Method for detecting vibrio parahaemolyticus by combining RPA-CRISPR / Cas12a with microfluidic biosensor

Abstract

The invention relates to the field of microbiological detection, in particular to a method for detecting vibrio parahaemolyticus by combining RPA-CRISPR / Cas12a with a microfluidic biosensor. According to the method, the preloaded freeze-drying reagent is combined with centrifugally driven liquid operation, so that rapid and closed system detection is realized, and the pollution risk is effectively reduced. According to the invention, quantitative detection of vibrio parahaemolyticus is realized in a centrifugal micro-fluidic chip by systematically optimizing an RPA amplification primer and crRNA. The detection method shows high specificity and high sensitivity to non-target vibrio and other food-borne bacteria, and the lower detection limit is 6.08 copies / mu L. In a word, the invention provides a rapid, portable and sensitive platform, can realize quantitative detection in a wide linear range, provides a practical tool for real-time monitoring of pathogens, and has application prospects in monitoring of aquaculture biosafety and marine product safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganism detection, and particularly relates to a method for detecting Vibrio parahaemolyticus by using RPA-CRISPR / Cas12a combined microfluidic biosensor. BACKGROUND

[0002] Seafood is an important component of human diet and global trade, providing high-quality protein, polyunsaturated fatty acids, vitamins, and minerals. With growing consumer demand and the increasing economic value of aquaculture, global seafood production continues to grow. However, this growth is accompanied by increasing microbial safety concerns. Seafood is highly susceptible to contamination by foodborne pathogens, which can endanger consumer health and disrupt aquaculture production and international trade. Among such pathogens, Vibrio parahaemolyticus is a major bacterial hazard associated with seafood worldwide. This halophilic, Gram-negative bacterium can colonize a variety of marine organisms and can enter at multiple stages of the aquatic production chain. Human consumption of raw or undercooked seafood contaminated with V. parahaemolyticus can cause acute gastroenteritis, with symptoms including diarrhea, abdominal pain, and vomiting; in severe cases, it can lead to septicemia and even death. In addition, V. parahaemolyticus can also infect aquatic animals, inducing diseases such as early mortality syndrome and acute hepatopancreas necrosis disease, causing devastating damage to the aquaculture industry. Therefore, reliable detection of V. parahaemolyticus is not only crucial for public health protection, but also a key to ensuring the sustainable development of global aquaculture. More importantly, detection methods should not only stop at the level of "presence / absence" identification, but also need to achieve quantification of bacterial load, as quantitative information is the basis for risk assessment, water quality monitoring, and process control.

[0003] Over the past few decades, researchers have applied a variety of methods to detect V. parahaemolyticus, including traditional culture-based plate counting, enzyme-linked immunosorbent assay, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, and polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR). Although these methods are accurate and reliable, they are usually time-consuming, taking hours to days, and rely on complex and sophisticated instruments, making it difficult to promote their application outside of centralized laboratories, especially quantitative detection methods that can be applied on-site are still very scarce. In practice, if on-site quantitative detection can be achieved, it can timely assess whether the microbial contamination in seafood reaches the threshold, monitor the water quality of aquaculture water in real time, and verify the sterilization efficiency in the processing process. Without these capabilities, early warning and risk management will be severely limited, highlighting the urgent need to develop a portable, sensitive, and quantitative detection platform.

[0004] In recent years, isothermal amplification technology has gradually become an important alternative solution for rapid pathogen detection. Among them, recombinase polymerase amplification (RPA) is considered to be very promising because it can complete amplification in a few minutes under low constant temperature conditions (25-42°C) and has very low requirements for instruments. RPA can be combined with gel electrophoresis, immunochromatographic test strips or fluorescence detection methods to provide qualitative results, and there have been many reports of RPA-based Vibrio detection tests. However, RPA itself has the problem of non-linearity of amplification kinetics, which makes it difficult to achieve accurate quantification, thereby limiting its application in on-site quantitative detection. Therefore, researchers have developed a single-tube RPA detection system assisted by CRISPR / Cas12a. In such detection, RPA first rapidly amplifies the target sequence, and then the Cas12a protein guided by crRNA specifically cleaves the amplification product, while activating its transcleavage activity on the reporter probe, producing a detectable fluorescence signal. Notably, the specificity of Cas12a can compensate for the non-linearity of RPA to some extent, opening up new avenues for sensitive and quantitative nucleic acid detection. In fact, recent studies have used single-tube RPA-CRISPR / Cas12a testing to achieve quantitative detection of viral and bacterial targets. However, despite its promising prospects, most existing systems have only been validated in controlled laboratory environments, still facing problems such as the risk of aerosol contamination, difficulty in achieving multiplex detection, and limited throughput, which seriously hinder the transformation of this technology into a reliable quantitative tool suitable for food safety monitoring.

[0005] Microfluidic technology provides a potential way to solve these bottlenecks. Microfluidic platforms, commonly known as "lab-on-a-chip", can miniaturize and partition reactions in a closed chamber, thereby simplifying the operation process, preventing cross-contamination, and supporting multiplex and high-throughput analysis. Several studies have demonstrated the application of microfluidics in pathogen detection, such as detecting E. coli, Salmonella and Staphylococcus aureus in combination with nanomaterials and smartphone reading technology, or distinguishing the virulence genes of Vibrio parahaemolyticus in combination with isothermal amplification.

[0006] However, the current combination of microfluidic technology and RPA-CRISPR has not yet achieved accurate quantitative detection of Vibrio parahaemolyticus, i.e., the linear relationship of quantification is not good and the detection sensitivity is not high, which may be because the appropriate RPA amplification primers and crRNA have not been found; or the appropriate RPA amplification primers and crRNA have not been found. combination. Quantitative detection is crucial in microbial risk assessment and disease prevention and control in aquaculture. SUMMARY

[0007] In order to solve the above problems, the application develops a closed microfluidic chip, and combines it with RPA and CRISPR / Cas12a fluorescence detection technology, to obtain a new RPA-CRISPR / Cas12a combined microfluidic biosensor detection method for Vibrio parahaemolyticus, named as ORCMB detection method. The application realizes quantitative detection of Vibrio parahaemolyticus in a centrifugal microfluidic chip by systematically optimizing RPA amplification primers and crRNA. By using the functions of amplification, recognition and signal generation integrated on the microfluidic chip, the method can quickly, sensitively, high-throughput and reliably complete quantitative analysis of target DNA, without the need of expensive and precise instruments and equipment. The detection method not only provides a new solution for seafood safety management, but also expands the application prospect of the RPA-CRISPR / Cas12a system in the field of quantitative biosensing. The steps of the detection method and comparison with traditional methods are shown in Figure 1 .

[0008] To achieve the above object, the technical scheme adopted by the application is as follows:

[0009] On the one hand, the application provides a centrifugal microfluidic chip for detecting Vibrio parahaemolyticus, wherein the microfluidic chip comprises embedded RPA-CRISPR / Cas12a reaction buffer, RPA amplification primers, crRNA, Cas12a protein and nucleic acid fluorescent probe.

[0010] Further, the RPA amplification primers are divided into upstream primers and downstream primers; the upstream primers comprise the sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; and the downstream primers comprise the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.

[0011] Further, the crRNA comprises the sequence shown in any one of SEQ ID NO: 8 to SEQ ID NO: 10.

[0012] Preferably, the RPA amplification primers comprise the sequence shown in SEQ ID NO: 2 and / or SEQ ID NO: 3; and the crRNA comprises the sequence shown in SEQ ID NO: 9.

[0013] The concentration of the RPA amplification primers is 100-1000nM, preferably 300nM; the concentration of the crRNA and Cas12a protein is 50-1000nM, preferably 100nM; and the concentration of the nucleic acid probe is 100-2000nM, preferably 500nM.

[0014] Furthermore, the detection module includes any one or more of the following: a sample chamber, an RPA-CRISPR / Cas12a reagent embedding chamber, a sample waste liquid chamber, a reaction chamber, a volume control chamber, and a vent.

[0015] The RPA-CRISPR / Cas12a reagent embedding cavity is used to preload the lyophilized powder of the RPA-CRISPR / Cas12a detection system.

[0016] Furthermore, the microfluidic chip is used in conjunction with a portable centrifugal microfluidic nucleic acid detection device.

[0017] On the other hand, the present invention provides a method for preparing a centrifugal microfluidic chip for quantitative detection of Vibrio parahaemolyticus, wherein the centrifugal microfluidic chip is as described above.

[0018] In some embodiments, the chip is modified from the chip described in "A Detection Device for a Centrifugal Microfluidic Chip" (CN118995400A). Then, the reagents of the above-mentioned RPA-CRISPR / Cas12a detection system are added to the RPA-CRISPR / Cas12a reagent embedding cavity of the chip, and the detection reagents are then lyophilized for subsequent detection.

[0019] On the other hand, the centrifugal microfluidic chip is used for the quantitative detection of Vibrio parahaemolyticus, as described above.

