Kit and detection method for detecting pathogens in sample

Abstract

The invention provides a primer group for specifically detecting pathogens in a sample. The primer group comprises at least one of a first primer group for specifically targeting a malB gene of escherichia coli, a second primer group for specifically targeting an invA gene of salmonella and a third primer group for specifically targeting an ipaH gene of shigella. The invention also provides a kit and a detection method for detecting pathogens in a sample by adopting the primer group.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to reagent kits, detection systems, and detection methods for detecting pathogens in samples. Background Technology

[0002] Various pathogenic microorganisms are widespread in the environment, typically originating from the environment or infected hosts (humans or other animals). Pathogenic microorganisms enter environmental media through the respiration of carriers, fecal excrement, and household waste, spreading and propagating through the air, sewage systems, and soil. Pollution of water bodies directly impacts drinking water quality. The emergence of biological pollution in water bodies presents new challenges to drinking water safety, as the presence of microorganisms, especially pathogens, in drinking water poses a threat to human life. Pathogenic microorganisms entering sewage systems may spread throughout the environment, posing a significant threat to public health. Therefore, it is crucial to adopt rapid, accurate, and effective methods to monitor pathogenic microorganisms in the environment to prevent their spread.

[0003] When water bodies are biologically polluted, the most common harm is the spread of waterborne infectious diseases through drinking and contact with the infected individuals. These diseases include intestinal infectious diseases such as cholera, typhoid fever, dementia, and hepatitis. The pathogens in the water mainly refer to infectious intestinal pathogens, such as Salmonella typhi, Shigella dysenteriae, Vibrio cholerae, and Escherichia coli. These pathogens are capable of causing illness in humans, and in severe cases, can even lead to death.

[0004] Currently, the most direct method for detecting pathogenic microorganisms such as bacteria and viruses is nucleic acid detection. Although PCR has high sensitivity and specificity, its complex sample processing, the need for specialized operators, large-scale instruments and equipment, data processing requirements, and long analysis cycles make it unsuitable for real-time and effective sample monitoring. Because the environment for detecting pathogenic microorganisms in the environment differs from that of clinical testing, the requirements for expensive instruments (requiring thermal cycling) and skilled personnel limit the field use of PCR. Relying on PCR for analysis makes obtaining results from environmental sampling both time-consuming and difficult. Therefore, developing effective analytical tools to rapidly and accurately track ultra-trace molecular levels of pathogenic microorganisms in the environment, enabling rapid and precise detection of pathogenic microorganisms even without a laboratory, is crucial. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this disclosure targets common pathogens in aquatic environments, such as Escherichia coli, Salmonella, and Shigella, and conducts research on rapid on-site detection of drinking water in potentially pathogen-contaminated areas, thereby quickly assessing the risk of drinking water contamination by pathogens.

[0006] According to one aspect of this disclosure, a primer set for specifically detecting pathogens in a sample is provided, said primer set comprising at least one of a first primer set specifically targeting the malB gene of Escherichia coli, a second primer set specifically targeting the invA gene of Salmonella, and a third primer set specifically targeting the ipaH gene of Shigella.

[0007] In this disclosure, the important pathogenic gene invA of Salmonella is selected. It is a highly conserved sequence related to virulence and invasiveness and can be used as a basis for gene detection and identification. It can detect all Salmonella and distinguish it from other pathogens. On the other hand, it can avoid the possibility of missed detection within the same genus.

[0008] In this disclosure, a gene fragment encoding the invasion plasmid-associated antigen H (ipaH) is selected. This fragment determines the ability of Shigella to invade the epithelial cells of the coli mucosa. At the same time, multiple copies exist on the chromosome and the invasion plasmid and are not lost with passage. This allows it to be distinguished from other pathogenic bacteria and can detect all four groups of Shigella.

[0009] In some embodiments, the primer set is used for loop-mediated isothermal amplification reactions, preferably for real-time loop-mediated isothermal amplification reactions.

[0010] In some embodiments, each primer set includes an upstream outer primer, a downstream outer primer, an upstream inner primer, and / or a downstream inner primer.

[0011] In some embodiments, the first primer set includes:

[0012] The first upstream outer primer F3 has the sequence shown in SEQ ID NO:1.

[0013] The first downstream outer primer B3 has the sequence shown in SEQ ID NO:2.

[0014] The first upstream inner primer, FIP, has the sequence shown in SEQ ID NO:3, and

[0015] The first downstream inner primer BIP has the sequence shown in SEQ ID NO:4.

[0016] In some embodiments, the second primer set includes:

[0017] The second upstream outer primer F3 has the sequence shown in SEQ ID NO:12.

[0018] The second downstream outer primer B3 has the sequence shown in SEQ ID NO:13.

[0019] The second upstream inner primer, FIP, has the sequence shown in SEQ ID NO:14, and

[0020] The second downstream inner primer BIP has the sequence shown in SEQ ID NO:15.

[0021] In some embodiments, the third primer set includes:

[0022] The third upstream primer F3 has the sequence shown in SEQ ID NO:32.

[0023] The third downstream outer primer B3 has the sequence shown in SEQ ID NO:33.

[0024] The third upstream inner primer, FIP, has the sequence shown in SEQ ID NO:34, and

[0025] The third downstream inner primer, BIP, has the sequence shown in SEQ ID NO:35.

[0026] In some embodiments, each primer set further includes an upstream loop primer and / or a downstream loop primer.

[0027] In some embodiments, the first primer set further includes:

[0028] The first upstream loop primer LF has the sequence shown in SEQ ID NO:5, and

[0029] The first downstream loop primer LB has the sequence shown in SEQ ID NO:6.

[0030] In some embodiments, the second primer set further includes:

[0031] The second upstream loop primer LF has the sequence shown in SEQ ID NO:16, and

[0032] The second downstream loop primer LB has the sequence shown in SEQ ID NO:17.

[0033] In some embodiments, the third primer set further includes:

[0034] The third upstream loop primer LF has the sequence shown in SEQ ID NO:36, and

[0035] The third downstream loop primer LB has the sequence shown in SEQ ID NO:37;

[0036] According to another aspect of this disclosure, a kit for specifically detecting pathogens in a sample is provided, the kit comprising the primer set described in this disclosure.

[0037] In some embodiments, the kit further includes dNTPs, DNA polymerase, calcein, and Mg. 2+ At least one of them.

[0038] In some embodiments, the kit also includes a microfluidic paper device.

[0039] In some embodiments, the microfluidic paper device includes a paper chip and a detection plate; the paper chip includes a loading layer, a drainage layer, and an adsorption layer made of a hydrophilic material coated with a hydrophobic material; wherein, the loading layer is provided with a plurality of hydrophilic loading regions; the drainage layer is provided with hydrophilic drainage channels, and when the drainage layer and the loading layer are stacked, the loading regions overlap with the drainage channels; the adsorption layer is provided with hydrophilic adsorption regions, and when the adsorption layer and the drainage layer are stacked, the adsorption regions overlap with the drainage channels; an adsorption component for adsorbing nucleic acids is provided at the adsorption region; the loading regions on the loading layer of the paper chip correspond to the detection regions on the detection plate.

[0040] In some embodiments, the paper chip further includes an absorbent layer made of a hydrophilic material coated with a hydrophobic material, the absorbent layer having hydrophilic absorbent regions, and the absorbent regions overlapping the adsorption regions when the absorbent layer is stacked with the adsorption layer.

[0041] In some implementations, the loading layer, drainage layer, adsorption layer, and absorbent layer are connected in a predetermined order or are independent of each other.

[0042] In some embodiments, the adsorption layer includes a first adsorption layer and a second adsorption layer connected together, wherein the first adsorption layer and the second adsorption layer are provided with adsorption regions at corresponding positions, and the adsorption component is disposed between the first adsorption layer and the second adsorption layer.

[0043] In some embodiments, the adsorption component is made of glass fiber.

[0044] In some embodiments, the loading layer, drainage layer, adsorption layer, and absorbent layer are made of wax-coated filter paper.

[0045] In some embodiments, the detection plate is made of acrylic material.

[0046] In some embodiments, the kit also includes a programmable nuclease, crRNA, and a reporter probe.

[0047] In some embodiments, the programmable nuclease is selected from Cas12, Cas13 or Cas14 nucleases, preferably enASCas12a.

[0048] In some embodiments, the crRNA includes at least one of a first crRNA specifically targeting the malB gene of *E. coli*, a second crRNA specifically targeting the invA gene of *Salmonella*, and a third crRNA specifically targeting the ipaH gene of *Shigella*; more preferably, the first crRNA has the sequence shown in SEQ ID NO.:19; more preferably, the second crRNA has the sequence shown in SEQ ID NO.:19; more preferably, the third crRNA has the sequence shown in SEQ ID NO.:19.

[0049] In some implementations, the reporter probe is a single-stranded nucleic acid sequence.

[0050] In some embodiments, the reporter probe includes a detection group and a quenching group located at both ends of the single-stranded nucleic acid sequence, wherein the detection group is selected from fluorescein, 6-fluorescein, IRDYE 700, TYE 665, AlexaFluor, or ATTO TM633, and the quenching group is selected from BHQ1, Iowa Black RQ, Iowa Black FQ, or BlackHole quencher.

[0051] According to another aspect of this disclosure, a system for specifically detecting pathogens in a sample is provided, the system comprising a LAMP system and a microfluidic paper device, the LAMP system comprising the primer set described in this disclosure.

[0052] In some embodiments, the LAMP system further includes dNTPs, DNA polymerase, a DNA template, and Mg. 2+ At least one of them.

[0053] In some embodiments, the concentration of the upstream and / or downstream outer primers in the LAMP system is 0.05-0.3 μM, preferably 0.1-0.15 μM. In some embodiments, the concentration of the upstream and / or downstream outer primers in the LAMP system is 0.05 μM, 0.1 μM, 0.12 μM, 0.15 μM, 0.18 μM, 0.2 μM, 0.25 μM, 0.3 μM, or any value between them.

[0054] In some embodiments, the concentration of inner primer FIP and / or inner primer BIP in the LAMP system is 0.5-2 μM, preferably 0.7-0.9 μM. In some embodiments, the concentration of inner primer FIP and / or inner primer BIP in the LAMP system is 0.5 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 1 μM, 1.5 μM, 2 μM, or any value between them.

[0055] In some embodiments, the concentration of circular primer LF and / or circular primer LB in the LAMP system is 0.1-0.8 μM, preferably 0.5-0.7 μM. In some embodiments, the concentration of circular primer LF and / or circular primer LB in the LAMP system is 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.8 μM, or any value between them.

[0056] In some embodiments, in the LAMP system, Mg 2+ The concentration is 4-12 mM, preferably 5-8 mM. In some embodiments, in the LAMP system, Mg 2+ The concentration is 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM or any value between them.

[0057] In some embodiments, the LAMP system further includes a fluorescent dye, preferably calcein.

[0058] In some embodiments, the microfluidic paper device includes a paper chip and a detection plate; the paper chip includes a loading layer, a drainage layer, and an adsorption layer made of a hydrophilic material coated with a hydrophobic material; wherein, the loading layer is provided with a plurality of hydrophilic loading regions; the drainage layer is provided with hydrophilic drainage channels, and when the drainage layer and the loading layer are stacked, the loading regions overlap with the drainage channels; the adsorption layer is provided with hydrophilic adsorption regions, and when the adsorption layer and the drainage layer are stacked, the adsorption regions overlap with the drainage channels; an adsorption component for adsorbing nucleic acids is provided at the adsorption region; the loading regions on the loading layer of the paper chip correspond to the detection regions on the detection plate.

[0059] In some embodiments, the paper chip further includes an absorbent layer made of a hydrophilic material coated with a hydrophobic material, the absorbent layer having hydrophilic absorbent regions, and the absorbent regions overlapping the adsorption regions when the absorbent layer is stacked with the adsorption layer.

