluorescence images:

[0021] The imaging analyzer is equipped with an optical dual-color fluorescence channel designed for FAM and Texas Red, which can detect dual signals emitted by each reaction well by switching the fluorescence channels; a CMOS detector is integrated to ensure accurate image capture; using the statistical framework of Poisson distribution, the data analysis software automatically calculates the initial copy number of the target molecule; the copy number of the template DNA is calculated by the following formula:

[0022] c(copies / μL)=-In(1-a / b) / v

[0023] Wherein, a represents the number of positive reaction wells, b represents the total number of effective reaction wells, v represents the volume of each reaction unit, and the ratio a / b represents the positive reaction rate.

[0024] 7. The detection method of the detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP according to claim 6, characterized in that the specific method of preparing the chip in step 4 comprises:

[0025] Preparation: The silicon chip is cleaned with acetone, anhydrous ethanol and deionized water in sequence, and then exposed to hexamethyldisilane vapor, the hydrophilic hydroxyl groups on the surface of the silicon wafer are replaced by hydrophilic groups, and then the silicon wafer is placed on a hot plate at 140°C for 10 minutes and spin-coated to form a 5-micron thick photoresist layer; the chip is pre-baked at 100°C for 5 minutes, then exposed to a photolithography machine and post-baked, a regular hexagonal reaction microwell array with a pitch of 20 microns and a diameter of 50 microns is patterned onto the photoresist, and the microwell array is formed by photoresist cleaning, inductively coupled plasma etching and diamond grinding processes;

[0026] Surface modification: The chip was ultrasonically cleaned with trichloroethylene, acetone, and ethanol, and then immersed in H 2 SO 4 :H 2 O 2 The method comprises the following steps: first, treating the chip with a volume ratio of 7:3 and treating at 60°C for 1.5 hours; then ultrasonically cleaning the chip with deionized water and ethanol twice and blowing dry with nitrogen; modifying the chip with silane by microcontact printing to form a hydrophobic surface; using a mold with a thickness of about 0.2 mm to be immersed in (1H,1H,2H,2H-perfluorodecyl)trimethoxysilane and drying it under a nitrogen flow for about 5 seconds to remove excess reagents; placing the mold on the chip, pressing it for 20 seconds, and then removing it to ensure that a hydrophobic seal is formed; heating the chip to 90°C for 30 minutes; rinsing the chip with deionized water and blowing nitrogen to remove residual reagents; and storing the manufactured chip in a sealed environment at room temperature.

[0027] The present invention is beneficial in that:

[0028] The present invention realizes dual detection by using a chip with a dual-color fluorescence channel carried by an imaging analyzer, which has higher sensitivity than qPCR. By verifying spiked food and actual samples, it is confirmed that the detection limits of two pathogens detected within 45 minutes are 2.0 CFU / mL and single copy respectively. The dynamic detection range spans 5 orders of magnitude.

[0029] The present invention found that only in 0.05% Tween-20, 8mM Mg 2+ Under the synergistic effect of 0.16U / μL Bst DNA polymerase, the parameters such as fluorescence image, number of effective wells, number of positive wells and signal difference are optimal, ensuring the sensitivity and accuracy of the detection.

[0030] The validated method of the present invention has high accuracy and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a working diagram of cdLAMP of the present invention and a structural diagram of the chip and the imaging analyzer;

[0032] Figure 2 Schematic diagram of the optimization results of cdLAMP of the present invention; (a) the effect of Tween-20 on cdLAMP; (b) Mg 2+ Effect of concentration on cdLAMP; (c) Effect of reaction time on cdLAMP; (d) Effect of ratio of outer primer to inner primer on cdLAMP; (e) Effect of enzyme concentration on cdLAMP. (f) Effect of temperature on cdLAMP;

[0033] Figure 3Specificity, sensitivity and precision of cdLAMP; (a) Specificity of cdLAMP; (b) Using 10 0 Up to 10 4 Fluorescence images were recorded at 10 dilutions of STA and SAL. NTC was a negative control; the concentration was 10 0 -10 4 The dispersion of templates on the chip in CFU / mL; (c) The accuracy of cdLAMP for STA determination; “Low”, “Medium” and “High” represent 10 0 CFU / mL, 10 2 CFU / mL and 10 4 CFU / mL concentration; each concentration was measured 3 times a day for 10 consecutive days; (d) The signal difference of STA at 5 concentrations; (e) The linear fitting curve of STA was obtained by plotting the measured concentration against the expected concentration. The dark area represents the 95% confidence interval. The light area represents the 95% prediction interval; (f) The accuracy of cdLAMP for SAL determination; (g) The signal difference of SAL at 5 concentrations; (h) The linear fitting curve of SAL was obtained by plotting the measured concentration against the expected concentration; the dark area represents the 95% confidence interval; the light area represents the 95% prediction interval;

