A dual-channel quadruple ddPCR detection method and kit for simultaneous detection of diarrheal bacteria in wastewater based on proportional fluorescent probe strategy.

Abstract

This invention discloses a dual-channel quadruple ddPCR detection method and kit for the simultaneous detection of diarrheal bacteria in wastewater based on a proportional fluorescent probe strategy. Compared with traditional quadruple detection methods using four different fluorescent channels, the quadruple ddPCR detection method of this invention achieves quadruple detection only through the difference in probe concentration within two fluorescent channels. Wastewater, as a key interface between excrement and the environment, is not only an important site for the storage and transmission of pathogens but also a sentinel for early warning. The dual-channel quadruple ddPCR method established in this invention can simultaneously detect Salmonella in wastewater. Salmonella spp., SM), Shigella ( Shigella spp., SH), Vibrio parahaemolyticus ( V.parahaemolyticus, VP) and Vibrio cholerae ( Vibrio cholerae, VC (Vacuum-Containing Microorganisms) not only improves wastewater monitoring technology and methods, but also enables the detection of potential epidemic risks before disease outbreaks occur, allowing for timely implementation of public health prevention and control measures.

Description

Technical Field

[0001] This invention relates to a quadruple ddPCR detection method and kit for the simultaneous detection of diarrheal bacteria in wastewater, and particularly to a quadruple ddPCR detection method and kit based on a proportional fluorescent probe strategy for the simultaneous detection of diarrheal bacteria in wastewater. This invention belongs to the field of biotechnology. Background Technology

[0002] Globally, bacterial infectious diarrhea is one of the leading causes of death in children under five years old, with Shigella spp. being a major culprit. Shigella spp., SH), Salmonella spp. Salmonella spp., SM), Vibrio parahaemolyticus ( V. parahaemolyticus, VP) and Vibrio cholerae ( Vibrio cholerae, VC (vitamin C) is a key pathogen. These pathogens are widely distributed and continuously spread in the environment, and coupled with increasing environmental pollution, the risk of infection remains high, particularly in low- and middle-income countries. Therefore, wastewater, which integrates information on numerous pathogens, has become a crucial medium reflecting the overall health status of the population.

[0003] Wastewater-based epidemiology (WBE) has become an effective method for tracking the spatiotemporal dynamics of infectious disease pathogens. It can provide near real-time, objective population health information and can supplement traditional clinical surveillance. Wastewater epidemiology has proven applicable to a variety of pathogenic microorganisms, including drug-resistant bacteria, Mycobacterium tuberculosis, and viral pathogens such as respiratory syncytial virus, influenza virus, and monkeypox virus. However, the concentration of pathogens in wastewater is usually low, posing challenges for detection and quantification. While traditional molecular methods are useful, their sensitivity may be limited in complex environmental matrices.

[0004] Digital PCR (ddPCR) offers accurate quantification and good inhibitor tolerance, enabling absolute quantification of low-abundance targets, thus effectively overcoming the limitations of traditional detection methods in environmental samples. Multiplex ddPCR technology further expands its application potential, allowing simultaneous detection of multiple pathogens while maintaining stable analytical performance. These advantages make ddPCR an ideal analytical platform for detecting low concentrations of pathogens in the environment, achieving early warning of outbreaks, and strengthening the prevention of waterborne and diarrheal diseases.

[0005] In China, Salmonella (SM) and Vibrio parahaemolyticus (VP) are among the most common pathogens causing acute diarrhea. In contrast, Shigella (SH) and Vibrio cholerae (VC) have lower detection rates in clinical diarrhea samples, with VC showing particularly low positivity rates in routine diagnosis. However, both SH and VC can cause severe diarrheal symptoms and have strong transmission potential, thus requiring continued surveillance. Currently, although various clinical detection methods for diarrheal pathogens exist, their application remains primarily limited to the individual level. Population or community-level surveillance efforts are still insufficient, and asymptomatic infections or undiagnosed cases are easily missed, potentially leading to an underestimation of transmission risk. Wastewater epidemiology, by capturing pathogen aggregation signals from mild / asymptomatic carriers, offers a way to overcome these limitations, thus providing a more comprehensive reflection of the infection burden at the community level.

[0006] Integrating wastewater monitoring into the epidemiological investigation of diarrheal diseases can not only provide early warnings before clinical cases appear, facilitating timely control measures, but also enable disinfection interventions to address wastewater contamination and prevent further transmission of pathogens. However, achieving real-time, multi-target monitoring of diarrheal pathogens remains challenging due to limitations in current technologies. While traditional culture methods are considered the "gold standard," their time-consuming nature and low sensitivity make them unsuitable for early warning needs before outbreaks. Real-time quantitative polymerase chain reaction (qPCR), despite its rapid detection advantage, relies on standard curves, is sensitive to inhibitors, and is affected by matrix effects, impacting the reliability of results.