[0060] In some implementations, the loading layer, drainage layer, adsorption layer, and absorbent layer are connected in a predetermined order or are independent of each other.

[0061] In some embodiments, the adsorption layer includes a first adsorption layer and a second adsorption layer connected together, wherein the first adsorption layer and the second adsorption layer are provided with adsorption regions at corresponding positions, and the adsorption component is disposed between the first adsorption layer and the second adsorption layer.

[0062] In some embodiments, the adsorption component is made of glass fiber.

[0063] In some embodiments, the loading layer, drainage layer, adsorption layer, and absorbent layer are made of wax-coated filter paper.

[0064] In some embodiments, the detection plate is made of acrylic material.

[0065] In some implementations, the system also includes a CRISPR architecture.

[0066] In some implementations, the CRISPR system includes a programmable nuclease, crRNA, and a reporter probe.

[0067] In some embodiments, the programmable nuclease is selected from Cas12, Cas13 or Cas14 nucleases, preferably enASCas12a.

[0068] In some embodiments, the concentration of the programmable nuclease in the CRISPR system is 80-200 nM, preferably 90-110 mM, for example 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 109 mM, 110 mM or any value between them.

[0069] In some embodiments, the crRNA includes at least one of a first crRNA specifically targeting the malB gene of Escherichia coli, a second crRNA specifically targeting the invA gene of Salmonella, and a third crRNA specifically targeting the ipaH gene of Shigella.

[0070] In some embodiments, the first crRNA has a sequence as shown in SEQ ID NO.:19.

[0071] In some embodiments, the second crRNA has a sequence as shown in SEQ ID NO.:19.

[0072] In some embodiments, the third crRNA has a sequence as shown in SEQ ID NO.:19.

[0073] In some embodiments, the concentration of each crRNA in the CRISPR system is 0.5-2 μM, preferably 0.8-1.2 μM. In some embodiments, the concentration of each crRNA in the CRISPR system is 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.5 μM, 1.8 μM, 2 μM, or any value between them.

[0074] In some implementations, the reporter probe is a single-stranded nucleic acid sequence.

[0075] In some embodiments, the reporter probe includes a detection group and a quenching group located at both ends of the single-stranded nucleic acid sequence, wherein the detection group is selected from fluorescein, 6-fluorescein, IRDYE 700, TYE 665, AlexaFluor, or ATTO TM633, and the quenching group is selected from BHQ1, Iowa Black RQ, Iowa Black FQ, or BlackHole quencher.

[0076] According to another aspect of this disclosure, a method for specifically detecting pathogens in a sample is provided, the method comprising detection using the primer set or the kit described in this disclosure; or detection using the system described in this disclosure.

[0077] In some embodiments, the method includes: (1-1) incubating the primer set described herein with the sample to perform a LAMP reaction to obtain the corresponding reaction products; (1-2) visually detecting the reaction products containing fluorescent dyes.

[0078] In some embodiments, the method includes: (2-1) incubating the primer set described herein with the sample to perform a LAMP reaction to obtain the corresponding reaction products; (2-2) adding a programmable nuclease, crRNA, and a reporter probe to the reaction products to perform a CRISPR reaction to obtain detection products; and (2-3) visually detecting the detection products.

[0079] In some embodiments, the LAMP reaction is carried out on the microfluidic paper device.

[0080] In some embodiments, the temperature of the LAMP reaction is 61-71°C, preferably 65-69°C. In some embodiments, the temperature of the LAMP reaction is 61°C, 63°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, or any value between them.

[0081] In some embodiments, the LAMP reaction time is 20-40 min, preferably 30-40 min. In some embodiments, the LAMP reaction time is 20 min, 25 min, 30 min, 32 min, 35 min, 40 min, or any value between them.

[0082] In some embodiments, the CRISPR reaction time is 20-40 min, preferably 30-40 min. In some embodiments, the CRISPR reaction time is 20 min, 25 min, 30 min, 32 min, 35 min, 40 min, or any value between them.

[0083] In some embodiments, the sample is an aqueous sample, preferably a drinking water sample.

[0084] This disclosure discloses a novel paper-based sensing device that combines paper folding technology with nucleic acid isothermal amplification technology. This device combines the high sensitivity of nucleic acid amplification detection with the simplicity, portability, and operability of paper-based devices, enabling simultaneous on-site detection of multiple pathogenic microorganisms in the environment (based on operability and practical needs, using Escherichia coli, Salmonella, and Shigella as model target pathogens; while ensuring the detection of model target pathogens, it can be flexibly modified for the detection of different pathogens). This improves and supplements the shortcomings of previous methods that required traditional PCR analysis or multiple separate procedures to obtain analytical results. Furthermore, it can be applied to high-risk areas with drinking water pathogen contamination, providing necessary analytical means and technical support for better research on the migration, diffusion, and transmission of pathogenic microorganisms in the environment, as well as for effectively preventing pathogen infection risks and protecting public health. Attached Figure Description

[0085] Figure 1 The fluorescence detection principle of the paper-based biosensor is demonstrated.

[0086] Figure 2 The results of real-time fluorescent LAMP amplification of E. coli are shown.

[0087] Figure 3 The image shows the real-time fluorescence LAMP amplification results of three sets of LAMP primers targeting Salmonella and Shigella. ac represents the real-time fluorescence results of the three sets of primers for Salmonella invA gene L1-L3, respectively; df represents the real-time fluorescence results of the three sets of primers for Shigella ipah gene L1-L3, respectively.

[0088] Figure 4 The results of LAMP amplification with different primer concentrations are shown. ad represents the LAMP amplification results of primer concentrations from group a to group d, respectively.

[0089] Figure 5 Different Mg 2+ LAMP amplification results under the specified conditions.

[0090] Figure 6 The images show the direct visual fluorescence visualization of LAMP at different temperatures.

[0091] Figure 7 The LAMP fluorescence curves at different reaction times are shown, where ae represents the LAMP fluorescence curves at reaction times of 20 min, 30 min, 40 min, 50 min, and 60 min, respectively.

[0092] Figure 8 This image shows the real-time LAMP detection of different concentrations of *E. coli*, *Shigella*, and *Salmonella*, as well as Shigella plasmid DNA. A, d, and g represent the real-time LAMP detection of 10-fold dilutions of *E. coli* (a), *Salmonella* (d), and *Shigella* (g) DNA at different concentrations. Following a ten-fold increase in concentration, the *E. coli* concentration gradient is 1.11 × 10⁻⁶. 1 -1.11×10 8 copies / μL; Salmonella concentration gradient was 1.87×10⁻⁶. 3 -1.87×10 10 copies / μL; Shigella concentration gradient was 1.95×10⁻⁶. 1 -1.95×10 9 copies / μL; b, e, h are quantitative curves of different target gene concentrations of the three pathogens and LAMP cycle threshold time (t); c, f, i are LAMP amplicon agarose gel electrophoresis images of Escherichia coli (c), Salmonella (f), and Shigella (i). Lanes 1-2 of c, f, i are DNA markers and negative controls. The lanes are arranged in order of increasing concentration by a factor of ten.

[0093] Figure 9 A schematic diagram of the site testing process for paper-based biosensors is shown.

[0094] Figure 10 The design drawing (a) and the actual product (b) of the paper-based biosensor are shown.

[0095] Figure 11 The detection completion time of the LAMP-based paper-based biosensor is shown.

[0096] Figure 12The image shows the specificity and precision tests of the LAMP-based paper-based biosensor. Figure 'ac' represents visualizations of LAMP reactions with different template samples, showing the addition of ddH2O, ermb plasmid DNA (200 pg), and three pathogen DNAs (1, 2, 3 representing *E. coli*, *Salmonella*, and *Shigella*, respectively, at concentrations of several hundred pg). Figure 'd' shows the arrangement of *E. coli*, *Salmonella*, and *Shigella* in the order shown in the image. 1, 2, 3 represent *E. coli*, *Salmonella*, and *Shigella*, respectively. Each experiment was repeated three times.

[0097] Figure 13 The sensitivity of the paper-based biosensor in analyzing three pathogens is shown. Image a shows the real-time visualization of the paper-based biosensor's sensitivity to different concentrations of DNA from the three pathogens, captured by a mobile phone. Images b and d are the normalized data of green fluorescence signal intensity for Escherichia coli, Salmonella, and Shigella, respectively (extracted using ImageJ software, normalized for positive and negative controls; positive controls were assigned an arbitrary unit of 1, and negative controls were assigned 0).

[0098] Figure 14 The results of qPCR detection of three pathogens in drinking water sources in Tongren City are shown. A, D, and G represent the qPCR quantitative curves of different concentrations of Escherichia coli, Salmonella, and Shigella DNA, respectively. B, E, and I are the qPCR fluorescence curves of Escherichia coli, Salmonella, and Shigella detected at 15 drinking water source sections in Tongren City, respectively. C, F, and H represent the concentrations of Escherichia coli, Salmonella, and Shigella at the 15 drinking water source sections in Tongren City, respectively. The Shigella negative control showed a fluorescence signal at 34 min. To distinguish it from the actual samples, the relative concentration was calculated as: relative concentration = sample concentration - negative control concentration.

[0099] Figure 15 The results of LAMP detection of three pathogens in drinking water sources in Tongren City are shown. Among them, a, d, and g are LAMP quantitative curves of different concentrations of Escherichia coli, Salmonella, and Shigella DNA, respectively; b, e, and h are LAMP fluorescence curves of Escherichia coli, Salmonella, and Shigella detected by real-time fluorescence LAMP at 15 drinking water source sections in Tongren City; c, f, and i are the concentrations of Escherichia coli, Salmonella, and Shigella at 15 drinking water source sections in Tongren City, respectively.

[0100] Figure 16 The results show the relative deviations between LAMP quantification and PCR quantification. Among them, a, b, and c are the relative deviations between LAMP quantification and PCR quantification of Escherichia coli, Salmonella, and Shigella at 15 drinking water source sections in Tongren City, respectively.

[0101] Figure 17Real-time fluorescence images are shown under different CRISPR / enASCas12a protease concentrations, where ac represents the real-time fluorescence curves of E. coli, Salmonella, and Shigella plasmid DNA cut by different concentrations of CRISPR / enASCas12a, respectively. Sterile enzyme-depleted water was used as a negative control.

[0102] Figure 18 Real-time fluorescence images are shown under different CRISPR / enASCas12a crRNA concentrations, where ac represents the real-time fluorescence curves of CRISPR / enASCas12a combined with different concentrations of crRNA. ac represents the cleavage of plasmid DNA from *E. coli*, *Salmonella*, and *Shigella*, respectively. Sterile enzyme-degraded water was used as a negative control.

[0103] Figure 19 The visualization results show the different reaction times of CRISPR / enASCas12a.

[0104] Figure 20 The detection process (a) and mechanism (b) of three pathogens in drinking water by an integrated CRISPR / cas12-based biosensor are shown.

[0105] Figure 21 This study demonstrates the feasibility analysis of an integrated CRISPR / enASCas12a-based biosensor for detecting pathogens in drinking water. Here, ac represents the feasibility of 100pM LAMP for *Escherichia coli* (primer set in Table 1), *Salmonella* (L2 primer set in Table 1), and *Shigella* (L3 primer set in Table 1); df represents the feasibility of CRISPR / enASCas12a cleavage activity for *E. coli*, *Salmonella*, and *Shigella*; and gh represents the feasibility of combined LAMP-CRISPR / enASCas12a cleavage activity for *E. coli*, *Salmonella*, and *Shigella*.

