RAA isothermal amplification method for brucella detection
By using the RAA isothermal amplification method, which utilizes cell lysis and nucleic acid protection agents to treat samples, combined with dual-labeled oligonucleotide probes and signal processing technology, the problems of long detection cycle and insufficient sensitivity of Brucella can be solved, achieving efficient and accurate on-site detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-19
- Publication Date
- 2026-03-27
AI Technical Summary
Existing Brucella detection methods suffer from problems such as long cycle time, cumbersome operation, high requirements for environment and skills, and insufficient signal detection sensitivity, making them difficult to widely apply, especially in field testing.
The RAA isothermal amplification method was used to obtain crude nucleic acid samples by co-incubating the test sample with cell lysis agents and nucleic acid protectants. The samples were then mixed with RAA isothermal amplification lyophilized microspheres and dual-labeled oligonucleotide probes in a closed system, and the amplification reaction was triggered under isothermal conditions. The fluorescence signal was monitored in real time and the signal was processed to output the detection results.
It achieves rapid molecular detection of Brucella with high sensitivity and specificity, eliminating the subjectivity of human interpretation and background interference, and is suitable for on-site detection.
Smart Images
Figure CN121737320A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pathogen detection technology, and in particular to the RAA isothermal amplification method for detecting Brucella. Background Technology
[0002] Pathogen detection of Brucella primarily relies on traditional bacterial isolation and culture, serological tests, and molecular diagnostic methods based on polymerase chain reaction (PCR). However, bacterial culture cycles are long and pose high biosafety risks, and serological tests are susceptible to cross-reactivity interference, leading to false positives. While conventional PCR technology offers good specificity, it relies on sophisticated thermal cycling equipment, is cumbersome and time-consuming, and demands high levels of expertise from both the testing environment and the operators, making it difficult to widely apply in field testing. Although techniques such as loop-mediated isothermal amplification (LAMP) simplify the amplification process to some extent, their primer design is complex and carries the risk of non-specific amplification. Recombinase-mediated isothermal amplification (RAA) technology holds promise for rapid and low-cost nucleic acid amplification, but its practical application still faces technical bottlenecks such as inhibitor interference in crude samples, unstable amplification efficiency, and insufficient signal detection sensitivity. Summary of the Invention
[0003] This invention overcomes the shortcomings of the prior art and provides a RAA isothermal amplification method for Brucella detection.
[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention discloses a RAA isothermal amplification method for Brucella detection, comprising the following steps: The sample to be tested was co-incubated with a sample pretreatment solution containing cell lysis agent and nucleic acid protectant to obtain crude nucleic acid samples for direct amplification. The crude nucleic acid sample, RAA isothermal amplification lyophilized microspheres, and dual-labeled oligonucleotide probes designed for Brucella specific gene sequences were mixed in a closed system to form a pre-prepared reaction system. The pre-set reaction system is placed in a constant temperature device to trigger the amplification reaction. Under isothermal conditions, the dual-labeled oligonucleotide probe is specifically cleaved during the amplification process to generate a measurable signal molecule. The fluorescence intensity of the measurable signal molecules in the reaction system is monitored in real time or at the endpoint and converted into raw electrical signal data; The raw electrical signal data is analyzed to output a positive result for Brucella.
[0005] Furthermore, the sample to be tested is co-incubated with a sample pretreatment solution containing cell lysis agent and nucleic acid protectant to obtain a crude nucleic acid sample for direct amplification, specifically: The sample to be tested is mixed with the sample pretreatment solution in a predetermined ratio to form a sample pretreatment mixture; The sample pretreatment mixture is incubated at a constant temperature for a predetermined time to allow the cell lysis agent to penetrate and disrupt the cell wall and cell membrane, releasing intracellular nucleic acids. Meanwhile, nucleic acid protectants stabilize nucleic acid molecules by chelating metal ions or inhibiting nuclease activity, forming a stable nucleic acid lysis buffer; The nucleic acid lysis buffer was subjected to solid-liquid separation treatment, and the liquid phase component was collected as a crude nucleic acid sample.
[0006] Further, the crude nucleic acid sample, RAA isothermal amplification lyophilized microspheres, and dual-labeled oligonucleotide probes designed for Brucella-specific gene sequences are mixed in a closed system to form a pre-prepared reaction system, specifically as follows: The crude nucleic acid sample was subjected to volume standardization to obtain a standard volume nucleic acid sample. The standard volume of nucleic acid sample is injected into a sealed reaction container containing the RAA isothermal amplification lyophilized microspheres, so that the RAA isothermal amplification lyophilized microspheres are fully reconstituted to form a homogeneous amplification reaction solution. The dual-labeled oligonucleotide probe is added to the amplification reaction solution, and the probe is uniformly dispersed and integrated into the amplification reaction solution through a closed pulse mixing operation to form a homogeneous pre-set reaction system.