[0020] On the other hand, the present invention provides a method for quantitative detection of Vibrio parahaemolyticus using an RPA-CRISPR / Cas12a combined microfluidic biosensor, employing the kit described above to quantitatively detect Vibrio parahaemolyticus.

[0021] Furthermore, the steps of the method are as follows:

[0022] A. Add the sample to the reaction chamber, place the microfluidic chip in the portable centrifugal microfluidic nucleic acid detection device, and set the centrifugation and reaction programs.

[0023] Furthermore, the centrifugation program is 1,000 rpm for 10 seconds and 500 rpm for 10 seconds; the reaction program is 39℃~45℃ for 45 min~60 min.

[0024] In some methods, the reaction procedure is to react at 39°C for 60 minutes.

[0025] B. After the reaction is complete, the portable centrifugal microfluidic nucleic acid detection device generates real-time fluorescence data.

[0026] C. Use second derivatives and Python to convert real-time fluorescence data into points of interest (POIs).

[0027] In some ways, the second derivative is

[0028] D. A standard curve is established by performing linear regression fitting calculations between the POI and DNA concentration of the standard.

[0029] In some methods, when the concentration of RPA amplification primers (RPA toxR-RPA-F1 / R1) in the detection system is 300 nM, the standard curve is y = -4.1466x + 22.5821(R). 2 =0.9734).

[0030] E. Substitute the POI of the sample into the standard curve to calculate the Vibrio parahaemolyticus content in the sample. The beneficial effects of this invention include:

[0031] 1. This invention develops and verifies a one-pot RPA-CRISPR / Cas12a detection method—ORCMB detection system, which can achieve rapid, sensitive and quantitative detection of Vibrio parahaemolyticus.

[0032] 2. This invention achieves a precise balance between RPA amplification and Cas12a cleavage activity by rationally optimizing the parameters of the RPA-CRISPR / Cas12a detection system (RPA primers, types and concentrations of crRNA, etc.), thus simultaneously possessing high sensitivity (detection limit of 6.08 copies / μL) and reliable quantitative performance (i.e., standard plasmid concentration range of 10). 0 -10 4 Good linearity within copies / μL).

[0033] 3. This invention develops an integrated co-lyophilization process. By introducing a three-dimensional stable framework based on natural polysaccharides, it successfully constructs a microenvironment that allows RPA and CRISPR systems to coexist harmoniously and stably, achieving one-time, high-performance co-lyophilization of all reaction components within a single chamber. This process solves the traditional problem of incompatibility between the two systems. Compared to traditional methods that require "physical separation," the "integrated" solution provided by this invention ensures that all reagents reach the optimal reaction ratio immediately upon reconstitution, eliminating all uncertainties introduced by the mixing step. Repeated verification shows that the lyophilized chip prepared using this process exhibits stability exceeding 30 days under accelerated aging conditions at 45°C. Its detection sensitivity and quantitative linearity are indistinguishable from liquid reagents, laying an irreplaceable reagent foundation for achieving highly robust on-site quantitative detection.

[0034] 4. This invention combines freeze-dried reagent pre-embedding technology, microfluidic chips, and portable centrifugal microfluidic detection devices to construct a robust, field-deployable platform suitable for real-time detection in aquaculture environments.

[0035] 5. Traditional RPA technology is difficult to quantify directly due to nonlinear amplification kinetics. This invention designs a piece of Python code based on real-time fluorescence data obtained from a portable centrifugal microfluidic detection device and the mathematical relationship between the inflection point time (Tt) and template concentration, thereby achieving a leap from qualitative to quantitative analysis. After the above algorithm is combined with microfluidic quantitative detection, the fluorescence acquisition data is directly input into the algorithm for real-time analysis, and the inflection point detection result is automatically converted into a concentration value, realizing a fully automated quantitative detection process of "sample in - result out".

[0036] 6. Compared with traditional qPCR technology, the ORCMB method demonstrates superior analytical sensitivity and specificity, and has successfully achieved quantitative detection of Vibrio parahaemolyticus in artificially spiked fish, shrimp, shellfish (oyster) samples and naturally infected shrimp samples. Furthermore, this platform is highly versatile; simply changing the primers and crRNA allows for flexible expansion to the quantitative detection of other foodborne pathogens.

[0037] 7. Highly integrated design for true "one-pot" detection: Nucleic acid amplification and CRISPR / Cas12a signal detection are completed continuously in the same closed reaction chamber, eliminating the need for sample transfer or multi-step operations, significantly reducing the risk of aerosol contamination, and ensuring that the detection process is fast, simple, and can be performed on-site.

[0038] 8. Modular and portable design enhances field application potential: The platform has a simple structure, high degree of automation, and is easy to mass-produce. It can be flexibly deployed in fields such as food safety, aquaculture, and public health to achieve rapid and low-cost on-site molecular detection.

[0039] Overall, the developed ORCMB detection system represents a new generation of promising point-of-care diagnostic tools that combines speed, sensitivity, quantitative capabilities, and portability. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1The flowchart illustrates the quantitative detection process for Vibrio parahaemolyticus, including traditional biochemical assays, qPCR, and ORCMB methods. Traditional biochemical assays and qPCR require 2 days or more, or 5 hours or more, respectively, to obtain quantitative results from sample collection (top and middle diagrams). In contrast, the ORCMB platform can complete the entire quantitative detection process from sample collection to result output within 1.5 hours (bottom diagram).

[0042] Figure 2 : Map of standard plasmid pGEM-T-Easy-toxR

[0043] Figure 3 The structure and analysis workflow of the ORCMB platform: (A) Physical diagram of the ORCMB microfluidic chip and schematic layout of the reaction chamber; (B) Schematic diagram of the multilayer structure of the chip; (C) Components of the portable centrifugal microfluidic nucleic acid detection device; (D) Workflow of the ORCMB platform for sensitive quantitative detection of Vibrio parahaemolyticus; (E) Flowchart of the reagent loading and release process in the ORCMB microfluidic chip.

[0044] Figure 4 Schematic diagram and detailed structure of the three-layer ORCMB chip

[0045] Figure 5 Two detection schemes for ORCMB chips: (A) the types and distribution of pre-added reagents to the chip during standard curve preparation; (B) the types and distribution of pre-added reagents to the chip during sample detection.

[0046] Figure 6 Optimization of the RPA-CRISPR / Cas12a detection system: (A) RPA primers, crRNA sequences, and their core recognition sites in the toxR gene; (B) Optimal RPA primer screening results for the toxR gene (agarose gel electrophoresis), M: DNADL2000 marker; 1-2: Amplification products using primer set toxR-RPA-F1 / R1 and negative control (NTC); 3-4: Amplification products using primer set toxR-RPA-F2 / R2 and NTC; 5-6: Amplification products using primer set toxR-RPA-F2 / R2 and NTC. (C) Amplification products of F3 / R3 and NTC; (D) Fluorescence intensity and POI time obtained using crRNA with typical PAM, suboptimal PAM, and PAM-free regions, PAM: typical PAM-guided crRNA group, sPAM: suboptimal PAM-guided crRNA group, PAM-free: PAM-free crRNA group; (E) Concentration optimization of ssDNA-FQ reporter molecule; (F) Optimization of Cas12a and crRNA concentrations at a 1:1 molar ratio of Cas12a to crRNA; All data are expressed as mean ± standard deviation of three replicates.

[0047] Figure 7 The optimization of RPA primer concentration in the ORCMB detection method was carried out, and fluorescence curves and the linear relationship between target DNA concentration and POI time (standard curves) were obtained at different primer concentrations: (A) 200 nM toxR-RPA-F1 / R1; (B) 300 nM RPA toxR-RPA-F1 / R1; (C) 500 nM RPA toxR-RPA-F1 / R1. All data are expressed as mean ± standard deviation of three replicates.

[0048] Figure 8 The specificity and sensitivity of the ORCMB detection method were evaluated. (A) Specificity analysis against Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio harveyi, Vibrio alginolyticus, Vibrio cholerae, Escherichia coli O157:H7, and Staphylococcus aureus; (B) Sensitivity assessment using serial dilutions of standard plasmid templates. All data are expressed as mean ± standard deviation of three replicates.

[0049] Figure 9 Comparison of ORCMB and qPCR detection methods in spiked and actual shrimp samples: (A) Amplification curves of ORCMB detection in spiked shrimp samples; (B) Amplification curves of qPCR detection in spiked shrimp samples; (C) ROC analysis of ORCMB detection of the toxR gene in actual shrimp samples; (D) Heatmap results of ORCMB and qPCR quantitative detection of the toxR gene in 50 freshly collected shrimp samples; All data are expressed as mean ± standard deviation of three replicates.

[0050] Figure 10 The quantitative performance of ORCMB in detecting Vibrio parahaemolyticus spiked samples is shown in the following figures: (A) Amplification curve of ORCMB in mackerel spiked samples; (B) Amplification curve of ORCMB in oyster spiked samples.

[0051] Figure 11 The quantitative performance of real-time fluorescence quantitative PCR in detecting Vibrio parahaemolyticus in spiked samples is shown in the following figures: (A) qPCR amplification curve of spiked mackerel sample; (B) qPCR amplification curve of spiked oyster sample.

