Multiplex digital PCR detection reagent, detection device and detection method for foodborne pathogenic bacteria

Abstract

The application relates to a foodborne pathogenic bacteria multiplex digital PCR detection reagent, a detection device and a detection method thereof. The application provides a super-multiplex foodborne pathogenic bacteria digital PCR detection system based on a multi-color coding technology. The system breaks through the physical limitation of a fluorescence channel through an innovative probe coding strategy and a signal decoding algorithm, realizes absolute quantitative detection of 7 or more foodborne pathogenic bacteria in a single reaction tube at the same time, and ensures high specificity, high accuracy, a wide linear range and good repeatability in a complex matrix.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to multiplex digital PCR detection reagents, detection devices and detection methods for foodborne pathogens. Background Technology

[0002] Foodborne pathogens (such as Salmonella, Staphylococcus aureus, Listeria monocytogenes, Vibrio parahaemolyticus, Vibrio cholerae, Cronobacter, and pathogenic Escherichia coli O157:H7) are important pathogens that cause food safety incidents, and rapid and accurate detection of them is key to preventing and controlling contamination.

[0003] Routine methods for detecting foodborne pathogens include: 1) Traditional culture methods: the gold standard, but cumbersome and time-consuming (usually 3-7 days), unsuitable for rapid detection and products with short shelf lives, and prone to missing detection of "live, non-culturable (VBNC)" bacteria. 2) Immunological methods: such as ELISA and colloidal gold test strips, which can achieve rapid screening, but are susceptible to interference from antibody cross-reactions, and have relatively low sensitivity and specificity. 3) Conventional PCR / real-time quantitative PCR (qPCR): shortens the detection cycle, but qPCR relies on a standard curve for relative quantification and is susceptible to interference from PCR inhibitors; conventional PCR is difficult to achieve accurate quantification. When performing multiplex detection, interference between fluorescence channels and primer competition are prominent issues, usually only allowing for the joint detection of a small number of targets. Summary of the Invention

[0004] Based on this, this application provides a multiplex digital PCR detection reagent, detection device, and detection method for foodborne pathogens. Using this detection reagent, multiple foodborne pathogens can be detected simultaneously in a single reaction tube, and it possesses both detection accuracy and specificity.

[0005] The first aspect of this application provides a multiplex digital PCR detection reagent for detecting foodborne pathogens according to one embodiment, comprising detection probes for detecting foodborne pathogens, wherein the foodborne pathogens include *Cronobacter* spp., *Staphylococcus aureus*, *Vibrio cholerae*, *Vibrio parahaemolyticus*, *Salmonella* spp., *Listeria monocytogenes*, and *Escherichia coli* O157:H7. The nucleotide sequences of the detection probes corresponding to *Cronobacter* spp., *Staphylococcus aureus*, *Vibrio cholerae*, *Vibrio parahaemolyticus*, *Salmonella* spp., *Listeria monocytogenes*, and *Escherichia coli* O157:H7 are shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.18, and SEQ ID NO.21, respectively. The detection probes are connected to fluorescent groups. The fluorescent group of the detection probe for Staphylococcus aureus is FAM, the fluorescent group of the detection probe for Cronobacter spp. is HEX, the fluorescent group of the detection probe for Listeria monocytogenes is ROX, the fluorescent group of the detection probe for Escherichia coli O157:H7 is Cy5, the fluorescent groups of the detection probe for Vibrio cholerae are FAM and HEX, the fluorescent groups of the detection probe for Vibrio parahaemolyticus are HEX and ROX, and the fluorescent groups of the detection probe for Salmonella spp. are HEX and Cy5.

[0006] The aforementioned multiplex digital PCR detection reagent is based on multiplex digital PCR detection technology, combined with multicolor fluorescent coding, and features probes with specific sequences labeled with specific fluorescent groups. This enables the simultaneous detection of multiple foodborne pathogens in a single reaction tube, while maintaining both accuracy and specificity. Experimental verification shows that the multiplex digital PCR detection reagent of this application can simultaneously detect at least seven foodborne pathogens in a single reaction tube, while maintaining both accuracy and specificity.

[0007] In some embodiments, the multiplex digital PCR detection reagent further includes amplification primers for detecting the foodborne pathogens. The amplification primers corresponding to the *Cronobacter* spp., the *Staphylococcus aureus*, the *Vibrio cholerae*, the *Vibrio parahaemolyticus*, the *Salmonella* spp., the *Listeria monocytogenes*, and the *Escherichia coli* O157:H7 are shown in SEQ ID NO.1-SEQ ID NO.2, SEQ ID NO.4-SEQ ID NO.5, SEQ ID NO.7-SEQ ID NO.8, SEQ ID NO.10-SEQ ID NO.11, SEQ ID NO.13-SEQ ID NO.14, SEQ ID NO.16-SEQ ID NO.17, and SEQ ID NO.19-SEQ ID NO.20, respectively.

[0008] In some embodiments, the multiplex digital PCR detection probe is further connected to a quenching group, the quenching group including BHQ1 and / or BHQ2.

[0009] In some embodiments, the multiplex digital PCR detection reagent further includes: dPCR buffer, DNA polymerase, and nuclease-free water.

[0010] The second aspect of this application provides a multiplex digital PCR detection device for detecting foodborne pathogens according to one embodiment, comprising: a digital PCR instrument and the multiplex PCR detection reagents described in the above embodiment.