[0034] Figure 4 Sample analysis by BAM, cdLAMP, and qPCR. (a) Consistency of cdLAMP and BAM tests on 150 actual samples. (b) Comparison of the consistency of 150 actual samples detected by cdLAMP and qPCR. (c) Comparative analysis of the results of 150 actual samples detected by cdLAMP and qPCR. The repeated average detection values ​​of each sample are indicated using a color scale, representing a total of 150 samples. Blue, STA positive; red, SAL positive. The yellow box (sample 1) indicates that qPCR only detected SAL, while cdLAMP detected a mixed infection. The red box (sample 4) indicates that the pathogen is non-viable. The green box (sample 5) indicates that the qPCR test was negative, while cdLAMP detected STA;

[0035] Figure 5 It is a fluorescence image result diagram of the cdLAMP reaction system optimization experiment of the present invention;

[0036] Figure 6 It is a fluorescent image of the enzyme concentration optimization of the cdLAMP reaction system of the present invention; Figure 7 This is the sensitivity result diagram of qPCR detection of STA and SAL. DETAILED DESCRIPTION

[0037] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0038] 1. Materials and Methods

[0039] 1.1. DNA preparation

[0040] Twelve bacterial strains were cultured in this study, and the relevant information is presented in Table 1. Vibrio parahaemolyticus strains, Vibrio vulnificus strains, and Vibrio mimicus strains were inoculated into alkaline peptone water (APW, Qingdao, China) containing 3% sodium chloride. The remaining strains were inoculated into Luria-Bertani (LB) broth (Sangon, Shanghai, China), and all strains were incubated at 37°C for 18 h. Subsequently, single colonies were picked and transferred to 3 ml of sterile LB medium and incubated at 37°C for another 4 h. Then, the bacterial suspension was diluted 10-fold using sterile phosphate-buffered saline (PBS), and the viable count was determined by the dilution plating technique. 0 to 10 4 Colony forming units per milliliter (CFU / mL) of bacterial solution were used, and bacterial DNA was extracted from these dilutions using a rapid bacterial genomic DNA extraction kit (Sangon, Shanghai, China). A260 and A280 absorbance values ​​were measured using a NanoDrop spectrophotometer (Beckman Coulter, USA), and the A260 / A280 ratio was calculated to assess the quality and quantity of the extracted DNA. The purified DNA was then stored at -20°C for subsequent applications.

[0041] Table 1 Bacterial strain information

[0042]

[0043] 1.2. Design of LAMP primers and probes

[0044] Primers were designed based on the nuc gene of Staphylococcus aureus (GenBank accession number EF529607.1) and the fimY gene of Salmonella (GenBank accession number JQ665438.1). FD (fluorescent dye-labeled primer) was manually designed to be complementary to the F1c sequence in FIP. The complete sequences of these primers and FIP probes are listed in detail in Table 2. Quenched FIP (Q-FIP) and fluorescein-labeled FD were evenly diluted to 100 μM. Then, equal volumes of Q-FIP and FD were mixed, and after thorough mixing, they were heated at 95°C for 10 minutes. After cooling to room temperature, a 50 μM FIP probe solution was obtained. All prepared probes were stored at -20°C for preservation.

[0045] Table 2 cdLAMP primers and FIP probe sequences for detecting Staphylococcus aureus (STA) and Salmonella enteritidis (SAL)

[0046]

[0047]

[0048] 1.3.cdLAMP reaction

[0049] In the cdLAMP reaction system, the reaction buffer contained 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO 4 、10mM(NH 4 )2SO 4 and 0.05% Tween-20 (Sigma-Aldrich, St.Louis, USA). 0.2 μM outer primers (STA-F3, STA-B3, SAL-F3 and SAL-B), 1.2 μM FIP (STA-FIP and SAL-FIP), 0.4 μM FIP probe (STA-FIP probe and SAL-FIP probe), 1.6 μM BIP (STA-BIP and SAL-BIP) and 0.8 μM loop primer (STA-LF, STA-LB and SAL-LF), 2.5 μL DNA template, 1.4 mM dNTP (Thermo Fisher Scientific, Waltham, USA), 0.16 U / μL Bst 2.0 WS DNA polymerase, and DEPC, a total of 20 μL. TE was used as a template in the negative control. After loading, the chip was placed on a hot platform, amplified at 65 ° C for 1 h, and then cooled to 4 ° C. The chip is read after the reaction and the data is recorded and analyzed.