[0052] Figure 12 : qPCR melting curve analysis for identifying Vibrio parahaemolyticus in spiked samples, including (A) spiked shrimp sample; (B) spiked mackerel sample; and (C) spiked oyster sample. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0055] Example 1: Materials and Methods

[0056] 1.1 Reagents and Materials

[0057] Vibrio parahaemolyticus ATCC 17802 was obtained from the China General Microbiological Culture Collection Center (Beijing, China). RPA primers (SEQ ID NO:2–SEQ ID NO:7), crRNAs (SEQ ID NO:8–SEQ ID NO:10), and ssDNA-FQ reporter probe (SEQ ID NO:11) were synthesized and purified by Genscript Biotech Inc. (Nanjing, China). Specific sequence information is shown in Table 1. The V. parahaemolyticus toxR standard plasmid (pGEM-T-Easy-toxR) was also used. Figure 2 The samples were previously stored in the laboratory. Bacterial genome extraction kits (centrifuge column type) and plasmid miniprep kits (centrifuge column type) were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Lachnospiraceae bacterium Cas12a (LbCas12a(Cpf1)) nuclease was provided by Tulugang Biotechnology Co., Ltd. The isothermal DNA amplification kit (basic type) was purchased from Changzhou Anpu Future Biotechnology Co., Ltd. The qPCR kit SupRealQ Ultra HunterSYBR qPCR Master Mix (U+) was purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). The DNA Marker DL2000 was purchased from Takara Bio Inc. (Dalian, China). 50×TAE buffer was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The microfluidic chip was manufactured on a high-precision computer numerical control (CNC) machine.

[0058] The sequences involved in this invention are shown in Table 1.

[0059] Table 1 Sequence Information

[0060]

[0061]

[0062] 1.2 Design and fabrication of microfluidic chip and portable centrifugal microfluidic nucleic acid detection device

[0063] The microfluidic chip developed in this invention adopts a disk-type structure design, consisting of eight independent detection modules radially distributed around a center. It can detect up to eight samples simultaneously, enabling simultaneous standard curve generation and actual sample detection. Each module comprises the following parts: a sample chamber, an RPA-CRISPR / Cas12a reagent embedding chamber, a waste chamber, three reaction chambers, a sacrificial chamber, and a vent hole. Figure 3 A and Figure 4 The sample chamber is circular with an 8.9 mm diameter. It is connected to the RPA-CRISPR / Cas12a reagent embedding chamber. The former is used to load the sample to be tested, while the latter is used to load the lyophilized reagent required for RPA-CRISPR / Cas12a detection. The volume control chamber to the right of the reagent chamber collects excess sample solution. The RPA-CRISPR / Cas12a reagent embedding chamber is connected to the reaction chamber area via a capillary tube. Each reaction chamber has a diameter of 6.5 mm. Setting up three detection chambers can increase the detection throughput, be used for embedding gradient concentration standard plasmids, or for three repetitions in actual sample detection, and reduce experimental errors. A volume control chamber is located after the three reaction chambers. The latter is used to collect excess reagent, so that no residual liquid remains in the capillary or microchannel, thereby effectively avoiding cross-contamination between the three reaction chambers. Vents are located after the sample waste liquid chamber and the volume control chamber, respectively. Their function is to balance the gas pressure and ensure that the reagent can enter the reaction chamber for reaction.

[0064] Depending on the experimental objective, the solutions pre-contained in different chambers of the chip vary. When the objective is to prepare a sample standard curve, the reagent chamber and reaction chamber are pre-embedded with lyophilized powder of the RPA-CRISPR / Cas12a detection system and the gradient standard plasmid pGEM-T-Easy-toxR, respectively. When it is necessary to detect whether the sample contains Vibrio parahaemolyticus, the reagent chamber is pre-embedded with lyophilized powder of the RPA-CRISPR / Cas12a detection system. In summary, this chip supports two different detection modes (RPA-CRISPR / Cas12a standard curve generation and actual sample detection), thus providing flexibility for experimental design. Figure 5 ).

[0065] The microfluidic chip is made of polycarbonate (PC). It is prefabricated through mechanical micromachining followed by chemical polishing. The chip's overall structure consists of three layers: an injection layer, a reaction layer, and a bottom layer. The detection module is located in the reaction layer. The injection layer and bottom layer are both 1 mm thick and are inert sealing films. The reaction layer is 3 mm thick and is composed of a high-precision PC substrate. Figure 3 B and Figure 4 ).

[0066] During chip fabrication, a CNC milling machine equipped with precision tools is used to etch microstructures onto a polycarbonate (PC) substrate. During assembly, the bottom layer of the chip is sealed with an inert film. Appropriate reagents are then added to the RPA-CRISPR / Cas12a reagent embedding chamber and reaction chamber, respectively, and the top layer is sealed with another inert film and freeze-dried.

[0067] Before use, the sample chamber and vent are manually punctured with a needle to equalize atmospheric pressure, allowing liquid to flow into the embedding chamber. The chip is then run on a portable centrifugal microfluidic nucleic acid testing device, which significantly reduces pipetting time and improves detection efficiency. During use, the sample solution is added to the sample chamber, then enters the RPA-CRISPR / Cas12a reagent embedding chamber to dissolve the lyophilized detection reagent. After brief centrifugation, the mixed solution enters the capillary channel due to surface tension and is then evenly distributed into the three reaction chambers for detection, yielding the results.

[0068] The portable centrifugal microfluidic nucleic acid detection device described herein is developed to achieve rapid on-site nucleic acid detection based on the aforementioned chip. This device is compact (300mm × 210mm × 150mm; weight < 5kg) and integrates centrifugation drive, isothermal heating, fluorescence signal acquisition, and automatic data analysis on a single platform. The system consists of multiple functional modules, including a power system, motor, human-machine interface, temperature control module, power module, and fluorescence detection module, thereby ensuring stable and efficient operation. The overall structural layout is shown in [details omitted]. Figure 3 C. The temperature control module of the portable centrifugal microfluidic nucleic acid detection device consists of a heating plate and a temperature sensor. Two heating plates are positioned below the top cover and the chip platform, respectively; the temperature sensor monitors the chip temperature in real time and provides feedback to maintain uniform heating. The drive module includes a motor driver and related sensors. Pulse signals from a stepper motor control the chip's rotation speed and angle, while the sensors monitor the operating status of the drive system. By calculating the product of the motor's steps and the rotation angle, different detection chambers on the microfluidic chip can be precisely positioned. The fluorescence detection module contains an LED emitting 470nm excitation light. The layout of the optical system components guides the light path through the optical fiber. A photomultiplier tube captures the emitted fluorescence signal and converts it into an electrical signal, which is then amplified by a low-noise circuit to expand the detection range and improve sensitivity. Finally, an optimized algorithm based on Fast Fourier Transform is used to analyze the real-time fluorescence data and generate an "S"-shaped amplification curve of relative fluorescence intensity.

[0069] Whether a centrifugal microfluidic chip can provide a closed, stable, and contamination-free microenvironment for its internal biochemical reactions is the physical basis for ensuring accurate, reliable, and reproducible nucleic acid test results. It is an essential verification step for the chip from design to successful application. Therefore, in order to evaluate the chip's sealing performance, this embodiment tested the chip under simulated heating environments such as a water bath, an oven, and a portable centrifugal microfluidic nucleic acid detection device.

[0070] Considering that RPA reactions typically run at 30-50℃ for about 30 minutes, the stress condition was set to 70℃ for 60 minutes. Each reaction chamber was filled with 25 μL of water for testing. The solution volume after heating was recorded, and the solution loss rate was calculated as (initial solution volume - final solution volume) / initial solution volume × 100%. The results show that the solution volume loss is small under both water bath and portable centrifugal microfluidic nucleic acid detection device conditions, especially in the water bath (Table 2). This indicates that the chip provides sufficient sealing to prevent aerosol leakage and ensure the reliability of the experimental results.

[0071] Table 2. Microfluidic chip sealing performance test under different heating methods.

[0072]

[0073] 1.3 Primer design, plasmid construction and DNA extraction

[0074] In this embodiment, three sets of RPA primers (SEQ ID NO:2-3, SEQ ID NO:4-5, SEQ ID NO:6-7) were designed based on the Vibrio parahaemolyticus toxR gene (GenBank: NC_004603.1, SEQ ID NO:1). The toxR gene was chosen as the detection target because it is highly conserved and has low homology with other Vibrio species. In addition, three sets of crRNAs were designed based on the RPA amplification products, including classic PAM (PAM, SEQ ID NO:8), suboptimal PAM (sPAM, SEQ ID NO:9), and PAM-free (PAM-free, SEQ ID NO:10). Single-stranded DNA (ssDNA, SEQ ID NO:11) labeled with FAM-BHQ1 was used as a fluorescent probe. All sequences are listed in Table 1.

