Multi-target detection system based on paraffin phase change and application thereof

Abstract

The invention belongs to the technical field of nucleic acid detection, and particularly discloses a multi-target detection system based on paraffin phase change and application of the multi-target detection system. The detection system comprises a micro-fluidic chip and a fluorescence detection module, the micro-fluidic chip integrates an RPA amplification reaction and a CRISPR reaction, the micro-fluidic chip comprises a CRISPR layer, a paraffin layer and an RPA layer which are arranged from top to bottom, the RPA amplification reaction and the CRISPR reaction are isolated through the paraffin layer, the RPA amplification reaction is operated at a certain temperature, paraffin is molten into a liquid state, the RPA layer and the CRISPR layer which have been reacted are mixed, and the fluorescence detection module is used for detecting the RPA layer and the CRISPR layer which have been reacted. The CRISPR reaction is carried out, and the reacted CRISPR layer is detected through the fluorescence detection module, so that multi-target detection is realized. The detection system can detect various sexually transmitted disease pathogens within 30 minutes, has the advantages of high detection speed, high sensitivity, low cost and portability, and is suitable for family detection and application in areas with limited medical resources.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a multi-target detection system based on paraffin phase change and application thereof. Background Art

[0002] CRISPR diagnostic (CRISPR-Dx) systems provide a rapid and efficient method for nucleic acid detection. These systems work by utilizing CRISPR RNA (crRNA) to pair with complementary DNA to activate the nuclease activity of CRISPR proteins, thereby cross-cutting non-target single-stranded RNA or DNA sequences, an activity that is only activated after cis-cutting. The DNA Nuclease Targeted CRISPR Cross-Cut Reporting System (DETECTR) combining CRISPR-Cas12a and recombinase polymerase amplification (RPA) has been successfully used to detect viruses including SARS-CoV-2 and HPV. Existing technologies combine RPA and CRISPR through microfluidics technology to achieve multiplex detection of HPV DNA. These detection methods require strict step-by-step operations in the laboratory to ensure that the RPA amplification reaction and the CRISPR cleavage reaction are separated in time and space to ensure smooth RPA amplification and avoid affecting the early amplification stage due to the presence of activated Cas12a, thereby avoiding the loss of sensitivity caused by mixing. However, these methods face several challenges in resource-limited areas, including: (i) the need for well-equipped testing facilities and specialized equipment; (ii) susceptibility to aerosol contamination, which may lead to erroneous results; and (iii) the procedures are complicated and require specialized technicians to operate.

[0003] To solve the problem of aerosol contamination caused by the strict operation of professionals and the two-step method, some researchers have tried to spatially separate the RPA amplification system and the CRISPR detection system. One notable design is the use of microfluidic chips. However, these methods need to be adapted to microfluidic chips, which requires complex instruments and is costly, making it difficult to promote in areas with scarce medical resources. Some research groups have introduced a special test tube that can accommodate both RPA and CRISPR reactions. This one-tube method achieves spatial isolation and avoids aerosol contamination and complex manual operations. However, temperature control and fluorescence quantification within the system still require the use of laboratory equipment. Some researchers have also tried to separate the RPA and CRISPR systems through a "one-pot method" based on time differences. Lu et al. achieved this by screening suboptimal PAM sequences (NTTV and TTNT) of Cas12a, which produce weaker and slower para-cleavage activity compared to classical PAM. These methods provide a larger operating window for the RPA amplification system and avoid excessive consumption caused by cis-cutting DNA templates and cross-cutting primers of the CRISPR system. Although these methods achieve "one-pot" detection, the process of screening the best PAM site (balancing reaction speed and intensity) requires a lot of preliminary work, and short nucleotides require additional chemical modifications to achieve the blocking effect on crRNA. In addition, suboptimal reaction effects limit the optimal improvement in sensitivity. To overcome the limitations of large laboratory equipment, many researchers have explored the use of lateral flow test strips (LFD) or visual observation of fluorescence intensity to interpret the results, using cost-effective and readily available heating sources to eliminate the need for dedicated equipment. However, relying on LFD for result interpretation lacks high sensitivity and can only perform qualitative or semi-quantitative detection, while visual observation may lead to misjudgment of results. In addition, the stability of the heating source in these methods may be affected by fluctuations in ambient temperature. Summary of the invention