[0050] 1.4.Workflow

[0051] The instrument used in this study consists of a preparation instrument, an amplification instrument, and an imaging analyzer. The sample distribution process is fully automated and requires the use of a disposable chip with a size of 1×1 cm, a scraper with a size of 10×43 mm, and a cover plate. The scraper base is a clip-on type that can be firmly attached to the sample preparation equipment. Figure 1As shown in a, 20 microliters of sample are introduced into the scraper, and the sample is fixed at the bottom of the scraper. When the preparation instrument is started, the sample is scraped into the microwells of the chip to ensure that the sample volume allocated to each microwell is the same. After the sample is loaded, the instrument is automatically oil-sealed, and the amount of sealing oil for each chip is 250μL to avoid fusion or cross-contamination of each micro-reaction system. Finally, cover the cover plate and the chip preparation is completed. The whole process can be completed within 150s. The chip is placed on the amplification instrument for reaction. After the amplification is completed, the chip is placed in the imaging analyzer, the imaging is completed within 1min, and the copy number concentration in the system is automatically calculated within 3min.

[0052] 1.5. Chip Manufacturing

[0053] The silicon-based nanoporous chip, fabricated using microelectromechanical systems (MEMS) technology, contains 20,000 microreaction chambers, each with a uniform volume. Figure 1 b and 1c show the three main components and microstructure of the chip.

[0054] The chip manufacturing process was based on Li's work. The silicon chip was cleaned with acetone, anhydrous ethanol, and deionized water in sequence to remove surface contaminants, and then exposed to hexamethyldisilane vapor to enhance adhesion. The hydrophilic hydroxyl groups on the surface of the silicon wafer were replaced by hydrophilic groups. The silicon wafer was then placed on a hot plate at 140°C for 10 minutes and spin-coated to form a 5-micron-thick photoresist layer. The chip was first pre-baked at 100°C for 5 minutes, then exposed to a photolithography machine and post-baked. Next, a regular hexagonal reaction microwell array (20 micron pitch, 50 micron diameter) was patterned onto the photoresist. Finally, the microwell array was formed by photoresist cleaning, inductively coupled plasma etching, and diamond grinding processes.

[0055] Subsequently, the chip surface was modified. The chip was ultrasonically cleaned with trichloroethylene, acetone, and ethanol. Then, it was immersed in (H 2 SO 4 :H 2 O 2 =7:3) solution at 60°C for 1.5 hours. Afterwards, it was ultrasonically cleaned twice with deionized water and ethanol and dried with nitrogen. It was modified with silane by microcontact printing to form a hydrophobic surface. A mold with a thickness of about 0.2 mm was immersed in (1H,1H,2H,2H-perfluorodecyl)trimethoxysilane and dried under a nitrogen flow for about 5 seconds to remove excess reagents. Subsequently, the mold was placed on the chip, pressed for 20 seconds, and then removed to ensure that a hydrophobic seal was formed. Next, the chip was heated to 90°C for 30 minutes. Finally, the chip was rinsed with deionized water and purged with nitrogen to remove residual reagents. The manufactured chip was stored in a sealed environment at room temperature.

[0056] 1.6. Capturing fluorescence images

[0057] Figure 1 d shows the internal structure of the imaging analyzer. The analyzer is equipped with an optical dual-color fluorescence channel designed for FAM and Texas Red, which is able to detect dual signals emitted by each reaction well by switching the fluorescence channels. A CMOS detector is integrated to ensure accurate image capture. Using the statistical framework of Poisson distribution, the data analysis software automatically calculates the initial copy number of the target molecule. The copy number of the template DNA is determined using the following formula:

[0058] c(copies / μL)=-In(1-a / b) / v

[0059] Where a represents the number of positive reaction wells, b represents the total number of effective reaction wells, and v represents the volume of each reaction unit. The ratio a / b represents the positive rate.

[0060] 1.7.cdLAMP optimization

[0061] In the field of digital nucleic acid detection, the number of positive wells and signal difference are key parameters. Previous studies have optimized the ratio of FIP probe to total FIP content (Du et al., 2022). Therefore, the current study focused on optimizing the following variables: Tween-20 (0, 0.025%, 0.05%, 0.075%, and 0.1%), Mg 2+ concentration (2mM, 4mM, 6mM, 8mM, 10mM and 12mM), reaction time (35min, 40min, 45min, 50min and 55min), ratio of inner and outer primers (1:4, 1:6, 1:8, 1:1 and 1:12), enzyme concentration (0.08U / μL, 0.16U / μL, 0.32U / μL, 0.64U / μL and 1.28U / μL), reaction temperature (59°C, 61°C, 63°C, 65°C and 67°C). 10 3 The concentration of CFU / mL was optimized. Each optimization experiment was repeated three times to ensure reliability.

[0062] 1.8. cdLAMP specificity and sensitivity

[0063] In this study, the optimized cdLAMP method was tested for specificity using 12 bacterial strains listed in Table 1. 3 The concentration of CFU / mL was used for specific experiments.

[0064] The concentration was 10 0 Up to 10 4STA and SAL with a CFU / mL range were mixed in equal volumes to test the sensitivity of cdLAMP assay. TE was used as a negative control. Each experiment was repeated three times to ensure the reliability of the results.