[0075] The full-length fragment of the toxR gene was cloned into the pGEM-T-Easy plasmid to construct a standard plasmid for quantitative analysis; the plasmid map (pGEM-T-Easy-toxR) can be found here. Figure 2 Genomic DNA of Vibrio parahaemolyticus was extracted using a commercial bacterial genomic DNA extraction kit according to the kit instructions; plasmid DNA was pre-purified using a plasmid mini-prep kit. After nucleic acid extraction, the OD of the obtained DNA was measured using a UV spectrophotometer. 260 / OD 280 The ratio; then the DNA molecule copy number is calculated using the following formula:

[0076]

[0077] Where C represents DNA concentration and n is the number of base pairs.

[0078] 1.4 ORCMB Detection Method

[0079] Based on the chip-based design, the RPA-CRISPR / Cas12a detection system was homogenized before adding the DNA standard plasmid (pGEM-T-Easy-toxR) / sample used to prepare the standard curve. To achieve fully integrated, rapid, and field-suitable detection, the optimized RPA-CRISPR / Cas12a reaction system was added to the RPA-CRISPR / Cas12a reagent embedding chamber and then lyophilized. During the preparation of the standard curve, different concentrations (6.08 × 10⁻⁶) were used. 0 Up to 6.08×10 4 DNA standard plasmids (copies / μL) were freeze-dried in the reaction chamber.

[0080] The RPA-CRISPR / Cas12a reagent comprises: 0.75 μL 10 μM RPA forward primer (preferably SEQ ID NO:2), 0.75 μL 10 μM RPA reverse primer (preferably SEQ ID NO:3), 14.7 μL RPA A buffer, 0.25 μL 10 μM MbCas12a, 0.25 μL 10 μM crRNA (preferably SEQ ID NO:9), and 1.25 μL 10 μM ssDNA-FQ fluorescent probe (SEQ ID NO:11); after premixing, it is added to the chamber and lyophilized to prepare the ORCMB chip.

[0081] The fabrication steps of the ORCMB chip for the pre-embedded detection system are as follows:

[0082] A. Accurately weigh 2.5g trehalose, 1.0g pullulan, 0.5g sodium glutamate, 0.25g mannitol, and 25mg PEG 8000, and place them in a 50mL sterile centrifuge tube; add approximately 40mL of nuclease-free water and vortex until completely dissolved; then, add 2.5mL of 1M HEPES-KOH buffer (pH 8.2) and 250μL of 1M DTT solution, and finally bring the volume to 50mL with nuclease-free water; the prepared 2× stock solution should be filtered through a 0.22μm filter membrane for sterilization, aliquoted, and stored at -20℃ for later use.

[0083] B. Preparation and dispensing of the master mixture: In a pre-chilled container, add the following components in the following order (taking a single-reaction 25μL system as an example): 14.7μL RPA A buffer, 0.75μL 10μM upstream primer, 0.75μL 10μM downstream primer, 0.25μL 10μM crRNA, 1.25μL 10μM ssDNA fluorescent reporter probe, and 0.25μL 10μM LbCas12a protein. After each addition, gently mix with a pipette. Place the prepared master mixture on ice and precisely dispense it into the centrifugal microfluidic chip chamber at a volume of 25μL / chamber using a non-contact nanoliter dispensing system.

[0084] C. Programmed Freeze-Vacuum Drying: Immediately transfer the packaged chips to the sample rack of a freeze dryer pre-cooled to 4°C and execute the following optimized three-step freeze-drying program: Deep freezing stage: reduce the sample temperature from 4°C to -45°C at a rate of 1.5°C / min and maintain at -45°C for 120 min; Main drying stage: activate the vacuum and maintain it at <100 mTorr, raise the sample rack temperature from -45°C to -20°C at a rate of 0.2°C / min and maintain for 360 min; Desorption drying stage: raise the rack temperature from -20°C to 0°C at a rate of 0.1°C / min and maintain for 480 min. After the freeze-drying program is completed, inject high-purity nitrogen into the chamber to atmospheric pressure while maintaining vacuum, quickly remove the chips, and heat-seal them with an aluminum foil composite film. Store the finished product in a sealed bag containing desiccant at 4°C or -20°C in the dark.

[0085] D. Quality Control and Validation: A qualified lyophilized product should be a dense, porous, white or slightly yellow cake-like substance without signs of collapse. After randomly selecting a chip and adding 25 μL of nuclease-free water, it should completely reconstitute within 1 minute, and the solution should be clear. Functional validation should be performed using samples containing known concentrations of Vibrio parahaemolyticus target DNA. The fluorescence growth curve and Ct value of the lyophilized chip should not differ significantly from those of the freshly prepared liquid phase.

[0086] The steps for using the ORCMB chip are as follows: During detection, add the sample to the sample chamber. The sample (total volume 25 μL) contains the following components: 1.25 μL RPA B buffer containing MgOAc, 2.5 μL 10×HOLMES buffer (for CRISPR / Cas12a reaction), 19.25 μL sterile deionized water, and 2 μL of the sample to be tested / sterile deionized water (negative control). After adding the sample, place the chip in a portable centrifugal microfluidic nucleic acid detection device and set an alternating high and low speed centrifugation program (1,000 rpm for 10 s → 500 rpm for 10 s), a reaction temperature of 39℃, and a reaction duration of 60 min until the reaction is complete. The fluorescent signal is continuously monitored in real time using the portable centrifugal microfluidic nucleic acid detection device, and the fluorescent signal is converted into the point of inflection (POI). The POI is calculated according to the following formula:

[0087]

[0088] Where F represents the fluorescence intensity at time t.

[0089] This invention employs a customized Python algorithm (Table 3) to obtain POI time data, then performs linear regression fitting calculations with DNA concentration to establish a standard curve; the detected sample fluorescence values ​​are then substituted into the standard curve (y(POI) = -4.1466x(concentration) + 22.5821, R0). 2 =0.9734), thus calculating the concentration / copy number of Vibrio parahaemolyticus in the sample, thereby achieving the purpose of quantitative detection.

[0090] Table 3 Python Algorithms

[0091]

[0092]

[0093]

[0094] 1.5 Specificity and Sensitivity Analysis

[0095] To comprehensively evaluate the specificity of the ORCMB detection method, this invention uses a commercially available DNA extraction kit to extract corresponding genomic DNA from various marine pathogenic Vibrio species and common foodborne pathogens, including: *V. vulnificus*, *V. harvestyi*, *V. alginolyticus*, *V. cholerae*, *Escherichia coli* O157:H7, and *Staphylococcus aureus*. During the evaluation process, *Vibrio parahaemolyticus* was used as a positive control, and sterile deionized water was used as a negative control.

[0096] In the sensitivity test, the standard plasmid (pGEM-T-Easy-toxR) containing the Vibrio parahaemolyticus toxR gene was serially diluted with Tris-HCl buffer to a final concentration of 6.08 × 10⁻⁶. 6 ~6.08×10 0 Copies / μL. Detection was performed under optimized reagent conditions, and the results were interpreted based on real-time fluorescence amplification curves.

[0097] 1.6 Testing of spiked samples and actual samples

[0098] To evaluate the performance and application prospects of the RPA-CRISPR / Cas12a-ORCMB method for detecting Vibrio parahaemolyticus, this invention tested fish, shrimp, and shellfish (oyster) samples artificially supplemented with Vibrio parahaemolyticus standards, as well as shrimp samples collected in the field, and compared the results with the traditional qPCR method.

[0099] Fresh mackerel, shrimp, and oyster samples were purchased from a local seafood market in Hangzhou, China. Before use, they were verified to be free of Vibrio parahaemolyticus contamination according to the Chinese national standard method (GB4789.7-2013). Specifically, whole mackerel / shrimp / oyster samples were taken, and 25g of each sample was immersed in 75% ethanol for 30 minutes. After rinsing with sterile water, the samples were irradiated with ultraviolet light under a biosafety cabinet for 30 minutes. The treated samples were then homogenized using a beater homogenizer for 2 minutes to prepare a 1:10 homogenate.

[0100] Vibrio parahaemolyticus cultures were counted using the colony forming unit (CFU) method and prepared into suspensions, which were then serially diluted to obtain a concentration of 10. 1 -10 6 A CFU / mL bacterial suspension was added to shrimp samples known to be free of Vibrio parahaemolyticus. After homogenization, genomic DNA was extracted using a previously reported optimized boiling lysis method: 1 μL of the target bacterial suspension was mixed with 30 μL of Tris buffer, boiled for 5 minutes, and then cooled on ice for 2 minutes; subsequently, it was centrifuged at 12,000 × g for 2 minutes, and the supernatant was collected as the DNA template. qPCR detection was performed according to the instructions of the Vazyme SuperReal PreMix Plus SYBR qPCR Master Mix and in accordance with the national industry standard "Dual Real-Time PCR Detection Method for Vibrio parahaemolyticus and Vibrio cholerae in Aquatic Animals" (SN / T5125-2019). The primer sequences used in the qPCR experiment were SEO·ID:NO:12~SEO·ID:NO:13.