[0007] ddPCR overcomes many of the aforementioned limitations by providing absolute quantification and improving relative tolerance to inhibitors; however, traditional ddPCR platforms can only detect one target per fluorescence channel, limiting their multipathogen detection capabilities. To overcome these challenges, there is an urgent need to develop innovative detection strategies that combine sensitivity, multiplexing capabilities, and cost-effectiveness to achieve multiplex detection of diarrheal bacteria in wastewater for diarrheal disease surveillance and early warning. Such methods will provide an economical and convenient solution for the sustainable surveillance of diarrheal bacteria in resource-scarce areas and offer necessary technical support for disease prevention.

[0008] Previous studies have confirmed the feasibility of detecting four diarrheal bacteria in wastewater using the QIAcuity platform; however, these methods require four independent fluorescence channels, which limits their applicability to other platforms. Therefore, this invention aims to develop and validate a ddPCR method based on the Bio-Rad platform that achieves quadruple detection using only two channels. Summary of the Invention

[0009] The purpose of this invention is to provide a dual-channel quadruple ddPCR method and kit for detecting diarrheal bacteria in wastewater based on a proportional fluorescent probe strategy.

[0010] To achieve the above objectives, the present invention employs the following technical means:

[0011] This invention discloses a dual-channel quadruple ddPCR method for detecting diarrheal bacteria in wastewater based on a proportional fluorescence probe strategy. The method is not intended for disease diagnosis or treatment. Instead, it employs a single-channel signal multiplexing strategy to achieve simultaneous quantification of multiple target diarrheal bacteria. The method includes the following steps:

[0012] (1) After extracting DNA from the sewage sample, it was resuspended in nuclease-free water for later use;

[0013] (2) Using the DNA obtained in step (1) as a template, a quadruple ddPCR detection kit for simultaneously detecting diarrheal bacteria in sewage was used for detection; the quadruple ddPCR detection kit contains reagents for detecting Salmonella ( Salmonella spp., SM), Shigella ( Shigella spp., SH), Vibrio parahaemolyticus ( V. parahaemolyticus, VP) and Vibrio cholerae ( Vibrio cholerae, Primer pairs and probes for VC; where:

[0014] The primer pair used for detecting Salmonella has the sequences of its upstream and downstream primers as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe sequence as shown in SEQ ID NO:3.

[0015] The primer pair used for detecting Shigella has the sequences of its upstream and downstream primers as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequence as shown in SEQ ID NO:6.

[0016] The primer pair used for detecting Vibrio parahaemolyticus has the sequences of its upstream and downstream primers as shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe sequence as shown in SEQ ID NO:9.

[0017] The primer pair used for detecting Vibrio cholerae has the sequences of its upstream and downstream primers as shown in SEQ ID NO:10 and SEQ ID NO:11, and the probe sequence as shown in SEQ ID NO:12.

[0018] The 5' ends of the specific probes for detecting SM and VP are labeled with 6-carboxyfluorescein (FAM); the 5' ends of the specific probes for detecting SH and VC are labeled with hexachlorofluorescein (HEX); the remaining probes are all quenched at the 3' end with Black Hole Quencher-1 (BHQ1); the specific probe for detecting VC uses the Zen dual quenching system, which, in addition to the conventional 3' end modification with the BHQ1 quenching group, introduces an additional Zen quenching group at the 9th base of the sequence, thus forming a dual quenching mechanism;

[0019] The detection was performed using a 20 µL reaction mixture, which contained: 10.0 µL of ddPCR ultramixture, 8 primers at a concentration of 750 nM, 4 probes, 2 µL of DNA template, and nuclease-free water. The probe concentrations were as follows: in the FAM channel, the probe concentrations for detecting SM and VP were 150 nM and 200 nM, respectively; in the HEX channel, the probe concentrations for detecting VC and SH were 200 nM and 250 nM, respectively.

[0020] The specific experimental procedure is as follows: 20 µL of the reaction mixture was dropped into a 96-well ddPCR plate and sealed. The mixture was generated using a QX200™ droplet generator and amplified on a T100 thermal cycler under the following conditions: initial denaturation at 95 °C for 10 min, denaturation at 95 °C for 30 s, annealing / extension at 57-59 °C for 80 s, for 45 cycles.

[0021] (1) Fluorescence signal detection was performed using a QX200™ droplet reader, and the results were determined by distinguishing between positive and negative droplets. In the FAM channel, positive droplets with high fluorescence intensity came from VP, while positive droplets with low fluorescence intensity came from SM. In the HEX channel, positive droplets with high fluorescence intensity came from VC, while positive droplets with low fluorescence intensity came from SH.

[0022] Preferably, the wastewater sample is incubated in a 65°C water bath for 20-40 minutes to eliminate the potential biological risks posed by live bacteria.

[0023] Furthermore, this invention also proposes a dual-channel quadruple ddPCR kit for detecting diarrheal bacteria in wastewater based on a proportional fluorescence probe strategy. The kit contains primer pairs and probes for detecting Salmonella, Shigella, Vibrio parahaemolyticus, and Vibrio cholerae; wherein:

[0024] The primer pair used for detecting Salmonella has the sequences of its upstream and downstream primers as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe sequence as shown in SEQ ID NO:3.