[0106] Figure 22 The results show the effectiveness of the CRISPR / enASCas12a single method in detecting Escherichia coli, Salmonella, and Shigella, with ac representing real-time fluorescence curves (copies / μL) of the sensitivity of CRISPR / enASCas12a in detecting these bacteria. Sterile deenzyme-free water was used as a negative control.

[0107] Figure 23The effectiveness of the LAMP-CRISPR / enASCas12a combination assay is evaluated. Here, ac represents the sensitivity of the LAMP-CRISPR / enASCas12a assay system for detecting *E. coli*, *Salmonella*, and *Shigella* (in copies / μL); df represents the fluorescence intensity characteristics of different concentrations of target endpoints for *E. coli*, *Salmonella*, and *Shigella* using LAMP-CRISPR / Cas12a; and gh represents the visualization detection using LAMP-CRISPR / Cas12a. The fluorescence signal-based visualization and normalization of *E. coli*, *Salmonella*, and *Shigella* were performed separately. ImageJ was used to quantify and normalize the fluorescence visual signals. Sterile enzyme-free water was used as a negative control.

[0108] Figure 24 The design and detection of a paper-based biosensor based on CRISPR / enASCas12a are shown, where a is a schematic diagram of the paper device design; b is a process for purifying pathogens using the paper device; c is a visualization of pathogen detection using the paper device; and d is a schematic diagram of the fluorescence detection results. Detailed Implementation

[0109] Contamination of drinking water by pathogenic microorganisms (pathogens) poses a significant threat to public health. Therefore, it is crucial to conduct research on rapid on-site testing of drinking water in potentially pathogen-contaminated areas to quickly assess the risk of pathogen contamination in drinking water.

[0110] Although numerous isothermal amplification analytical methods have been developed and applied in disease diagnosis, food safety, forensic analysis, and environmental monitoring, current analytical methods still fall far short of meeting the needs of pathogen detection in the environment. This is due to the diverse range of pathogenic microorganisms and complex environmental substrates (such as wastewater), coupled with the requirement for simple, rapid, effective, and cost-effective detection methods. Therefore, the continued design and optimization of highly sensitive, accurate, interference-resistant, simple, and cost-effective nucleic acid isothermal amplification detection methods to meet the needs of detecting single or multiple pathogens simultaneously in various complex environments remains of great significance.

[0111] Furthermore, isothermal amplification for pathogen detection has a significant drawback: the analysis requires a series of processes, including sample extraction, enrichment, purification, elution, amplification, and analysis. These cumbersome procedures, along with the different but essential reagents involved in each step, limit its rapid and effective application in the field.

[0112] This problem can be solved by using paper folding technology. The principle is to design a paper folding device with different functional areas and use different folding methods of the paper folding device to realize the various steps required for nucleic acid isothermal amplification detection, thereby achieving the goal of integrating various operation processes and avoiding the separate performance of multiple processes. This allows the entire detection work to be completed with a simple paper device.

[0113] Integrated paper microfluidic devices (paper devices) serve as an effective tool, combining nucleic acid extraction and amplification via mobile phones, lateral flow testing, and even visual detection or quantification using the naked eye. Furthermore, adding samples to the microfluidic environment reduces the risk of sample contamination and minimizes the sample volume required for detection. The combination of LAMP technology's advantage in enabling rapid on-site nucleic acid detection, along with the rapid nucleic acid extraction capabilities of paper microfluidics, significantly improves the speed, accuracy, and portability of on-site nucleic acid testing. Therefore, LAMP-based paper-based biosensing technology enables high-resolution on-site screening and diagnosis of pathogens in the field.

[0114] This disclosure presents a novel paper-based analytical device that combines paper folding technology with isothermal nucleic acid amplification technology. This device offers the advantages of high sensitivity in nucleic acid amplification detection and the simplicity, portability, and ease of use of paper-based equipment. This enables simultaneous on-site detection of multiple pathogenic microorganisms in the environment, thus addressing the shortcomings of traditional PCR analysis methods or multi-step procedures required to obtain results. It can be applied to high-risk areas with drinking water pathogen contamination, providing necessary analytical tools and technical support for better research on the migration, diffusion, and transmission of pathogenic microorganisms in the environment during epidemics, and for effectively preventing environmental risks and protecting public health. In the exemplary embodiment, *Escherichia coli*, *Salmonella*, and *Shigella* were used as model target pathogens for detection. However, those skilled in the art will understand that the method of this disclosure can be flexibly modified for the detection of different pathogenic microorganisms.

[0115] In some embodiments, this disclosure provides a method for fluorescent detection of pathogens using a paper-based biosensor. In this method, calcein in the reaction system initially reacts with Mn... 2+ Binding, fluorescence quenching; when the target sequence is added, Mn... 2+ With P2O7 4- The resulting complex contains Mn in the reaction mixture. 2+ The fluorescence was enhanced, and the concentration of pathogen nucleic acids was detected by measuring the fluorescence intensity. Figure 1In some implementations, but not limited to, a handheld UV lamp can be used to visualize and read the results for qualitative analysis, while a mobile phone can be used to capture the intensity of the green fluorescence signal and extract it using ImageJ software for quantitative analysis.

[0116] In some implementations, nucleic acids extracted via a paper apparatus are used to uniformly distribute DNA into the reaction chamber through a paper microfluidic channel, followed by the addition of various reagents required for the LAMP reaction. The paper apparatus is then removed, and the chamber is sealed with an acetate film to prevent liquid evaporation during the isothermal amplification process. Finally, a plastic plate with the sealed reaction chamber is placed in a 65°C insulated water cup to carry out the LAMP reaction.

[0117] Escherichia coli (E. coli) is also known as Escherichia coli. Most E. coli are normal flora in the intestines of humans and animals and are generally harmless. However, of the 140 known serotypes of E. coli, 11 serotypes can cause gastrointestinal diseases in humans. Based on their causative agents, they can be divided into five groups: enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC), and enteroadhesive Escherichia coli (EAEC). All E. coli are transmitted via the fecal-oral route.

[0118] The main serotypes of Escherichia coli (EHEC) are O157:H7 and O111, both of which can cause diarrhea of ​​varying degrees. Serotype O157:H7 can produce Shigella-like toxin (SLT), leading to bloody dysentery in infants. If left untreated, it can cause fatal hemolytic uremic syndrome, with a mortality rate as high as 3-5%. EHEC is much more infectious than other Escherichia coli strains; fewer than 100 bacteria can cause infection. ETEC causes disease by producing heat-stable and heat-sensitive enterotoxins. Symptoms of infection include watery diarrhea, abdominal cramps, nausea, and headache, and it is a significant factor in causing diarrhea, especially in young children. EPEC infection can cause severe diarrhea, vomiting, and fever. EIEC causes watery diarrhea, occasionally leading to bloody stools. EAEC can cause acute or chronic diarrhea with dehydration in infants, but the diarrhea symptoms are milder than those of EPEC.

[0119] Humans are the primary host for pathogenic Escherichia coli, especially EPEC, ETEC, and EIEC. Approximately 2–8% of Escherichia coli in aquatic environments are EPEC. EHEC has a wider range of sources; livestock and poultry are major sources of EHEC infection, and even raw vegetables can carry EHEC. These pathogenic Escherichia coli can be detected in various environmental water bodies.

[0120] Salmonella is a large group of Gram-negative bacteria with similar morphology, biochemical characteristics, and antigenic structure. Salmonella is widely distributed in the environment, but certain species or serotypes exhibit host specificity. Although Salmonella paratyphi occasionally infects livestock, the primary host of both Salmonella typhi and paratyphi is humans. Many serotypes of Salmonella, including Salmonella typhimurium and Salmonella enteritidis, can infect humans and most animals, including poultry, cattle, pigs, birds, and even reptiles. Pathogens are typically introduced into water systems through sewage discharge or the feces of livestock and wild animals. Salmonella has now been detected in many types of food.

[0121] Salmonella belongs to the Enterobacteriaceae family and can cause various syndromes, including gastroenteritis, typhoid fever, sepsis, and focal extraintestinal infections, collectively known as Salmonella infection (or salmonellosis). These syndromes can sometimes overlap. Infections caused by typhoid and paratyphoid bacilli are not included. The primary route of Salmonella infection in humans is the fecal-oral route. Infections with non-typhoid Salmonella species are mainly related to person-to-person contact, ingestion of contaminated food, and contact with animals. Typhoid Salmonella infection is primarily through the ingestion of contaminated food or water; direct person-to-person transmission is uncommon. After passing through the stomach, Salmonella typhi and other Salmonella species multiply in the intestines. Subsequently, Salmonella typhi enters the bloodstream and colonizes the periosteum, liver, gallbladder, bone marrow, spleen, or kidneys. Salmonella typhi can cause meningitis or pneumonia. In fatal cases, late-stage complications are not the primary cause of death, but rather the initial stages of intestinal infection.

[0122] Shigella is a genus of Gram-negative bacilli, the most common pathogen causing bacterial shigia in humans. It is a rod-shaped bacterium, hence its common name, shigella bacilli. Shigella-induced intestinal infectious diseases, also known as shigellosis, are among the most common intestinal infectious diseases in summer and autumn. The main clinical manifestations are abdominal pain, diarrhea, tenesmus, and bloody mucus stools, which may be accompanied by fever and systemic toxemia symptoms. Severe cases may result in septic shock and / or toxic encephalopathy. Shigella is an intestinal pathogen primarily transmitted through the fecal-oral route via person-to-person contact and ingestion of contaminated food and water. Because these bacteria are not particularly stable in water, their presence in drinking water indicates recent contamination by human feces. However, currently used detection techniques have relatively low sensitivity and reliability, and data used in water supply often underestimate the concentration of Shigella.

[0123] Shigella infection is often caused by foodborne outbreaks or is transmitted via water. Shigella can spread rapidly under crowded and unsanitary conditions. The main causes of foodborne Shigella outbreaks are food processing workers with bacillary dysentery or carriers contaminating food, poor personal hygiene among food handlers, and improper storage temperatures for contaminated food.

[0124] This disclosure targets Escherichia coli, Salmonella, and Shigella as pathogens, constructing a paper-based biosensor based on paper microfluidic devices and LAMP. This combines the advantages of paper devices—simplicity, portability, and ease of operation—with the high specificity and sensitivity of nucleic acid amplification detection. The technology is then used for on-site detection of pathogens in drinking water, and validated and optimized using traditional PCR techniques. Finally, this disclosure also assesses the risk of pathogen contamination in drinking water sources in Tongren, Guizhou. The detection reagents, kits, and methods disclosed in this disclosure improve and supplement previous methods that required traditional PCR analysis or multiple separate procedures to obtain results. This provides a basis and technical support for rapid on-site monitoring, early warning, and assessment of the transmission patterns and contamination risks of pathogenic microorganisms in drinking water.

[0125] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0126] The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0127] Experimental materials:

[0128] Reagents: Bst-DNA polymerase and NEB Buffer 2.1 (B7202S) were purchased from New England Biolabs (UK); MgSO4 was purchased from OptiGene Ltd; gel extraction kit (D4002) was purchased from Zymo Research; ALFA-water DNA mini-extraction kit was purchased from Guangzhou Ark Biotechnology Co., Ltd.; high-purity plasmid mini-extraction kit (TIANGEN, Beijing); Maker D2000+ was purchased from Bomeide Biotechnology; calcein was purchased from Sigma; SYBR Gold nucleic acid gel staining agent was purchased from Thermo Scientific; kanamycin sulfate (K8020) was purchased from Solarbio (China); Green-2-Go 2X qPCR-Low-ROX premix, 10 mmol / L dNTP, NaOAc, 70% v / v ethanol, Tris, EDTA, TE... Buffer, enzyme-free sterile water, and TAE buffer were purchased from Sangon Biotech (Shanghai); glass fiber (1825-047) was purchased from Whatman; paraffin was purchased from Xerox. Gene plasmids for Escherichia coli, Salmonella, and Shigella were synthesized by Genewiz Biotechnology Co., Ltd. (Suzhou, China), and all primers were synthesized by Sangon Biotech (Shanghai, China).