[0007] Furthermore, the pre-prepared reaction system is placed in a thermostat to trigger the amplification reaction. Under isothermal conditions, the dual-labeled oligonucleotide probe is specifically cleaved during amplification to generate a measurable signal molecule, specifically as follows: The pre-set reaction system is placed in a constant temperature device, and the RAA amplification reaction is triggered under preset isothermal conditions to start the enzyme-catalyzed chain displacement polymerization process and generate amplification products. During the amplification process, the dual-labeled oligonucleotide probe specifically hybridizes with the target gene sequence in the amplification product to form a probe-template hybrid; DNA polymerase recognizes the probe-template hybrid during the chain extension stage and specifically cleaves the probe through the 5'→3' exonuclease activity of DNA polymerase to produce cleavage fragments; the cleavage fragments contain isolated fluorescent reporter groups, and their fluorescence intensity increases with the accumulation of cleavage fragments, generating measurable signal molecules; Real-time monitoring of fluorescence intensity changes, and differential calculation between fluorescence intensity values and initial background values to obtain fluorescence intensity increment sequences; When the fluorescence intensity increment exceeds a preset threshold, a probe cleavage event is determined to have occurred, confirming the generation of measurable signal molecules.
[0008] Furthermore, the fluorescence intensity of the measurable signal molecules in the reaction system is monitored in real time or at the endpoint and converted into raw electrical signal data, specifically as follows: The time-domain fluorescence pulse train generated by measurable signal molecules under specific excitation light is collected, and the time-domain fluorescence pulse train is converted into a primary current signal; The primary current signal is processed, and the target fluorescence signal component synchronized with the excitation light frequency is extracted by mixing and phase-sensitive detection, while suppressing broadband background noise. The target fluorescence signal component is input into an adaptive sliding window. The adaptive sliding window adjusts its width according to the change of the first derivative of the signal to perform mean filtering during the signal stationary period and retain the original sampling rate during the signal rising period, thereby outputting a denoised fluorescence trajectory. Based on the denoised fluorescence trajectory in the early stage of the reaction, the background fluorescence quenching factor is calculated by nonlinear fitting, and the background fluorescence quenching factor is used to perform background correction on the entire subsequent denoised fluorescence trajectory to generate an apparent fluorescence intensity sequence. The apparent fluorescence intensity sequence is aligned and encapsulated with the timestamp generated by the timer to generate structured raw electrical signal data.
[0009] Specifically, the primary current signal is processed to extract the target fluorescence signal component synchronized with the excitation light frequency through frequency mixing and phase-sensitive detection, while suppressing broadband background noise. The primary current signal is converted into a primary voltage signal by a transimpedance amplifier; The primary voltage signal is synchronously mixed with the quadrature local oscillator signal pair generated by the excitation light modulation frequency. The quadrature local oscillator signal pair includes an in-phase component and a quadrature component. The in-phase modulation signal and the quadrature modulation signal are generated by multiplication operation, respectively. The in-phase modulation signal and the quadrature modulation signal are respectively input into a pair of matched low-pass filters for integral smoothing to filter out high-frequency noise, thereby outputting the DC in-phase component and the DC quadrature component. The sum of squares of the DC in-phase component and the DC quadrature component is calculated to determine their combined vector amplitude, thereby generating the target fluorescence signal component. The target fluorescence signal component is compared with a preset discrimination threshold. When the amplitude of the signal component continuously exceeds the preset discrimination threshold, it is determined that the effective fluorescence signal is extracted, and the final optimized target fluorescence signal component is output.
[0010] Specifically, the target fluorescence signal component is input into an adaptive sliding window. The adaptive sliding window adjusts its width according to the change of the first derivative of the signal to perform mean filtering during the signal stationary period and retain the original sampling rate during the signal rising period, thereby outputting a denoised fluorescence trajectory. The target fluorescence signal component is input into an adaptive sliding window, and the continuous first derivative sequence of the target fluorescence signal component is calculated in real time. The absolute value of the first derivative sequence is compared with a preset derivative threshold, and the width of the adaptive sliding window is adjusted according to the comparison result: when the absolute value of the first derivative is less than the preset derivative threshold, the signal is determined to be in a stable period, and the window width is expanded to a preset maximum width value. At the same time, an arithmetic mean operation is performed on the data points contained in the window to generate a mean-filtered output sequence; when the absolute value of the first derivative is greater than or equal to the preset derivative threshold, the signal is determined to be in an rising period, and the window width is shrunk to a unit width, directly retaining the data points at the original sampling rate as the original output sequence. The mean-filtered output sequence and the original output sequence are seamlessly spliced and integrated in chronological order to form a continuous denoised fluorescence trajectory.