[0025] The primer pair used for detecting Shigella has the sequences of its upstream and downstream primers as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequence as shown in SEQ ID NO:6.

[0026] The primer pair used for detecting Vibrio parahaemolyticus has the sequences of its upstream and downstream primers as shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe sequence as shown in SEQ ID NO:9.

[0027] The primer pair used for detecting Vibrio cholerae has the sequences of its upstream and downstream primers as shown in SEQ ID NO:10 and SEQ ID NO:11, and the probe sequence as shown in SEQ ID NO:12.

[0028] The 5' ends of the specific probes for detecting SM and VP are labeled with 6-carboxyfluorescein (FAM); the 5' ends of the specific probes for detecting SH and VC are labeled with hexachlorofluorescein (HEX); the remaining probes are all quenched at the 3' end with Black Hole Quencher-1 (BHQ1). The specific probe for detecting VC uses the Zen dual quenching system, which, in addition to the conventional BHQ1 quenching group modified at the 3' end, introduces an additional Zen quenching group at the 9th base of the sequence, thus forming a dual quenching mechanism.

[0029] Furthermore, this invention also proposes a dual-channel quadruple ddPCR reaction mixture for the simultaneous detection of diarrheal bacteria in wastewater based on a proportional fluorescence probe strategy. The dual-channel quadruple ddPCR reaction mixture contains: 10.0 µL of ddPCR supermixture, 8 primers at a concentration of 750 nM, 4 probes, 2 µL of DNA template, and nuclease-free water. The probe concentrations are as follows: in the FAM channel, the probe concentrations for detecting SM and VP are 150 nM and 200 nM, respectively; in the HEX channel, the probe concentrations for detecting VC and SH are 200 nM and 250 nM, respectively. The sequences of the primers and probes are as described above.

[0030] Furthermore, the present invention also proposes the application of the kit or the quadruple ddPCR reaction mixture described herein in the preparation of reagents for the simultaneous detection of Salmonella, Shigella, Vibrio parahaemolyticus and Vibrio cholerae.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] This invention applies a proportional probe strategy to wastewater monitoring. Compared to the traditional quadruple detection method with four different fluorescence channels [PITTON M, MCLEOD RE, CADUFF L, et al. A six-plex digital PCR assay for monitoring respiratory viruses in wastewater [J]. Nature Water, 2025:1-13. http: / / dx.doi.org / DOl], the dual-channel quadruple ddPCR detection method of this invention utilizes only the difference in probe concentration within two fluorescence channels to achieve quadruple detection.

[0033] Currently, ddPCR and qPCR are the two most widely used techniques for pathogen nucleic acid detection. qPCR is extremely fast, but its quantification depends on a standard curve and is susceptible to inhibitor interference in complex matrices such as wastewater, potentially affecting accuracy. In contrast, ddPCR achieves absolute quantification without relying on a standard curve and is relatively more tolerant of inhibitors. Our results show that the constructed quadruple ddPCR exhibits comparable quantification performance to single ddPCR at two nucleic acid dilutions, demonstrating the independence of multiple primer-probe interactions. Notably, this method can reliably detect low concentrations of pathogens, further highlighting its potential application in early epidemic surveillance.

[0034] Wastewater, as the confluence of human excrement and the environment, is not only a key channel for pathogen transmission but also plays the role of a public health early warning "sentinel." The dual-channel quadruple ddPCR method developed in this invention can simultaneously detect four types of diarrheal bacteria—VC, SM, VP, and SH—in wastewater. This method can capture potential risks at the community level before clinical cases appear, providing precise early warning and decision-making basis for proactive intervention at the population level, interrupting transmission chains, and reducing infection risks. Attached Figure Description

[0035] Figure 1 Optimize fluorescence intensity and annealing temperature for quadruple ddPCR detection;

[0036] Among them, (A, B) results of singlet ddPCR detection of four bacterial targets under temperature gradient optimization (from 60℃ to 55℃); (CF) fluorescence intensity of (C) SM, (D) VP, (E) SH and (F) VC in the FAM and HEX channels in quadruple ddPCR; (G) a representative two-dimensional droplet plot showing the separation of different clusters of each target under optimized conditions; negative results are represented by the gray droplet cluster in the lower left corner; SH, Shigella; SM, Salmonella; VC, Vibrio cholerae; VP, Vibrio parahaemolyticus;

[0037] Figure 2 This is to assess the stability of the quadruple ddPCR method and the actual sample detection results;

[0038] Among them, (A) shows the linear dynamic range of the four strains under successive 10-fold dilutions, with the x-axis representing log10. (CFU / mL) The y-axis is log10 (Copies / µL reaction) The bacterial counts were as follows: Salmonella, Shigella, Vibrio parahaemolyticus, and Vibrio cholerae; (B) Differences between quadruple and single ddPCR detection values; Two-way ANOVA was used to compare the detection results of quadruple ddPCR and single ddPCR under two dilution factors, and the differences were not statistically significant (P < 0.05); (C) Concentration of diarrheal bacteria detected in actual sewage samples; Quadruple ddPCR was performed on 60 sewage samples (7 samples from farmers' markets, 8 samples from hospitals, and 45 samples from communities). Blue represents communities, red represents hospitals, and gray represents farmers' markets; all experiments were repeated three times; the unit is Copies / 100 mL sewage.

