POCT system and method thereof

Abstract

The invention discloses a POCT (point-of-care testing) system and a method thereof, and relates to the technical field of biological medicine. The present invention comprises: a sample processing module for processing a plurality of sample types, including swabs, blood, milk and urine; the nucleic acid amplification module adopts an enzymatic isothermal amplification (ERA) technology; the target recognition module is used for carrying out nucleic acid recognition by utilizing a CRISPR / Cas12a system and has specificity of a single base level; and the result reading module can realize visualization of nucleic acid reaction signals and automatically judge whether the nucleic acid reaction signals are negative or positive. According to the present invention, a variety of animal infectious disease pathogens can be simultaneously detected, the detection requirements in the actual production can be met, the detection sensitivity is high, the low-concentration pathogen nucleic acid can be detected within the short time, the specificity is good, the false positive result can be effectively avoided, the operation is simple, the automation degree is high, the operation is not required by the professional technician, and the cost is low. The kit is suitable for on-site rapid detection, and provides powerful support for early diagnosis and prevention and control of animal infectious diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a POCT system and method thereof. Background Art

[0002] With the continuous development of the global breeding industry, the prevention and control of animal infectious diseases has become a key link in ensuring the sustainable development of the breeding industry and public health safety. Various animal infectious diseases not only lead to the death of a large number of farmed animals and the decline of production performance, causing serious economic losses to farmers, but may also be transmitted to humans through the food chain, direct contact and other channels, causing public health crises. Therefore, rapid and accurate detection of animal infectious pathogens is of vital importance for early diagnosis, timely prevention and control, and effective treatment.

[0003] Traditional animal infectious disease detection methods mainly rely on large-scale laboratory instruments and equipment, such as PCR (polymerase chain reaction) technology. Although it has high detection sensitivity and specificity, it has the following limitations:

[0004] The existing equipment has a long testing cycle: it usually takes hours or even days from sample collection and transportation to the laboratory to completion of testing and issuance of reports. This makes it difficult to meet the needs of rapid on-site diagnosis and can easily delay disease prevention and control.

[0005] Complex operation: Professional technicians are required to operate, and strict requirements are placed on sample processing, nucleic acid extraction, amplification reaction and other steps. It also involves the use of multiple reagents and consumables. The operation process is cumbersome and it is easy to cause detection errors due to human factors;

[0006] Most POCT systems can only detect a single or a few pathogens, and cannot meet the needs of simultaneous detection of multiple animal infectious diseases in actual production. When multiple pathogens need to be detected, multiple different detection devices or test kits are often required, which increases the detection cost and operational complexity.

[0007] Therefore, the present invention is a POCT system and method that can simultaneously detect multiple animal infectious disease pathogens, has high sensitivity and specificity, is easy to operate, has a high degree of automation, and is suitable for rapid on-site detection. It has become a technical problem that needs to be urgently solved in the current field of animal infectious disease detection, and is also an important requirement for promoting the healthy development of the breeding industry and ensuring public health safety.

[0008] Therefore, a POCT system and method are proposed. Summary of the invention

[0009] The purpose of the present invention is to solve the problems raised in the above background technology. The present invention provides a POCT system and method thereof.

[0010] In order to achieve the above-mentioned purpose, the present invention specifically adopts the following technical solutions:

[0011] A POCT system, comprising:

[0012] Sample processing modules for processing a variety of sample types, including swabs, blood, milk, and urine;

[0013] A nucleic acid amplification module, wherein the nucleic acid amplification module adopts enzymatic isothermal amplification technology (ERA);

[0014] The target recognition module uses the CRISPR / Cas12a system for nucleic acid recognition with single-base specificity;

[0015] The result readout module can visualize the nucleic acid reaction signal and automatically determine the positive and negative.

[0016] Data transmission module, used to automatically upload the test results to the cloud monitoring and early warning system in real time;

[0017] The multiple ERA-CRI SPR / Cas12a microfluidic chip, in conjunction with the above modules, realizes automated processing and detection of samples. It can detect more than 8 pathogens at the same time and can be expanded to 16 / 24 / 48 pathogens.