[0011] Specifically, based on the denoised fluorescence trajectory in the initial stage of the reaction, a background fluorescence quenching factor is calculated through nonlinear fitting. This background fluorescence quenching factor is then used to perform background correction on the entire subsequent denoised fluorescence trajectory, generating an apparent fluorescence intensity sequence. Based on the denoised fluorescence trajectory, the initial reaction period is defined as the time interval from the start of the reaction until the absolute value of the first derivative of the denoised fluorescence trajectory first exceeds the preset stability threshold. Data points within this time interval are extracted to form the initial trajectory segment. Nonlinear least squares fitting is performed on the initial trajectory segment to generate a fitting curve and a corresponding set of fitting parameters. The background fluorescence quenching factor is extracted from the set of fitting parameters as a key correction parameter; The background fluorescence quenching factor is convolved with the time axis of the entire denoised fluorescence trajectory to generate a dynamic background fluorescence estimation sequence. The denoised fluorescence trajectory is subjected to point-by-point difference operation with the dynamic background fluorescence estimation sequence to obtain the background-corrected fluorescence intensity value. When the background-corrected fluorescence intensity value is lower than the preset minimum fluorescence threshold, it is clamped to the minimum fluorescence threshold; All processed fluorescence intensity values were integrated in chronological order to generate an apparent fluorescence intensity sequence.
[0012] Furthermore, the raw electrical signal data is analyzed to output a positive Brucella detection conclusion, specifically as follows: The apparent fluorescence intensity sequence is parsed from the original electrical signal data; the apparent fluorescence intensity sequence is differentiated to generate a fluorescence change rate sequence; Local extrema detection is performed based on the fluorescence rate change sequence to identify amplification inflection points; Using the amplification inflection point as the boundary, the apparent fluorescence intensity sequence is divided into a baseline phase and a growth phase; The cumulative increase in fluorescence intensity during the growth period is calculated as the signal amplification. The signal amplification is compared with a preset positive threshold. If the signal amplification exceeds the preset positive threshold, a positive Brucella detection conclusion is output.
[0013] The present invention also discloses a RAA isothermal amplification system for Brucella detection, comprising a memory and a processor. The memory stores a RAA isothermal amplification method program for Brucella detection. When the RAA isothermal amplification method program for Brucella detection is executed by the processor, the steps of the RAA isothermal amplification method for Brucella detection described in any one of the present invention are implemented.
[0014] This invention addresses the technical deficiencies in the prior art and has the following beneficial effects: By monitoring the fluorescence signal generated by probe cutting in real time and combining it with advanced signal processing and objective algorithms for automatic interpretation, this invention effectively eliminates the subjectivity and background interference of human interpretation, thereby achieving high sensitivity and high specificity for rapid molecular detection of Brucella. Attached Figure Description
[0015] 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 embodiments can be obtained from these drawings without creative effort.
[0016] Figure 1 This is a flowchart of the RAA isothermal amplification method for Brucella detection; Figure 2 This is a block diagram of the RAA isothermal amplification system used for Brucella detection. Detailed Implementation
[0017] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.
[0018] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.
[0019] like Figure 1 As shown, this invention discloses a RAA isothermal amplification method for Brucella detection, comprising the following steps: The sample to be tested was co-incubated with a sample pretreatment solution containing cell lysis agent and nucleic acid protectant to obtain crude nucleic acid samples for direct amplification. The crude nucleic acid sample, RAA isothermal amplification lyophilized microspheres, and dual-labeled oligonucleotide probes designed for Brucella specific gene sequences were mixed in a closed system to form a pre-prepared reaction system. The pre-set reaction system is placed in a constant temperature device to trigger the amplification reaction. Under isothermal conditions, the dual-labeled oligonucleotide probe is specifically cleaved during the amplification process to generate a measurable signal molecule. The fluorescence intensity of the measurable signal molecules in the reaction system is monitored in real time or at the endpoint and converted into raw electrical signal data; The raw electrical signal data is analyzed to output a positive result for Brucella.
[0020] Furthermore, the sample to be tested is co-incubated with a sample pretreatment solution containing cell lysis agent and nucleic acid protectant to obtain a crude nucleic acid sample for direct amplification, specifically: The sample to be tested is mixed with the sample pretreatment solution in a predetermined ratio to form a sample pretreatment mixture; The sample pretreatment mixture is incubated at a constant temperature for a predetermined time to allow the cell lysis agent to penetrate and disrupt the cell wall and cell membrane, releasing intracellular nucleic acids. Meanwhile, nucleic acid protectants stabilize nucleic acid molecules by chelating metal ions or inhibiting nuclease activity, forming a stable nucleic acid lysis buffer; The nucleic acid lysis buffer was subjected to solid-liquid separation treatment, and the liquid phase component was collected as a crude nucleic acid sample.