[0039] Figure 3 The results show the difference in inhibition between humic acid-spiked singlet qPCR and quadruple ddPCR.

[0040] Among them, (A) single qPCR; (B) quadruple ddPCR; the horizontal axis represents the humic acid concentration in units of (μg / mL), with a 10-fold serial dilution, for a total of four gradients; the vertical axis represents the actual value detected at different humic acid concentration gradients / the standard value without humic acid addition. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0042] Example 1: Establishment of a quadruple ddPCR detection method for simultaneous detection of diarrheal bacteria in wastewater based on proportional fluorescent probes.

[0043] method:

[0044] 1. Bacterial strains

[0045] Positive control strains were Shigella (SH1), Salmonella (LT2), Vibrio parahaemolyticus (ICDC-VP1329) and Vibrio cholerae (N16961) [SUN H, YAO Q, ZHAO X, et al. A Quadruplex Digital PCR Assay for the Simultaneous Detection of Four Intestinal Bacterial Pathogens and Its Application in Wastewater Samples [J]. China CDC Weekly, 2025, 7(12):393. http: / / dx.doi.org / DOl;MCCLELLAND M, SANDERSON KE, SPIETH J, et al. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2 [J]. Nature, 2001, 413(6858): 852-6. http: / / dx.doi.org / DOl;HEIDELBERG JF, EISEN JA, NELSON WC, et al. DNA sequence of both chromosomes of the cholerapathogen Vibrio cholerae [J]. Nature, 2000, 406(6795): 477-83.http: / / dx.doi.org / DOl].

[0046] 2. Primers and probes

[0047] The four primer-probe sets used in this study are shown in Table 1. Salmonella ( Salmonella spp., SM), Vibrio parahaemolyticus ( V. parahaemolyticus, The 5' end of the VP-specific probe is labeled with 6-carboxyfluorescein (FAM); Shigella ( Shigella spp., SH), Vibrio cholerae ( Vibrio cholerae,The 5' end of the VC-specific probe is labeled with hexachlorofluorocarbon (HEX). When detecting low-abundance environmental pathogens, high background fluorescence signals often mask positive signals. Therefore, adding an internal quencher group helps reduce fluorescence leakage and significantly lowers the background fluorescence signal. The VC probe employs a Zen dual-quenching system. In addition to the conventional BHQ1 quencher group modified at the 3' end, an additional Zen quencher group is introduced at a specific position (the 9th base), forming a dual quenching mechanism, which can be represented as 5'-HEX / Zen / BHQ1-3'. The probe was synthesized by Kunshan Puno Biotechnology Co., Ltd. (Jiangsu, China). The remaining probes were quenched at the 3' end using Black HoleQuencher-1 (BHQ1).

[0048] Table 1 Information on the four pairs of primers and probes

[0049]

[0050] 3. ddPCR reaction

[0051] Quadruple ddPCR and single ddPCR detection were performed using a QX200 AutoDG droplet digital PCR system (Bio-Rad, USA). For the quadruple ddPCR reaction, 20 µL of reaction mixture contained: 10.0 µL of ddPCR supermix (Bio-Rad, ddPCRSupermix for Probes, 500 Rxns), 8 primers at 750 nM, 4 probes, 2 µL of DNA template, and nuclease-free water. The specific probe concentrations were as follows: in the FAM channel, the probe concentrations for SM and VP were 150 nM and 200 nM, respectively; in the HEX channel, the probe concentrations for VC and SH were 200 nM and 250 nM, respectively. 20 µL of the reaction mixture was added to a 96-well ddPCR plate (Bio-Rad, USA), sealed, and then processed using a QX200 system. TM Droplets were generated using a Bio-Rad (USA) generator and amplified on a T100 thermal cycler (Bio-Rad (USA)) under the following conditions: initial denaturation at 95°C for 10 min, denaturation at 95°C for 30 s, annealing / extension at 57-59°C for 80 s, for 45 cycles. Fluorescence signal detection was performed using a QX200™ droplet reader (Bio-Rad (USA),) and results were determined by distinguishing between positive and negative droplets.