[0004] The purpose of the present invention is to provide a multi-target detection system based on paraffin phase change and its application in view of the above-mentioned deficiencies in the prior art. The system separates and mixes recombinase polymerase amplification (RPA) and CRISPR reaction through solid-liquid phase change of paraffin. Combined with portable detection equipment, it is possible to detect a variety of sexually transmitted disease pathogens within 30 minutes, including HPV16, HPV18, Chlamydia (CT), Mycoplasma (MG), Ureaplasma (UU) and Neisseria gonorrhoeae (NG). The system has the advantages of fast detection speed, high sensitivity, low cost and portability, and is suitable for home detection and application in areas with limited medical resources.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first object of the present invention is to provide a multi-target detection system based on paraffin phase change, including a microfluidic chip and a fluorescence detection module that integrate RPA amplification reaction and CRISPR reaction, the microfluidic chip including a CRISPR layer, a paraffin layer and an RPA layer arranged from top to bottom, the RPA amplification reaction and the CRISPR reaction are isolated by the paraffin layer, at a certain temperature, the RPA amplification reaction is run and the paraffin is melted into a liquid state to achieve mixing of the reacted RPA layer and the CRISPR layer, the CRISPR reaction is run, and the reacted CRISPR layer is detected by the fluorescence detection module to achieve multi-target detection.

[0007] Furthermore, the microfluidic chip consists of a CRISPR storage area, a paraffin isolation area and an RPA reaction area, the CRISPR storage area includes a cover layer and a CRISPR layer, the RPA reaction area includes an airway layer, a liquid channel layer, an RPA layer and a base layer, the CRISPR layer is arranged under the cover plate, the cover plate is provided with an injection hole, the paraffin isolation area is arranged under the CRISPR layer, and the RPA reaction area is arranged under the paraffin isolation area.

[0008] Furthermore, the CRISPR layer includes CRISPR reaction chambers that are arranged in sequence at a periphery away from the center.

[0009] Furthermore, the paraffin isolation area is provided with wax isolation components corresponding one to one with the CRISPR reaction chambers, and the wax isolation components are in contact with the reaction liquid of the CRISPR reaction chambers.

[0010] Furthermore, the RPA reaction area includes the air channel layer, liquid channel layer, RPA layer and base layer arranged in sequence from top to bottom, the air channel layer is arranged below the paraffin isolation area, the air channel layer includes first channels arranged in sequence at intervals on the periphery away from the center, and each of the first channels is connected to an exhaust hole; the liquid channel layer includes second channels arranged in sequence at intervals on the periphery away from the center, and each of the second channels is connected to a microfluidic channel, and one ends of multiple microfluidic channels converge at the center of the liquid channel layer; the RPA layer includes RPA reaction chambers arranged in sequence at intervals on the periphery away from the center; the first channels, the second channels and the RPA reaction chambers correspond one to one.

[0011] Furthermore, the microfluidic chip is disc-shaped.

[0012] The second object of the present invention is to provide a portable detection device, comprising the above-mentioned multi-target detection system.

[0013] Furthermore, the portable detection device also includes a heating module and a vibration module.

[0014] The third object of the present invention is to provide a multi-target pathogen detection method, wherein a PRA solution containing a nucleic acid sample is added to the above-mentioned microfluidic chip and processed for 30 to 35 minutes, and then the detection result is obtained through the fluorescence detection module.

[0015] Furthermore, the pathogens include HPV 16, HPV 18, CT, UU, MG and NG.

[0016] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0017] (1) The multi-target detection system based on paraffin phase change provided by the present invention comprises a microfluidic chip and a fluorescence detection module that integrate RPA amplification reaction and CRISPR reaction. The microfluidic chip comprises a CRISPR layer, a paraffin layer and an RPA layer arranged from top to bottom. The RPA amplification reaction and the CRISPR reaction are isolated by the paraffin layer. At a certain temperature, the RPA amplification reaction is run and the paraffin is melted into a liquid state to achieve mixing of the reacted RPA layer and the CRISPR layer. The CRISPR reaction is run and the reacted CRISPR layer is detected by the fluorescence detection module to achieve multi-target detection.

[0018] (2) The multi-target detection system provided by the present invention can detect multiple sexually transmitted disease pathogens within 30 minutes, including HPV16, HPV18, Chlamydia (CT), Mycoplasma (MG), Ureaplasma (UU) and Neisseria gonorrhoeae (NG).