[0101] In addition, in collaboration with a certified diagnostics company, 50 actual shrimp samples were collected from aquaculture ponds with and without vibrio outbreaks. Samples were placed in sterile sampling bags, transported to the laboratory on ice, and processed under sterile conditions. A simplified procedure was as follows: 25g of shrimp sample was placed in 225mL APW (3% NaCl), homogenized using a tapping homogenizer for 2 min, followed by centrifugation at 8,000g for 2 min, discarding as much supernatant as possible; the precipitate was resuspended in 100μL phosphate buffer, 20μL of deactivated nuclease was added, and the mixture was incubated at 37℃ for 15–30 min, followed by heating at 95℃ for 10 min to inactivate the enzyme. After DNA extraction, *Vibrio parahaemolyticus* was detected using ORCMB and qPCR methods for comparative evaluation.

[0102] 1.7 Data Processing and Analysis

[0103] All experiments were performed in triplicate. Data visualization was performed using Origin 2024 software, and statistical analysis was conducted using SPSS software.

[0104] Example 2: Overview of Microfluidic Chips and Portable Centrifugal Microfluidic Nucleic Acid Detection Device

[0105] Figure 3 This paper demonstrates the design and workflow of a microfluidic chip and a portable centrifugal microfluidic nucleic acid detection device. The microfluidic chip employs a radial structure containing eight independent detection modules, enabling independent RPA-CRISPR / Cas12a reactions for sensitive quantification of Vibrio parahaemolyticus. Each independent detection module's sample chamber is connected to its corresponding RPA-CRISPR / Cas12a reagent embedding chamber via a 600 μm wide microchannel. The reagent embedding chamber contains pre-lyophilized and embedded RPAA buffer, LbCas12a, RPA primers, crRNA, and ssDNA-FQ probes. This reagent chamber connects three reaction chambers to a waste chamber via a 100 μm capillary channel and a narrow microchannel. To ensure a final reaction volume of 6.25 μL within each reaction chamber, each module is designed with a volume control chamber to collect excess reagent, preventing residual liquid in the capillary or microchannel and effectively avoiding cross-contamination between the three reaction chambers. Figure 3 A).

[0106] Next, according to the experimental objectives, RPA-CRISPR / Cas12a reagent and standard plasmids pGEM-T-Easy-toxR at different concentrations were pre-embedded into the reagent chamber and reaction chamber using lyophilization technology. Specifically, when preparing the standard curve, the reaction chamber was pre-embedded with standard plasmid pGEM-T-Easy-toxR (100-10... 4(copies / μL). The six reaction chambers in the two independent detection modules each contain five gradient concentrations of standard plasmids and one template-free control (NTC). By adding samples in this manner, three replicates can be set for each concentration of standard plasmid through the six independent modules, thus constructing a standard curve. The remaining two independent modules do not contain pre-added standard plasmids and are used to detect real samples. Figure 5 A). When the chip is used to detect samples, the reaction chamber no longer contains standard plasmids, and each chip can detect up to 8 samples simultaneously. Figure 5 B). After preloading is completed, the inlets of each cavity are sealed by the injection membrane. Finally, a leakage test is used to verify the sealing performance of the chip. Only chips that pass the sealing test can be used in subsequent testing experiments.

[0107] During testing, sample reagents (including 1.25 μL of RPA B buffer containing MgOAc, 2.5 μL of 10×HOLMES buffer, 19.25 μL of sterile deionized water, and 2 μL of the sample to be tested / sterile deionized water (negative control)) were added to the sample chamber of the finished chip. The chip was then placed in a portable microfluidic centrifuge device, and a preset program was started (alternating high and low speed centrifugation program (1,000 rpm 10 s → 500 rpm 10 s), with the reaction temperature set to 39°C and the reaction duration 60 min). It is important to understand that centrifugation speed and time are crucial for ensuring the complete dissolution of the lyophilized reagents. The microfluidic device was designed and manufactured in our laboratory, as detailed below. Figure 3 As shown in C.

[0108] After the detection solution (i.e. the sample reagent mentioned above) is introduced into the injection layer, the chip is placed in the matching portable centrifugal microfluidic nucleic acid detection device. Through the centrifugal force generated by different rotation speeds and the capillary action of the microfluidic structure, the detection solution enters the reagent chamber and mixes with the lyophilized reagent. Then it flows into the reaction chamber to start the RPA-CRISPR / Cas12a detection reaction.

[0109] To verify whether the liquid flow in the chip meets expectations, this embodiment uses ferric chloride solution (yellow) to simulate the reagent and copper sulfate solution (blue) to simulate the detection solution. After high-speed centrifugation at 1,300 rpm for 15 seconds, the detection solution enters the reagent chamber and dissolves the lyophilized reagent, turning the solution color green; subsequently, after low-speed centrifugation at 300 rpm for 15 seconds, the mixture enters the reaction chamber through a capillary tube and narrow channel, initiating the reaction. Figure 3 D~ Figure 3 E). The experimental results fully demonstrate that the liquid in the chip flows into the reagent chamber and the reaction chamber successively under different centrifugation conditions, which is in line with expectations and lays the foundation for detecting the reaction.

[0110] Based on this, after adding the detection solution, a high-low speed alternating centrifugation program was set (1,000 rpm 10 s → 500 rpm 10 s), and the reaction temperature was set to 39℃. The reaction lasted for 60 min until the reaction was complete. The integrated fluorescence detection module uses a light-emitting diode (LED, λ = 470 nm) as the excitation source. The excitation light illuminates the reaction chamber, causing the cleaved ssDNA-FQ probe to emit fluorescence (λ = 525 nm). The emitted signal is received by a photodetector and converted into an electrical signal, which is transmitted to the control board. Subsequently, the signal is processed by the onboard algorithm to generate real-time fluorescence curves for each reaction chamber.

[0111] Example 3: Optimization of the RPA-CRISPR / Cas12a reaction system

[0112] In the RPA-CRISPR / Cas12a detection system, its sensitivity and quantitative accuracy depend on the balance between RPA amplification efficiency and Cas12a protein cleavage activity. To establish a dynamic balance in this system, this example uses 6.08 × 10⁻⁶... 6 Using the standard plasmid copies / μLpGEM-T-Easy-toxR as the detection target, multiple sets of RPA primers (SEQ ID NO:2-3, SEQ ID NO:4-5, SEQ ID NO:6-7) and crRNA (SEQ ID NO:8-10) were systematically designed and evaluated to optimize amplification and cleavage. The specific steps are the same as described in Example 1.

[0113] Figure 6 A demonstrates the design principles of the primers and crRNA. Three primer pairs targeting the toxR gene were tested under standard RPA conditions, with the specific experimental methods described in Example 1. Agarose gel electrophoresis results showed that ( Figure 6 (B) Primer pair toxR-RPA-F1 / R1 (lane 1) amplified a single, well-defined, and brightest band (179 bp), while the negative control (lane 2) showed no nonspecific amplification, demonstrating high specificity and amplification efficiency. In contrast, the other two primer pairs (186 bp and 224 bp) produced weaker bands (lanes 3-6) and were therefore not used for subsequent integration. In summary, when detecting Vibrio parahaemolyticus, the amplification primers toxR-RPA-F1 / R1 (SEQ ID NO: 2-3) are recommended.

[0114] In the dynamic reaction, the RPA amplification reaction continuously generates target templates to promote the activation of the Cas12a protein. However, the Cas12a protein, with its cis-cleavage and trans-cleavage properties, may degrade the template and primers, thereby inhibiting subsequent amplification. Therefore, it is necessary to regulate the cleavage kinetics of the Cas12a protein to avoid premature inhibition of amplification. Based on this, this example uses 6.08 × 10⁻⁶... 6 Using pGEM-T-Easy-toxR copies / μL as the detection target, three types of crRNAs were designed: targeting typical PAM sites (PAM, SEQ ID NO:8), suboptimal PAM (sPAM, SEQ ID NO:9), and PAM-free (PAM-free, SEQ ID NO:10), in order to regulate the cleavage activity of Cas12a protein and improve detection sensitivity. The specific steps are the same as described in Example 1.

[0115] like Figure 6 As shown in Figure C, the three types of crRNA produced different fluorescence kinetic characteristics. Among them, sPAM showed significantly better overall performance than PAM and PAM-free. Specifically, sPAM activated the Cas12a protein most quickly, with the fluorescence signal reaching its inflection point around 10 minutes, and the final fluorescence signal intensity was also much higher than the other two groups. In summary, the suboptimal PAM (SEQ ID NO:9) achieved the optimal balance between amplification and cleavage in the reaction system, laying the foundation for shortening the detection time and improving detection sensitivity. Therefore, it was selected for subsequent experimental systems.

[0116] Furthermore, the fluorescence signal was generated due to the non-specific cleavage of the FAM-BHQ1-labeled ssDNA-FQ probe (SEQ ID NO: 11) by the Cas12a protein after target activation. To determine the optimal concentration, this example used 6.08 × 10⁻⁶. 6 Using copies / μLpGEM-T-Easy-toxR as the detection target, the detection effect of probes with different concentrations (250–2,000 nM) was tested. The specific steps were the same as described in Example 1. Figure 6 As shown in D, when the probe concentration is between 250 and 500 nM, the fluorescence intensity reaches the inflection point more quickly as the probe concentration increases, and the final fluorescence value is also greater. However, when the probe concentration is higher than 500 nM, the fluorescence value takes longer to reach the plateau phase, and the fluorescence value also decreases. Therefore, the probe concentration is preferably between 250 and 500 nM, and more preferably, 500 nM is selected as the optimal concentration for use, which takes into account signal strength, inflection point time, and cost.