[0052] 4. Establishment of quadruple ddPCR

[0053] First, DNA from four positive reference strains was used as templates for ddPCR. Annealing / extension temperatures were optimized using gradient PCR, within a range of 55℃ to 60℃. The optimal temperature for clearly separating positive and negative signals was determined to be 57-59℃. Figure 1 A, B), and then used in all ddPCR experiments. DNA samples containing the four positive targets were diluted to appropriate concentrations within the instrument's detection range, and primer / probe concentrations were systematically optimized to achieve concentration and equilibrium. DNA was serially diluted 5 times at 10-fold, log10 (CFU / mL) For the x-axis, log10 (Copies / µLreaction) The y-axis was used to assess linearity. Medium concentrations of DNA template were detected using quadruple and singlet ddPCR (each test was repeated three times), and the results were compared. All DNA samples were referenced and stored at -80°C for further analysis.

[0054] 5. Manually spiked wastewater samples

[0055] The sensitivity of this method was evaluated using artificially spiked wastewater samples. The specific methods are as follows: 1) Spreading: Overnight cultures were serially diluted 10-fold, and 10 µl of each dilution was spread onto solid culture medium to determine bacterial count. 2) DNA preparation: Positive reference bacterial nucleic acid was serially diluted 10-fold. An equal volume (1 mL) of each bacterial dilution was added to 100 mL of wastewater (the wastewater was previously tested negative for all four target pathogens). DNA was extracted immediately using the Fast DNA SPIN Kit for Soil (MP Biomedicals) according to the manufacturer's instructions. 3) The DNA was resuspended in 100 µL of nuclease-free water, with 2 µL used as a ddPCR template. Each dilution was tested three times in a 96-well plate. The limit of detection (LOD) was defined as the lowest CFU concentration that produced a positive amplified signal in three replicates. The coefficient of variation (CV) was determined by three measurements for each spiked sample. DNA from positive strains was used as a positive control, and nuclease-free water as a negative control. All DNA samples were referenced and stored at -80°C.

[0056] 6. qPCR

[0057] Both qPCR and quadruple ddPCR assays used the same probes, primers, and DNA template. For qPCR, only one target was detected per reaction. A 20 µL reaction mixture consisted of 10 µL premix (Takara, Japan), primers and probes at a final concentration of 250 nM, 2 µL DNA template, and nuclease-free water, for a final volume of 20 µL. (Source: QuantStudio) TM 5Amplification was performed on a system (Thermo Fisher Scientific, USA) under the following cycling conditions: 95°C for 60 seconds, followed by denaturation at 95°C for 10 seconds, and annealing / extension at 57-59°C for 30 seconds, for a total of 40 cycles. DNA from artificially added wastewater was serially diluted 10-fold, for a total of four dilutions. These dilutions were used as templates for generating a standard curve for qPCR and determining the level of detail (LOD). All measurements were performed in triplicate. (Log10) CFU A linear regression model is established for X and Cq value y, using the slope of the standard curve (E=10). -1 / slope-1 Calculate the amplification efficiency of each primer and probe.

[0058] 7. Differences in humic acid inhibition between singlet qPCR and quadruple ddPCR

[0059] Four target nucleic acids were extracted using the same method as when establishing the quadruple ddPCR method, and set aside. 0.05 g of humic acid was dissolved in 10 mL of nuclease-free water and diluted to concentrations of 0, 100, 200, 300, and 400 μg / mL, and set aside. Samples were diluted 10 times under different humic acid concentrations. 0 -10 5 Simultaneous detection of singlet qPCR and quadruple ddPCR was performed at different folds. The ratio of spiked humic acid detection value to unspiked humic acid detection value was used as the result to assess its stability against inhibitors.

[0060] 8. Wastewater samples

[0061] Sixty wastewater samples (7 from farmers' markets, 8 from hospitals, and 45 from communities) were collected from hospitals, communities, and farmers' markets in five districts of Beijing: Haidian, Daxing, Changping, Fengtai, and Chaoyang. All samples were collected on April 14 and June 4, 2025, and stored at 4°C. The samples were categorized as follows: 7 from farmers' markets, 8 from hospitals, and 41 from communities. Each 100 mL wastewater sample was incubated in a 65°C water bath for 30 minutes to eliminate potential biological risks from live bacteria. DNA was extracted from the wastewater samples using the same method as for artificially spiked wastewater samples. The extracted DNA was resuspended in 100 µL of nuclease-free water and analyzed by qPCR and ddPCR. All DNA samples were referenced and stored at -80°C.

[0062] 9. Statistical Analysis

[0063] All collected data were recorded in Microsoft Excel 365. Two-way ANOVA was performed using GraphPad Prism 10 software to evaluate the statistical differences in detection values ​​between quadruple ddPCR and singlet qPCR at the two dilutions. R4.4.3 was used to display the results of quadruple ddPCR samples. AI was used to enhance and stitch all images.