[0019] (3) The multi-target detection system based on paraffin phase change provided by the present invention is suitable for widespread use in underdeveloped medical areas and home testing due to its rapid, sensitive, multiple, easy-to-use and low-cost characteristics. It can quickly detect common gynecological STI pathogens, help block the spread of sexually transmitted diseases, reduce the incidence of cervical cancer, and protect women's reproductive health. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of the structure of a microfluidic chip provided by the present invention;

[0021] Figure 2 A physical picture of the structure assembled together with the CRISPR storage area, paraffin isolation area, and RPA reaction area;

[0022] Figure 3 Dimensions of a microfluidic chip used in an embodiment of the present invention;

[0023] Figure 4 The microfluidic chip provided by the present invention is suitable for efficient batch production and assembly demonstration diagram;

[0024] Figure 5A The microfluidic chip is shown in different states at various stages of use;

[0025] Figure 5B To quantify the liquid volume in each reaction well of the chip after adding the RPA reaction solution, all values ​​represent the mean ± standard deviation (SD), n = 3 biological independent experiments;

[0026] Figure 5C To evaluate the discrimination ability of the system by measuring the fluorescence values ​​of serially diluted samples, all values ​​represent the mean ± standard deviation (SD), n = 3 biologically independent experiments;

[0027] Figure 6 The present invention provides a structural schematic diagram of a portable detection device, in which A is a schematic diagram of the detection principle of the portable device and a perspective view of the internal structure, B is a display diagram of three modules and all components of the portable detection device, and C is a display diagram of the appearance of the portable device and its internal structure;

[0028] Figure 7 Calibration curves using different primers and corresponding crRNA for different targets, detection of different concentrations of targets in PCR tubes, detection using different primers and corresponding crRNA, and the lowest limit of detection (LOD) calculated based on 3σ / slope, where σ is the standard deviation of three blank samples, all values ​​represent mean ± standard deviation (SD), n = 3 biologically independent experiments;

[0029] Figure 8 B is an agarose gel image showing the final concentration of each primer in the system at different primer pool concentrations and the final concentration of each primer at 10 -18 The RPA reaction results for different targets at M concentration; C is the CRISPR cleavage reaction for the RPA product in B;

[0030] Fig. 9 Figure 1 is a graph showing the performance results of single-plex and six-plex RPA. Figure A is an agarose gel image showing the performance of different targets at 10 -14 Single-plex RPA results at M concentration, B is cross-validation of different target crRNAs for the RPA product in A, C is quantitative analysis of fluorescence intensity for each tube in B, all values ​​represent mean ± standard deviation (SD), n = 3 biological independent experiments, D is six-plex target joint detection in the background containing vaginal secretions, the reaction and fluorescence intensity readings were performed by qPCR, and a false negative result of the MG target was observed only in one sample (in the red box);

[0031] Fig.10The calibration curves of different concentrations of targets in the multi-target detection system were obtained. The reactions were performed by a PCR thermal cycler, and the fluorescence intensity quantitative analysis was completed using a microplate reader. The lowest limit of detection (LOD) was calculated based on 3σ / slope, where σ is the standard deviation of three blank samples. All values ​​represent mean ± standard deviation (SD), n = 3 biological independent experiments;

[0032] Fig.11 C in the middle is the detection result of the multi-target detection system at different reaction temperatures, and paraffin wax with melting points suitable for different reaction temperatures is selected for separation; D is the detection result of the multi-target detection system at different reaction times, all detection times include RPA and CRISPR stages, and the duration of each stage is 1:1; E is the detection result of the multi-target detection system at different system ratios; for the 50μL detection system, different volumes of RPA and CRISPR solutions were used for testing; F is the multi-target detection system used to detect target plasmids of different concentrations in PCR tubes, and the quantitative analysis of the detection results was performed by a microplate reader, and all values ​​are expressed as mean ± standard deviation (SD), n = 3 biological independent experiments;

[0033] Fig.12 The schematic diagram and operation flow chart of the multi-target detection system, in which A is a patient's vaginal secretion sample that releases nucleic acid through a cell lysis buffer, and then the RPA system containing nucleic acid is added to the chip B; C is the chip placed in a portable device for reaction and obtaining results, the reaction procedure is sequentially carried out at 40°C for 15 minutes, followed by vibration mixing, and then another CRISPR reaction at 40°C for 15 minutes, and the positive target is marked by green fluorescence in the observation port; D shows a schematic diagram of the molecular reaction process, (i-iv) shows the four stages of the automatic system;