[0065] 1.9. Accuracy of cdLAMP

[0066] The accuracy of cdLAMP was evaluated by using three different concentrations of STA and SAL. STA and SAL were selected at low (10 0 CFU / mL), medium (10 2 CFU / mL), high (10 4 The accuracy of the detection system was evaluated by three concentrations of 1000 CFU / mL. The samples were tested in batches every day, and each batch was repeated three times. The test was performed by different personnel in the same laboratory for 10 consecutive days. Finally, the average coefficient of variation (CV) within and between batches was calculated based on the test results to evaluate the repeatability and accuracy of the cdLAMP method.

[0067] 1.10. Application of cdLAMP in spiked food samples

[0068] A total of 18 food samples, including milk, pork, and eggs, were purchased from a local supermarket in Hangzhou, China. 25.0 ± 0.1 ml or 25.0 ± 0.1 g of each sample were prepared so that the concentration range of the food samples was 10 0 -10 4 CFU / mL. After sampling, centrifuge at 12000×g for 1 minute. The precipitate was then resuspended in 250 μL of lysis buffer (Uelandy, Suzhou, China) and incubated at 100°C for 10 minutes for cell lysis. The supernatant of the lysate was used as a template for cdLAMP and qPCR kits (Takara, Tokyo, Japan). Each concentration was tested three times.

[0069] 1.11. Testing of on-site samples

[0070] In this study, 150 seafood samples were comprehensively analyzed, including six different species: shrimp, crab, clam, scallop, fish, and squid. Each sample was accurately weighed to 25.0 ± 0.1 g and then immersed in 225 mL of buffered peptone water (BPW). The mixture was thoroughly homogenized and incubated at 37 °C with a rotation speed of 200 rpm for 18 h. After this enrichment step, genomic DNA was extracted using a DNA extraction kit (Sangji, Shanghai, China). Subsequently, the samples were tested using cdLAMP, qPCR, and BAM methods.

[0071] Data analysis

[0072] Data analysis and schematic representation were performed using Microsoft Excel 2021 and PowerPoint 2021 (Microsoft Corporation, Redmond, WA, USA). Experimental data were analyzed and processed using GraphPad Prism 7 (GraphPad Software and, USA) and Origin 2021 (Origin Lab, Northampton, USA).

[0073] 2. Results

[0074] 2.1 Detection principle

[0075] Figure 1 a shows the process of cdLAMP detection method. Two sets of LAMP primers and FIP probes were used, each equipped with a different fluorophore-quencher pair. The target genes include the nuc gene of STA (labeled with Texas Red / BHQ2) and the fimY gene of SAL (labeled with FAM / BHQ1). After adding the templates of STA and SAL, the corresponding primers and probes will recognize them. Under the action of the strand displacement activity of Bst DNA polymerase, the probes are separated and produce specific fluorescent signals, which can be captured by the imaging analyzer. The amplification of each microwell is independent. The imaging analyzer can display the fluorescence signals of the two channels of Texas Red and FAM for each reaction well. The fluorescence signal of each channel corresponds to the detection result of a foodborne pathogen.

[0076] 2.2 Optimization of cdLAMP reaction

[0077] Considering the hydrophobic surface and hydrophilic interior of the chip, it is necessary to optimize the final concentration of Tween-20. The dispersion and amplification effects of Tween-20 on the chip at 0%, 0.025%, 0.05%, 0.075%, and 0.1% concentrations need to be evaluated. Figure 2 a shows that in the absence of Tween-20, the system becomes unstable, signal detection is negligible, and the reaction process is hindered. At a final concentration of 0.025% Tween-20, the generated signal did not reach the peak, resulting in a decrease in the signal difference. When the final concentration of Tween-20 exceeds 0.05%, aggregation of the reaction solution can be observed on the chip, indicating uneven distribution in the microchamber, thus affecting the amplification process. Therefore, a concentration of 0.05% was selected as the optimal condition to ensure uniform distribution and effective amplification.

[0078] Magnesium ion (Mg 2+) not only helps stabilize the DNA double helix structure, but also has an important impact on the specificity and yield of the amplification process (Mukherjee & Bhattacharyya, 2013). 2+ When the concentration is too low, the activity of the polymerase may be reduced, resulting in a decrease in the amount of reaction products. 2+ Therefore, strict control of Mg 2+ The concentration is critical to the efficiency of the amplification reaction. Six final Mg 2+ The concentration gradient was 2mM, 4mM, 6mM, 8mM, 10mM and 12mM to evaluate the amplification results. Figure 2 As shown in Figure 2, the positive detection rates (defined as the percentage of positive wells to the total number of valid wells) were 2.27%, 3.77%, 6.18%, 13.4%, 14.3%, and 7.52%, respectively. It is worth noting that at a concentration of 8 mM, the positive signal reached a peak, thereby increasing the difference between the negative and positive signals. 2+ The concentration has been determined to be 8 mM. This concentration is within the 7-15 mM range previously optimized by Jin et al., indicating that our findings are consistent with previous studies.