[0018] Furthermore, the sample processing module can directly add samples without manual operation, and is suitable for field experimental environments.

[0019] Furthermore, the ERA technology of the nucleic acid amplification module does not require complicated temperature cycling equipment and can perform amplification under constant temperature conditions.

[0020] Furthermore, the CRISPR / Cas12a system of the target recognition module can effectively solve the false positive risk brought by the constant temperature nucleic acid amplification system.

[0021] Furthermore, the automated interpretation program of the result reading module can automatically determine the positivity and positivity, and upload the results to the cloud in real time without the need for manual repeated reporting.

[0022] Furthermore, the minimum detection limit of the multiple ERA-CRI SPR / Cas12a microfluidic chip is 300 copies / ml, the true positive rate is 100%, it is not affected by the feeding conditions, and there is no cross-influence between various pathogens.

[0023] A detection method of a POCT system comprises the following steps:

[0024] Step S1: Collect samples, including swabs, blood, milk or urine;

[0025] Step S2: adding the sample to the multiplex ERA-CRI SPR / Cas12a microfluidic chip;

[0026] Step S3: Processing the sample through a sample processing module;

[0027] Step S4: performing enzymatic isothermal amplification using a nucleic acid amplification module;

[0028] Step S5: performing nucleic acid recognition through a target recognition module;

[0029] Step S6: judging the test result through the result reading module;

[0030] Step S7: Upload the results to the cloud monitoring and early warning system through the data transmission module.

[0031] Furthermore, the processing process of the sample processing module includes automated operations without the need for human intervention.

[0032] Furthermore, the amplification process of the nucleic acid amplification module is carried out under constant temperature conditions for 20 minutes.

[0033] Furthermore, the nucleic acid recognition process of the target recognition module has single-base level specificity, which can effectively avoid false positive results.

[0034] The beneficial effects of the present invention are as follows:

[0035] It can detect a variety of animal infectious pathogens at the same time to meet the detection needs in actual production. It has high detection sensitivity and can detect low concentrations of pathogen nucleic acids in a short time. It has good specificity and can effectively avoid the occurrence of false positive results. It is easy to operate and has a high degree of automation. It does not require professional technicians to operate. It is suitable for on-site rapid detection and provides strong support for the early diagnosis and prevention and control of animal infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the structure of the POCT system of the present invention;

[0037] Figure 2 It is a schematic diagram of a flow chart of the detection method of the present invention;

[0038] Figure 3 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0040] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] It should be noted that similar reference numerals and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings. In addition, the terms "first", "second", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0042] In the description of the embodiments of the present invention, it should be noted that the terms "inside", "outside", "upper", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, or are the orientations or positional relationships in which the inventive product is usually placed when used. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0043] like Figures 1 to 3 As shown, a POCT system comprises:

[0044] Sample processing modules for processing a variety of sample types, including swabs, blood, milk, and urine;

[0045] A nucleic acid amplification module, wherein the nucleic acid amplification module uses enzymatic isothermal amplification technology (ERA) and can amplify as few as 1 to 10 copies of nucleic acid within 20 minutes;

[0046] The target recognition module uses the CRISPR / Cas12a system for nucleic acid recognition with single-base specificity;

[0047] The result readout module can visualize the nucleic acid reaction signal and automatically determine the positive and negative.

[0048] Data transmission module, used to automatically upload the test results to the cloud monitoring and early warning system in real time;

[0049] The multiplex ERA-CRI SPR / Cas12a microfluidic chip, in conjunction with the above modules, realizes automated processing and detection of samples. It has high sensitivity, high specificity and high scalability. It can detect more than 8 pathogens at the same time and can be expanded to 16 / 24 / 48 pathogens.

[0050] The output end of the sample processing module is connected to the input end of the nucleic acid amplification module, and the processed sample is passed to the nucleic acid amplification module for the next amplification reaction.