[0021] For example, the sample to be tested, such as serum or whole blood, is precisely mixed with a sample pretreatment solution at a volume ratio of 1:1 to 1:5 to form a homogeneous sample pretreatment mixture. This mixture is then incubated at a constant temperature of 25°C to 37°C for 5 to 15 minutes. During incubation, a cell lysis agent, selected from nonionic surfactants (such as Triton X-100) or anionic surfactants (such as sodium dodecyl sulfate SDS), at a concentration ranging from 0.1% to 2% (w / v), effectively penetrates and disrupts the cell wall and cell membrane structure of Brucella, thereby promoting the release of cell contents, including target nucleic acids, into the solution. Meanwhile, nucleic acid protectants, which can be selected from metal ion chelating agents (such as EDTA, with a concentration range of 1 mM to 10 mM) or nuclease inhibitors (such as RNase inhibitors or modified oligonucleotides), prevent the released nucleic acids from being degraded by specifically chelating divalent metal ions such as Mg²⁺ and Ca²⁺ in the solution or directly inhibiting the catalytic activity of nucleases, thereby forming a stable nucleic acid lysis buffer. Finally, the nucleic acid lysis buffer is centrifuged for 5 to 10 minutes to separate the solid and liquid, and the supernatant is then aspirated to obtain the crude nucleic acid sample.
[0022] Further, the crude nucleic acid sample, RAA isothermal amplification lyophilized microspheres, and dual-labeled oligonucleotide probes designed for Brucella-specific gene sequences are mixed in a closed system to form a pre-prepared reaction system, specifically as follows: The crude nucleic acid sample was subjected to volume standardization to obtain a standard volume nucleic acid sample. The standard volume of nucleic acid sample is injected into a sealed reaction container containing the RAA isothermal amplification lyophilized microspheres, so that the RAA isothermal amplification lyophilized microspheres are fully reconstituted to form a homogeneous amplification reaction solution. The dual-labeled oligonucleotide probe is added to the amplification reaction solution, and the probe is uniformly dispersed and integrated into the amplification reaction solution through a closed pulse mixing operation to form a homogeneous pre-set reaction system.
[0023] For example, the crude nucleic acid sample obtained in the preceding steps is volume-standardized, for instance, by precisely measuring 2-10 μL of sample using a micropipette to obtain a standard volume of nucleic acid sample. This aims to eliminate the interference of sample loading volume errors on subsequent amplification efficiency. Subsequently, the standard volume of nucleic acid sample is injected entirely into a sealed reaction container (such as a 0.2 mL eight-tube tube or a specially designed microfluidic chip chamber) pre-filled with the RAA isothermal amplification lyophilized microspheres. The lyophilized microspheres contain all the biochemical components necessary for amplification, including recombinase, single-stranded DNA-binding protein, DNA polymerase, dNTPs, and reaction buffer salts, which have been solidified using freeze-drying technology. After loading, the mixture is allowed to stand or briefly centrifuged to bring the liquid into contact with the microspheres, and then allowed to stand at room temperature for 1-2 minutes. Gentle finger-splashing or low-speed vortexing is used to ensure that the lyophilized microspheres are fully reconstituted, forming a homogeneous and clear amplification reaction solution. The dual-labeled oligonucleotide probe (with a fluorescent reporter group and a quencher group modified at its 5' and 3' ends, respectively, such as the FAM / BHQ1 combination) is added to the amplification reaction solution in a volume of 1-5 μL through the pre-reserved sample loading port on the reaction tube cap or using a precision syringe. After sample loading, a closed-loop pulse mixing operation is immediately performed on the sealed reaction container. Specifically, this operation involves three pulsed oscillations at a speed of 2000-3000 rpm for 2 seconds each, with a 1-second interval between each pulse. This ensures that the probe is uniformly dispersed in the amplification reaction solution and fully integrated with other components of the system, while maintaining the airtightness of the reaction system to prevent aerosol contamination. The result is a homogeneous pre-set reaction system that can be used to directly trigger the amplification reaction.
[0024] Furthermore, the pre-prepared reaction system is placed in a thermostat to trigger the amplification reaction. Under isothermal conditions, the dual-labeled oligonucleotide probe is specifically cleaved during amplification to generate a measurable signal molecule, specifically as follows: The pre-set reaction system is placed in a constant temperature device, and the RAA amplification reaction is triggered under preset isothermal conditions to start the enzyme-catalyzed chain displacement polymerization process and generate amplification products. During the amplification process, the dual-labeled oligonucleotide probe specifically hybridizes with the target gene sequence in the amplification product to form a probe-template hybrid; DNA polymerase recognizes the probe-template hybrid during the chain extension stage and specifically cleaves the probe through the 5'→3' exonuclease activity of DNA polymerase to produce cleavage fragments; the cleavage fragments contain isolated fluorescent reporter groups, and their fluorescence intensity increases with the accumulation of cleavage fragments, generating measurable signal molecules; Real-time monitoring of fluorescence intensity changes, and differential calculation between fluorescence intensity values and initial background values to obtain fluorescence intensity increment sequences; When the fluorescence intensity increment exceeds a preset threshold, a probe cleavage event is determined to have occurred, confirming the generation of measurable signal molecules.