[0011] In some embodiments, the digital PCR instrument is used to perform a PCR reaction between the nucleic acid sample to be tested and the multiplex PCR detection reagent; and after the PCR reaction is completed, multicolor fluorescence encoding and decoding technology, combined with a quantitative formula, is used to calculate the absolute copy number concentration of each target gene in the nucleic acid sample to be tested.

[0012] A third aspect of this application provides a method for multiplex PCR detection of foodborne pathogens, comprising the following steps: The nucleic acid sample to be tested and the multiplex digital PCR detection reagent described in the above embodiments are added to a digital PCR instrument for PCR reaction; After the PCR reaction, multicolor fluorescence encoding and decoding technology, combined with quantitative formulas, was used to calculate the absolute copy number concentration of each target gene in the nucleic acid sample to be tested.

[0013] In some embodiments, The step of calculating the absolute copy number concentration of each target gene in the nucleic acid sample to be tested using multicolor fluorescence encoding and decoding technology and combined with quantitative formulas includes: The fluorescence signal of each reaction chamber in each fluorescence detection channel is read, and the images of each fluorescence detection channel are spatially aligned so that the index number of each reaction chamber is consistent under different fluorescence detection channels; Identify the positive or negative status of each of the reaction chambers in each of the fluorescence detection channels; For foodborne pathogens that require labeling with two fluorescent groups, when both corresponding fluorescent detection channels are positive in the reaction chamber with the same index number, the chamber is determined to be a positive droplet of the pathogen. Based on the number of positive droplets corresponding to each of the foodborne pathogen genes, and using a quantitative formula, the absolute copy number concentration of each of the foodborne pathogen genes in the nucleic acid sample to be tested is calculated.

[0014] In some embodiments, the quantitative formula is as follows: ; ; Where λ is the average molecular copy number, Pn is the corrected positive rate, Ti is the positive rate of the i-th fluorescence detection channel, and Tj ∩ Tk is the co-localization positive rate when the j-th and k-th fluorescence detection channels are simultaneously positive.

[0015] In some embodiments, the reaction conditions for PCR reaction performed by the digital PCR instrument include: the concentration of each primer is 400 nM, the concentration of each detection probe is 100 nM-200 nM, and the annealing temperature is 60°C. Attached Figure Description

[0016] Figure 1 This is a flowchart of the technical solution of this application; Figure 2 This is a schematic diagram illustrating the principle of multicolor fluorescent coding. Figure 3 This is a schematic diagram illustrating the principle of chamber index consistency decoding. Figure 4 This is a partial view of a one-dimensional scatter plot for the detection of single targets of seven pathogenic bacteria. Figure 5 One-dimensional scatter plot of Staphylococcus aureus, Cronobacter, Listeria monocytogenes, and Escherichia coli O157; Figure 6 Two-dimensional scatter plot and raw fluorescence image for the detection of Vibrio cholerae (two-color coding); Figure 7 Two-dimensional graphs for the detection of Vibrio parahaemolyticus and general-purpose two-dimensional graphs for Salmonella; Figure 8 A one-dimensional scatter plot for specific testing of multiple systems under complex matrix interference. Detailed Implementation

[0017] The technical solution of this application will be further described in detail below with reference to specific embodiments. This application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0019] As used herein, the terms “and / or,” “or / and,” and “and / or” may include any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.

[0020] In this article, "one or at least one" means any one, any two, or any two or more of the listed items.

[0021] In this application, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, terms such as "first," "second," "third," and "fourth" serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0022] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0023] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0024] Unless otherwise specified, the percentage content mentioned in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0025] Unless otherwise specified, all percentage concentrations mentioned in this application refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0026] Unless otherwise specified, the temperature parameters in this application may be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows for temperature fluctuations within the precision range controlled by the instrument.

[0027] This application addresses the challenge of achieving single-tube, simultaneous, rapid, and absolute quantitative detection of multiple foodborne pathogens in complex matrices such as food, pharmaceuticals, environmental samples, and clinical samples. Specifically, it aims to overcome the technical bottlenecks of existing detection technologies (such as traditional culture methods, immunological methods, conventional PCR, and real-time quantitative PCR), including low throughput, long processing times, inability to achieve absolute quantification, poor resistance to interference in complex matrices, and difficulty in distinguishing coexisting signals of multiple targets. Based on this, the application provides a multiplex digital PCR detection reagent, detection device, and detection method for foodborne pathogens. Using this detection reagent, at least seven foodborne pathogens can be detected simultaneously in a single reaction tube, exhibiting both accuracy and specificity.

[0028] The first aspect of this application provides a multiplex digital PCR detection reagent for detecting foodborne pathogens according to one embodiment, comprising detection probes for detecting foodborne pathogens, wherein the foodborne pathogens include *Cronobacter* spp., *Staphylococcus aureus*, *Vibrio cholerae*, *Vibrio parahaemolyticus*, *Salmonella* spp., *Listeria monocytogenes*, and *Escherichia coli* O157:H7. The nucleotide sequences of the detection probes corresponding to *Cronobacter* spp., *Staphylococcus aureus*, *Vibrio cholerae*, *Vibrio parahaemolyticus*, *Salmonella* spp., *Listeria monocytogenes*, and *Escherichia coli* O157:H7 are shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.18, and SEQ ID NO.21, respectively. The detection probes are connected to fluorescent groups. The fluorescent group of the detection probe for Staphylococcus aureus is FAM, the fluorescent group of the detection probe for Cronobacter spp. is HEX, the fluorescent group of the detection probe for Listeria monocytogenes is ROX, the fluorescent group of the detection probe for Escherichia coli O157:H7 is Cy5, the fluorescent groups of the detection probe for Vibrio cholerae are FAM and HEX, the fluorescent groups of the detection probe for Vibrio parahaemolyticus are HEX and ROX, and the fluorescent groups of the detection probe for Salmonella spp. are HEX and Cy5.