[0129] Instruments: ABI 7500 Real-Time PCR System, Qubit 4.0 Nucleic Acid / Protein Quantitative Analyzer (Invitrogen, USA), Electrophoresis System (Bio-Rad, USA), Gel Imaging System (Tanon, China).

[0130] Example 1: Design, optimization, and evaluation of a loop-mediated isothermal amplification detection system for typical foodborne pathogens.

[0131] 1. Screening of LAMP primers

[0132] (1) Design and optimization of LAMP primers

[0133] Based on the gene sequences of *Escherichia coli* (malB, GenBank: MZ934649.1), *Salmonella* (invA, GenBank: MK017936.1), and *Shigella* (ipah, GenBank: LC111495.1), multiple sets of LAMP primers were designed. Each set of primers includes two outer primers (F3, B3), two inner primers (FIP, BIP), and two loop primers (LF, LB). Primer synthesis and purification were performed by Shanghai Sangon Biotech Co., Ltd.

[0134] Through extensive analysis and comparison, taking into account factors such as reaction medium, sensitivity, detection limit, and stability, and based on the detection performance of each primer set, the inventors initially screened out detection primer sets for Escherichia coli (malB), Salmonella (invA), and Shigella (ipah) pathogens (as shown in Table 1) for further validation in LAMP.

[0135] Table 1 Primers and probes

[0136]

[0137]

[0138] (2) Construction of LAMP reaction

[0139] The reagents required for LAMP amplification include 2 μL of 10X Isothermal Amplification Buffer and 0.32 U / μL LBst 2.0. DNA polymerase, 1.6 μM inner primers (FIP and BIP), 0.4 μM loop primers (LF and LB), 0.2 μM outer primers (F3 and B3), 0.4 mM dNTPs, 4.0 mM magnesium sulfate, 25 μM calcein, 500 μM Mn 2+ 2 μL DNA template, brought to a final volume of 20 μL with water. 1X Isothermal Amplification Buffer contains 20 mM Tris-HCl (pH 8.8 @ 25℃), 50 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, and 0.1% Tween-20. The DNA template is plasmid DNA, with gene plasmids for *E. coli*, *Salmonella*, and *Shigella* synthesized by Genewiz Biotechnology Co., Ltd. (Suzhou, China). The plasmid vector is Puc57-kan. The *E. coli* malB gene is available in GenBank: MZ934649.1; the *Salmonella* invA gene in Bank: MK017936.1; and the *Shigella* ipah gene in Bank: LC111495.1. All primers were synthesized by Sangon Biotech (Shanghai, China).

[0140] In the experiment, a positive control was introduced using the ermb resistance gene as a template, and a negative control was introduced using ddH2O as a template. After adding the target compound, the LAMP method was used for detection and optimization on a real-time quantitative PCR instrument. The LAMP reaction was carried out at 65℃ for 40 min. Three replicates were performed for each of the negative and positive controls and the sample group. The fluorescence color development results were observed at a UV wavelength of 365 nm; negative results showed brownish-yellow, and positive results showed fluorescent green. 5 μL of the amplification product was subjected to agarose gel electrophoresis, and imaging was performed using a Tianneng 3500B.

[0141] Real-time fluorescent LAMP amplification was performed on a real-time fluorescent PCR instrument using the primer sets listed in Table 1, following the methods described above. The results showed that the *E. coli* LAMP primers could be successfully amplified (see...). Figure 2 For Salmonella and Shigella, among the three primer sets, only the Salmonella invA gene primer set L2 (… Figure 3 a-3c) and Shigella ipah gene primer set L3 ( Figure 3 (d-3f) showed better amplification in fluorescence analysis, with amplicones observed within the first 20 minutes. In summary, the LAMP method can rapidly amplify Escherichia coli, Salmonella, and Shigella, successfully amplifying the target signal.

[0142] 2. Optimization of LAMP detection conditions

[0143] The key factors affecting the LAMP reaction are primer concentration, magnesium ion concentration, enzyme activity, and reaction time. Therefore, this embodiment takes the E. coli malB primer set in Table 1 as an example to optimize parameters such as enzyme activity, reaction temperature, and magnesium ion concentration for the LAMP reaction, thereby determining suitable reaction conditions to achieve efficient and rapid isothermal amplification and detection to meet the detection requirements.

[0144] a. Primer concentration optimization

[0145] Primer concentration is one of the important factors affecting LAMP amplification reactions. The primer concentration significantly impacts the specificity of the amplification reaction. Too high a primer concentration can lead to non-specific binding between the primers and the template, or the formation of primer dimers, resulting in false positives and affecting experimental results. Too low a primer concentration may reduce the amplification rate or the amount of amplified product, resulting in false negatives and affecting experimental results. This experiment set up a series of primer concentrations and conducted optimization experiments. With other components and conditions in the system remaining constant, the primer concentrations are shown in Table 2 to select the optimal primer set concentration.

[0146] Table 2 LAMP primer concentration information

[0147]

[0148]

[0149] The results are as follows Figure 4 As shown in a-4d, the amplification effect of primer group b was better than that of group a, and the difference between group b and group c was only about one CT. Considering the concentration of different primer groups and the total primer concentration, the concentration of primer group b was the best. Therefore, the optimal primer ratio concentration was determined to be 0.1 μM each for F3 and B3, 0.8 μM each for FIP and BIP, and 0.6 μM each for LF and LB.

[0150] b.Mg 2+ Concentration optimization

[0151] During the LAMP reaction, dNTPs generate a large amount of pyrophosphate, and the Mg added to the system... 2+ It forms a white precipitate of magnesium pyrophosphate with pyrophosphate, which is an indicator of LAMP amplification. 2+ Concentration significantly affects the amplification effect of Bst chain displacement enzyme. To determine the concentration of Mg in the system... 2+ To determine the optimal concentration, five experimental groups were established. Under the condition that other components and conditions in the system remained constant, the concentration of Mg in the system was determined. 2+ The concentrations were 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM, respectively.

[0152] Test results are as follows Figure 5 As shown, the reaction exhibits optimal activity at a magnesium ion concentration of 4 mM. Since the 1X Isothermal Amplification Buffer contains 2 mM MgSO4, the reaction exhibits optimal activity at a total magnesium ion concentration of 6 mM.

[0153] c. Optimization of enzyme activity

[0154] As an enzymatic amplification reaction, the LAMP reaction is highly sensitive to temperature, which affects enzyme activity and ultimately the outcome. Therefore, the reaction can only be carried out at temperatures where the enzyme has high activity. To select a suitable temperature for amplification, amplification reactions were conducted at different temperatures while keeping other components and conditions constant.

[0155] The reaction results are as follows Figure 6 As shown, green indicates a positive result, and yellowish-brown indicates a negative result. The results indicate that the LAMP reaction has high activity at 61-71℃.

[0156] d. Optimization of reaction time

[0157] Reaction time affects the yield of reaction products. Too short a time results in insufficient amplified product yield, making it impossible to distinguish by electrophoresis or the naked eye. Too long a reaction time leads to non-specific amplification. With other components and conditions remaining constant, this experiment set reaction times of 20 min, 30 min, 40 min, 50 min, and 60 min, with corresponding negative controls, to explore the shortest time to detect LAMP products while avoiding non-specific amplification.

[0158] The reaction results are as follows Figure 7 As shown in a-7e, LAMP amplification occurred within 20-60 min. Before 40 min, all negative controls showed no nonspecific amplification, while after 40 min, amplified fluorescence appeared in the negative controls. These results indicate that, with the aim of saving time, the LAMP reaction can detect the target sequence within 40 min, shorter than the at least 2-hour reaction time of qPCR, reducing the reaction time by at least two-thirds.

[0159] 3. Establishment of a Visualized LAMP Reaction System

[0160] By selecting appropriate primer sets and optimizing enzymes, temperature, magnesium ions, etc., a reaction system as shown in Table 3 was constructed. This system can detect pathogens within 1 hour and allows for visual observation of experimental results, providing a good laboratory foundation for subsequent paper-based biosensing.

[0161] Table 3 LAMP reaction system

[0162] reagents Final concentration 10×Isothermal Amplification Buffer <![CDATA[1×(containing 2 mM MgSO4)]]> <![CDATA[MgSO4(100mM)]]> 4mM dNTP Mix 1.4mM each F3 / B3 0.1μM each FIP / BIP 0.8μM each LF / LB 0.6μM each Bst enzyme 0.32 U / μL <![CDATA[Mn 2+ ]]> 500μM Calcein 25μM DNA sample (plasmid DNA sample or genomic DNA sample extracted from an actual sample) 2μL Nuclease-free Water Make up to 20 μL

[0163] 4. LAMP sensitivity characterization

[0164] To evaluate the sensitivity of LAMP detection, plasmid DNA from three pathogens was serially diluted and subjected to LAMP reaction at 65°C for 40 min for real-time fluorescent LAMP. Simultaneously, enzyme-free sterile water was used as a negative control to determine the reliability of the amplification results.

[0165] The LAMP amplification results were verified using agarose gel electrophoresis. Figure 8 As can be seen from a, 8d, and 8g, with increasing target nucleic acid concentration, the fluorescence curve shows an exponential increase earlier and reaches the plateau phase sooner; simultaneously, the detection limits for Escherichia coli, Shigella, and Salmonella are 1.11 × 10⁻⁶, respectively. 2 copies / μL (converted to concentration units: 2.32 × 10⁻⁶) -11 μg / μL), 1.87×10 4 copies / μL (converted to concentration units: 3.8 × 10⁻⁶) -9 μg / μL), 1.95×102 copies / μL (converted to concentration units: 5.54 × 10⁻⁶) -11 μg / μL)(see Table 4). For example... Figure 8 As shown in b, 8e, and 8h, there is a good linear relationship between the concentration of the target DNA of the three pathogens and CT, with the linear range spanning 6-7 orders of magnitude, indicating that LAMP has good real-time quantification capabilities.

[0166] The results were further verified by agarose gel electrophoresis. When the concentration was greater than or equal to the LOD, ladder-like stripes were produced because the specific amplicons of LAMP are a mixture of various lengths and structures, including stem loop DNA of different stem lengths and multi-loop cauliflower-like structures. The bands in lanes 1 and 2 of the agarose gel electrophoresis represent the DNA marker and the negative control, respectively, followed by LAMP amplification products at low to high concentrations of the DNA template. Figure 8 As shown in c, 8f, and 8i, Escherichia coli, Salmonella, and Shigella show stepwise stripes starting from lanes 4, 4, and 4, respectively, indicating that this method is effective at concentrations as low as 1.11 × 10⁻⁶. 2 copies / μL (i.e., 2.32 × 10⁻⁶) -11 μg / μL), 1.87×10 4 copies / μL (i.e., 3.8 × 10⁻⁶) -9 μg / μL), 1.95×10 2 copies / μL (i.e., 5.54 × 10⁻⁶) -11 Escherichia coli, Salmonella, and Shigella could be detected at a concentration of μg / μL. The results were consistent with those obtained from real-time LAMP fluorescence detection at LOD.

[0167] Table 4. Detection limits for typical pathogens based on LAMP.