[0034] Fig.13 For quantitative analysis of on-chip fluorescence images using ImageJ, the cutoff value was set at 35 A.U;

[0035] Fig.14 Figure 1 shows the detection results of six targets in clinical samples. Figure A shows the detection results of the multi-target detection system (original images and positive / negative results) (left) and the clinical detection results (right). The red boxes in the figure show the inconsistencies between the two detection methods (S34: UU; S36: NG; S97: HPV 16); Figure B shows the detailed analysis of each pathogen by comparing the multi-target detection system and clinical detection results; Figure C shows the positive prediction agreement (PPA), negative prediction agreement (NPA), sensitivity and specificity of the multi-target detection system in detecting six pathogens in clinical samples;

[0036] Fig.15Receiver operating characteristic curves for six targets testing 100 clinical samples for the multi-target detection system.

[0037] In the figure: 1. Cover plate; 11. Injection hole; 2. CRISPR layer; 21. CRISPR reaction chamber; 3. Paraffin isolation area; 31. Wax isolation assembly; 4. Airway layer; 41. First channel; 42. Exhaust hole; 5. Liquid channel layer; 51. Second channel; 52. Microfluidic channel; 6. RPA layer; 61. RPA reaction chamber; 7. Base layer. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present invention more clear, the specific implementation methods of the present invention are further described in detail below in conjunction with specific embodiments and drawings.

[0039] The principle of the multi-target detection system based on paraffin phase change provided by the present invention is to utilize the phase change characteristics of low-melting-point paraffin, and effectively isolate and automatically synthesize the RPA amplification and CRISPR detection reactions by controlling the reaction temperature. The entire detection process includes the following steps: After the sample is collected, the sample is added to a microfluidic chip pre-filled with RPA and CRISPR reaction components. The chip is heated to 39-40°C, the paraffin begins to phase change, the RPA reaction is completed first, and then the paraffin floats and mixes with the CRISPR reaction system to complete the multi-target CRISPR detection.

[0040] refer to Figure 1, which is a schematic diagram of the structure of a microfluidic chip provided by the present invention. The microfluidic chip consists of a CRISPR storage area, a paraffin isolation area and an RPA reaction area. The CRISPR storage area includes a cover layer 1 and a CRISPR layer 2. The RPA reaction area includes an airway layer 4, a liquid channel layer 5, an RPA layer 6 and a base layer 7. The CRISPR layer 2 is arranged below the cover plate 1, and an injection hole 11 is arranged on the cover plate. The paraffin isolation area 3 is arranged below the CRISPR layer 2, and the RPA reaction area is arranged below the paraffin isolation area. The CRISPR layer 2 includes CRISPR reaction chambers 21 arranged in sequence at intervals on the periphery away from the center. The paraffin isolation area 3 is provided with wax isolation components 31 corresponding to the CRISPR reaction chambers 21 one by one. The wax isolation components 31 are arranged with the CRISPR reaction chambers 21. The PR reaction chamber 21 is in contact with the reaction liquid; the RPA reaction area includes an airway layer 4, a liquid channel layer 5, an RPA layer 6 and a base layer 7 arranged in sequence from top to bottom, the airway layer 4 is arranged below the paraffin isolation area 3, and the airway layer 4 includes first channels 41 arranged in sequence at intervals on the periphery away from the center, and each first channel is connected to an exhaust hole 42; the liquid channel layer 5 includes second channels 51 arranged in sequence at intervals on the periphery away from the center, and each second channel 51 is connected to a microchannel 52, and one end of multiple microchannels 52 converges at the center of the liquid channel layer 5; the RPA layer 6 includes an RPA reaction chamber 61 arranged in sequence at intervals on the periphery away from the center; the first channel 41, the second channel 51 and the RPA reaction chamber 61 correspond one to one.

[0041] refer to Figure 2 , a physical picture of the structure of the CRISPR storage area, paraffin isolation area and RPA reaction area assembled together. There is no limit to the way the microfluidic chip is manufactured. It can be made of seven layers of PMMA plates of different thicknesses and processed by CO2 laser cutting.