[0117] Furthermore, since Cas12a and crRNA typically form the complex RNP in a 1:1 molar ratio, this invention also uses 6.08 × 10 6 Using pGEM-T-Easy-toxR copies / μL as the detection target, the effects of different concentrations (50-400 nM) of Cas12a protein and crRNA on the detection results were evaluated when the molar ratio of Cas12a protein to crRNA was 1:1. The specific steps were the same as those described in Example 1.

[0118] like Figure 6 As shown in Figure E, the detection systems performed best at Cas12a protein and crRNA concentrations of 100 nM and 200 nM, respectively, with the inflection point appearing around 5 minutes and strong fluorescence intensity. The 100 nM concentration showed the best overall performance. Concentrations of Cas12a protein and crRNA that are too high or too low are detrimental to detection. For example, at a concentration of 50 nM, the signal is sluggish; at a concentration of 400 nM, the signal weakens due to excessively rapid product cleavage. Therefore, the optimal concentration of Cas12a and crRNA is 100 nM.

[0119] In RPA amplification reactions, excessively low primer concentrations may reduce amplification efficiency, while excessively high concentrations may cause a competitive effect, interfering with target amplification and affecting quantitative accuracy. Therefore, by adjusting the concentrations of RPA primers, Cas12a, and crRNA, the reaction can be made more quantitative. This invention establishes a linear regression curve between DNA concentration and POI, and calculates different coefficients of determination R. 2 R 2 The closer the value is to 1.0, the closer the system is to a quantifiable state.

[0120] The results show ( Figure 7 With both Cas12a and crRNA concentrations at 100 nM, the RPA primer concentrations were set to 200 nM, 300 nM, and 500 nM, respectively. When the primer concentration was 200 nM, R… 2 =0.9520; the linear correlation was best at a primer concentration of 300 nM, R 2 =0.9734; R when primer concentration is increased to 500 nM 2 This then decreases to 0.9494. In summary, an RPA primer concentration of 300 nM is optimal, and a concentration of 6.08 × 10⁻⁶ is achieved. 0 -6.08×10 4 Reliable quantification is achieved within the range of copies / μL.

[0121] Example 4: Specificity and sensitivity of ORCMB detection method

[0122] To improve detection efficiency, Example 3 optimized the parameters in the RPA-CRISPR / Cas12a detection system. Based on the optimized scheme, this example systematically evaluated the specificity and sensitivity of the ORCMB method for detecting Vibrio parahaemolyticus, with the specific steps being the same as described in Example 1.

[0123] In the specificity test, the ORCMB method was used to detect Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio harveyi, Vibrio alginolyticus, Vibrio cholerae, Escherichia coli O157:H7, and Staphylococcus aureus, with a negative control (NTC) using sterile deionized water instead of DNA. The results showed that only Vibrio parahaemolyticus produced a significant fluorescent signal; the others showed no amplification, indicating that the system has excellent detection specificity. Figure 8 A).

[0124] In sensitivity testing, the pGEM-T-Easy-toxR standard plasmid was serially diluted to 6.08 × 10⁻⁶. 6 -6.08 copies / μL, with an NTC (deionized water) group as a negative control. The results showed that ( Figure 8 B) As the plasmid concentration decreased, the detected fluorescence signal weakened and the inflection point appeared at a longer time; NTC did not generate a signal. Furthermore, a detection signal was obtained at a plasmid concentration of 6.08 copies / μL, therefore the limit of detection (LOD) of this method is 6.08 copies / μL.

[0125] Compared with other known methods for detecting Vibrio parahaemolyticus, the ORCMB detection method provided by this invention exhibits comparable or even higher sensitivity, and can complete quantitative detection on-site within 30 minutes and obtain full-process results within 1.5 hours, showing good potential for on-site application (Table 4).

[0126] Table 4 Comparison of different detection methods

[0127]

[0128]

[0129] Furthermore, the ORCMB detection method provided by this invention exhibits quantitative performance distinct from previously reported RPA-CRISPR / Cas12a methods (Lu et al., 2022; Shen et al., 2023; Hu et al., 2025), which primarily provide rapid and sensitive qualitative detection. Recent research indicates that regulating the dynamic balance between RPA and CRISPR / Cas12a reactions is crucial for achieving nucleic acid quantification in a single-tube system (Yao et al., 2024). This detection method employs suboptimal PAM crRNA to inhibit Cas12a cleavage activity, thereby minimizing its inhibition of RPA amplification and achieving accurate quantification of target DNA. By further optimizing primer or Cas12a concentrations, a dynamic equilibrium reaction (R...) between the two systems was established. 2 >0.9), ensuring sensitive and accurate detection over a wide concentration range. Unlike traditional single-tube assays, which are prone to variability due to open operation and manual handling, ORCMB assays utilize a closed-loop centrifugal microfluidic platform with lyophilized reagents, effectively eliminating aerosol contamination and ensuring stable reaction initiation. This integrated design achieves a well-defined "quantifiable state" (R0.9). 2 >0.97), in a wide linear range (10 0 -10 4 Maintaining stable quantitative performance within copies / μL, it propels the RPA-CRISPR platform toward becoming a reliable, pollution-free, and practical on-site quantitative diagnostic tool.

[0130] Example 5: Practical Application of ORCMB Detection Method

[0131] To investigate the effectiveness of the ORCMB method in detecting real samples, this embodiment tested manually spiked (10) samples. 6 -10 1 Shrimp (CFU / mL), mackerel, shellfish (oysters), and farmed shrimp samples collected in the field were used, with the specific steps as described in Example 1.

[0132] The results showed that the ORCMB method can accurately identify different concentrations of Vibrio parahaemolyticus in shrimp samples. Figure 9 A); In contrast, although the detection results of qPCR also showed a linear response, at low concentrations (10... 1 -10 2 Nonspecific amplification occurred at CFU / mL, i.e., detection of 10 1 The fluorescence response ratio for detecting Vibrio parahaemolyticus at CFU / mL was 10. 2 For CFU / mL, it needs to be faster ( Figure 9 B), this result shows that when the target species is shrimp, the detection limit of the ORCMB method is better than that of qPCR.

[0133] Besides shrimp, the ORCMB method also showed consistent results with qPCR in detecting spiked mackerel and oysters, and could accurately quantify them. Figures 10-12 This indicates that the ORCMB detection method has a wide range of applications and is suitable for detecting Vibrio parahaemolyticus in a variety of aquatic products.

[0134] Results of ORCMB and qPCR tests on 50 shrimp samples collected in the field (20 from diseased ponds (theoretically positive), 30 from healthy ponds (theoretically negative)) are shown below. Figure 9 C~ Figure 9 D. Table 5. Table 5 shows that the ORCMB method detected 21 positive samples and 29 negative samples; the qPCR method detected 20 positive samples and 30 negative samples. The positive result concordance rate of the two detection methods was 95.24%, and the negative result concordance rate was 100%; the specificity was 96.67%, and the sensitivity reached 100%. ROC analysis results showed that AUC = 0.984 (p < 0.0001), and Youden's index was 0.968 (p < 0.0001). Figure 9 C) shows that the ORCMB method has extremely high statistical reliability.

[0135] Table 5 Comparison of results from ORCMB and qPCR methods in detecting field-collected shrimp samples.

[0136]

[0137] Figure 9 D presents the specific results of the two methods described above on 50 field-collected shrimp samples. The results indicate that the toxR gene copy number in diseased pond shrimp samples often exceeds 10. 2 Copies / μL; 29 samples from healthy ponds were negative using both methods, with only one sample testing positive using the ORCMB method but negative using qPCR. This is because this sample was a potential carrier of Vibrio parahaemolyticus, detectable only by methods with lower detection limits, meaning the ORCMB method is more sensitive for low abundance detection. In summary, the ORCMB detection platform demonstrated high specificity and accuracy in both spiked and actual samples, showing significant value not only in pathogen monitoring in shrimp farming but also potentially for water quality monitoring, food safety, and clinical pathogen diagnosis. Its closed-loop microfluidic design, rapid detection process, and high sensitivity make it a promising candidate for on-site early warning, early disease diagnosis, and public health monitoring in public environmental waters (coastal waters, recreational waters, seafood markets).

[0138] In summary, to achieve sensitive and quantitative detection of Vibrio parahaemolyticus and meet the needs of field applications, this invention has overcome a series of key technical challenges from molecular design to reagent engineering and system integration.

[0139] 1. Systematic screening and optimization establish a quantitative basis:

[0140] This invention first involved multiple rounds of primer design and large-scale parallel screening to select the optimal primer sequences that exhibited both excellent amplification efficiency and specificity. Based on this, a series of meticulous concentration optimization experiments were conducted to precisely balance the concentrations of RPA primers, crRNA and Cas12a protein, and reporter nucleic acid probes. This systematic engineering optimization is a prerequisite for ensuring that the entire RPA-CRISPR reaction system reaches dynamic equilibrium, thereby successfully transforming the nonlinear amplification process into a stable and reliable quantitative signal output.