[0051] The ERA-CRI SPR / Cas12a microfluidic chip works closely together, using the microchannels and microstructures in the chip to automatically process samples, improving processing efficiency and accuracy. The output end of the nucleic acid amplification module is connected to the input end of the target recognition module, and the amplified nucleic acid product is passed to the target recognition module for specific recognition. At the same time, the nucleic acid amplification module is combined with the multiple ERA-CRI SPR / Cas12a microfluidic chip, and the reaction chamber and temperature control elements in the chip are used to achieve precise temperature control and efficient amplification reaction. The output end of the target recognition module is connected to the input end of the result readout module, and the identified signal is passed to the result readout module for visualization and result judgment. At the same time, the target recognition module is closely integrated with the multiple ERA-CRI SPR / Cas12a microfluidic chip, and the signal conversion elements and detection probes in the chip are used to realize real-time monitoring and amplification of the signal. The output end of the result readout module is connected to the input end of the data transmission module, and the judged result is passed to the data transmission module for uploading and storage. At the same time, the result readout module is connected to the multiple ERA-CRI The SPR / Cas12a microfluidic chip is used to collect and convert signals using the optical elements and circuit elements in the chip. The data transmission module is connected to the external network and cloud system of the system, responsible for uploading and downloading data, and providing support for data sharing and remote operation of the system. The microfluidic chip, as an integrated platform, runs through the entire detection process, providing physical support and chemical environment for each functional module, so that sample processing, nucleic acid amplification, target identification and result reading can be carried out efficiently and accurately inside the chip, improving the integration and stability of the system.

[0052] It solves the problems of rapid on-site detection of major cattle infectious diseases such as infectious rhinotracheitis virus, bluetongue virus, bovine viral diarrhea virus, Brucella, Listeria, bovine tuberculosis, Leptospira, and Neospora, as well as the difficulties in collecting information on sick cattle and providing early warning of regional infectious diseases.

[0053] The sample processing module can directly add samples without manual operation and is suitable for field experimental environments.

[0054] The ERA technology of the nucleic acid amplification module does not require complicated temperature cycling equipment and can perform amplification under constant temperature conditions.

[0055] The CRISPR / Cas12a system of the target recognition module can effectively solve the false positive risk brought by the constant temperature nucleic acid amplification system.

[0056] The automated interpretation program of the result reading module can automatically judge the positive or negative, and upload the results to the cloud in real time without the need for manual repeated reporting.

[0057] The multiplex ERA-CRI SPR / Cas12a microfluidic chip has a minimum detection limit of 300 copies / ml, a true positive rate of 100%, is not affected by feeding conditions, and has no cross-influence between various pathogens.

[0058] A detection method of a POCT system comprises the following steps:

[0059] Step S1: Collect samples, including swabs, blood, milk or urine;

[0060] Step S2: adding the sample to the multiplex ERA-CRI SPR / Cas12a microfluidic chip;

[0061] Step S3: Processing the sample through a sample processing module;

[0062] Step S4: performing enzymatic isothermal amplification using a nucleic acid amplification module;

[0063] Step S5: performing nucleic acid recognition through a target recognition module;

[0064] Step S6: judging the test result through the result reading module;

[0065] Step S7: Upload the results to the cloud monitoring and early warning system through the data transmission module.

[0066] The processing process of the sample processing module includes automated operations and does not require human intervention.

[0067] The amplification process of the nucleic acid amplification module is carried out under constant temperature conditions for 20 minutes.

[0068] The nucleic acid recognition process of the target recognition module has single-base level specificity, which can effectively avoid false positive results.

[0069] Embodiment 1:

[0070] In a certain farm, cattle need to be tested for infectious diseases. First, collect swab samples from cattle and add them directly to the multiple ERA-CRISPR / Cas12a microfluidic chip. The sample processing module automatically processes the samples without human intervention. Then, the nucleic acid amplification module uses ERA technology to amplify the nucleic acid in the sample under constant temperature conditions. The amplification time is 20 minutes, and it can amplify as few as 1 to 10 copies of nucleic acid. Next, the target recognition module uses the CRISPR / Cas12a system to identify the amplified nucleic acid, which has single-base level specificity and effectively avoids false positive results. The result readout module realizes the visualization of nucleic acid reaction signals and automatically determines the positive and negative. Finally, the data transmission module automatically uploads the test results to the cloud monitoring and early warning system in real time. Farmers can query the test results through the mobile client and take corresponding prevention and control measures in time.