[0168] Pathogens Method detection limit (μg / μL) Method detection range (μg / μL) E. coli <![CDATA[2.32×10 -11 ]]> <![CDATA[2.32×10 -11 -2.32×10 -5 ]]> salmonella <![CDATA[3.8×10 -9 ]]> <![CDATA[3.8×10 -9 -3.8×10 -3 ]]> Shigella <![CDATA[5.54×10 -11 ]]> <![CDATA[5.54×10 -11 -5.54×10 -4 ]]>

[0169] Example 2: Paper Equipment and Characterization

[0170] For sample enrichment, a certain amount of water (approximately 50 ml) can be pressurized and passed through a 0.22 μm filter membrane using a syringe to enrich the microorganisms on the membrane. Then, heavy salt is added for incubation to lyse and release DNA, which is then purified using a microfluidic paper device. For this purpose, a paper-based sensor is introduced in this embodiment. A schematic diagram of the paper device site testing process is shown below. Figure 9 As shown in the diagram, the paper equipment is as follows: Figure 10 As shown.

[0171] 1. Design of paper processing equipment and reaction chamber

[0172] First, the paper equipment was designed using CorelDRAW software. Then, it was printed using a paraffin printer, where hot wax printed the channels and holes from the design onto the paper. The printed wax was then heated on a hot plate to melt the wax, allowing it to diffuse across the entire thickness of the paper, creating a hydrophobic pattern of channels and holes. A fiberglass disc was directly embedded into a 4mm hole in the printing panel to connect to the paper equipment (see...). Figure 10 a).

[0173] The paper-based biosensor comprises three components (see...) Figure 10 b), namely: a paper-based device based on filter paper – in which fluid is formed by printing hydrophobic wax to create microfluidic channels; a glass fiber disc – embedded in the sample preparation area of ​​the paper device for enriching and purifying nucleic acids, which is readily available; and a plastic plate sealed with a single-sided vinyl acetate film – with holes consistent with the detection area to form the LAMP reaction chamber. The reaction chamber is made by cutting the plastic plate using a laser cutter.

[0174] To ensure accuracy and avoid false positives, the paper-based testing device is designed with 5 channels (negative control channel, positive control channel, and 3 sample channels), enabling simultaneous on-site detection of 3 random pathogens. This device integrates the nucleic acid extraction process (enrichment, purification, and elution) onto inexpensive paper. The entire detection process only requires folding the paper-based device according to specific steps to complete the nucleic acid extraction, completely overcoming the limitations of PCR reactions, avoiding multiple temperature cycles, and eliminating the need for specialized instruments. This achieves highly sensitive, multi-channel, rapid, real-time, and in-situ detection.

[0175] A schematic diagram of the paper equipment is shown below. Figure 10 As shown in Figure a, the paper apparatus consists of five parts labeled 1 to 5. The black area of ​​the filter paper is a hydrophobic region created by wax printing diffusion, the white area is a non-wax hydrophilic channel or a perforated area, and the three central white areas are glass fiber discs. Part 1 consists of a large hydrophilic disc and a wax-printed hydrophobic region. Part 2 utilizes small holes created by a perforator for liquid delivery. Part 3 consists of a glass fiber disc for DNA adsorption and a hydrophobic wax-printed region. Part 4 uses rectangular channels to dispense elution buffer into small hydrophilic paper discs in Part 5. Part 5 includes three paper discs for DNA sample loading and two paper discs for negative and positive controls. Furthermore, the reaction chamber is a cavity created by a microfluidic plate laser cutter, and the microfluidic plate is sealed with an acetate film. The diameter of the hydrophilic circular channels in the paper apparatus is 4 mm, and the diameter of the glass fiber is 4 mm.

[0176] Paper equipment usage methods such as Figure 9As shown, first fold the glass fiber disc No. 3 above region No. 2, aligning the glass fiber of No. 3 with the pores of No. 2. Then fold the absorbent disc No. 1 below region No. 2. Add the lysis buffer to the glass fiber disc No. 3. After the lysis buffer flows through, add 100 μL of washing buffer directly to the glass fiber disc No. 3 to rinse away cell residues and purify DNA. Wash 2-3 times. After the glass fiber of No. 3 dries, unfold No. 1, then place No. 2 tightly against No. 3. Fold No. 4 and No. 5 below region No. 3, aligning the glass fibers with the intersection of No. 4 and No. 5. Add 40 μL of elution buffer and allow it to spread evenly to each well. Then open the disc, align it with the microfluidic plate, and use a punch to create hydrophilic circular holes in each reaction chamber of the microfluidic plate. Add reagents, seal the plate, and heat in a water bath to initiate the reaction.

[0177] 2. Paper-based equipment for extracting pathogen DNA from drinking water

[0178] The process for extracting and testing genomic DNA from pathogens in wastewater using a paper-based biosensor is as follows: First, 50 mL of water sample is forced through a 0.22 μm pore size filter using a syringe. Due to their larger size, bacteria are retained on the filter membrane. Then, 40 μL of lysis buffer (1.20 g / mL GuSCN, 0.1 M Tris-hydrochloride, 0.04 M EDTA, pH adjusted to 8.0 with NaOH, followed by 26 mg mL⁻¹ Triton X-100) is added to the filter membrane and incubated at room temperature for 5 min. The DNA-containing lysis buffer is then pipetted onto the paper device, where it permeates into the glass fibers and is absorbed by the surrounding absorbent paper via capillary action. The DNA in the lysis buffer is captured and adsorbed onto the glass fibers during flow. Subsequently, 100 μL of washing buffer (30 mm NaOAc, 70% v / v ethanol) is added to remove impurities and rinse away cell residues for DNA purification. Then, the folded paper device was used for elution, with 40 μL of elution buffer (10 mM TE buffer, pH = 8.0) added to the elution zone. As the elution buffer was added, DNA was released from the glass fiber and evenly distributed to the detection zone through the interconnected paper microfluidic channels, then entered the corresponding LAMP reaction chamber. LAMP reaction reagents were then added, and the chamber was sealed with an acetate film to prevent evaporation during amplification. The chamber was then placed in an insulated container and reacted at 65°C for 40 min. Color development was achieved by excitation with a handheld UV flashlight at 365 nm; the sample was colorless under negative conditions and fluorescent green under positive conditions. The positive rate in the samples was calculated, and the results were compared with those of the PCR method. Finally, the samples allocated to the reaction chambers were subjected to qPCR testing to analyze the sample extraction recovery rate and the uniformity of sample distribution in different reaction chambers.

[0179] 3. LAMP reaction in paper-based biosensors

[0180] Nucleic acid extracted using the paper apparatus is evenly distributed into the reaction chamber via a paper microfluidic channel, followed by the addition of various reagents required for the LAMP reaction. The paper apparatus is then removed, and the chamber is sealed with an acetate film to prevent liquid evaporation during the isothermal amplification process. Finally, a plastic plate with the sealed reaction chamber is placed in a 65°C insulated water cup for the LAMP reaction. The results are visualized and read using a handheld UV lamp for qualitative analysis, and the intensity of the green fluorescence signal is captured using a mobile phone camera and extracted using ImageJ software for quantitative analysis.

[0181] 4. Performance Testing of LAMP-Based Paper-Based Biosensors

[0182] After completing the LAMP reaction construction, reaction performance testing, and paper-based microfluidic device design, the paper-based biosensor construction was basically completed. To further verify that the LAMP system constructed in Example 1 can be used to perform detection with high specificity, excellent sensitivity, and superior precision, the following experiments were conducted.

[0183] Following the paper-based biosensor detection procedure (see...) Figure 9 As shown, the detection performance was evaluated: the detection time from sample introduction to completion of detection by the paper-based biosensor was assessed; target sequences of different concentration gradients were prepared, and the detection range and limit of detection of the paper-based biosensor were evaluated; the precision of the sensor was evaluated by detecting three groups of target sequences of the same concentration using three different paper-based biosensors; and the DNA recovery rate of the paper-based biosensor was verified using qPCR. DNA of different concentrations was added to the sample, and DNA was extracted using a paper device for PCR detection. The extraction and elution procedures were strictly the same as those of the LAMP method, except that the DNA transfer step in the PCR analysis was performed manually.

[0184] Quantitative real-time PCR: The paper-based biosensor was validated using standard PCR methods. DNA extraction from drinking water was performed using a commercially available DNA extraction kit. After determining the DNA concentration and quality of the extracted nucleic acid samples, they were stored at -80°C. PCR reactions were incubated using an ABI 7500, with LAMP F3 and B3 primers as primers. The reaction system consisted of 4 μM F primer, 4 μM R primer, 1X Green-2-Go 2X qPCR-Low-ROX premix, and 2 μL of drinking water genomic sample, which was then brought to a final volume of 20 μL with enzyme-free sterile water. Each sample was tested in triplicate, and at least three blanks were used for each pathogen per test to ensure contamination-free operation. Finally, the target fragment was extracted using a Zymoclean DNA gel extraction kit, and DNA concentration was quantified using Qubite 4.0. This value was used for subsequent standard samples. A quantitative standard curve for the target gene was prepared by serially diluting the PCR products 10-fold. The DNA solution, diluted 10-fold with ultrapure water, was then analyzed using real-time quantitative PCR to quantify the nucleic acid concentration of the pathogen being tested.

[0185] a. Assessment of test completion time

[0186] The process of paper-based biosensors from sample introduction to detection includes: sample pretreatment (sample enrichment and lysis, DNA extraction and washing, DNA elution), LAMP reaction, and fluorescence visualization detection. Based on the optimization of LAMP reaction time conditions, the required detection throughout the process is as follows: Figure 11 As shown, the total time is approximately 51 minutes (<1 hour), which is much shorter than the traditional gold standard method qPCR (approximately 2-3 hours).

[0187] b. Specificity and precision testing

[0188] To verify the detection specificity of the paper-based biosensor, ddH2O, ermb resistance gene plasmid DNA (purchased from Genewiz Biotechnology Co., Ltd., GenBank: JN607214.1, vector PUC57-amp), and plasmid DNA of three pathogens (Escherichia coli, Salmonella, and Shigella) (the same as used in 1.(2) of Example 1) were added as templates, respectively. The ermb resistance gene plasmid DNA served as a positive control, and ddH2O served as a negative control. Specific primers were used for the reaction, and the colorimetric results were observed using a visualization-LAMP reaction to evaluate the specificity of the system. In addition, the same concentration of the target sequences was detected using three different paper-based biosensors to evaluate the sensor precision.

[0189] Visualization results showed that green (see image) was observed in the chambers containing the positive control and those containing E. coli, Salmonella, and Shigella DNA, respectively. Figure 12The result of a-12c was positive, while the other non-target DNA and negative control tubes remained colorless, indicating negative results. Figure 12 Cross-sample LAMP amplification showed extremely high specificity, indicating that paper-based biosensors possess high specificity. Furthermore, the visualization results of the three paper-based biosensors in each group exhibited the same trend, indicating that paper-based biosensors have high precision. Figure 12 a-12d).

[0190] c Sensitivity Test

[0191] The sensitivity of the origami detection device was evaluated by conducting experiments on a series of target DNA concentration gradients. The limit of detection (LOD) and detection range were assessed by visually analyzing the color intensities of the reaction chamber. The E. coli concentration gradient was 1.11 × 10⁻⁶. 7 1.11×10 6 1.11×10 5 1.11×10 4 1.11×10 3 1.11×10 2 1.11×10 1 (Unit: copies / μL); Salmonella concentration gradient is 1.87×10⁻⁶. 9 1.87×10 8 1.87×10 7 1.87×10 6 1.87×10 5 1.87×10 4 1.87×10 3 (Unit: copies / μL); Shigella concentration gradient is 1.95×10⁻⁶. 7 1.95×10 6 1.95×10 5 1.95×10 4 1.95×10 3 1.95×10 2 1.95×10 1 (Unit: copies / μL). Five reaction wells contain a negative control, a positive control, and the remaining three contain reaction solutions with primers for E. coli, Salmonella, and Shigella, respectively. Taking E. coli as an example, when adding a positive sample and different concentrations of E. coli, the positive wells remain unchanged; only the E. coli sample wells are observed. The remaining sample wells serve as controls to determine if false positives occur.