[0029] The above-mentioned detection reagents are based on multiplex digital PCR detection technology, combined with multicolor fluorescent coding, and designed probes with specific sequences. The probes are labeled with specific fluorescent groups, which can realize the simultaneous detection of at least 7 foodborne pathogens in a single reaction tube, and have both detection accuracy and specificity.

[0030] In this application, a unique "fluorescent barcode" is designed for each target using a limited number of fluorescence channels (e.g., 4 colors: FAM, HEX, ROX, Cy5) through single-channel labeling and pairwise combinations of different channels. For example, target A uses a single FAM label, target B uses a single HEX label, and target C uses a dual FAM+HEX label. Thus, theoretically, N fluorescence channels can encode 2... N -1 different target (15 can be encoded when N=4).

[0031] In some embodiments, the multiplex digital PCR detection reagent further includes amplification primers for detecting the foodborne pathogens. The amplification primers corresponding to *Cronobacter* spp., *Staphylococcus aureus*, *Vibrio cholerae*, *Vibrio parahaemolyticus*, *Salmonella* spp., *Listeria monocytogenes*, and *Escherichia coli* O157:H7 are shown in SEQ ID NO.1-SEQ ID NO.2, SEQ ID NO.4-SEQ ID NO.5, SEQ ID NO.7-SEQ ID NO.8, SEQ ID NO.10-SEQ ID NO.11, SEQ ID NO.13-SEQ ID NO.14, SEQ ID NO.16-SEQ ID NO.17, and SEQ ID NO.19-SEQ ID NO.20, respectively. Using the amplification primers with the above specific sequences enables the simultaneous detection of at least seven foodborne pathogens in a single reaction tube, while maintaining both detection accuracy and specificity.

[0032] In some embodiments, the multiplex digital PCR detection probe is further connected to a quenching group, the quenching group including BHQ1 and / or BHQ2.

[0033] In some embodiments, the multiplex digital PCR detection reagent further includes: dPCR buffer, DNA polymerase, and nuclease-free water.

[0034] Multiplex detection technology based on digital PCR (dPCR). dPCR achieves absolute quantification by dividing the reaction system into a large number of micro-reaction units, and has the advantages of high sensitivity and strong anti-interference ability. Its specific implementation scheme is: for 2-4 different targets, TaqMan probes labeled with different fluorescent groups (such as FAM, VIC, ROX, Cy5) are designed. Each target corresponds to a unique monochromatic fluorescence. After amplification on the dPCR platform, the positive signal of each droplet in different fluorescence channels is read to determine which target is present in the sample, and its absolute copy number is calculated according to the Poisson distribution. Currently, the mainstream dPCR platforms on the market are usually configured with 2-4 fluorescence channels, so their multiplex detection capability is also limited to 4. The existing dPCR technology has the following problems: (1) Low detection throughput: limited by the number of fluorescence channels of the instrument (usually 2-4), the existing dPCR technology can only detect 4 different pathogens in the same reaction at most. When facing multiple pathogens that may exist in the actual sample (such as the 7 in this invention), multiple reactions are required, which increases reagent cost, sample consumption and operation time. (2) Limited signal discrimination capability: When attempting to detect more targets, if fluorescence combination is used (such as one target simultaneously labeled with two fluorescences), new problems will be introduced: when two different single-labeled targets coexist with one dual-labeled target, the instrument cannot distinguish between "positive droplets of two single-labeled targets" and "positive droplets of one dual-labeled target", resulting in signal crosstalk and decoding errors. (3) Difficult to meet actual needs: In food safety monitoring and emergency response to public health events, it is often necessary to conduct batch and rapid screening of multiple potential pathogens. The insufficient throughput of existing technologies has become a bottleneck for their application. In view of the above-mentioned shortcomings of existing technologies, this application provides a super-multiplex digital PCR detection system for foodborne pathogens based on multicolor coding technology. This system breaks through the physical limitations of fluorescence channels through innovative probe coding strategies and signal decoding algorithms, realizing the simultaneous absolute quantitative detection of 7 or more foodborne pathogens in a single reaction tube, and ensuring high specificity, high accuracy, wide linear range and good repeatability in complex matrices.

[0035] The core technical solution of this application is: "multicolor fluorescence encoding" + "compartment index consistency decoding" + "digital PCR absolute quantification".

[0036] (1) Multicolor fluorescence encoding: Utilizing a limited number of fluorescence channels (e.g., 4 colors: FAM, HEX, ROX, Cy5), a unique "fluorescent barcode" is designed for each target through single-channel labeling and pairwise combinations of different channels. For example, target A uses a single FAM label, target B uses a single HEX label, and target C uses a dual FAM+HEX label. In this way, theoretically, N fluorescence channels can encode 2... N -1 different target (15 can be encoded when N=4).