[0141] 2. Overcoming the dual design challenges of high specificity and dynamic equilibrium:

[0142] Accurate detection of Vibrio parahaemolyticus (Vp) faces two major challenges: First, there is the challenge of interspecies specificity. Vp has a large number of closely related species (such as Vibrio cholerae), whose genomes contain highly homologous sequences. This requires primers and crRNA to accurately identify the "unique" target fragment within an extremely similar genetic background. Conventional designs are prone to off-target amplification and non-specific recognition, leading to false positives. Second, there is the kinetic challenge of quantitative applications. In the RPA-CRISPR combined system, if the "optimal" PAM site with the strongest cleavage activity is used, CRISPR / Cas12a will prematurely cleave the RPA amplification product, effectively "cutting off the fuel supply," causing the amplification system to be interrupted before reaching stable exponential growth. The reaction kinetics exhibit highly nonlinearity, making it impossible to establish a standard curve for quantitative analysis.

[0143] To this end, this invention precisely located specific sites in conserved regions through whole-genome alignment and innovatively selected a "suboptimal" PAM site. This strategy is like setting a precise speed controller for the Cas12a "molecular scissors," matching its cutting rate with the RPA amplification rate, allowing the two systems to operate collaboratively rather than compete with each other, ultimately successfully transforming nonlinear amplification into an excellent quantitative linear relationship.

[0144] 3. Experimental verification and scientific mechanism explanation:

[0145] To objectively evaluate the superiority of the design strategy of this invention, primer and crRNA combinations targeting the same Vibrio parahaemolyticus (Vp) gene from the literature were selected as controls. The crRNA was designed strictly according to the "optimal PAM" (5'-TTTN-3') principle. However, experimental results showed that the Cas12a protein exhibited extremely high trans-cleavage activity under the guidance of the "optimal PAM," resulting in a cleavage rate much higher than that of RPA amplification. This kinetic imbalance caused newly generated amplicones from RPA to be degraded prematurely and too quickly, failing to accumulate and form stable exponential amplification, ultimately leading to poor quantitative linearity and a detection sensitivity one order of magnitude lower than our system.

[0146] In contrast, the design of this invention actively avoids the "optimal PAM". Our crRNA has a PAM site of [5'-CTTG-3'].

[0147] The chosen "suboptimal" PAM exhibits relatively mild Cas12a cleavage activity. This crucial modification creates an ideal kinetic balance at the scientific level: the RPA amplification system continuously generates sufficient amplicones, while the Cas12a system cleaves and reports fluorescence signals at a matched rate that does not "overdraw" the substrate. This allows the entire reaction to establish and maintain a stable and predictable correspondence between the initial template concentration and the final fluorescence signal, thus achieving excellent quantitative linearity and higher detection sensitivity.

[0148] 4. Integrated microfluidic chip improves performance:

[0149] This invention successfully integrates the optimized reaction system into a self-developed centrifugal microfluidic chip. Pre-dispensed reagents within the chip achieve automated liquid control through precision flow channels, completely avoiding aerosol contamination. The enclosed environment also significantly improves the reproducibility and stability of the reaction. Thanks to the precise control of the reaction volume and efficient mixing within the chip, the system achieves a lower limit of detection.

[0150] 5. Pioneering integrated freeze-drying process: Overcoming the technical barriers to synergistic stability

[0151] Conventional RPA-CRISPR lyophilization protocols typically employ spatial separation or stepwise lyophilization strategies to maintain the activity of each enzyme component. For example, RPA reaction components and the CRISPR / Cas12a reporter system are lyophilized separately in different chambers of a chip or on different reagent sheets. During use, they must be mixed via liquid flow or manual manipulation. This method not only increases the complexity of the chip structure and the number of operational steps but also introduces a high risk of aerosol contamination during mixing. Furthermore, the mixing efficiency may affect the uniformity and synchronicity of the reaction, thus posing a challenge to quantitative reproducibility.

[0152] To solve this fundamental problem, this invention has developed and optimized an innovative integrated, one-time encapsulation freeze-drying process, the core steps of which are as follows:

[0153] Optimized Protectant Formulation: This invention abandons the conventional single protectant system and develops a proprietary composite freeze-drying protectant. This system includes trehalose, dextran, and other structural protectants, forming a stable amorphous glassy matrix during freeze-drying to "immobilize" various enzymes and nucleic acid molecules. Simultaneously, it is compounded with specific polyols and metal ions to maintain the correct conformation of the enzyme's active site at the microscopic level, particularly addressing the problem of Cas12a protein's easy inactivation during dehydration.

[0154] Premixing and dispensing: The optimized concentrations of RPA enzyme, primers, Cas12a protein, crRNA and fluorescent reporter probe are precisely premixed in the same buffer system to ensure uniform distribution of each component.

[0155] Rapid vacuum freeze-drying: After the mixture is precisely dispensed into the chip reaction chamber, our optimized "three-step" freeze-drying procedure is used (rapidly cooling to -45℃ and maintaining for 2 hours, followed by step sublimation and desorption drying at two platforms of -20℃ and 0℃). This ensures that the ice crystals are small and do not damage the protein structure, while efficiently removing moisture and finally forming a stable porous solid reagent cake.

[0156] Scientific Mechanism and Advantages:

[0157] The key to the success of this invention lies in the fact that the composite protectant simultaneously creates a stable microenvironment at the molecular level for both RPA enzymes (mainly BST polymerase) and Cas12a protein during the freeze-drying process, effectively preventing protein denaturation, aggregation, and nucleic acid degradation caused by dehydration. More importantly, the integrated freeze-drying process ensures that the RPA and CRISPR systems reach their optimal working concentration and ratio immediately upon reconstitution, avoiding the reaction initiation delays and efficiency fluctuations caused by differences in dissolution efficiency and uneven mixing in stepwise freeze-drying. This allows the freeze-dried reagent to maintain a high sensitivity and quantitative linearity comparable to the liquid reaction after reconstitution (R0). 2 >0.99), and its stability was confirmed to exceed 30 days in high-temperature accelerated testing, providing core technical support for achieving stable, reliable and easy-to-use on-site quantitative detection.

[0158] 6. Primer anti-interference ability ensures detection robustness:

[0159] Finally, we verified the reliability of the detection system when faced with complex real-world samples. The designed primers and crRNA maintained excellent detection performance and specificity even under simulated aquatic product matrix, high-salt background, and interference from common coexisting microorganisms, demonstrating its strong anti-interference capability and ability to meet the practical needs of food safety monitoring.

[0160] Summary of integrated advantages: Through systematic primer screening and reaction system optimization, combined with a "suboptimal" PAM balancing strategy, a pioneering integrated freeze-drying process, centrifugal microfluidic chip integration, and highly anti-interference molecular design, we have successfully constructed a stable, sensitive, quantitative, and field-suitable integrated Vp detection platform, solving the long-standing pain points in this field such as difficulty in quantification, cumbersome operation, and susceptibility to contamination.

[0161] References

[0162] Ajay,G.,Vishnuraj,MR,Kumar,NA,Chauhan,A.,Rawool,DB,Barbuddhe,SB,2025.Anovel duplex qPCR-HRMA technique for simultaneous detection of Listeria monocytogenes and Salmonella typhimuriumin meat products.FoodChem.474,143245.https: / / doi.org / 10.1016 / j.foodchem.2025.143245

[0163] Chen,Q.,Wang,H.,Xu,HY,Peng,YB,Yao,BB,Chen,ZR,Yang,JN,Adeloju,S.,Chen,W.,2025.One-pot RPA-CRISPR / Cas12a integrated dual-modeelectrochemical lateral flow strip for ultrasensitive and precise detectionof Salmonella.Biosens.Bioelectron.285,117529.https: / / doi.org / 10.1016 / j.bios.2025.117529

[0164] Ge,R.,Dai,H,J.,Zhang,S.M.,Wei,J.,Jiao,T.H.,Chen,Q.M.,Chen,Q.S.,Chen,X.M.,2023.Acollection of RPA-Based photoelectrochemical assays for theportabledetection of multiple pathogens.Anal.Chem.95,7379-7386.https: / / doi.org / 10.1021 / acs.analchem.3c01006

[0165] Lang,Z.Y.,Lin,S.J.,Li,J.M.,Xie,Z.R.,Cao,H.M.,2025.A microfluidicbased stem-loop probes mediated isothermal amplification for multiplexdetection of Vibrio parahaemolyticus.Food chemistry.492,145571.Advance onlinepublication.https: / / doi.org / 10.1016 / j.foodchem.2025.145571

[0166] Lang,Z.Y.,Ye,X.,Liu,J.,Lin,S.J.,Li,Y.F.,Chen,Q.,Cao,H.M.,2024.Rapidand simultaneous detection of virulence factors of Vibrio parahaemolyticus bymultiplex microfluidic-LAMP(MMLAMP).Microchem.J.201,110738.https: / / doi.org / 10.1016 / j.microc.2024.110738