[0064] result:

[0065] 1. qPCR reaction system and sensitivity

[0066] Each qPCR reaction contained only one template. We first used the DNA from a single template within the reaction system to assess amplification efficiency. Genomic DNA from four positive strains was serially diluted 10-fold to create four gradients. -1 and 10 -5 The amplification efficiency among the dilution factors was relatively high (90%–110%), exhibiting a strong linear correlation (R² > 0.99) (Table 2). The level of difference (LOD) was further evaluated by serially diluting DNA extracted from artificially spiked wastewater samples by 10-fold dilution. The LODs for singleton qPCR detection were 81, 13, 50, and 25 CFU per 100 mL of wastewater for VC, SH, VP, and SM, respectively. The results indicate that the amplification results are linearly stable and highly sensitive.

[0067] Table 2 Standard curve of singlet qPCR and LOD in simulated wastewater

[0068]

[0069] Note: using log10 CFU A linear regression model is established for X and Cq value y, using the slope of the standard curve (E=10). -1 / slope-1 Calculate the amplification efficiency of each primer and probe. The standard curve is plotted with CT value on the ordinate and the logarithm of the number of colonies (CFU) added in each reaction on the abscissa. CFU / mL wastewater: Bacterial concentration calculated from colony count results. LOD: Lowest concentration of CFU with three consecutive positive tests. * Three replicate negative wells are defined as LOD. LOD, Limit of Detection; CFU, Colony Forming Units.

[0070] 2. Optimization of quadruple ddPCR reaction conditions and probe ratios

[0071] Next, we optimized the probe concentrations in the quadruple ddPCR reaction mixture to improve recognition capability. First, the concentration of each probe in the quadruple ddPCR reaction was fixed at 230 nm, and only one template DNA was added to measure the fluorescence intensity of each target gene positive droplet. In the FAM channel, high fluorescence intensity positive droplets (FAM-hi) corresponded to VP, while low fluorescence intensity positive droplets (FAM-lo) corresponded to SM. In the HEX channel, high fluorescence intensity positive droplets (HEX-hi) corresponded to VC, and low fluorescence intensity positive droplets (HEX-lo) corresponded to SH. Figure 1 CF).

[0072] In a quadruple ddPCR reaction, four template DNAs are added simultaneously, and the probe concentration is adjusted within the range of 100-300 nM. The optimal probe concentration for clearly distinguishing positive droplets between two target genes within the same fluorescence channel is determined as follows: for the FAM channel, the probe concentrations for SM and VP are 150 nM and 200 nM, respectively; while for the HEX channel, the probe concentrations for SH and VC are 250 nM and 200 nM, respectively. Figure 1 G).

[0073] When detecting four target DNAs simultaneously, the DNA molecules are randomly dispersed into 20,000 droplets. DNAs with two or more targets may be present in these droplets, which enhances fluorescence and produces additional positive clusters. Ultimately, the positive and negative droplets are clearly separated into 2... 4 (16) clusters. However, the fluorescence amplitudes of VC and SH on the x-axis ranged from 3500-4500 to 3000-4000, while the fluorescence amplitudes of VP and SM on the y-axis were approximately 10000-12000 and 6000-10000, respectively. The positions of the positive clusters remained essentially unchanged across different template concentration ranges.

[0074] 3. Analytical quadruple ddPCR: linearity, precision, and sensitivity

[0075] Within the reaction concentration range, SM: 3.80-3.00×10 4 Copy / reaction, VP: 5.80-2.01×10 4 Copy / reaction, SH: 3.67-1.25×10 5 Copy / reaction, VC: 7.27-6.42×10 4 Copy / reaction, all targets showed good linearity (R²). 2 > 0.99). (Table 3, Figure 2 A). As template concentration decreases, CV values ​​increase, but accuracy decreases (Table 3). At 10... -4In the diluted sample, the concentration of SM (Mean ± SD) was 4.53 × 10⁻⁶. 2 ±50.33 copies / reaction (CV 11.1%), SH concentration (Mean ± SD) was 6.73 × 10⁻⁶. 2 ±110.15 copies / reaction (CV 16.5%), VP concentration (Mean ± SD) was 2.14 × 10⁻⁶. 2 ±25.71 copies / reaction (CV 14.9%), VC concentration (Mean ± SD) was 9.75 × 10⁻⁶. 2 ±86.49 copies / reaction (CV 8.9%). Using 10-fold dilutions of spiked wastewater DNA, the LODs of quadruple ddPCR were (VC) 81, (SH) 13, (VP) 50, and (SM) 31 CFU / 100 mL, respectively.

[0076] Table 3. LOD of quadruple ddPCR in simulated spiked wastewater

[0077]

[0078] Note: CFU / mL wastewater: Bacterial concentration calculated from colony count results. LOD: Lowest CFU concentration with three consecutive positive tests. copies / µL: Concentration of extracted DNA detected in spiked wastewater. * Three replicate negative wells are defined as LOD. CV: Coefficient of variation; SD: Standard deviation; LOD: Limit of detection; CFU: Colony forming unit.