[0037] (2) Chamber Index Consistency Decoding: To address the potential signal confusion that may occur when multiple targets coexist (such as the "FAM+HEX" signal generated by the simultaneous presence of targets A and B, which cannot be distinguished from the dual-label signal of target C itself), this invention utilizes the physical partitioning characteristics of digital PCR for decoding. Since the position of each reaction chamber is fixed, by comparing the signals of chambers at the same position under different fluorescence channels, it is possible to distinguish: if a chamber is positive in both the FAM and HEX channels, and the "index" of the chamber is completely consistent in the images of the two channels, it is determined to be dual-labeled target C; if FAM-positive and HEX-positive signals appear in chambers with different indices, it is determined to be the coexistence of targets A and B. This principle precisely solves the signal crosstalk problem in fluorescence combination encoding.

[0038] (3) Absolute quantification by digital PCR: Finally, based on the number of positive droplets of each target after decoding, combined with the Poisson distribution formula, the absolute copy number concentration of each target in the original sample is calculated to achieve accurate quantification.

[0039] refer to Figure 1 ( Figure 1 (This is a flowchart of the technical solution of this application). The technical solution of this application generally includes the following steps: (1) Target selection and primer / probe design: Seven target foodborne pathogens were selected: *Cronobacter* spp., *Staphylococcus aureus*, *Vibrio cholerae*, *Vibrio parahaemolyticus*, *Salmonella* spp., *Listeria monocytogenes*, and *Escherichia coli* O157:H7. Species-specific conserved genes for each species were screened from the NCBI database. Specific primers and TaqMan probes were designed, and bioinformatics alignments were performed to ensure no cross-reactivity with the human genome or common background microbiota.

[0040] The probe's 5' end is labeled with different fluorescent groups (FAM, HEX, ROX, Cy5) according to a preset encoding scheme, and the 3' end is labeled with a quenching group (such as BHQ).

[0041] (2) Establish a multicolor fluorescent coding table like Figure 2 ( Figure 2 As shown in the schematic diagram of the multicolor fluorescence coding principle, four fluorescence channels are used: FAM, HEX, ROX, and Cy5. The coding rules are as follows: Staphylococcus aureus: FAM single label; Cronobacter spp.: HEX single label; Listeria monocytogenes: ROX single label; Escherichia coli O157:H7: Cy5 single label; Vibrio cholerae: FAM+HEX double label; Vibrio parahaemolyticus: HEX+ROX double label; Salmonella spp.: HEX+Cy5 double label.

[0042] (3) Construction of a super-multiplex digital PCR reaction system: Mix the above 7 pairs of primers and 7 labeled probes at optimized concentrations to form a primer-probe mixture. Prepare a 30 μL reaction system containing dPCR buffer, DNA polymerase, primer-probe mixture, and DNA from the sample to be tested.

[0043] (4) Digital PCR amplification and signal reading: The reaction system was loaded into a digital PCR instrument, which automatically performed chip partitioning and PCR amplification (annealing temperature 60℃, 45 cycles). After PCR, the instrument read the fluorescence signals of each reaction chamber in the four channels of FAM, HEX, ROX, and Cy5.

[0044] (5) Signal decoding and quantification based on chamber index consistency (see appendix) Figure 3 (Schematic diagram of chamber index consistency decoding principle) Data alignment: The software spatially aligns the images of the four channels to ensure that the index ID of each reaction chamber is consistent across different channels.

[0045] Signal recognition: Identify the positive / negative status of each chamber in each channel.

[0046] Decoding and Quantification: Conventional digital PCR typically uses the Poisson distribution formula to calculate copy number concentration. For multicolor fluorescence encoding and decoding technology, the conventional digital PCR formula is applicable when a single target is present, but when multiple targets coexist, a multiplexed formula for multiple decoding is required for calculation.

[0047]

[0048]

[0049] : Average molecular copy number; Pn: Corrected positive rate; Ti is the positive rate of the i-th fluorescence detection channel, and Tj ∩ Tk is the co-localization positive rate when the j-th and k-th fluorescence detection channels are simultaneously positive.

[0050] ① Single-target analysis: Count the number of chambers that are positive only in the FAM channel, substitute the results into the Poisson distribution formula, and calculate the concentration of Staphylococcus aureus. Similarly, calculate the concentrations of Cronobacterium, Listeria monocytogenes, and Escherichia coli O157:H7 based on the single-positive chambers of HEX, ROX, and Cy5, respectively.

[0051] ② Dual-target analysis: For Vibrio cholerae (encoded FAM+HEX), the number of cells where both FAM and HEX channels are positive and both positive signals appear in the same indexed chamber (i.e., co-localized positive chambers) is counted to calculate the Vibrio cholerae concentration. Similarly, the concentrations of Vibrio parahaemolyticus and Salmonella are calculated based on the co-localized positive chambers of HEX+ROX and HEX+Cy5, respectively.

[0052] ③ Signal differentiation based on index consistency: If FAM-positive and HEX-positive results appear in chambers with different indices, they are correctly classified as mixed contamination of Staphylococcus aureus and Cronobacter, and will not be incorrectly classified as Vibrio cholerae. This judgment logic is the key innovation of this invention, perfectly solving the crosstalk problem of mixed single-label and double-label signals in multicolor fluorescent coding.

[0053] Based on the above, a second aspect of this application provides a multiplex digital PCR detection device for detecting foodborne pathogens, comprising: a digital PCR instrument and the multiplex PCR detection reagents described in the above embodiment.

[0054] The aforementioned multiplex digital PCR detection device can simultaneously detect at least seven foodborne pathogens in a single reaction tube, while also possessing both detection accuracy and specificity.