[0192] The results showed that the reaction chamber glowed fluorescent green with increasing genomic DNA concentration. When the concentrations of the three pathogen DNAs were high, the reaction chamber was brighter; when the concentrations were below the detection limit, the color was very weak, making it difficult to interpret (e.g., Figure 13 (as shown in a-13d). Therefore, the detection limit for Escherichia coli is 1.11 × 10⁻⁶. 2 copies / μL (converted to concentration units: 2.32 × 10⁻⁶) -11 The detection range is 1.11 × 10 μg / μL. 2 -1.11×10 7 copies / μL (converted to concentration units: 2.32 × 10⁻⁶) -11 -2.32×10 -6 μg / μL); the detection limit for Salmonella was 1.87 × 10 μg / μL. 4 copies / μL (3.80×10) -9 The detection range is 1.87 × 10 μg / μL. 4 -1.87×10 9 copies / μL (converted to concentration units: 3.80 × 10⁻⁶) -9 -3.80×10 -4 The detection limit for Shigella (μg / μL) was 1.95 × 10⁻⁶. 2 copies / μL (5.54×10) -11 The detection range is 1.95 × 10 μg / μL. 2 -1.95×10 7 copies / μL (converted to concentration units: 5.54 × 10⁻⁶) -11 -5.54×10 -6 The detection limits for the three pathogens (as shown in Table 5 below) are basically consistent with those of the LAMP method, indicating that the paper-based device does not have a significant impact on the sensitivity of LAMP.

[0193] Table 5. Detection limits for typical pathogens based on LAMP paper-based biosensors.

[0194] Pathogens Method detection limit (μg / μL) Method detection range (μg / μL) E. coli <![CDATA[2.32×10 -11 ]]> <![CDATA[2.32×10 -11 -2.32×10 -6 ]]> salmonella <![CDATA[3.8×10 -9 ]]> <![CDATA[3.8×10 -9 -3.8×10 -4 ]]> Shigella <![CDATA[5.54×10 -11 ]]> <![CDATA[5.54×10 -11 -5.54×10 -6 ]]>

[0195] Example 3: Validation of Paper-Based Biosensor and Risk Assessment of Pathogen Contamination in Tongren Drinking Water Source

[0196] This embodiment uses the LAMP detection system constructed in Example 1 and the paper-based microfluidic device constructed in Example 2 to test 15 surface water drinking water sources in Tongren City, which fully demonstrates that the device has high specificity, excellent sensitivity and excellent precision.

[0197] This embodiment utilizes the LAMP detection system constructed in Example 1 and the paper-based microfluidic device constructed in Example 2 to detect and analyze pathogenic microorganisms in 15 national and provincial control sections of surface water drinking water sources within Tongren City, as well as in drinking water near these sections. The detection results are shown in Table 6.

[0198] In this embodiment, positive, uncertain, and negative are defined as follows: Positive means that compared to the negative control (ddH2O), the sample signal is stronger and appears earlier, indicating a higher concentration of pathogens in the drinking water. Negative means that the sample signal is the same as or even lower than the negative control, indicating the absence of the pathogen in the wastewater. Uncertain means that during monitoring, the sample signal is difficult to distinguish from the negative control signal, indicating that the pathogen concentration may be extremely low or non-existent. Based on the above criteria, a total of 15 drinking water samples were monitored, with 45 tests conducted for the three pathogens. Among them, there were 33 positive samples, 4 uncertain samples, and 8 negative samples. Positive (P) samples accounted for 73.33%, negative (N) samples accounted for 17.78%, and uncertain (Not sure) samples accounted for 8.89%.

[0199] Table 6. Detection results of paper-based biosensors at drinking water source sections in Tongren City

[0200]

[0201]

[0202] To ensure the accuracy of the analysis and prediction, this embodiment was compared with the standard methods—quantitative PCR (qPCR) and real-time fluorescent LAMP. The qPCR results are as follows: Figure 14 As shown, the real-time fluorescence LAMP results are as follows: Figure 15 As shown. Combined with PCR amplification results (see...) Figure 14 Overall, except for Salmonella, the results for both methods (a-14h) are largely consistent, with a sample detection accuracy of 77.78%, indicating that the paper-based biosensor method is generally feasible. This is further supported by the real-time fluorescence LAMP amplification results (see...). Figure 15 (a-15h) The detection results of the paper-based biosensor and the qPCR were completely consistent. Therefore, the paper-based biosensor is generally feasible for actual sample detection. Meanwhile, compared with the real-time fluorescence LAMP quantification results, the relative deviations between the qPCR and real-time fluorescence LAMP quantification results, except for Salmonella, were as high as 31.39% and as low as -15.19% for the other two pathogens (see [link to qPCR results]). Figure 16 For strains a-16c, the overall magnitudes are not significantly different, making LAMP detection a feasible method. However, Salmonella may exhibit higher LAMP quantification results due to nonspecific amplification or other factors.

[0203] The main reason for the low accuracy of paper-based biosensors may be that Salmonella, due to the high detection limit of LAMP, is located in the range of 0–3.8 × 10⁻⁶. -9 Salmonella was not detected in the μg / μL range. However, despite numerous attempts using LAMP primers from other literature and our own designed LAMP primer pairs, Salmonella was still not detected. Therefore, the primers used in this application are the optimal choice. Alternatively, the concentration of Salmonella itself in drinking water sources may be very low, leading to undetectable LAMP levels. However, this does not negatively impact the results of drinking water pathogen monitoring, as such extremely low concentrations of pathogens are often naturally occurring, similar to background levels, and therefore do not affect the assessment of health risks to drinking water. In such cases, the LAMP reaction results are often more meaningful. This result demonstrates that the paper-based biosensor developed in this application possesses suitable sensitivity, high specificity, and extremely high precision, making it suitable for drinking water monitoring.

[0204] In summary, this application presents a LAMP-based paper-based sensing device for the detection of typical pathogens in drinking water. This paper-based sensing device not only successfully solves the challenge of nucleic acid purification under suitable site conditions, but also achieves rapid detection and rapid visual risk assessment by combining it with the LAMP reaction. The detection process, from sampling to results, takes less than one hour and requires no complex equipment. The detection limits of the paper-based sensing device are 2.32 × 10⁻⁶ for Escherichia coli. -11 μg / μL, 3.8×10 -9 μg / μL, 5.54×10 -11 μg / μL, detection range 2.32×10 -11 -2.32×10 -6 μg / μL, 3.8×10 -9 -3.8×10 -4 μg / μL, 5.54×10 -11 -5.54×10 -6 μg / μL. Finally, the paper-based sensing device was used to detect typical pathogen contamination in drinking water in Tongren, and the results showed that there was a certain risk of pathogen contamination and infection.

[0205] Example 4: Detection of drinking water pathogens by combining nucleic acid isothermal amplification with gene editing.

[0206] By coupling loop-mediated isothermal amplification (LAMP) with CRISPR / Cas12a, a portable fluorescence detection system based on LAMP-CRISPR / Cas12a was established to achieve rapid, sensitive, and visualized on-site detection of pathogenic microorganisms in drinking water.

[0207] 1. Optimization of CRISPR / enASCas12a-HF detection method conditions

[0208] The reaction system and conditions for the CRISPR / enASCas12a-HF standard reaction are as follows: Prepare an 18 μL premix of 100 nM Cas12a, 125 nM crRNA, 500 nM fluorescent probe, and 1×NEBuffer 2.1. Then add 2 μL of DNA template and incubate at 37°C for 60 min. Fluorescence data are collected using an ABI 7500.

[0209] The enAsCas12a-HF1 protein was prepared according to the following method.

[0210] a. First, the pET-28b-T7-henAsCas12a-HF1 plasmid was introduced into Rosetta 2(DE3) competent cells via heat shock transformation (the coding sequence of henAsCas12a comes from Addgene plasmid 107942). Then, the cells were inoculated into LB agar containing kanamycin and chloramphenicol, and cultured overnight at 37°C. Single colonies were selected and inoculated into 100 mL of liquid LB agar containing kanamycin and chloramphenicol, and cultured overnight at 37°C and 220 rpm until saturation. 25 mL of the saturated bacterial culture was added to 1 L of liquid LB agar containing kanamycin and chloramphenicol, and cultured at 37°C until mid-logarithmic growth (OD2). 600 After adding IPTG (final concentration 0.1 mM) to a concentration of approximately 0.4–0.6, the cells were expressed overnight at 18°C. Finally, the cells were centrifuged at 5000 rpm for 10 minutes at 4°C, the supernatant was discarded, and the cells were stored at -80°C for later use.

[0211] b. Thaw the bacterial cells on ice, and add lysis buffer (20mM HEPES pH=7.5, 300mM NaCl, 2mM MgCl2, 20mM imidazole, 0.5mM TCEP, 0.1% Triton X-100, 0.25mg / mL lysozyme, 1:100 protease inhibitor) to the cells at a ratio of 1:5 and sonicate. After lysis, centrifuge at 40000g for 1 hour and 30 minutes at 4°C. The supernatant was loaded onto Histrap HP, and then linearly eluted in AKTA pure 25M (Cytiva, USA) using Buffer A (50 mM Tris-HCl, pH 8.0, 20 mM imidazole, 300 mM NaCl, 0.5 mM TCEP) and Buffer B (20 mM Tris-HCl, pH 8.0, 300 mM imidazole, 300 mM NaCl, 0.5 mM TCEP), and the elution peaks were collected. The buffer was then replaced with Buffer E (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10% (v / v) glycerol) using a desalting column, and protein concentration was measured using Qubit 4.0. The protein was then flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0212] The specific sequences of the fluorescent probe and crRNA are shown in Table 7.

[0213] Table 7 Probe and crRNA sequences

[0214]

[0215] The main parameters of the established CRISPR / enASCas12a-HF detection method were optimized, primarily focusing on the concentration of enASCas12a-HF protein, crRNA concentration, and reaction time.

[0216] (1) concentration of enASCas12a-HF protease

[0217] Based on the CRISPR / enASCas12a-HF standard reaction, plasmid DNA of three pathogens (the same as that used in 1.(2) of Example 1, 10 nM), fluorescent probe (500 nM), crRNA (final concentration 100 nM) and different concentrations of Cas12a (0-100 nM) were reacted at 37°C for 60 min.

[0218] The measured fluorescence results are as follows Figure 17 As shown in a-17c, the results show that the fluorescence intensity is high only when the final concentration of Cas12a protein is 100 nM. In order to save detection costs, 100 nM was selected as the optimal concentration of Cas12a protein.

[0219] (2) crRNA concentration

[0220] Based on the CRISPR / enASCas12a-HF standard reaction, different concentrations of crRNA were added to the system while keeping other components and conditions constant. The optimal concentration was selected based on the fluorescence value to make the detection reaction more sensitive.

[0221] according to Figure 18 As shown in a-18c, the fluorescence values ​​of *E. coli* and *Shigella* crRNA showed an increasing trend at concentrations of 0.2 μM and 1 μM, but stopped increasing after reaching 1 μM; while *Salmonella* only showed fluorescence at 1 μM. In summary, the activity of *E. coli*, *Salmonella*, and *Shigella* crRNA was high at a concentration of 1 μM.

[0222] (3) Reaction time

[0223] Based on the CRISPR / enASCas12a-HF standard reaction, Salmonella with a high detection limit (10 nm) was selected as a template, and different incubation times were set at 37°C. Figure 19 As shown, the CRISPR / enASCas12a detection method demonstrates the effect of fluorescence signal changes over time, with the fluorescence signal gradually increasing over time, reaching its peak after 30 minutes. Considering the need for rapid and sensitive detection, a detection time of 30 minutes was chosen to obtain an appropriate fluorescence signal.