[0079] 4. Wastewater sample testing

[0080] Analysis of 60 wastewater samples (7 from farmers' markets, 8 from hospitals, and 45 from communities) showed that the positive detection rate of quadruple ddPCR was consistently higher than that of qPCR (Table 4). The results are summarized as follows: the detection rates (SM) of quadruple ddPCR and qPCR were 56 / 60 (93.3%) and 53 / 60 (88.3%), respectively; the SH values ​​were 46 / 60 (76.7%) and 44 / 60 (73.3%), respectively; and the VP values ​​were 20 / 60 (33.3%) and 19 / 60 (31.7%), respectively. VC was not detected by either method (0 / 60, 0.0%). The concentration ranges detected by quadruple ddPCR were SM 10.8 ± 15.4 copies / µL, SH 4.5 ± 9.3 copies / µL, and VP 6.7 ± 17.5 copies / µL. Figure 2 A). VC, SH, VP, and SM were analyzed separately, and the results are shown in Table 4. The LOD of quadruple ddPCR and single qPCR was similar. (In 10...) -4 and 10 -5At the dilution, there was no difference in the detection results of the four targets between the quadruple ddPCR experiment and the single experiment (P > 0.05). Figure 2 B).

[0081] Table 3. Detection rate of diarrheal bacteria in actual sewage samples from hospitals, communities, and farmers' markets.

[0082]

[0083] 5. Differences in inhibition between singlet qPCR and multiplex ddPCR

[0084] After adding humic acid, the results were as follows Figure 3 As shown in the figure, the results indicate that singleton qPCR showed a slight decreasing trend in the detection values ​​of SM, SH, and VC, but an increasing trend in the detection value of VP. Multiplex ddPCR showed greater stability than singleton qPCR in the detection of different concentrations of humic acid, but was more prone to fluctuations at lower pathogen concentrations.

[0085] Example 2: A quadruple ddPCR detection kit for simultaneous detection of diarrheal bacteria in wastewater based on proportional fluorescent probes.

[0086] The kit contains ingredients for detecting Salmonella (…). Salmonella spp., SM), Shigella ( Shigella spp., SH), Vibrio parahaemolyticus ( V. parahaemolyticus, VP) and Vibrio cholerae ( Vibrio cholerae, Primer pairs and probes for VC; where:

[0087] The primer pair used for detecting Salmonella has the sequences of its upstream and downstream primers as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe sequence as shown in SEQ ID NO:3.

[0088] The primer pair used for detecting Shigella has the sequences of its upstream and downstream primers as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequence as shown in SEQ ID NO:6.

[0089] The primer pair used for detecting Vibrio parahaemolyticus has the sequences of its upstream and downstream primers as shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe sequence as shown in SEQ ID NO:9.

[0090] The primer pair used for detecting Vibrio cholerae has the sequences of its upstream and downstream primers as shown in SEQ ID NO:10 and SEQ ID NO:11, and the probe sequence as shown in SEQ ID NO:12.

[0091] The 5' ends of the specific probes for detecting SM and VP are labeled with 6-carboxyfluorescein (FAM); the 5' ends of the specific probes for detecting SH and VC are labeled with hexachlorofluorescein (HEX); the remaining probes are quenched at the 3' end with Black Hole Quencher-1 (BHQ1). The VC probe employs a Zen dual quenching system, which, in addition to the conventional 3' end modification with a BHQ1 quenching group, introduces an additional Zen quenching group at a specific internal position (the 9th base), thus forming a dual quenching mechanism.

[0092] When the kit is used to simultaneously detect diarrheal bacteria in sewage, a 20 µL reaction mixture contains: 10.0 µL of ddPCR ultramixture, 8 primers at a concentration of 750 nM, 2 µL of DNA template, and nuclease-free water. The probe concentration ranges are as follows: in the FAM channel, the probe concentrations of SM and VP are 150 nM and 200 nM, respectively; in the HEX channel, the probe concentrations of VC and SH are 200 nM and 250 nM, respectively.

[0093] Add 20 µL of the reaction mixture to a 96-well ddPCR plate and seal. Use QX200. TM Droplets were generated using a droplet generator and amplified on a T100 thermal cycler under the following conditions: initial denaturation at 95°C for 10 min, denaturation at 95°C for 30 s, annealing / extension at 57-59°C for 80 s, for 45 cycles.

Claims

1. A dual-channel quadruple ddPCR method for detecting diarrheal bacteria in wastewater based on a proportional fluorescent probe strategy, characterized in that, The method employs a single-channel signal multiplexing strategy to achieve simultaneous quantification of multiple target diarrheal bacteria. The method includes the following steps: (1) After extracting DNA from the sewage sample, it was resuspended in nuclease-free water for later use; (2) Using the DNA obtained in step (1) as a template, a quadruple ddPCR detection kit for simultaneously detecting diarrheal bacteria in sewage is used for detection; the quadruple ddPCR detection kit contains reagents for detecting Salmonella ( Salmonella spp., SM), Shigella ( Shigella spp., SH), Vibrio parahaemolyticus ( V. parahaemolyticus, VP) and Vibrio cholerae ( Vibrio cholerae, Primer pairs and probes for VC; where: The primer pair used for detecting Salmonella has the sequences of its upstream and downstream primers as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe sequence as shown in SEQ ID NO:

3. The primer pair used for detecting Shigella has the sequences of its upstream and downstream primers as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequence as shown in SEQ ID NO:

6. The primer pair used for detecting Vibrio parahaemolyticus has the sequences of its upstream and downstream primers as shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe sequence as shown in SEQ ID NO:

9. The primer pair used for detecting Vibrio cholerae has the sequences of its upstream and downstream primers as shown in SEQ ID NO:10 and SEQ ID NO:11, and the probe sequence as shown in SEQ ID NO:

12. The 5' ends of the specific probes for detecting SM and VP are labeled with 6-carboxyfluorescein (FAM); the 5' ends of the specific probes for detecting SH and VC are labeled with hexachlorofluorescein (HEX); the remaining probes are all quenched at the 3' end with Black Hole Quencher-1 (BHQ1); the specific probe for detecting VC uses the Zen dual quenching system, which, in addition to the conventional 3' end modification with the BHQ1 quenching group, introduces an additional Zen quenching group at the 9th base of the sequence, thus forming a dual quenching mechanism; The detection was performed using a 20 µL reaction mixture, which contained: 10.0 µL of ddPCR ultramixture, 8 primers at a concentration of 750 nM, 4 probes, 2 µL of DNA template, and nuclease-free water. The probe concentrations were as follows: in the FAM channel, the probe concentrations for detecting SM and VP were 150 nM and 200 nM, respectively; in the HEX channel, the probe concentrations for detecting VC and SH were 200 nM and 250 nM, respectively. The specific experimental procedure is as follows: 20 µL of the reaction mixture was dropped into a 96-well ddPCR plate and sealed. The mixture was generated using a QX200™ droplet generator and amplified on a T100 thermal cycler under the following conditions: initial denaturation at 95 °C for 10 min, denaturation at 95 °C for 30 s, annealing / extension at 57-59 °C for 80 s, for 45 cycles. (3) Fluorescence signal detection was performed using QX200. TM The droplet reader determines the result by distinguishing between positive and negative droplets; in the FAM channel, positive droplets with high fluorescence intensity come from VP, while positive droplets with low fluorescence intensity come from SM; in the HEX channel, positive droplets with high fluorescence intensity come from VC, while positive droplets with low fluorescence intensity come from SH.

2. The method as described in claim 1, characterized in that, Wastewater samples were incubated in a 65°C water bath for 20-40 minutes to eliminate potential biological risks posed by live bacteria.

3. A dual-channel quadruple ddPCR kit for detecting diarrheal bacteria in wastewater based on a proportional fluorescent probe strategy, characterized in that, The kit contains primer pairs and probes for detecting Salmonella, Shigella, Vibrio parahaemolyticus, and Vibrio cholerae; wherein: The primer pair used for detecting Salmonella has the sequences of its upstream and downstream primers as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe sequence as shown in SEQ ID NO:

3. The primer pair used for detecting Shigella has the sequences of its upstream and downstream primers as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequence as shown in SEQ ID NO:

6. The primer pair used for detecting Vibrio parahaemolyticus has the sequences of its upstream and downstream primers as shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe sequence as shown in SEQ ID NO:

9. The primer pair used for detecting Vibrio cholerae has the sequences of its upstream and downstream primers as shown in SEQ ID NO:10 and SEQ ID NO:11, and the probe sequence as shown in SEQ ID NO:

12. The 5' ends of the specific probes for detecting SM and VP are labeled with 6-carboxyfluorescein (FAM); the 5' ends of the specific probes for detecting SH and VC are labeled with hexachlorofluorescein (HEX); the remaining probes are all quenched at the 3' end with Black Hole Quencher-1 (BHQ1). The specific probe for detecting VC uses the Zen dual quenching system, which, in addition to the conventional BHQ1 quenching group modified at the 3' end, introduces an additional Zen quenching group at the 9th base of the sequence, thus forming a dual quenching mechanism.

4. A dual-channel quadruple ddPCR reaction mixture for simultaneous detection of diarrheal bacteria in wastewater based on a proportional fluorescent probe strategy, characterized in that, The dual-channel quadruple ddPCR reaction mixture contains: 10.0 µL of ddPCR supermixture, 8 primers at a concentration of 750 nM, 4 probes, 2 µL of DNA template, and nuclease-free water. The probe concentrations are as follows: in the FAM channel, the probe concentrations for detecting SM and VP are 150 nM and 200 nM, respectively; in the HEX channel, the probe concentrations for detecting VC and SH are 200 nM and 250 nM, respectively. The primers and probes are as described in claim 3.

5. The use of the kit of claim 3 or the quadruple ddPCR reaction mixture of claim 4 in the preparation of reagents for the simultaneous detection of Salmonella, Shigella, Vibrio parahaemolyticus and Vibrio cholerae.

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