[0025] For example, the pre-set reaction system is placed in a thermostat (such as a thermostat metal bath or temperature control module) and incubated at a preset isothermal condition of 39°C to trigger the RAA amplification reaction. This process then initiates an enzymatic chain displacement polymerization process in which recombinase, single-stranded DNA binding protein, and chain displacement DNA polymerase (e.g., Bsu DNA polymerase) work synergistically to specifically generate a large number of amplification products containing the Brucella target gene sequence. During the amplification process, the pre-added dual-labeled oligonucleotide probe (with a fluorescent reporter group such as FAM at its 5' end and a quencher group such as BHQ1 at its 3' end) specifically hybridizes with the complementary sequence in the amplification product to form a stable probe-template hybrid. Subsequently, the DNA polymerase undergoing chain extension recognizes this hybrid and uses its inherent 5'→3' exonuclease activity to specifically cleave and degrade the hybridized probe. This enzymatic cleavage reaction causes the fluorescent reporter group and the quencher group to be permanently separated in space, disrupting the previous fluorescence resonance energy transfer effect, thereby allowing the reporter group to release a detectable fluorescent signal after excitation.
[0026] As the amplification cycle progresses, the cleavage fragments accumulate, and the overall fluorescence intensity of the system increases accordingly, thereby generating a measurable signal molecule. To objectively determine signal generation, the fluorescence intensity changes are monitored in real time every 30 seconds using a fluorescence detection device. The real-time fluorescence intensity value at each monitoring time point is differentially calculated with the average fluorescence intensity value (i.e., the initial background value) of the initial reaction stage (e.g., the first 10 monitoring cycles) to obtain a fluorescence intensity increment sequence over time. A specific judgment threshold is set: when the fluorescence intensity increment continuously exceeds three times the standard deviation of the initial background value or the absolute value is greater than 500 RFU (relative fluorescence units), a valid probe cleavage event is determined to have occurred, thus confirming that the measurable signal molecule has been successfully generated, marking the specific amplification of the target nucleic acid.
[0027] Furthermore, the fluorescence intensity of the measurable signal molecules in the reaction system is monitored in real time or at the endpoint and converted into raw electrical signal data, specifically as follows: The time-domain fluorescence pulse train generated by measurable signal molecules under specific excitation light is collected, and the time-domain fluorescence pulse train is converted into a primary current signal; The primary current signal is processed, and the target fluorescence signal component synchronized with the excitation light frequency is extracted by mixing and phase-sensitive detection, while suppressing broadband background noise. The target fluorescence signal component is input into an adaptive sliding window. The adaptive sliding window adjusts its width according to the change of the first derivative of the signal to perform mean filtering during the signal stationary period and retain the original sampling rate during the signal rising period, thereby outputting a denoised fluorescence trajectory. Based on the denoised fluorescence trajectory in the early stage of the reaction, the background fluorescence quenching factor is calculated by nonlinear fitting, and the background fluorescence quenching factor is used to perform background correction on the entire subsequent denoised fluorescence trajectory to generate an apparent fluorescence intensity sequence. The apparent fluorescence intensity sequence is aligned and encapsulated with the timestamp generated by the timer to generate structured raw electrical signal data.
[0028] The primary current signal is processed by mixing and phase-sensitive detection to extract the target fluorescence signal component synchronized with the excitation light frequency, while suppressing broadband background noise. Specifically, the primary current signal is converted into a primary voltage signal through a transimpedance amplifier; the primary voltage signal is synchronously mixed with a quadrature local oscillator signal pair generated by the excitation light modulation frequency, the quadrature local oscillator signal pair including an in-phase component and a quadrature component, which are multiplied to generate an in-phase modulation signal and a quadrature modulation signal respectively; the in-phase modulation signal and the quadrature modulation signal are respectively input into a pair of matched low-pass filters for integral smoothing to filter out high-frequency noise, thereby outputting a DC in-phase component and a DC quadrature component; the sum of squares of the DC in-phase component and the DC quadrature component is performed to calculate their combined vector amplitude, generating the target fluorescence signal component; the target fluorescence signal component is compared with a preset discrimination threshold, and when the signal component amplitude continuously exceeds the preset discrimination threshold, a valid fluorescence signal is determined to be extracted, and the final optimized target fluorescence signal component is output.