[0055] In some embodiments, the digital PCR instrument is used to perform a PCR reaction between the nucleic acid sample to be tested and the above-mentioned multiplex PCR detection reagent; after the PCR reaction is completed, multicolor fluorescence encoding and decoding technology is used, combined with a quantitative formula, to calculate the absolute copy number concentration of each target gene in the nucleic acid sample to be tested.

[0056] The step of calculating the absolute copy number concentration of each target gene in the nucleic acid sample to be tested using multicolor fluorescence encoding and decoding technology and combined with quantitative formulas includes: The fluorescence signal of each reaction chamber in each fluorescence detection channel is read, and the images of each fluorescence detection channel are spatially aligned so that the index number of each reaction chamber is consistent under different fluorescence detection channels; Identify the positive or negative status of each of the reaction chambers in each of the fluorescence detection channels; For foodborne pathogens that require labeling with two fluorescent groups, when both corresponding fluorescent detection channels are positive in the reaction chamber with the same index number, the chamber is determined to be a positive droplet of the pathogen. Based on the number of positive droplets corresponding to each of the foodborne pathogen genes, and using a quantitative formula, the absolute copy number concentration of each of the foodborne pathogen genes in the nucleic acid sample to be tested is calculated.

[0057] The quantitative formula is as follows: ; ; Where λ is the average molecular copy number, Pn is the corrected positive rate, Ti is the positive rate of the i-th fluorescence detection channel, and Tj ∩ Tk is the co-localization positive rate when the j-th and k-th fluorescence detection channels are simultaneously positive.

[0058] The reaction conditions for the PCR reaction performed by the digital PCR instrument include: the concentration of each primer is 400 nM, the concentration of each detection probe is 100 nM-200 nM, and the annealing temperature is 60°C.

[0059] A third aspect of this application provides a method for multiplex PCR detection of foodborne pathogens, comprising the following steps: The nucleic acid sample to be tested and the multiplex PCR detection reagent described in the above embodiments are added to a digital PCR instrument for PCR reaction; After the PCR reaction, multicolor fluorescence encoding and decoding technology, combined with quantitative formulas, was used to calculate the absolute copy number concentration of each target gene in the nucleic acid sample to be tested.

[0060] In some embodiments, the step of calculating the absolute copy number concentration of each target gene in the nucleic acid sample to be tested using multicolor fluorescence encoding and decoding technology combined with a quantitative formula includes: The fluorescence signal of each reaction chamber in each fluorescence detection channel is read, and the images of each fluorescence detection channel are spatially aligned so that the index number of each reaction chamber is consistent under different fluorescence detection channels; Identify the positive or negative status of each of the reaction chambers in each of the fluorescence detection channels; For foodborne pathogens that require labeling with two fluorescent groups, when both corresponding fluorescent detection channels are positive in the reaction chamber with the same index number, the chamber is determined to be a positive droplet of the pathogen. Based on the number of positive droplets corresponding to each of the foodborne pathogen genes, and using a quantitative formula, the absolute copy number concentration of each of the foodborne pathogen genes in the nucleic acid sample to be tested is calculated.

[0061] In some embodiments, the quantitative formula is as follows: ; ; Where λ is the average molecular copy number, Pn is the corrected positive rate, Ti is the positive rate of the i-th fluorescence detection channel, and Tj ∩ Tk is the co-localization positive rate when the j-th and k-th fluorescence detection channels are simultaneously positive.

[0062] In some embodiments, the reaction conditions for PCR in the digital PCR instrument include: a concentration of 400 nM for each primer, a concentration of 100 nM-200 nM for each detection probe, and an annealing temperature of 60°C.

[0063] Compared with the prior art, the technical solution of this application has at least the following advantages:

[0064] The following is a specific embodiment.

[0065] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0066] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0067] Unless otherwise specified, the materials and methods in the following embodiments are as follows: (1) Materials and reagents Standard strain of Salmonella ( Salmonella typhi (CMCC(B) 50094), Staphylococcus aureus ( Staphylococcus aureus (CMCC(B) 26003)(China Medical Bacteriology Preservation and Management Center), Listeria monocytogenes ( Listeria monocytogenes (CICC 21635), Vibrio parahaemolyticus ( Vibrio Parahaemolyticus CICC 21617), Vibrio cholerae ( Vibrio cholerae (CICC 23794), Enterohemorrhagic Escherichia coli ( Escherichia coil O157:H7)(CICC 21530), Cronobacter spp. ( Cronobactersp.)(CICC 24097) (China Industrial Microbial Culture Collection Center). Sodium chloride (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); tryptic soy broth (TSB), tryptic soy digest agar (TSA) (Guangdong Huankai Microbial Technology Co., Ltd.); bacterial DNA extraction kit (Beijing Tiangen Biotech Co., Ltd.); fecal nucleic acid extraction kit (BeaverBeads® Wastewater Nucleic Acid Extraction Kit) (Suzhou Beaver Biomedical Engineering Co., Ltd.); digital PCR detection reagent (Shanghai Little Turtle Technology Co., Ltd.).

[0068] (2) Instruments and equipment BSP-150 Biochemical Incubator (Shanghai Boxun Medical Bio-Instrument Co., Ltd.); Kylin-Bell Vortex Shaker (Haimen Qilin Bell Instrument Manufacturing Co., Ltd.); Microfuge® 20R Centrifuge (Beckmancoulter, USA); HHW21.600 Water Bath (Beijing Yongguangming Medical Instrument Co., Ltd.); 1374 Biosafety Cabinet (Thermo Fisher Scientific (Suzhou) Instrument Co., Ltd.); SCI Digital PRO Digital PCR Instrument (including digital PCR chip loading, droplet preparation, PCR amplification, signal analysis, and multicolor encoded detection result output functions) (Shanghai Xiaohaigui Technology Co., Ltd.