[0224] 2. Construction by LAMP-CRISPR / enASCas12a reaction

[0225] The principle of LAMP-CRISPR / enASCas12a is as follows: Figure 20 As shown in Figure b, a CRISPR / enASCas12a-based biosensor was constructed to monitor three pathogens in drinking water sources in the field. The detection process of this biosensor is as follows: Figure 20As shown in Figure a, lysis buffer was first added to a 0.22 μm filter membrane, and sample DNA was extracted using a paper-based biosensor, completing rapid pretreatment of the wastewater sample within 10 min. Then, the extracted DNA template was pre-amplified using LAMP, generating a large number of amplicones in the presence of the target. The LAMP product was added to the Cas12a-crRNA complex. After the target DNA was specifically recognized under the guidance of crRNA, the Cas12a trans-cleavage activity was activated, and the ssDNA probe was non-specifically cleaved, producing an observable signal. For fluorescence-based detection, the ssDNA reporter gene was labeled with a fluorophore / quencher pair (FAM-BHQ). In the presence of Cas12a trans-cleavage, the ssDNA fluorescent probe dissociated, releasing the quencher and restoring the fluorescence signal. The results can be visually read using a fluorescence reading device or excitation by a specific light source. Conversely, if the ssDNA probe is not dissociated and the two fragments remain ligated, no fluorescence signal is generated.

[0226] As described in Example 1, LAMP detection of three pathogen genes was performed using different primers on a real-time fluorescence PCR instrument. The results showed good amplification at a target concentration of 100 pM, with signals observable after 20 minutes, while the negative control showed no fluorescence signal. Figure 21 a-21c) indicates that LAMP detection can rapidly and specifically amplify three pathogens and successfully amplify the target signal.

[0227] The system described in the optimization of the CRISPR / enASCas12a-HF detection method conditions in Example 4 is as follows: Figure 21 As shown in d-21f, the fluorescence signal containing the target DNA is significantly higher than that without the target, indicating that the CRISPR / Cas12a detection system can be specifically activated by the target DNA and produce observable fluorescence.

[0228] (1) Construction of the coupled LAMP-CRISPR / enASCas12a-HF detection system

[0229] First, the pathogen DNA fragment was amplified using the LAMP method, as follows:

[0230] LAMP primers targeting *Escherichia coli* (malB gene), *Salmonella* (invA gene), and *Shigella* (ipah gene) are shown in Table 1. The LAMP reaction was performed in a 20 μL system containing 0.2 μM F3, B3 (1:1), 1.6 μM FIP, BIP (1:1), 0.4 μM LF, LB (1:1), 2 μL 10X Isothermal Amplification Buffer, and 0.32 U / μL Bst 2.0. DNA polymerase, 0.4 mM dNTPs, 4.0 mM magnesium sulfate, 500 μM Mn 2+ 2 μL DNA template. No chromogenic reagent should be added to the LAMP-CRISPR / enASCas12a-HF coupled reaction, as it will interfere with the FAM fluorescence signal. The reaction is performed at 65°C for 40 min.

[0231] Then, an 18 μL premix containing 100 nM Cas12a, 125 nM crRNA, 500 nM fluorescent probe, and 1×NEBuffer 2.1 was prepared, followed by the addition of 2 μL of LAMP product (DNA template), and incubation at 37°C for 30 min. Fluorescence data were collected using an ABI 7500.

[0232] (2) Visualized detection

[0233] Fluorescence detection: After the reaction, visualization was performed under 480nm blue light. The solution exhibited green fluorescence in the presence of a positive sample and was colorless in the presence of a negative sample. Fluorescence intensity was extracted using ImageJ.

[0234] (3) Sensitivity detection of coupled LAMP-CRISPR / enASCas12a-HF

[0235] First, the *E. coli* malB gene, *Salmonella* invA gene, and *Shigella* ipah gene were serially diluted, and then a LAMP-CRISPR conjugation experiment was performed. The limit of detection of the detection method was evaluated by visually analyzing the changes in the intensity of the fluorescence color.

[0236] Based on the two existing detection methods, a joint detection method was constructed by combining LAMP with CRISPR / enASCas12a recognition. For example... Figure 21 As shown in g-21h, the signal in the experimental group was significantly higher than that in the control group, indicating that the system successfully detected the target gene. The experimental results demonstrate the successful establishment of a CRISPR / enASCas12a-based biosensor.

[0237] 3. Evaluation of the effectiveness of LAMP and CRISPR / enASCas12a single method detection

[0238] (1) Evaluation of the effectiveness of CRISPR / enASCas12a single method detection

[0239] To verify the effectiveness of the CRISPR / enASCas12a single method, the DNA concentrations of three pathogens were diluted at different folds. Figure 22As shown in a-22c, only higher DNA concentrations promoted the cleavage reaction of the Cas12a-crRNA complex, and the detection limit of the E. coli plasmid was 1.11 × 10⁻⁶. 9 The limit of detection for Salmonella was 3.74 × 10⁻⁶ copies / μL. 9 The detection limit for Shigella is 1.95 × 10⁻⁶ copies / μL. 9 copies / μL. This shows that CRISPR's detection sensitivity is insufficient.

[0240] (2) Evaluation of the detection effect of LAMP single method

[0241] LAMP is a necessary condition for signal amplification and initial target identification in coupling methods. Furthermore, the sensitivity and specificity of LAMP have a significant impact on the coupling method.

[0242] According to the "LAMP sensitivity characterization" in Example 1, the fluorescence LAMP successfully detected the LOD of the three pathogens malB, invA, and ipah genes, which were 1.11 × 10⁻⁶. 2 copies / μL, 1.87×10 4 copies / μL, 1.95×10 2 copies / μL; using 25% of the maximum fluorescence intensity as a threshold, the time to reach the threshold was fitted with the logarithm of the corresponding target concentration, invA gene R 2 The results showed that the values ​​reached above 0.97, and even reached 0.99 for the malB and ipah genes, indicating that real-time LAMP has excellent real-time quantification capabilities and the linear range can reach 6-7 orders of magnitude.

[0243] However, in qualitative site testing, false positives caused by nonspecific amplification can interfere with the detection process. For example... Figure 8 As shown in c, 8f, and 8i, bands resembling dimers also appear in the amplification of negative controls (channels 2-3) and samples below the detection limit. Channel 4 in c, f, and i exhibits severe blurring and corresponding fluorescence signals, suggesting non-specific amplification. While dimer-induced fluorescence signals can be distinguished using fusion curves and electrophoresis under laboratory conditions, non-specific identification methods such as calcein and turbidimetry, commonly used in field settings, cannot distinguish false positives. Therefore, distinguishing specific amplified signals from false positives, such as dimers, under field conditions is crucial for the field application of LAMP. This application introduces the Cas12a system in subsequent reactions to construct a LAMP-CRISPR / enASCas12a detection platform, further optimizing the detection method, as shown in "5. Evaluation of LAMP-CRISPR / enASCas12a Detection Performance" below.

[0244] 4. Evaluation of LAMP-CRISPR / enASCas12a detection effectiveness

[0245] First, ten pathogen DNA fragments, each diluted 10-fold, were used to assess whether the introduction of CRISPR / Cas12a could improve the sensitivity of LAMP. Real-time fluorescence results showed that the introduction of Cas12a achieved efficient signal transduction, and the designed CRISPR / enASCas12a system could detect as low as 111 copies / μL (E. coli), 18700 copies / μL (Salmonella), and 195 copies / μL (Shigella) (see [link to study]). Figure 23 The detection limit (a-23c) is the same as that of LAMP, and the sensitivity for E. coli, Salmonella, and Shigella genes is not affected. Furthermore, no signal was generated in the negative control, indicating that the LAMP-coupled CRISPR / enASCas12a system successfully avoids potential false positives.

[0246] Next, to address the various visualization requirements under complex on-site conditions, fluorescence was used for visual detection and analysis. The operation of the fluorescence method remained the same as above, except that a fluorescence reading instrument was no longer used for the final signal observation; instead, a 480nm portable flashlight was used to excite the color development. For example... Figure 23 As shown in g-23h, when the target concentration under blue light is higher than the detection limit, the solution exhibits green fluorescence, while the negative control shows no fluorescence. Next, ImageJ was used to extract the colorimetric information, and the results were completely consistent with the visual observations. This indicates that the reaction can yield reliable results without the aid of instruments, simply by observing with the naked eye.

[0247] Furthermore, analysis of the endpoint fluorescence intensity revealed no significant correlation between target DNA concentration and fluorescence intensity. Figure 23 d-23f), fluorescence intensity is not easily distinguishable to the naked eye using the fluorescence method. Figure 23 The reason for the failure of the visual detection platform (g-23h) is likely due to the "plateau effect" of the LAMP product cut by CRISPR / enASCas12a during amplification. Therefore, the platform can only be used for qualitative analysis and not for quantitative analysis.

[0248] 5. Evaluation of the detection performance of paper-based detection devices for three pathogen gene fragments

[0249] Paper equipment design and usage methods, such as Figure 24 As shown in a-24c, please refer to Example 3 for details.

[0250] Fabrication of the paper-based sensing device: The paper chip was fabricated using a Colorqube 8570 and a heating plate. Paraffin wax was applied to qualitative filter paper to create hydrophobic and hydrophilic regions, thereby controlling the flow of fluid. First, the corresponding pattern was printed on the filter paper using a paraffin wax printer. Then, the filter paper was baked at 120°C for 1-2 minutes using a heating plate until the paraffin wax was completely impregnated. Glass fibers were then added at specific locations, and DNA sample purification was achieved through appropriate folding. The design and folding method are detailed in [link to design details]. Figure 24 a. Then, 50 μL ddH2O was added to the paper equipment to test the flow rate under different pore sizes and optimize it.

[0251] The microfluidic board was printed using a laser cutting machine (Shandong Leapion Machine Co., Ltd.; LC-1390). The board material is white acrylic, 5mm thick, and the cutting power is 94% of maximum power, with a cutting rate of 0.48mm / s. The pore size is the same as the hydrophilic pore size of the paper chip.

[0252] To evaluate the detection performance of the paper processing equipment, a spiking experiment was conducted in this embodiment. Plasmid samples of the three genes malB, invA, and ipah were prepared in the same manner as in Example 1, with concentrations of 1.11 × 10⁻⁶. 2 copies, 1.87×10 4 copies, 1.95×10 2 Copies were then added to a collected 0.22 μm filtered drinking water sample. Detection was performed using the LAMP-CRISPR / enASCas12a reaction constructed as described above. Figure 24 As shown in d, the detection limits for the three genes malB, invA, and ipah can reach 1.11 × 10⁻⁶, respectively. 2 copies, 1.87×10 4 copies, 1.95×10 2 copies.

[0253] Considering the losses incurred during pathogen enrichment, lysis, DNA extraction and purification, and sample distribution, the introduction of a paper-based device did not significantly lower the detection limit. Therefore, the paper-based device exhibits good compatibility with the LAMP-CRISPR / enASCas12a coupling platform. False positives and other issues did not occur with the paper-based device. This biosensor provides a potential analytical method for detecting pathogens in drinking water.

[0254] Example 5: LAMP-CRISPR / enASCas12a detection of real samples

[0255] Starting from February 24, 2025, the developed paper equipment was used to conduct a week-long actual sample test on 15 national and provincial control sections of surface water drinking water sources in Tongren City.

[0256] The LAMP-CRISPR / enASCas12a detection system constructed according to Example 4 was tested using a paper-based device (Example 3.6), and the results are shown in Table 8. A total of 15 drinking water samples were tested, with a total of 90 tests. 60 samples were positive (P) and 30 were negative (N). Combined with the PCR amplification results, the detection accuracy for positive samples reached 66.6%. The main reason for the lower accuracy may be that the detection limit of Salmonella LAMP is relatively high, resulting in a detection range of 0–3.8 × 10⁻⁶. -9 Not detectable in the μg / μL range. Furthermore, comparison with the LAMP results of the previous examples demonstrates that the introduction of CRISPR / enASCas12a has largely and successfully avoided potential false positives. On the other hand, it is possible that Salmonella itself has a very low concentration in drinking water sources. These results indicate that the paper-based biosensor developed in this application possesses high specificity and extremely high precision, making it suitable for drinking water monitoring.