[0079] Subsequently, the amplification effects of cdLAMP at 35, 40, 45, 50, and 55 minutes were compared. The data showed that the positive signal gradually increased from 35 to 45 minutes and reached a peak at 45 minutes (see Figure 2 c). The positive detection rates of the five amplification durations were 6.96%, 8.05%, 10%, 10.3%, and 9.76%, respectively. After more than 45 minutes, the positive detection rate remained basically unchanged. Therefore, 45 minutes was selected as the optimal reaction duration. This is consistent with Jin's temperature optimization results (the reaction reached a plateau after 45 minutes). Compared with qPCR, the cdLAMP method is one hour faster, thereby improving detection efficiency.

[0080] The design, stoichiometry, and concentration of LAMP primers are key determinants of assay sensitivity. During the initiation phase of replication, double-stranded DNA is in a dynamic equilibrium under isothermal conditions. The internal primers first bind to their complementary sequences within the target gene, thereby initiating the reaction and affecting the amplification rate. In analyzing the amplification efficiency of the F3 / B3 and FIP / BIP primer ratios (1:4, 1:6, 1:8, 1:10, and 1:12), the highest positive detection rate was observed when the F3 / B3:FIP / BIP ratio was 1:8 for STA and SAL samples, which was 12.9% and 10.2%, respectively. In addition, the signal contrast of this ratio group was also better than that of the other groups, indicating that this was the best amplification effect. This emphasizes the importance of primer ratio optimization in achieving reliable and sensitive cdLAMP detection.

[0081] Bst DNA polymerase has strong polymerase activity and strand displacement ability, which contributes to efficient template amplification under isothermal conditions, thus serving as the driving force of the reaction. Therefore, Bst DNA polymerase is the core of the isothermal amplification process. Five enzyme concentration gradients ranging from 0.08U / μL to 1.28U / μL (see Figure 2 e). By observing the chip, an enzyme concentration exceeding 0.32U / μL will cause the reaction solution to aggregate on the chip, which reduces the number of effective wells and affects the analysis results (see Figure 6 ). The best detection effect was achieved at an enzyme concentration of 0.16 U / μL, which had the highest positive detection rate and significantly improved signal contrast, making it most suitable for detection purposes. This emphasizes the importance of primer ratio optimization in achieving reliable and sensitive cdLAMP detection.

[0082] Finally, the amplification efficiency of the two pathogenic bacteria was optimized in the temperature range of 59°C to 67°C. Figure 2 f shows that the signal contrast gradually increased when the temperature varied between 59°C and 65°C. In addition, at 65°C, the positive detection rates of STA and SAL strains were the highest, 9.94% and 10.5%, respectively. Therefore, 65°C was determined to be the optimal temperature for cdLAMP detection in this study, ensuring the most reliable and sensitive detection of the target pathogens.

[0083] 2.3 Specificity and sensitivity of cdLAMP

[0084] Using the above optimized conditions, STA and SAL primers were used to specifically analyze 10 other pathogenic bacterial species (selected 10 3 CFU / mL as experimental concentration). Only STA and SAL strains produced detectable signals, while the other strains did not show any signals (see Figure 3 a). This result confirms the high specificity of the primers in distinguishing the target pathogen from other closely related species.

[0085] The sensitivity of cdLAMP was tested at 10 0 Up to 10 4 The results were carried out over a range of CFU / mL, and each concentration level was tested three times to ensure the reliability of the results. TE was used as a negative control. The results showed that the negative control showed no signal. As the concentration decreased, the number of positive signal wells decreased accordingly (see Figure 3 b) The detection method can detect as low as 10 0 The concentration of pathogenic bacteria in CFU / mL. Based on the genome size, this is equivalent to 10 0 copies, which is consistent with the single-copy gene concentration predicted by the data analysis software. 0 Up to 10 1 Within the concentration range of CFU / mL, although the signal intensity difference decreased significantly, it was still above the threshold (100), thus ensuring the reliability of the results (see Figure 3 d and 3g). However, qPCR was able to detect up to 10 1 CFU / mL concentration, while at 10 0 CFU / mL, no fluorescence signal was observed (see Figure 7 ). Therefore, the sensitivity of cdLAMP was ten times higher than that of qPCR. cdLAMP has a superior sensitivity. Linear correlation was observed by logarithmic transformation of theoretical and measured concentrations and subsequent curve fitting. The coefficient of determination (R 2 ) are 0.9974 and 0.9903 respectively (see Figure 3 e and 3h). This indicates that the cdLAMP method provides reliable results for foodborne pathogen detection.