[0029] The process involves inputting the target fluorescence signal component into an adaptive sliding window. This window adjusts its width based on changes in the signal's first derivative to perform mean filtering during signal stabilization and retain the original sampling rate during signal rise, thereby outputting a denoised fluorescence trajectory. Specifically, the process involves: inputting the target fluorescence signal component into an adaptive sliding window and calculating a continuous first derivative sequence of the target fluorescence signal component in real time; comparing the absolute value of the first derivative sequence with a preset derivative threshold and adjusting the width of the adaptive sliding window based on the comparison result: when the absolute value of the first derivative is less than the preset derivative threshold, the signal is considered to be in a stabilization period, and the window width is expanded to a preset maximum width value. Simultaneously, an arithmetic average is performed on the data points contained within the window to generate a mean-filtered output sequence; when the absolute value of the first derivative is greater than or equal to the preset derivative threshold, the signal is considered to be in a rise period, and the window width is shrunk to a unit width, directly retaining the data points at the original sampling rate as the original output sequence; and seamlessly splicing and integrating the mean-filtered output sequence and the original output sequence in chronological order to form a continuous denoised fluorescence trajectory.
[0030] Specifically, based on the denoised fluorescence trajectory in the initial stage of the reaction, a background fluorescence quenching factor is calculated through nonlinear fitting. This background fluorescence quenching factor is then used to perform background correction on the entire subsequent denoised fluorescence trajectory, generating an apparent fluorescence intensity sequence. The process involves: defining the initial stage of the reaction as the time interval from the start of the reaction until the absolute value of the first derivative of the denoised fluorescence trajectory first exceeds a preset stability threshold; extracting data points within this time interval to form an initial trajectory segment; performing nonlinear least-squares fitting on the initial trajectory segment to generate a fitting curve and a corresponding set of fitting parameters; extracting the background fluorescence quenching factor from the set of fitting parameters as a key correction parameter; convolving the background fluorescence quenching factor with the time axis of the entire denoised fluorescence trajectory to generate a dynamic background fluorescence estimation sequence; performing point-by-point difference operations on the denoised fluorescence trajectory and the dynamic background fluorescence estimation sequence to obtain the background-corrected fluorescence intensity value; clamping the background-corrected fluorescence intensity value to the preset minimum fluorescence threshold when it is lower than the minimum fluorescence threshold; and integrating all processed fluorescence intensity values in chronological order to generate an apparent fluorescence intensity sequence.
[0031] For example, a time-domain fluorescence pulse train generated by a measurable signal molecule (i.e., a free fluorescent reporter group) under modulated excitation light at a specific frequency (e.g., 1 kHz) is acquired and converted into a primary current signal at the nanoampere level by a photodiode. This signal is then converted into a primary voltage signal by a transimpedance amplifier with a gain of 1 MΩ. To achieve noise suppression, the primary voltage signal is synchronously mixed with a quadrature local oscillator signal generated by the same excitation source at the same modulation frequency (1 kHz). The resulting in-phase and quadrature modulation signals are then integrally smoothed by a pair of low-pass filters with a cutoff frequency of 10 Hz, thereby filtering out all high-frequency noise that is out of sync with the excitation light frequency, and outputting a DC in-phase component (I) and a DC quadrature component (Q). The composite vector amplitude (Q) is calculated. The target fluorescence signal component is generated. This component is compared with a preset discrimination threshold (e.g., 50mV). When the signal amplitude continuously exceeds the threshold for 5 consecutive sampling cycles, the valid fluorescence signal is determined to have been successfully extracted.
[0032] The target fluorescence signal components are input into an adaptive sliding window. This window calculates the first derivative of the signal in real time and compares its absolute value with a preset derivative threshold (e.g., 5 RFU / sec). When the signal is in a stationary phase (absolute derivative value less than 5 RFU / sec), the window automatically expands to its maximum width (e.g., accommodating 10 data points) and performs an arithmetic mean on all points within the window to generate a mean-filtered output sequence, which effectively smooths random noise. When the signal enters an exponential growth phase (absolute derivative value greater than or equal to 5 RFU / sec), the window immediately shrinks to a unit width (i.e., 1 data point), directly outputting the original sampled value, thus fully preserving the rapid rise dynamics of the signal and avoiding phase delay and amplitude distortion caused by mean filtering. Finally, the mean sequence of the stationary phase is seamlessly spliced with the original sequence of the rising phase to form a denoised fluorescence trajectory.