[0042] Example 1

[0043] The ability of the microfluidic chip provided by the present invention to correctly distinguish different fluorescence intensities was examined.

[0044] The size design of the microfluidic chip can be based on the actual detection amount, refer to Figure 3 The diameter of the reaction holes in the CRISPR storage area and the RPA reaction area are both 5 mm, and the depths are 1.5 mm and 1.6 mm, respectively. In view of the decrease in stability of phase-change paraffin when it changes from solid to liquid, using paraffin alone as an isolation layer may lead to premature mixing of the upper and lower layers before vibration. Therefore, the inventors designed to reduce the pore size of the paraffin layer to provide additional support to ensure stable isolation between the upper and lower layers of the reaction system. This design effectively prevents accidental mixing of the CRISPR and RPA systems during the conversion of paraffin from solid to liquid. The simple design of the chip makes it suitable for efficient mass production and assembly. The manufacturing process based on CO2 laser cutting is fast and low-cost, and is suitable for high-throughput production, such as Figure 4 shown.

[0045] Microfluidic chips exhibit different states at different stages of use, such as Figure 5A As shown. "Blank chip" refers to the original chip without any reagents loaded; "preloaded chip" refers to the state before testing, when the CRISPR storage area has been preloaded in the upper layer, and paraffin has been preset in the paraffin isolation area to isolate the upper CRISPR layer. In addition, the lower layer of the chip is preloaded with reagents in the positive and negative control wells to form a positive control (PC) system and a negative control (NC) system, respectively. After the user injects the RPA system containing clinical samples into the chip through the injection hole, the chip enters the "loaded chip" state, at which time the entire lower layer is filled with the RPA system solution, while the paraffin remains isolated. Subsequently, the chip is placed in a portable device and heated to 40°C for 15 minutes to run the RPA reaction and melt the paraffin into a liquid state. After vibration, the CRISPR and RPA systems are completely mixed in each reaction well ( Figure 5A To further quantitatively evaluate the chip characteristics, a precision Hamilton syringe was used to measure the liquid volume of each reaction well after the final injection. Since the CRISPR layer was pre-installed, the volume of each reaction well was 30 μL ( Figure 5A ). The injected RPA reaction solution was evenly distributed in the six target RPA reaction wells, and its volume was consistent with the pre-loaded CRISPR volume, maintaining the optimal 1:1 system ratio determined previously.

[0046] like Figure 5C As shown, the concentration used is 10 -8 Using HPV 16 plasmid from M as a template, fluorescent solutions were prepared from the positive control and diluted with enzyme-free water at ratios of 9:1, 7:3, 5:5, 3:7, 2:8, and 1:9. These dilutions were then added to different reaction wells (#1-#6) and observed under fluorescent illumination. All sampling wells were able to effectively distinguish the negative control (NC).

[0047] Example 2

[0048] This embodiment provides a portable detection device.

[0049] refer to Figure 6, Figure A shows a portable detection device, and Figure B is a diagram of the various components in Figure A. The portable detection device includes a fluorescence optical reading module, a heating module, and an oscillation module. The fluorescence optical reading module contains a 488nm laser emitter and an optical lens with a reflectivity of nearly 95% for 488nm excitation light, which guides the light to the chip. This excitation causes the fluorescent solution in the positive well to emit light at 518nm. The emitted light is read through a filter on the top of the device, which has a transmittance of 90% for light in the range of 505-545nm, effectively blocking other wavelengths. The initial light intensity of the laser diode and the light intensity after mirror reflection are measured using a light intensity meter to ensure that the illumination is sufficient to excite fluorescence in order to interpret the detection results. The results show that even after mirror reflection, the light intensity is still higher than that of the commercially available high-sensitivity blue LED transilluminator. The heating module contains a chip holder and a heating film. The test results confirmed that the heating film can reach and stabilize at the required test temperature of about 40°C within 5min. The oscillation module treasure house rotation motor and oscillation stopper connected to the motor. These components ensure that the reagents are fully mixed during the test. The device housing is made of ABS engineering plastic using 3D printing technology ( Figure 6 All components of the portable device, including the 488nm laser emitter, heating membrane, rotation motor, optical lenses and filters, are commercially available low-cost parts, while the housing is custom designed and 3D printed. The device is designed for long-term repeated use and is suitable for home self-testing and application in resource-limited environments.