[0069] (3) Strains culture and nucleic acid extraction The laboratory-preserved bacterial strains were incubated with TSB at 36°C for 24 h. 1 mL of the bacterial suspension (approximately 10⁻⁶ ppm) was then taken. 8 Extract genomic DNA from each bacterium (CFU / mL) according to the instructions of the bacterial genomic DNA extraction kit.

[0070] (4) Nucleic acid extraction and purification of interfering substances Fresh human fecal samples were randomly collected, and nucleic acids were extracted according to the instructions of the fecal nucleic acid extraction kit. The purified fecal nucleic acid was stored at -20°C.

[0071] (5) The testing conditions for the multiplex digital PCR detection system are as follows: Thoroughly mix the nucleic acid sample to be tested, primers, probes, reaction buffer, and enzymes. Prepare a multiplex digital PCR detection system using the following ratio: 10 μL Buffer, 0.5 μL Taq enzyme, 8.5 μL mixed primers / probes, 2–5 μL template, and bring the total volume to 30 μL with nuclease-free water. The primer concentration is 400 nM, the probe concentration is 200 nM, and the digital PCR buffer is 3×Maxuseful dPCR Buffer. Place the prepared reaction solution, other consumables, and oil phase at the designated station of the fully automated digital PCR machine and perform detection using the fully automated digital PCR system. Following the set detection program, the equipment automatically completes droplet preparation, PCR amplification, signal reading, and outputs quantitative results based on the multicolor encoding / decoding formula. The amplification reaction program consists of three stages: Stage 1: 50℃ curing for 5 min; Stage 2: 90℃ pre-denaturation for 5 min; Stage 3: 95℃ denaturation for 10 s, 60℃ annealing / extension for 40 s, for 45 cycles.

[0072] Example 1: Primer and probe design and multicolor coding scheme 1.1 Primer and probe sequences Primers and TaqMan probes were designed for species-specific genes of seven foodborne pathogens, as shown in Table 1. The 5' end of the probe was labeled with the corresponding fluorescent group according to the preset coding scheme, and the 3' end was labeled with the matching BHQ quencher group (FAM / HEX label corresponds to BHQ1, ROX / Cy5 label corresponds to BHQ2).

[0073] Table 1. Base sequences of primers and probes for detecting seven foodborne pathogens.

[0074] 1.2 Multicolor Fluorescent Encoding Scheme Four fluorescence channels, FAM, HEX, ROX, and Cy5, were used and coded as shown in Table 2 below.

[0075] Table 2. Types of fluorescent groups labeled with the detection probes for 7 foodborne pathogens.

[0076] Note: + indicates that the fluorescence is labeled; / indicates that it is not labeled.

[0077] Example 2: Establishment of a multiplex digital PCR detection system 2.1 The reaction system (30 μL) is prepared as shown in Table 3 below: Table 3 Multiplex Digital PCR Detection Reaction System

[0078] 2.2 The amplification procedure is shown in Table 4 below: Table 4 Multiplex Digital PCR Amplification Program

[0079] Thoroughly mix the nucleic acid sample to be tested, primer-probe combination, reaction buffer, and enzyme. The detection system has a primer concentration of 400 nM, a probe concentration of 100–200 nM, and a 3× Maxuseful dPCR Buffer. Place the prepared reaction solution, other consumables, and oil phase together at the designated station of the fully automated digital PCR machine. Set the detection program, and the fully automated digital PCR machine will automatically complete droplet preparation, PCR amplification, and signal reading. Finally, it will output the quantitative results according to the multicolor encoding and decoding formula. The specific steps are as follows: (6) Digital PCR amplification and signal reading: The reaction system was loaded into a digital PCR instrument, which automatically performed chip partitioning and PCR amplification (annealing temperature 60℃, 45 cycles). After PCR, the instrument read the fluorescence signals of each reaction chamber in the four channels of FAM, HEX, ROX, and Cy5.

[0080] (7) Signal decoding and quantification based on chamber index consistency (see schematic diagram of chamber index consistency decoding principle) Figure 3 ): Data alignment: The software spatially aligns the images of the four channels to ensure that the index ID of each reaction chamber is consistent across different channels.

[0081] Signal recognition: Identify the positive / negative status of each chamber in each channel.

[0082] Decoding and quantification: The following formula for multiple decoding is used for calculation.

[0083]

[0084]

[0085] : Average molecular copy number; Pn: Corrected positive rate; Ti: Positive rate of the i-th fluorescence detection channel; Tj ∩ Tk: Co-localization positive rate when the j-th and k-th fluorescence detection channels are simultaneously positive.

[0086] ① Single-target analysis: Count the number of chambers that are positive only in the FAM channel, substitute the results into the Poisson distribution formula, and calculate the concentration of Staphylococcus aureus. Similarly, calculate the concentrations of Cronobacter, Listeria monocytogenes, and Escherichia coli O157 based on the single-positive chambers of HEX, ROX, and Cy5, respectively.

[0087] ② Dual-target analysis: The number of chambers where both the FAM and HEX channels are positive and have identical indices (double-positive chambers) is counted to calculate the concentration of Vibrio cholerae. Similarly, the concentrations of Vibrio parahaemolyticus and Salmonella are calculated based on the double-positive chambers of HEX+ROX and HEX+Cy5, respectively.