[0086] 2.4 Accuracy of cdLAMP

[0087] like Figure 3 As shown in Figures 3c and 3f, the test results at the three concentration levels always fell within the confidence interval (indicated by the dashed area), thus confirming the reliability of the results. Subsequently, the intra- and inter-batch coefficients of variation (CV) for the two target pathogens were determined. 0 The intra-assay and inter-assay CV values ​​were 4.81% and 2.83%, respectively. 2 CFU / mL STA, the corresponding values ​​were 2.24% and 1.05%, respectively. 4 CFU / mL STA, these values ​​were 2.89% and 3.26%, respectively. In addition, for 100 CFU / mL SAL, the intra- and inter-batch CV values ​​were 2.15% and 3.09%, respectively.2 CFU / mL SAL, they were 1.55% and 2.78%, respectively. 4 CFU / mL SAL, and the CV values ​​were 3.64% and 2.38%, respectively. Overall, all CVs were below 5%, indicating that the cdLAMP method has high precision and reproducibility.

[0088] 2.5 Testing of spiked food samples

[0089] The lowest concentration that produced a positive result was determined as the limit of detection (LOD). cdLAMP was able to detect 0 CFU / mL concentration, and the test results were consistent in the three types of food. qPCR was able to detect the concentration range of 10 1 Up to 10 4 CFU / μL of foodborne pathogens. 0 One milk sample tested positive for CFU / mL, but the cycle threshold (Ct) exceeded 35, indicating that the result was unreliable (Table 3). The recoveries of the spiked foods are shown in Table 4.

[0090] Table 3 Comparison of sensitivity of qPCR and cdLAMP in detecting STA and SAL in milk, pork and eggs

[0091]

[0092]

[0093] Recovery of STA and SAL in food samples

[0094]

[0095] 2.6 Testing of on-site samples

[0096] To verify the accuracy of cdLAMP, 150 seafood samples were tested using both cdLAMP and BAM analysis methods (see Figure 4 a and Table 5). The experimental results showed a high consistency between the two analytical methods. However, sample No. 4 (red box) showed a false positive in the cdLAMP test of STA, indicating that the pathogenic bacteria may have lost their activity. By calculation, the true positive (TP) was 6, the true negative (TN) was 143, the false positive (FP) was 1, and the false negative (FN) was 0.

[0097] Table 5 Comparison of cdLAMP detection of field samples and the Bacteriological Analysis Manual (BAM) method

[0098]

[0099]

[0100] At the same time, these samples were also tested by qPCR (see Figure 4 c). The positive detection rates of cdLAMP and qPCR were 4.7% and 4.0%, respectively. Overall, the positive detection rate of cdLAMP was higher than that of qPCR. Specifically, the infection rates of STA, SAL, and mixed infection detected by cdLAMP were 2.0% (3 samples), 1.3% (2 samples), and 1.3% (2 samples), respectively. In contrast, the infection rates of the same pathogens detected by qPCR were 1.3% (2 samples) for STA, 2.0% (3 samples) for SAL, and 0.7% (1 sample) for mixed infection. It is worth noting that in sample No. 1 (yellow box), qPCR only detected SAL, while cdLAMP showed the presence of a trace amount of STA co-infection. In sample No. 5 (green box), qPCR was negative, but cdLAMP detected STA infection. This difference may be attributed to the concentration of STA being below the detection limit of qPCR. The detection results of the two methods for the remaining samples were consistent. qPCR was unable to detect concentrations as low as 100 copies / μL, whereas cdLAMP was able to detect signals at this concentration. Therefore, cdLAMP showed higher sensitivity, especially in samples with lower infection levels, thus reducing the possibility of false negatives. These results are consistent with previous studies. In addition, the correlation coefficient between cdLAMP and qPCR was 0.9799, indicating a strong correlation between the two methods (see Figure 4 b).

[0101] STA (Staphylococcus aureus) and SAL (Salmonella) are common foodborne pathogens that can cause infection even at low concentrations. Therefore, accurate and sensitive identification and quantification of these pathogens are critical to ensure food safety and prevent foodborne illness.

[0102] In the present study, Tween-20 (0, 0.025%, 0.05%, 0.075% and 0.1%), Mg 2+ When optimizing the concentrations (2mM, 4mM, 6mM, 8mM, 10mM, and 12mM) and enzyme concentrations (0.08U / μL, 0.16U / μL, 0.32U / μL, 0.64U / μL, and 1.28U / μL), it was observed that at certain concentrations, the reagents could not enter the microwells, the reaction solution clumped on the chip, or the fluorescence image showed regular changes.