[0050] Example 3

[0051] This embodiment provides methods for detecting six pathogens, including human papillomavirus (HPV 16 / 18), Chlamydia (CT), Mycoplasma (MG), Ureaplasma urealyticum (UU), and Neisseria gonorrhoeae (NG).

[0052] Screening and optimization of detection primers and RNA:

[0053] Specific plasmids were designed as detection templates for the six pathogens. The L1 fragment of HPV encodes the major capsid protein and is highly conserved. The L1 nucleotide sequence of the HPV subtype is at least 10% different from that of any other subtype. Therefore, the HPV 16 / 18 plasmid template contains the corresponding L1 fragment as a target for subsequent primer and crRNA design. For bacterial DNA, the 16S ribosomal RNA gene is a highly conserved region and is often used as the basis for distinguishing bacteria in PCR detection methods. Therefore, the 16S rRNA genes of MG, NG, CT and UU were designed as plasmid templates. The 16S rRNA gene sequences of the six pathogens are shown in Table 1, all from the NCBI database.

[0054] Table 1. Sequence information of plasmid templates of six pathogens

[0055]

[0056] In order to optimize the amplification efficiency of different pathogens, five pairs of candidate primers were designed for each target, with primer lengths controlled at 30-32 bp and amplification product lengths between 100-300 bp. Capillary electrophoresis was used to evaluate the amplification effect of each primer, and the amplification efficiency was judged based on the matching degree of the amplification product size with the theoretical value and the significance of the main peak.

[0057] For HPV 16, HPV 18, MG, NG, CT and UU, the present invention determined at least 2 pairs of candidate primers for the next stage of screening (as shown in Table 2). All selected primers contain crRNA with PAM sequence (TTTV), and the final fluorescence value is measured using RPA products with different template concentrations as substrates. Thus, the minimum detection limit (LOD) of each pair of primers and crRNA combination is calculated. The results show that the detection limit of all combinations can reach 10 -18 M(such as Figure 7 For each target, the best primer / crRNA combination that produced relatively high fluorescence intensity was selected. The final optimized primers and crRNAs for the six targets are shown in Table 3.

[0058] Table 2. Sequence information of candidate primers and crRNA (second round)

[0059]

[0060] Table 3. Sequence information of primers and crRNA of the six targets finally selected

[0061]

[0062] Given that different primer concentrations in the primer pool significantly affect the amplification efficiency of the template, the primer concentrations were optimized during the study (the forward / reverse primer concentrations for each target were 0.5, 1, and 2 μM, respectively). Gel electrophoresis comparison showed that the final primer concentration of 0.5 μM presented the clearest and brightest bands, while higher concentrations resulted in fewer effective products and more primer dimers (such as Figure 8 Similarly, the CRISPR reaction was performed using RPA products amplified with different primer pool concentrations, and the results also showed that a primer concentration of 0.5 μM produced the brightest fluorescence result (as shown in Figure 2B). Figure 8 Therefore, the final concentration of the optimal primer pool for the six targets was determined to be 0.5 μM.

[0063] Furthermore, the six pairs of best primers were mixed in equal proportions (0.5 μM) to form a primer pool, and RPA amplification was performed on each individual plasmid of the six targets. The gel electrophoresis results showed that the primer pool could effectively amplify the six target templates, such as Fig. 9 As shown in Figure A. In addition, the single RPA products in Figure A were tested using selected crRNAs to cross-validate specificity. Fluorescence results confirmed that crRNAs were able to specifically distinguish six targets ( Fig. 9 B and C in the figure).

[0064] To further test the performance of RPA primers and crRNAs, we mixed the plasmids of the six targets in equal proportions (10 - 18 M) was mixed with the primer pool and added to 30 normal female vaginal secretion samples to simulate multiplex detection in a real sample environment. The results showed that in the test of these 30 samples, the six-plex detection effect was good, and only one sample had a false negative for the MG target (such as Fig. 9 These results indicate that the RPA primer pool and crRNAs are efficient and highly specific.