[0033] To eliminate background drift during the reaction process, correction parameters are calculated based on the denoised fluorescence trajectory at the initial stage of the reaction. The initial stage of the reaction is defined as the time interval from the start time until the absolute value of the first derivative of the trajectory first exceeds a preset stability threshold (e.g., 1.5 RFU / s) (typically the first 2-5 minutes). This initial trajectory segment is extracted, and a nonlinear least-squares fitting is performed using an exponential decay function to extract the background fluorescence quenching factor as a key correction parameter. This quenching factor is then convolved with the entire reaction time axis to generate a dynamic background fluorescence estimation sequence. The original denoised fluorescence trajectory is then differentially analyzed point-by-point with this estimation sequence. To ensure data validity, a minimum fluorescence threshold (e.g., 10 RFU) is set, and all fluorescence intensity values below this threshold after differential analysis are clamped to this threshold. Finally, all processed values are integrated in chronological order to generate an apparent fluorescence intensity sequence, which is then encapsulated with a timestamp generated by a high-precision timer to form structured raw electrical signal data.
[0034] Furthermore, the raw electrical signal data is analyzed to output a positive Brucella detection conclusion, specifically as follows: The apparent fluorescence intensity sequence is parsed from the original electrical signal data; the apparent fluorescence intensity sequence is differentiated to generate a fluorescence change rate sequence; Local extrema detection is performed based on the fluorescence rate change sequence to identify amplification inflection points; Using the amplification inflection point as the boundary, the apparent fluorescence intensity sequence is divided into a baseline phase and a growth phase; The cumulative increase in fluorescence intensity during the growth period is calculated as the signal amplification. The signal amplification is compared with a preset positive threshold. If the signal amplification exceeds the preset positive threshold, a positive Brucella detection conclusion is output.
[0035] For example, from the structured raw electrical signal data generated by the aforementioned signal processing steps, the apparent fluorescence intensity sequence, which is the core analysis object, is parsed. Differential operations are then performed on this sequence; specifically, the first derivative at each time point can be calculated using the five-point central difference method, thereby generating a fluorescence rate of change sequence characterizing the instantaneous rate of change of the signal. Based on this fluorescence rate of change sequence, local extremum detection is performed by finding the global maximum point of the sequence and its corresponding time point (t). ip The value is identified as the amplification inflection point of this amplification reaction. To ensure the validity of this inflection point, an auxiliary judgment condition is set, namely, the maximum value must be more than 3 times the average change rate in the early stage of the reaction (such as the first 5 minutes).
[0036] After accurately locating the amplification inflection point, the entire epifluorescence intensity sequence is automatically divided into two characteristic phases using this point as the boundary: the baseline phase before the inflection point (from the start of the reaction to t).ip ) and the growth period after the inflection point (from t) ip The reaction continues until either completion or a pre-defined detection endpoint, such as 30 minutes. Afterward, the cumulative increase in fluorescence intensity during the growth period is calculated as the signal amplification (ΔS). This calculation is performed by taking the endpoint of the growth period (t). end The fluorescence intensity value F(t) end The fluorescence intensity value F(t) at the amplification inflection point ip The difference is used to achieve this, i.e., ΔS = F(t) end ) -F(t ip Finally, the calculated signal amplification (ΔS) is compared with a preset positive threshold (e.g., 500 RFU or 8 times the standard deviation of baseline fluorescence intensity) after verification with a large number of positive and negative samples. If the signal amplification (ΔS) exceeds the preset positive threshold, the test result of "Brucella positive" is automatically determined and output; otherwise, a negative or invalid result is output.
[0037] This embodiment also includes: The original temperature fluctuation signal of the pre-set reaction system during the amplification process is collected in real time, and a real-time thermal power sequence of the system is generated by differential calculation with the reference temperature of the reaction environment. The real-time thermal power sequence of the system was subjected to mutation point detection based on the first derivative, and characteristic exothermic inflection points caused by enzyme-catalyzed reaction initiation and chain displacement process were identified. The time position of the characteristic exothermic inflection point is spatiotemporally aligned and correlated with the amplified fluorescence inflection point obtained by fluorescence monitoring to generate a thermal-optical linkage consistency coefficient. The real-time thermal power sequence and fluorescence signal intensity sequence of the system are convolved within the same reaction time window, and the ratio of the main peak area in the convolution spectrum is extracted to calculate the energy-signal coupling efficiency index. The thermal-optical linkage consistency coefficient and the energy-signal coupling efficiency index are input into a pre-trained linear discriminant model, which outputs a quantified response fidelity score. When the fidelity score of the reaction is lower than a preset score threshold, an early failure warning signal is triggered for this reaction.