[0071] Embodiment 2:

[0072] At the Animal Disease Control Center, a variety of animal infectious diseases need to be monitored. Collect blood, milk, urine and other samples from different animals and add them to multiple ERA-CRI SPR / Cas12a microfluidic chips. The sample processing module automatically processes different types of samples, and the nucleic acid amplification module and the target recognition module work together to achieve simultaneous detection of multiple pathogens. The test results are uploaded to the cloud through the data transmission module. The CDC staff can analyze and warn of the epidemic based on the cloud data, providing a scientific basis for the prevention and control of animal infectious diseases.

[0073] To sum up: it can detect multiple animal infectious pathogens at the same time, meet the detection needs in actual production, has high detection sensitivity, can detect low concentrations of pathogen nucleic acids in a short time, has good specificity, effectively avoids the occurrence of false positive results, is easy to operate, has a high degree of automation, does not require professional technicians to operate, is suitable for on-site rapid detection, and provides strong support for the early diagnosis and prevention and control of animal infectious diseases.

[0074] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. A POCT system, characterized in that: include: Sample processing modules for processing a variety of sample types, including swabs, blood, milk, and urine; A nucleic acid amplification module, wherein the nucleic acid amplification module adopts enzymatic isothermal amplification technology (ERA); The target recognition module uses the CRISPR / Cas12a system for nucleic acid recognition with single-base specificity; The result readout module can visualize the nucleic acid reaction signal and automatically determine the positive and negative. Data transmission module, used to automatically upload the test results to the cloud monitoring and early warning system in real time; The multiple ERA-CRISPR / Cas12a microfluidic chip, in conjunction with the above modules, enables automated processing and detection of samples. It can detect more than 8 pathogens at the same time and can be expanded to 16 / 24 / 48 pathogens.

2. A POCT system according to claim 1, characterized in that: The sample processing module can directly add samples without manual operation and is suitable for field experimental environments.

3. A POCT system according to claim 1, characterized in that: ERA of the nucleic acid amplification module The technology does not require complicated temperature cycling equipment and can perform amplification under constant temperature conditions.

4. A POCT system according to claim 1, characterized in that: The CRISPR / Cas12a system of the target recognition module can effectively solve the false positive risk brought by the constant temperature nucleic acid amplification system.

5. A POCT system according to claim 1, characterized in that: The automated interpretation program of the result reading module can automatically judge the positive or negative, and upload the results to the cloud in real time without the need for manual repeated reporting.

6. A POCT system according to claim 1, characterized in that: The multiple The minimum detection limit of the ERA-CRISPR / Cas12a microfluidic chip is 300 copies / ml, the true positive rate is 100%, it is not affected by the breeding conditions, and there is no cross-influence between various pathogens.

7. A detection method for a POCT system according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step S1: Collect samples, including swabs, blood, milk or urine; Step S2: adding the sample to the multiplex ERA-CRISPR / Cas12a microfluidic chip; Step S3: Processing the sample through a sample processing module; Step S4: performing enzymatic isothermal amplification using a nucleic acid amplification module; Step S5: performing nucleic acid recognition through a target recognition module; Step S6: judging the test result through the result reading module; Step S7: Upload the results to the cloud monitoring and early warning system through the data transmission module.

8. The detection method of a POCT system according to claim 7, characterized in that: The processing process of the sample processing module includes automated operations and does not require human intervention.

9. The detection method of a POCT system according to claim 7, characterized in that: The amplification process of the nucleic acid amplification module is carried out under constant temperature conditions for 20 minutes.

10. The detection method of a POCT system according to claim 7, characterized in that: The nucleic acid recognition process of the target recognition module has single-base level specificity, which can effectively avoid false positive results.

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