[0065] Optimization of multi-target detection system:

[0066] In order to optimize the reaction conditions of the system, a series of experiments were performed to determine the optimal parameters of temperature, reaction time and reagent ratio. -14 The results showed that the reaction effect was best at 40°C, and the fluorescence intensity was the highest (e.g. Fig.11 (as shown in Figure C). Tests conducted at a temperature range of 25°C, 37°C, 40°C, and 42°C confirmed that 40°C consistently produced the strongest results, with performance significantly reduced at temperatures below or above this. Further optimization focused on the reaction times of the RPA and CRISPR stages. The total reaction time was adjusted from 5 min to 30 min, with the RPA and CRISPR stages each accounting for half of the total time. The results showed that the best solution was a total reaction time of 30 min, 15 min before mixing in the RPA stage, and 15 min after mixing in the CRISPR stage (as shown in Figure C). Fig.11 This balanced allocation of time ensures complete target recognition and signal amplification, demonstrating the effectiveness of this approach.

[0067] In addition to time and temperature, the ratio of RPA to CRISPR reagents is also critical for achieving optimal performance. We tested different volume ratios and found that a 1:1 ratio produced the brightest fluorescent signal, indicating the highest efficiency of target detection (e.g. Fig.11Deviations from this ratio can result in inadequate mixing and reduced signal intensity, emphasizing the importance of maintaining this delicate balance.

[0068] The effectiveness of the optimized multi-target detection system was then evaluated by testing different plasmid concentrations of the six targets in PCR tubes (e.g. Fig.11 The results consistently showed that the system could achieve a limit of detection (LOD) of 10 for all six targets. -18 M(such as Fig.10 ), highlighting its strong stability and suitability for practical applications. By carefully optimizing each parameter, it is possible to ensure that the multi-target detection system has high efficiency and reliability, making it a powerful tool for home self-testing and application in resource-limited settings.

[0069] Example 4

[0070] This embodiment provides a portable device according to Embodiment 2 for detecting common STI pathogens in female vaginal secretions, including MG, NG, CT, UU, HPV 16 and HPV 18.

[0071] like Fig.12 As shown in Figure A, the patient's vaginal secretions are treated with a lysis buffer to release nucleic acids. Subsequently, the RPA solution containing the nucleic acid sample is injected into a disposable pre-loaded chip and processed in a compatible portable device for 30 minutes. Finally, the fluorescence detection results are obtained through the observation port of the device. Specifically, the low-melting point paraffin pre-cured in the reaction well acts as a physical barrier to effectively separate the upper CRISPR layer from the lower RPA layer, as shown in Figure 1. Fig.12 As shown in B. The reaction process in the portable device (such as Fig.12 (As shown in C) The RPA reaction is first initiated by heating the chip to 40°C for 15 minutes. During this process, the paraffin melts into a liquid state. Subsequently, the vibration system of the device mixes the upper CRISPR layer with the reacted lower RPA layer. Heating continues to initiate the CRISPR / Cas12a reaction (40°C, 15 minutes). After the reaction is completed, the fluorescent imaging system is started and the results are read through the observation port of the device to determine the status of each target. The eight reaction wells of the chip correspond to the positive control, negative control and target pathogens HPV 16, HPV 18, MG, NG, CT and UU, respectively. As shown Fig.12 As shown in Figure D, the principle of the RPA and CRISPR reaction process is demonstrated. The entire detection process takes 30 minutes and only requires one sample addition, avoiding aerosol contamination caused by amplicon transfer in the two-step method. The disposable pre-packaged chip is used in conjunction with a portable device, which is particularly suitable for patients who self-examine at home and in areas with limited medical resources.

[0072] Performance optimization and clinical validation: To verify the performance of the multi-target detection system, vaginal secretion swabs were collected from 100 clinical patients who had previously been tested for HPV and other vaginal pathogens at Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology. After the swabs were collected and treated with 1 mL of lysis buffer, the system was used to detect and obtain the fluorescence results of six targets. Fluorescence images from different targets were collected and analyzed, and the fluorescence intensity was measured using ImageJ. According to the overall intensity of the negative samples, 35 arbitrary units (AU) were set as the threshold for distinguishing between positive and negative results (such as Fig.13 shown).

[0073] When comparing the results of the multi-target detection system with the clinical test results, only three of the 100 clinical samples were found to be different: two false negatives and one false positive. The false negative targets were UU and NG, while the false positive targets were HPV16 (such as Fig.14 A and B in the figure).