[0167] Li,J.M.,Lin,S.J.,Lang,Z.Y.,Xing,G.W.,Jia,Y.W.,Cao,H.M.,2025a.Ahairpin reporters-based one-pot LAMP-CRISPR / Cas12a assay for the highlysensitive and visual detection of Vibrio parahaemolyticus.Food Control.172,111184.https: / / doi.org / 10.1016 / j.foodcont.2025.111184

[0168] Li,L.Y.,Fu,J.,Chiang,E.L.C.,Yoo,J.,Bae,S.,2025b.A 3D-printedsmartphone-based platform for in-situ and rapid monitoring of aquaculturepathogens using polydimethylsiloxane(PDMS)microchip with multiplex loop-mediated isothermal amplification(M-LAMP).Biosens.Bioelectron.:X,25,100635.https: / / doi.org / 10.1016 / j.biosx.2025.100635

[0169] Li,Y.,Lin,S.,Xue,Y.X.,Jia,Q.Y.,Wang,Y.,Xie,Y.Q.,Shi,C.,Ma,C.P.,2023.Boron nitride nanoplate-based improvement of the specificity andsensitivity in loop-mediated isothermal amplification for Vibrioparahaemolyticus detection.Anal.Chim.Acta.1280,341851.https: / / doi.org / 10.1016 / j.aca.2023.341851

[0170] Lin,Q.J.,Jia,K.Y.,Gou,H.C.,He,H.S.,Wen,J.P.,Shen,H.Y.,Chwn,K.F.,Wu,Y.Q.,Lu,B.J.,Liao,M.,Han,Y.X.,Zhang,J.M.,2022.A smartphone-assisted high-throughput integrated color-sensing platform for the rapid detection ofCampylobacter coli.LWT--Food Sci.Technol.167,113790.https: / / doi.org / 10.1016 / j.lwt.2022.113790

[0171] Liu,Y.J.,Liu,H.,Yu,G.L.,Sun,W.B.,Aizaz,M.,Yang,G.W.,Chen,L.,2023.One-tube RPA-CRISPR Cas12a / Cas13a rapid detection of methicillin-resistantStaphylococcus aureus.Anal.Chim.Acta.1278,341757.https: / / doi.org / 10.1016 / j.aca.2023.341757

[0172] Su,Y.,Li,X.T.,Zhu,L.J.,Chu,H.S.,Zhang,Y.Z.,Tian,J.J.,Lyu,S.X.,Xu,W.T.,2022.MSN / NA-doped nanoflower enhancing isothermal fluorescent sensorwith a portable PCR tube fluorescence reader for the on-site detection ofVibrio parahaemolyticus.Anal.Chim.Acta.1200,339448.https: / / doi.org / 10.1016 / j.aca.2022.339448

[0173] Xu,J.H.,Ma,J.F.,Li,Y.W.,Kang,L.,Yuan,B.,Li,S.Q.,Chao,J.,Wang,J.L.,Su,S.,Yuan,Y.,2022.A general RPA-CRISPR / Cas12a sensing platform for Brucellaspp.detection in blood and milk samples.Sens.Actuators,B.364,131864.https: / / doi.org / 10.1016 / j.snb.2022.131864

[0174] Yuan,J.R.,Wang,L.,Huang,L.,He,K.,Wang,H.M.,Xu,X.H.,Su,B.,Wang,J.,2024.CRISPR-Cas12a-Mediated hue-recognition lateral flow assay for point-of-need detection of Salmonella.Anal.Chem.96,220-228.https: / / doi.org / 10.1021 / acs.analchem.3c03753

[0175] Zhang,Y.L.,Chen,X.H.,Ouyang,G.F.,Wang,J.P.,Sun,Y.C.,Lai,Y.L.,Zhang,P.,Guo,F.,Yang,S.J.,Mao,R.,2024.Development of closed dumbbell mediatedisothermal amplification assay for rapid and on-site detection of Vibrioparahaemolyticus.Microchem.J.15,1435010. https: / / doi.org / 10.1016 / j.microc.2024.111892

[0176] Lu,SH,Tong,XH,Han,Y.,Zhang,K.,Zhang,YZ,Chen,QB,Duan,JY,Lei,XL,Huang,MH,Qiu,Y.,Zhang,DY,Zhou,X.,Zhang,Y.,Yin,H.,2022.Fast andsensitive detection of SARS-2 proRNAV proto-space using optimal adjacentmotifs for Cas12a.Nat.Biomed.Eng.6,286-297. https: / / doi.org / 10.1038 / s41551- 022-00861-x

[0177] Lu,SH,Tong,XH,Han,Y.,Zhang,K.,Zhang,YZ,Chen,QB,Duan,JY,Lei,XL,Huang,MH,Qiu,Y.,Zhang,DY,Zhou,X.,Zhang,Y.,Yin,H.,2022.Fast andsensitive detection of SARS-2 proRNAV proto-space using optimal adjacentmotifs for Cas12a.Nat.Biomed.Eng.6,286-297.https: / / doi.org / 10.1038 / s41551-022-00861-x

[0178] Shen,JN,Chen,Z.,Xie,RB,Li,JF,Liu,CY,He,YQ,Ma,XP,Yang,H.,Xie,ZJ,2023.CRISPR / Cas12a-Assisted isothermal amplification for rapid andspecific diagnosis of respiratory virus on a microfluidic platform.Biosens.Bioelectron.237,115523.https: / / doi.org / 10.1016 / j.bios.2023.115523

[0179] Hu, F., Zhang, YY, Yang, Y., Peng, LX, Cui, SH, Ma, Q., Wang, FN, Wang, XC, 2025. Arapid and ultrasensitive RPA-assisted CRISPR-Cas12a / Cas13a nucleicacid diagnostic platform with asmartphone-based portabledevice.Biosens.Bioelectron.280,117428.https: / / doi.org / 10.1016 / j.bios.2025.117428

[0180] Yao,ZH,He,KY,Wang,HM,Feng,SY,Ding,XQ,Xu,Y.,Wang,Q.,Xu,XH,Wu,Q.,Wang,L., 2024. Tuning the dynamic reaction balance of CRISPR / Cas12a andRPA in one pot: a key to switch nucleic acid quantification.ACS Sens.9,3511-3519.https: / / doi.org / 10.1021 / acssensors.3c02485

[0181] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A centrifugal microfluidic chip for detecting Vibrio parahaemolyticus, characterized in that, The microfluidic chip contains embedded RPA-CRISPR / Cas12a reaction buffer, RPA amplification primers, crRNA, Cas12a protein, and nucleic acid fluorescent probes.

2. The chip as described in claim 1, characterized in that, The RPA amplification primers are divided into upstream primers and downstream primers; the upstream primer contains any one of the sequences shown in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; the downstream primer contains any one of the sequences shown in SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:

7.

3. The chip as described in claim 1, characterized in that, The crRNA contains any one of the sequences shown in SEQ ID NO:8 to SEQ ID NO:

10.

4. The chip as described in claim 1, characterized in that, The RPA amplification primers contain the sequences described in SEQ ID NO:2 and / or SEQ ID NO:3; the crRNA contains the sequence shown in SEQ ID NO:

9.

5. The chip as described in claim 1, characterized in that, The concentration of the RPA amplification primers is 100–1000 nM; the concentration of the crRNA and Cas12a protein is 50–1000 nM; and the concentration of the nucleic acid probe is 100–2000 nM.

6. The chip as described in claim 1, characterized in that, The detection module includes any one or more of the following: a sample chamber, an RPA-CRISPR / Cas12a reagent embedding chamber, a sample waste liquid chamber, a reaction chamber, a volume control chamber, and a vent.

7. A method for preparing a centrifugal microfluidic chip for detecting Vibrio parahaemolyticus, characterized in that, The centrifugal microfluidic chip is as described in any one of claims 1 to 6.

8. An application of a centrifugal microfluidic chip for the quantitative detection of Vibrio parahaemolyticus, characterized in that, The centrifugal microfluidic chip is as described in any one of claims 1 to 6.

9. A method for quantitative detection of Vibrio parahaemolyticus using an RPA-CRISPR / Cas12a combined microfluidic biosensor, characterized in that, The chip described in any one of claims 1 to 6 is used to quantitatively detect Vibrio parahaemolyticus.

10. The method as described in claim 9, characterized in that, The steps of the method are as follows: A. Add the sample to the reaction chamber, place the microfluidic chip in the portable centrifugal microfluidic nucleic acid detection device, and set the centrifugation and reaction programs; B. After the reaction is complete, the portable centrifugal microfluidic nucleic acid detection device generates real-time fluorescence data; C. Convert real-time fluorescence data into points of interest (POIs) using second derivatives and Python; D. A standard curve is established by performing linear regression fitting calculations between the POI of the standard and the DNA concentration of the standard; E. Substitute the POI of the sample into the standard curve to calculate the Vibrio parahaemolyticus content in the sample.

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