[0257] Table 8. Actual samples detected by LAMP-CRISPR / enASCas12a.

[0258]

[0259]

[0260] In summary, this embodiment constructs a paper-based sensing device combining CRISPR / Cas12a and LAMP for the detection of typical foodborne pathogens in drinking water. The detection process, from sampling to results, takes no more than 2 hours and requires no complex equipment. Because the paper device has good compatibility with the CRISPR-LAMP method, the analytical performance of the integrated paper device is not significantly reduced, with detection limits of 1.11 × 10⁻⁶. 2 copies, 1.87×10 4 copies, 1.95×10 2 Finally, the paper-based sensing device was used to detect typical pathogen contamination in drinking water in Tongren, and the results showed that there was a certain risk of pathogen contamination. The construction of integrated paper-based sensing devices is versatile; by simply changing the LAMP primers and crRNA according to specific pathogens, its application can be extended to the detection of other pathogens, making it a powerful tool for rapid screening of pathogen contamination in drinking water in the future.

[0261] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A primer set for the specific detection of pathogens in a sample, said primer set comprising at least one of a first primer set specifically targeting the malB gene of Escherichia coli, a second primer set specifically targeting the invA gene of Salmonella, and a third primer set specifically targeting the ipaH gene of Shigella.

2. The system according to claim 1, characterized in that, The primer set is used for loop-mediated isothermal amplification reaction, preferably for real-time loop-mediated isothermal amplification reaction; Preferably, each primer set includes an upstream outer primer, a downstream outer primer, an upstream inner primer, and / or a downstream inner primer. More preferably, the first primer set includes: The first upstream outer primer F3 has the sequence shown in SEQ ID NO:

1. The first downstream outer primer B3 has the sequence shown in SEQ ID NO:

2. The first upstream inner primer, FIP, has the sequence shown in SEQ ID NO:3, and The first downstream inner primer BIP has the sequence shown in SEQ ID NO:4; More preferably, the second primer set includes: The second upstream outer primer F3 has the sequence shown in SEQ ID NO:

12. The second downstream outer primer B3 has the sequence shown in SEQ ID NO:

13. The second upstream inner primer, FIP, has the sequence shown in SEQ ID NO:14, and The second downstream inner primer BIP has the sequence shown in SEQ ID NO:15; More preferably, the third primer set includes: The third upstream primer F3 has the sequence shown in SEQ ID NO:

32. The third downstream outer primer B3 has the sequence shown in SEQ ID NO:

33. The third upstream inner primer, FIP, has the sequence shown in SEQ ID NO:34, and The third downstream inner primer, BIP, has the sequence shown in SEQ ID NO:

35.

3. The primer set according to claim 1 or 2, characterized in that, Each primer set further includes an upstream loop primer and / or a downstream loop primer; Preferably, the first primer set further includes: The first upstream loop primer LF has the sequence shown in SEQ ID NO:5, and The first downstream loop primer LB has the sequence shown in SEQ ID NO:6; Preferably, the second primer set further includes: The second upstream loop primer LF has the sequence shown in SEQ ID NO:16, and The second downstream loop primer LB has the sequence shown in SEQ ID NO:17; Preferably, the third primer set further includes: The third upstream loop primer LF has the sequence shown in SEQ ID NO:36, and The third downstream loop primer LB has the sequence shown in SEQ ID NO:

37.

4. A kit for specifically detecting pathogens in a sample, said kit comprising the primer set according to any one of claims 1-3; Preferably, the kit further includes dNTPs, DNA polymerase, calcein, and Mg. 2+ At least one of them; Preferably, the kit further includes a microfluidic paper device; More preferably, the microfluidic paper device includes a paper chip and a detection plate; the paper chip includes a loading layer, a drainage layer, and an adsorption layer made of a hydrophilic material coated with a hydrophobic material; wherein, The loading layer has multiple hydrophilic loading areas; the drainage layer has hydrophilic drainage channels, and when the drainage layer and the loading layer are stacked, the loading areas and the drainage channels overlap; the adsorption layer has hydrophilic adsorption areas, and when the adsorption layer and the drainage layer are stacked, the adsorption areas and the drainage channels overlap; the adsorption areas are provided with adsorption components for adsorbing nucleic acids; the loading areas on the loading layer of the paper chip correspond to the detection areas on the detection plate; Preferably, the paper chip further includes an absorbent layer made of a hydrophilic material coated with a hydrophobic material, the absorbent layer having a hydrophilic absorbent region, and the absorbent region overlapping the adsorption region when the absorbent layer is stacked with the adsorption layer. Preferably, the loading layer, drainage layer, adsorption layer, and absorbent layer are connected in a predetermined order or are independent of each other; Preferably, the adsorption layer includes a first adsorption layer and a second adsorption layer connected to each other, the first adsorption layer and the second adsorption layer are provided with adsorption regions at corresponding positions, and the adsorption component is disposed between the first adsorption layer and the second adsorption layer. Preferably, the adsorption component is made of glass fiber; Preferably, the loading layer, the drainage layer, the adsorption layer, and the absorbent layer are made of wax-coated filter paper; preferably, the detection plate is made of acrylic material. Preferably, the kit further includes a programmable nuclease, crRNA, and a reporter probe; More preferably, the programmable nuclease is selected from Cas12, Cas13 or Cas14 nucleases, and is preferably enASCas12a; More preferably, the crRNA includes at least one of a first crRNA specifically targeting the malB gene of *Escherichia coli*, a second crRNA specifically targeting the invA gene of *Salmonella*, and a third crRNA specifically targeting the ipaH gene of *Shigella*; further preferably, the first crRNA has the sequence shown in SEQ ID NO.:19; further preferably, the second crRNA has the sequence shown in SEQ ID NO.:19; further preferably, the third crRNA has the sequence shown in SEQ ID NO.:

19. More preferably, the reporter probe is a single-stranded nucleic acid sequence; even more preferably, the reporter probe includes a detection group and a quenching group located at both ends of the single-stranded nucleic acid sequence, wherein the detection group is selected from fluorescein, 6-fluorescein, IRDYE 700, TYE 665, Alexa Fluor, or ATTO TM633, and the quenching group is selected from BHQ1, IowaBlack RQ, Iowa Black FQ, or Black Hole quencher.

5. A system for specifically detecting pathogens in a sample, the system comprising a LAMP system and a microfluidic paper device, the LAMP system comprising the primer set according to any one of claims 1-3.

6. The system according to claim 5, characterized in that, The LAMP system also includes dNTPs, DNA polymerase, DNA template, and Mg. 2+ At least one of them, Preferably, in the LAMP system, the concentration of the upstream and / or downstream outer primers is 0.05-0.3 μM, more preferably 0.1-0.15 μM; Preferably, in the LAMP system, the concentration of inner primer FIP and / or inner primer BIP is 0.5-2 μM, more preferably 0.7-0.9 μM; Preferably, in the LAMP system, the concentration of the circular primer LF and / or the circular primer LB is 0.1-0.8 μM, more preferably 0.5-0.7 μM; Preferably, in the LAMP system, Mg 2+ The concentration is 4-12 mM, preferably 5-8 mM; Preferably, the LAMP system further includes a fluorescent dye, preferably calcein.

7. The system according to claim 5 or 6, characterized in that, The microfluidic paper device includes a paper chip and a detection board; The paper chip comprises a loading layer, a drainage layer, and an adsorption layer made of a hydrophilic material coated with a hydrophobic material; wherein... The loading layer is provided with multiple hydrophilic loading zones; The drainage layer is provided with a hydrophilic drainage channel. When the drainage layer is superimposed on the loading layer, the loading area overlaps with the drainage channel. The adsorption layer is provided with a hydrophilic adsorption region. When the adsorption layer is superimposed on the drainage layer, the adsorption region (310) overlaps with the drainage channel. An adsorption component for adsorbing nucleic acids is provided in the adsorption region; The loading area on the loading layer of the paper chip corresponds to the detection area on the detection board; Preferably, the paper chip further includes an absorbent layer made of a hydrophilic material coated with a hydrophobic material, the absorbent layer having a hydrophilic absorbent region, and the absorbent region overlapping the adsorption region when the absorbent layer is stacked with the adsorption layer. Preferably, the loading layer, drainage layer, adsorption layer, and absorbent layer are connected in a predetermined order or are independent of each other; Preferably, the adsorption layer includes a first adsorption layer and a second adsorption layer connected to each other, the first adsorption layer and the second adsorption layer are provided with adsorption regions at corresponding positions, and the adsorption component is disposed between the first adsorption layer and the second adsorption layer. Preferably, the adsorption component is made of glass fiber; Preferably, the loading layer, the drainage layer, the adsorption layer, and the absorbent layer are made of filter paper coated with wax; Preferably, the detection plate is made of acrylic material.

8. The system according to any one of claims 5-7, characterized in that, The system also includes the CRISPR architecture. Preferably, the CRISPR system includes a programmable nuclease, crRNA, and a reporter probe. More preferably, the programmable nuclease is selected from Cas12, Cas13 or Cas14 nucleases, and is preferably enASCas12a; More preferably, in the CRISPR system, the concentration of the programmable nuclease is 80-200 nM, preferably 90-110 mM; More preferably, the crRNA includes at least one of a first crRNA specifically targeting the malB gene of Escherichia coli, a second crRNA specifically targeting the invA gene of Salmonella, and a third crRNA specifically targeting the ipaH gene of Shigella. More preferably, the first crRNA has the sequence shown in SEQ ID NO.:19; More preferably, the second crRNA has the sequence shown in SEQ ID NO.:19; More preferably, the third crRNA has the sequence shown in SEQ ID NO.:19; More preferably, in the CRISPR system, the concentration of each crRNA is 0.5-2 μM, preferably 0.8-1.2 μM; Preferably, the reporter probe is a single-stranded nucleic acid sequence; More preferably, the reporter probe includes a detection group and a quenching group located at both ends of the single-stranded nucleic acid sequence, wherein the detection group is selected from fluorescein, 6-fluorescein, IRDYE 700, TYE 665, Alexa Fluor, or ATTOTM633, and the quenching group is selected from BHQ1, Iowa Black RQ, Iowa Black FQ, or Black Hole quencher.

9. A method for specifically detecting pathogens in a sample, the method comprising detection using the primer set according to any one of claims 1-3 or the kit according to claim 4; or detection using the system according to any one of claims 5-8.

10. The method according to claim 9, characterized in that, The method includes: (1-1) incubating the primer set according to any one of claims 1 to 3 with the sample to perform a LAMP reaction, thereby obtaining the corresponding reaction products; (1-2) visually detecting the reaction products containing fluorescent dyes; or... The method includes: (2-1) incubating the primer set according to any one of claims 1 to 3 with the sample to perform a LAMP reaction and obtain the corresponding reaction products; (2-2) adding a programmable nuclease, crRNA and a reporter probe to the reaction products to perform a CRISPR reaction and obtain a detection product; (2-3) visually detecting the detection product. Preferably, the LAMP reaction is carried out on the microfluidic paper device; Preferably, the temperature of the LAMP reaction is 61-71°C, and more preferably 65-69°C; Preferably, the LAMP reaction time is 20-40 min, more preferably 30-40 min; Preferably, the CRISPR reaction time is 20-40 min, more preferably 30-40 min; Preferably, the sample is a water-containing sample, and more preferably a drinking water sample.

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