[0074] Statistical analysis of the results showed that the multi-target detection system provided reliable detection capabilities for six targets (HPV 16, HPV 18, MG, NG, CT, and UU). Overall, the positive predictive value (PPV) and negative predictive value (NPV), as well as the sensitivity and specificity were calculated to be 98.4%, 94.7%, 96.8%, and 97.3%, respectively (Figure 2). Fig.14 The fluorescence results of the three abnormal samples are close to the critical value defined in the present invention and the critical value derived from the receiver operating characteristic curve (ROC) (as shown in FIG. Fig.15 shown).

[0075] In summary, the multi-target detection system provided by the present invention is an integrated home detection chip, which simplifies the interpretation of results by setting fixed critical values ​​for six target chips. With the advancement of artificial intelligence technology, different critical values ​​can be set for each target in the future to make the integration of test results more accurate. In the test of 100 clinical samples, 62 patients were found to be positive, including 17 cases of multiple infections. Among these patients, one patient was infected with HPV16, HPV18 and UU at the same time, and 16 patients were doubly infected. This result shows that co-infection of multiple common sexually transmitted pathogens in the population is very common. In addition, among all patients infected with MG, NG, CT or UU, 9 were simultaneously infected with HPV16 and 4 were simultaneously infected with HPV18. This emphasizes the necessity of simultaneous multiple detection of common sexually transmitted pathogens and HPV.

[0076] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A multi-target detection system based on paraffin phase change, characterized in that: It includes a microfluidic chip and a fluorescence detection module that integrate RPA amplification reaction and CRISPR reaction. The microfluidic chip includes a CRISPR layer, a paraffin layer and an RPA layer arranged from top to bottom. The RPA amplification reaction and the CRISPR reaction are isolated by the paraffin layer. At a certain temperature, the RPA amplification reaction is run and the paraffin is melted into a liquid state to achieve mixing of the reacted RPA layer and the CRISPR layer. The CRISPR reaction is run, and the reacted CRISPR layer is detected by the fluorescence detection module to achieve multi-target detection.

2. The multi-target detection system according to claim 1, characterized in that: The microfluidic chip consists of a CRISPR storage area, a paraffin isolation area and an RPA reaction area, the CRISPR storage area includes a cover layer and a CRISPR layer, the RPA reaction area includes an airway layer, a liquid channel layer, an RPA layer and a base layer, the CRISPR layer is arranged below the cover plate, an injection hole is arranged on the cover plate, the paraffin isolation area is arranged below the CRISPR layer, and the RPA reaction area is arranged below the paraffin isolation area.

3. The multi-target detection system according to claim 2, characterized in that: The CRISPR layer includes CRISPR reaction chambers that are arranged in sequence at a periphery away from the center.

4. The multi-target detection system according to claim 3, characterized in that: The paraffin isolation area is provided with wax isolation components corresponding to the CRISPR reaction chambers one by one, and the wax isolation components are in contact with the reaction liquid of the CRISPR reaction chambers.

5. The multi-target detection system according to claim 3, characterized in that: The RPA reaction area includes the air channel layer, the liquid channel layer, the RPA layer and the base layer arranged in sequence from top to bottom, the air channel layer is arranged below the paraffin isolation area, the air channel layer includes first channels arranged in sequence at intervals on the periphery away from the center, and each of the first channels is connected to an exhaust hole; the liquid channel layer includes second channels arranged in sequence at intervals on the periphery away from the center, and each of the second channels is connected to a microfluidic channel, and one ends of a plurality of the microfluidic channels converge at the center of the liquid channel layer; the RPA layer includes RPA reaction chambers arranged in sequence at intervals on the periphery away from the center; the first channels, the second channels and the RPA reaction chambers correspond one to one.

6. The multi-target detection system according to claim 1, characterized in that: The microfluidic chip is in a disc shape.

7. A portable detection device, characterized in that: Comprising a multi-target detection system as described in any one of claims 1-6.

8. The portable detection device according to claim 7, characterized in that: It also includes a heating module and a vibration module.

9. A multi-target pathogen detection method, characterized in that: The PRA solution containing the nucleic acid sample is added to the microfluidic chip according to any one of claims 1 to 6 and treated for 30 to 35 minutes, and then the detection result is obtained through the fluorescence detection module.

10. The detection method according to claim 9, characterized in that: The pathogens include HPV 16, HPV 18, CT, UU, MG and NG.

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