[0088] ③ Eliminate false positives: If FAM positive and HEX positive appear in chambers of different indices, they are correctly classified as mixed contamination of Staphylococcus aureus and Cronobacter, and will not be mistakenly classified as Vibrio cholerae.

[0089] 2.3 Verification Experiment: Each pathogen was individually tested using a digital PCR instrument, and Vibrio cholerae, Vibrio parahaemolyticus, and Salmonella were detected using a dual-channel method. The test results are as follows: Figures 4 to 7 As shown. Figure 4 This is a partial view of a one-dimensional scatter plot for the detection of single targets of seven pathogenic bacteria. Figure 5 One-dimensional scatter plot of Staphylococcus aureus, Cronobacter, Listeria monocytogenes, and Escherichia coli O157; Figure 6 Two-dimensional scatter plot and raw fluorescence image for Vibrio cholerae (two-color coding) detection. In the figure, A: FAM coding channel, B: HEX coding channel, C: two-dimensional plot, D: raw spectrum. Figure 7 Two-dimensional plots for the detection of Vibrio parahaemolyticus (A, ROX and HEX coding channels) and a general two-dimensional plot for Salmonella (B, Cy5 and HEX coding channels).

[0090] from Figure 4 It can be seen that when the seven pathogenic bacteria targets were tested individually, the positive and negative signals were clearly distinguishable, the signal-to-noise ratio of digital PCR was >4, and there was no non-specific amplification signal.

[0091] from Figure 5 It can be seen that when detecting dual-target bacteria (Vibrio cholerae, Vibrio parahaemolyticus, and Salmonella), the positive chamber indexes of the two channels are completely consistent, which is in line with the coding design expectations.

[0092] Example 3: Specificity Test Under interference from human genomic DNA (15 ng / μL) and fecal nucleic acid (100 ng / μL), the multiplex PCR detection systems described in Examples 1 and 2 of this application were used. Specifically, a single-particle digital PCR detection system was prepared to detect individual target pathogens, verifying the signal discrimination of the multiplex detection system for each pathogenic bacterium. A mixed sample of seven bacterial genomes was used as a positive control. Simultaneously, 3 μL each of human genomic DNA (15 ng / μL) and fecal nucleic acid (100 ng / μL) were added to the original multiplex digital PCR detection system before volume adjustment. Nuclease-free water was then added to a final volume of 30 μL to prepare a mixed multiplex digital PCR detection system containing interference. The specificity of the multiplex digital PCR detection system was then tested. The detection results are as follows: Figure 8 As shown.

[0093] from Figure 8 It was found that no non-specific signals were observed in any channel of the negative and interference groups. Only the positive control (mixed DNA from 7 bacterial strains) showed the expected encoded positive droplets, indicating that the system has high specificity in complex matrices and no cross-reactivity. The negative group sample was water; the interference group sample consisted of human genomic DNA (15 ng / μL) and fecal nucleic acid (100 ng / μL). Example 4: Quantitative Accuracy Test The plasmid DNA of the seven pathogenic bacteria was diluted to approximately 10³ copies / μL and quantitatively detected using singlet dPCR and the multiplex PCR systems described in Examples 1-2 of this application (n=4). Specifically, the concentration of pathogen plasmids was adjusted to 10³ copies / μL. 3 Multiplex PCR was performed on a copy / μL scale to detect multiplex foodborne pathogens and their corresponding singlet target bacteria. The quantitative values ​​were compared to evaluate the accuracy of the multiplex quantification system. The results are shown in Table 5 below. Table 57 Comparison of Quantitative Results of Singleton PCR and Multiple PCR for Pathogenic Bacteria

[0094] As shown in Table 5, the quantitative results of the multiplex PCR system and the singlex PCR system are highly consistent, proving that the multiplex PCR quantitative detection system of this application is accurate and reliable.

[0095] Example 5: Linear Range and Repeatability Test 5.1 Linear Range Seven plasmids were serially diluted 10-fold to four concentrations (approximately 10). 4 The samples were diluted to 10³, 10², and 10¹ copies / μL, with each gradient repeated three times. Multiplex PCR was performed on the diluted samples following the procedures in Examples 1-2, and the results are shown in Table 6 below.

[0096] Table 6. Linear Range Detection Results

[0097] As shown in Table 6, the linear correlation coefficient R² of the detection results of each target is greater than 0.999, and the linear relationship is excellent within a four-order-of-magnitude concentration range, covering the conventional detection concentration range of pathogenic bacteria in food samples.

[0098] 5.2 Repeatability Test The 10³ copies / μL concentration samples obtained by dilution in “5.1 Linear Range” were subjected to multiplex PCR detection according to the procedures in Examples 1-2, and the detection was repeated 4 times. The detection results are shown in Table 7.

[0099] As shown in Table 7, the relative standard deviation (RSD) of the quantitative results of the seven bacteria is ≤5%, indicating that the system has good repeatability and the results are stable and reliable.