[0103] like Figure 5a. When Tween-20 is not added to the system, the reagent cannot enter the microwells and the number of positive wells is almost zero. When the final concentration of Tween-20 exceeds 0.05%, the reaction solution aggregates on the chip and the number of effective wells is only 10916±10, which is about 1000 less than normal, affecting the experimental results. Figure 5 b, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM and 12 mM Mg 2+ The positive rates (defined as the percentage of positive wells to the total effective wells) were 2.27%, 3.77%, 6.18%, 13.4%, 14.3% and 7.52%, respectively. 2+ The concentration is less than 8mM, and the number of effective wells and positive wells increases with Mg 2+ The signal difference at other concentrations was less than 10 except for 8 mM. 2+ When the concentration is 8mM, the number of effective wells is 12398±10, the number of positive wells is 1926±9, the positive signal reaches a peak value of 250, and the difference between the positive signal and the background signal is more than 185. In this case, the captured fluorescence image is the best and the detection result is more reliable. Figure 5 c. When the enzyme concentration exceeds 0.16U / μL, the reaction solution will aggregate on the chip. The effective number of STA holes is only 10687±486, and the effective number of SAL holes is only 10076±694, which is about 2000 less than normal, affecting the experimental results. Therefore, the chip structure will affect the Tween-20, Mg 2+ and Bst DNA polymerase concentration. Only in the presence of 0.05% Tween-20, 8mM Mg 2+ Under the synergistic effect of 0.16U / μL Bst DNA polymerase, the parameters such as fluorescence image, number of effective wells, number of positive wells and signal difference were optimal.

[0104] Compared with qPCR, dLAMP provides higher accuracy, sensitivity, specificity, and inhibitor tolerance. The detection limit of the cdLAMP method of the present invention for STA and SAL is 2.0 CFU / mL. The results are better than those obtained by Xie, who used DMF-LAMP to detect STA with a detection limit of 2.6×10 3 CFU / mL, the detection limit of SAL is 3.5×10 3CFU / mL. This is because the DMF-LAMP reaction volume is only 2μL, while the chip in this study can accommodate 20μL, so the sensitivity is higher. Tao's dLAMP method for detecting Mycobacterium bovis in milk has a detection limit of 14CFU / mL within 2 hours. The experiment generated approximately 18,400 reaction units in 8 microwells. Fluorescence imaging also requires an inverted fluorescence microscope, and the image is analyzed and the positive droplets are counted using ImageJ software, and the template concentration is then calculated based on the Poisson distribution. Obtaining the detection results requires a series of processes, which leads to longer detection times, which also explains why the detection time is prolonged in many current studies. In contrast, this study involved 20,000 reaction units, and the imaging analyzer integrated image capture and calculation, which simplified the experimental process and shortened the detection time.

[0105] The present invention successfully established a cdLAMP detection method for detecting foodborne pathogens. The method achieved a low detection limit of 2.0 CFU / mL for STA and SAL. This cdLAMP technology provides a dynamic detection range spanning 5 orders of magnitude. During the optimization process, the present invention surprisingly found that only 0.05% Tween-20, 8mM Mg 2+ In synergy with 0.16U / μL Bst DNA polymerase, parameters such as fluorescence images, number of effective wells, number of positive wells, and signal difference are optimal, and the cdLAMP of the present invention shows higher sensitivity than qPCR. The cdLAMP method is a powerful platform for detecting and quantifying foodborne pathogens, providing more reliable insights for food safety and epidemiological investigations. Looking forward, the application of this system can be expanded to include other pathogens, highlighting its potential as a very promising detection technology.

[0106] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP, characterized in that: include: A silicon-based nanoporous chip, a primer-probe combination, 20 mM Tris-HCl, 10 mM KCl, 8 mM MgSO4, 10 mM (NH4)2SO4, 0.05% Tween-20, 2.5 μL DNA template, 1.4 mM dNTP, 0.16 U / μL Bst DNA polymerase and DEPC; the primer-probe combination includes: a primer probe designed based on the nuc gene of Staphylococcus aureus: GenBank accession number EF529607.1, and a primer probe designed based on the fimY gene of Salmonella: GenBank accession number JQ665438.

1.

2. The detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP according to claim 1, characterized in that: The primer-probe combination includes: 0.2 μM outer primer, 1.2 μM FIP, 0.4 μM FIP probe, 1.6μM BIP and 0.8μM loop primer; the outer primers include: STA-F3, STA-B3, SAL-F3 and SAL-B3; FIP includes: STA-FIP and SAL-FIP; FIP probes include: STA-FIP probe and SAL-FIP probe; BIP includes: STA-BIP and SAL-BIP; loop primers include: STA-LF, STA-LB and SAL-LF.

3. The detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP according to claim 2, characterized in that: The sequence of the STA-F3 is SEQ ID No: 01, the sequence of the STA-B3 is SEQ ID No: 02, the sequence of the STA-FIP is SEQ ID No: 03, the sequence of the STA-BIP is SEQ ID No: 04, the sequence of the STA-LF is SEQ ID No: 05, the sequence of the STA-LB is SEQ ID No: 06, the sequence of the STA-FD is SEQ ID No: 07, the sequence of the STA-Probe is SEQ ID No: 08, the sequence of the SAL-F3 is SEQ ID No: 09, the sequence of the SAL-B3 is SEQ ID No: 10, the sequence of the SAL-FIP is SEQ ID No: 11, the sequence of the SAL-BIP is SEQ ID No: 12, the sequence of the SAL-LF is SEQ ID No: 13, the sequence of the SAL-FD is SEQ ID No: 14, and the sequence of the SAL-Probe is SEQ ID No:

15.

4. The detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP according to claim 1, characterized in that: The silicon-based nanoporous chip contains 20,000 micropores, each of which has a uniform volume and a pore size of 50 μm. The micropores include: micropore side walls, micropore surfaces, nano-hydrophilic layers, and nano-hydrophobic layers modified on the micropore surfaces.

5. The detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP according to claim 4, characterized in that: The nano-hydrophobic layer is modified with silane by microcontact printing, and the specific method includes: the chip is ultrasonically cleaned with trichloroethylene, acetone and ethanol in sequence, and then immersed in a solution of H2SO4:H2O2 in a volume ratio of 7:3, treated at 60°C for 1.5 hours, and then ultrasonically cleaned twice with deionized water and ethanol, and blown dry with nitrogen; a mold with a thickness of about 0.2 mm is immersed in (1H, 1H, 2H, 2H-perfluorodecyl) trimethoxysilane, and dried under a nitrogen flow to remove excess reagents; the mold is placed on the chip, pressed for 20 seconds, and then removed; the chip is heated to 90°C for 30 minutes; the chip is rinsed with deionized water, and the residual reagents are blown away with nitrogen.

6. A detection method for a detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP, characterized in that: The detection method of the detection kit according to claim 1 comprises the following steps: Step 1, DNA preparation; Step 2: Design of LAMP primers and probes; Step 3, preparing the cdLAMP reaction system; Step 4, preparing the chip; Step 5: Sample allocation: Using a disposable chip with a size of 1×1 cm, a scraper with a size of 10×43 mm and a cover plate, 20 μL of sample is introduced into the scraper, and the sample is fixed at the bottom of the scraper; when the preparation instrument is started, the sample is scraped into the micropores of the chip; after the sample is loaded, the instrument automatically seals with oil, and the amount of sealing oil for each chip is 250 μL; finally, the cover plate is covered, and the chip preparation is completed; the chip is placed on the amplification instrument for reaction, and after the amplification is completed, the chip is placed in the imaging analyzer; Step 6: Capture fluorescence images: The imaging analyzer is equipped with an optical dual-color fluorescence channel designed for FAM and Texas Red, which can detect dual signals emitted by each reaction well by switching the fluorescence channels; a CMOS detector is integrated to ensure accurate image capture; using the statistical principle of Poisson distribution, the data analysis software automatically calculates the initial copy number of the target molecule; the copy number of the template DNA is calculated by the following formula: c(copies / μL)=-In(1-a / b) / v Wherein, a represents the number of positive reaction wells, b represents the total number of effective reaction wells, v represents the volume of each reaction unit, and the ratio a / b represents the positive rate.

7. The detection method of the detection kit for simultaneous detection of Staphylococcus aureus and Salmonella based on cdLAMP according to claim 6, characterized in that: In step 4, the specific method of preparing the chip includes: Preparation: The silicon chip is cleaned with acetone, anhydrous ethanol and deionized water in sequence, and then exposed to hexamethyldisilane vapor, the hydrophilic hydroxyl groups on the surface of the silicon wafer are replaced by hydrophilic groups, and then the silicon wafer is placed on a hot plate at 140°C for 10 minutes and spin-coated to form a 5-micron thick photoresist layer; the chip is pre-baked at 100°C for 5 minutes, then exposed to a photolithography machine and post-baked, a regular hexagonal reaction microwell array with a pitch of 20 microns and a diameter of 50 microns is patterned onto the photoresist, and the microwell array is formed by photoresist cleaning, inductively coupled plasma etching and diamond grinding processes; Surface modification: The chip was ultrasonically cleaned with trichloroethylene, acetone and ethanol in sequence, and then immersed in a solution of H2SO4:H2O2 with a volume ratio of 7:3 and treated at 60°C for 1.5 hours; it was ultrasonically cleaned twice with deionized water and ethanol and blown dry with nitrogen; it was modified with silane by microcontact printing to form a hydrophobic surface; a mold with a thickness of about 0.2 mm was immersed in (1H,1H,2H,2H-perfluorodecyl)trimethoxysilane and dried under a nitrogen flow for about 5 seconds to remove excess reagents; the mold was placed on the chip, pressed for 20 seconds, and then removed to ensure a hydrophobic seal; the chip was heated to 90°C for 30 minutes; the chip was rinsed with deionized water and purged with nitrogen to remove residual reagents; the manufactured chip was stored in a sealed environment at room temperature.

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