[0100] Table 7 Repeatability Test Results

[0101] ▲ Standard deviation / mean × 100% In summary, the technical solution of this application enables single-reaction detection of 7 targets (expandable to 15), significantly increasing throughput and truly achieving ultra-multiplex detection, greatly reducing detection costs and time. Utilizing the consistency of chamber indexes, it perfectly distinguishes all encoded signals, ensuring accurate decoding and fundamentally solving the signal crosstalk problem when multiple targets coexist. Only one reaction is required, increasing efficiency by more than 100%, simplifying operation, and saving samples and reagents. In addition to the advantages of dPCR, the specific primer and probe design ensures no cross-reaction in complex matrices such as the human genome and fecal nucleic acid, resulting in higher specificity and greater suitability for complex samples in real-world environments.

[0102] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A multiplex digital PCR detection reagent for detecting foodborne pathogens, characterized in that, The invention includes detection probes for detecting foodborne pathogens, including *Cronobacter* spp., *Staphylococcus aureus*, *Vibrio cholerae*, *Vibrio parahaemolyticus*, *Salmonella* spp., *Listeria monocytogenes*, and *Escherichia coli* O157:H7. The nucleotide sequences of the detection probes corresponding to *Cronobacter* spp., *Staphylococcus aureus*, *Vibrio cholerae*, *Vibrio parahaemolyticus*, *Salmonella* spp., *Listeria monocytogenes*, and *Escherichia coli* O157:H7 are shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.18, and SEQ ID NO.21, respectively. The detection probes are connected to fluorescent groups. The fluorescent group of the detection probe for Staphylococcus aureus is FAM, the fluorescent group of the detection probe for Cronobacter spp. is HEX, the fluorescent group of the detection probe for Listeria monocytogenes is ROX, the fluorescent group of the detection probe for Escherichia coli O157:H7 is Cy5, the fluorescent groups of the detection probe for Vibrio cholerae are FAM and HEX, the fluorescent groups of the detection probe for Vibrio parahaemolyticus are HEX and ROX, and the fluorescent groups of the detection probe for Salmonella spp. are HEX and Cy5.

2. The multiplex digital PCR detection reagent according to claim 1, characterized in that, The multiplex digital PCR detection reagent also includes amplification primers for detecting the foodborne pathogens. The amplification primers corresponding to the *Cronobacter* spp., *Staphylococcus aureus*, *Vibrio cholerae*, *Vibrio parahaemolyticus*, *Salmonella* spp., *Listeria monocytogenes*, and *Escherichia coli* O157:H7 are shown in SEQ ID NO.1-SEQ ID NO.2, SEQ ID NO.4-SEQ ID NO.5, SEQ ID NO.7-SEQ ID NO.8, SEQ ID NO.10-SEQ ID NO.11, SEQ ID NO.13-SEQ ID NO.14, SEQ ID NO.16-SEQ ID NO.17, and SEQ ID NO.19-SEQ ID NO.20, respectively.

3. The multiplex digital PCR detection reagent according to claim 1, characterized in that, The multiplex digital PCR detection probe is also linked to a quenching group, which includes BHQ1 and / or BHQ2.

4. The multiplex digital PCR detection reagent according to any one of claims 1-3, characterized in that, The multiplex digital PCR detection reagent also includes: dPCR buffer, DNA polymerase, and nuclease-free water.

5. A multiplex digital PCR detection device for detecting foodborne pathogens, characterized in that, include: Digital PCR instrument and multiplex PCR detection reagent as described in any one of claims 1-4.

6. The multiplex digital PCR detection device according to claim 5, characterized in that, The digital PCR instrument is used to perform PCR reactions on the nucleic acid sample to be tested and the multiplex PCR detection reagent; and after the PCR reaction is completed, multicolor fluorescence encoding and decoding technology, combined with quantitative formulas, is used to calculate the absolute copy number concentration of each target gene in the nucleic acid sample to be tested.

7. A multiplex PCR detection method for foodborne pathogens, characterized in that, Includes the following steps: The nucleic acid sample to be tested and the multiplex digital PCR detection reagent according to any one of claims 1-4 are added to a digital PCR instrument to perform a PCR reaction; After the PCR reaction, multicolor fluorescence encoding and decoding technology, combined with quantitative formulas, was used to calculate the absolute copy number concentration of each target gene in the nucleic acid sample to be tested.

8. The detection method according to claim 7, characterized in that, The step of calculating the absolute copy number concentration of each target gene in the nucleic acid sample to be tested using multicolor fluorescence encoding and decoding technology and combined with quantitative formulas includes: The fluorescence signal of each reaction chamber in each fluorescence detection channel is read, and the images of each fluorescence detection channel are spatially aligned so that the index number of each reaction chamber is consistent under different fluorescence detection channels; Identify the positive or negative status of each of the reaction chambers in each of the fluorescence detection channels; For foodborne pathogens that require labeling with two fluorescent groups, when both corresponding fluorescent detection channels are positive in the reaction chamber with the same index number, the chamber is determined to be a positive droplet of the pathogen. Based on the number of positive droplets corresponding to each of the foodborne pathogen genes, and using a quantitative formula, the absolute copy number concentration of each of the foodborne pathogen genes in the nucleic acid sample to be tested is calculated.

9. The detection method according to claim 8, characterized in that, The quantitative formula is as follows: ； ； Where λ is the average molecular copy number, Pn is the corrected positive rate, Ti is the positive rate of the i-th fluorescence detection channel, and Tj∩Tk is the co-localization positive rate when the j-th and k-th fluorescence detection channels are simultaneously positive.

10. The detection method according to any one of claims 7-9, characterized in that, The reaction conditions for PCR using the digital PCR instrument include: a concentration of 400 nM for each primer, a concentration of 100 nM-200 nM for each detection probe, and an annealing temperature of 60°C.

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