[0038] Specifically, the raw temperature fluctuation signals of the pre-set reaction system during the amplification process are acquired in real time using a thermistor (such as a negative temperature coefficient thermistor). A reference point, unaffected by the reaction and placed in the same isothermal device, is used as the baseline temperature. Real-time differential calculations are then performed to eliminate environmental background thermal noise, thereby generating a real-time thermal power sequence reflecting the thermal dynamics of the reaction system itself. This thermal power sequence is then subjected to first-derivative-based mutation point detection to identify characteristic exothermic inflection points caused by the large-scale initiation of enzymatic reactions and chain displacement processes. For multimodal signal cross-validation, the time position of the exothermic inflection point is spatiotemporally aligned with the amplification fluorescence inflection point obtained through the fluorescence monitoring channel, and the Pearson correlation coefficient of the time difference between the two is calculated to generate a thermo-optical linkage consistency coefficient.
[0039] Furthermore, the real-time thermal power sequence and fluorescence signal intensity sequence of the system are convolved within the same time window from the start of the reaction to the appearance of the fluorescence inflection point. The convolution spectrum is analyzed, and the ratio of the main peak area to the total area is extracted to obtain the energy-signal coupling efficiency index, which characterizes the efficiency of chemical energy conversion into the detection signal. The thermal-optical linkage consistency coefficient and the energy-signal coupling efficiency index are used as two key feature vectors and input into a linear discriminant analysis model pre-trained with a large number of known positive and negative samples. This model, through weighted calculation, outputs a quantified reaction fidelity score ranging from 0 to 1. A preset scoring threshold (e.g., 0.65) is set. When the calculated reaction fidelity score is lower than this threshold, it is determined that the reaction may be abnormal due to factors such as the presence of inhibitors, enzyme inactivation, or non-specific amplification. An early failure warning signal is then triggered, indicating to the operator that the detection result is unreliable and a retest is required, thereby effectively improving the reliability and credibility of the entire detection system.
[0040] This embodiment also includes: The apparent fluorescence intensity sequence acquired in real time is subjected to continuous first-order and second-order differential operations to generate a fluorescence rate of change sequence and a fluorescence acceleration sequence. Based on the fluorescence acceleration sequence, the early amplification initiation point is identified by detecting its local maxima. Using the early amplification initiation point as the lower limit of integration, the apparent fluorescence intensity sequence is numerically integrated to calculate the early signal amplification, and an early positive threshold is preset. When the early signal amplification exceeds the early positive threshold, a reaction termination command is immediately generated to forcibly terminate the amplification reaction. Based on the time difference between the early amplification initiation point and the reaction termination point, an early positive determination conclusion is output.
[0041] Specifically, the apparent fluorescence intensity sequence, acquired in real-time and corrected for background, undergoes continuous differential operations: first, a first-order differential is performed to generate a fluorescence rate of change sequence, characterizing the rate of change in fluorescence intensity; then, a second-order differential is performed to generate a fluorescence acceleration sequence, characterizing the acceleration of the rate of change. The fluorescence acceleration sequence is then analyzed in real-time, identifying the early amplification initiation point—marking the transition from the linear phase to the early exponential growth phase—by detecting the first local maximum point. After identifying this initiation point, the apparent fluorescence intensity sequence is numerically integrated using this initiation point as the lower limit of integration and the current time point as the upper limit of integration, calculating the early signal amplification accumulated from the initiation point. This early signal amplification is then compared in real-time with a pre-set early positive threshold. Once the system determines that the early signal amplification exceeds the early positive threshold, it immediately sends a reaction termination command to the thermostat, forcibly terminating the amplification reaction by cutting off the heating power or removing the reaction tube. Simultaneously, the time difference from the early amplification initiation point to the reaction termination point is recorded, and the positive result within this short period is output as the early positive determination conclusion. This embodiment, by monitoring the second-order kinetic characteristics of the fluorescence signal, can capture the amplification initiation event before the apparent fluorescence curve reaches a significant steep rise, thus shortening the detection time and saving reagent consumption while ensuring detection accuracy.
[0042] In summary, this invention achieves rapid molecular detection of Brucella with high sensitivity and high specificity by real-time monitoring of the fluorescence signal generated by probe cutting and combining advanced signal processing and objective algorithms for automatic interpretation, effectively eliminating the subjectivity and background interference of human interpretation.
[0043] like Figure 2 As shown, the present invention also discloses a RAA isothermal amplification system for Brucella detection, including a memory and a processor. The memory stores a RAA isothermal amplification method program for Brucella detection. When the RAA isothermal amplification method program for Brucella detection is executed by the processor, the steps of the RAA isothermal amplification method for Brucella detection described in any one of the claims are implemented.
[0044] In the several embodiments provided in this application, it should be understood that the disclosed devices and methods can be implemented in other ways. The device embodiments described above are merely illustrative. For example, the division of units is only a logical functional division, and in actual implementation, there may be other division methods, such as: multiple units or components can be combined, or integrated into another system, or some features can be ignored or not executed. In addition, the coupling, direct coupling, or communication connection between the various components shown or discussed can be through some interfaces, and the indirect coupling or communication connection between devices or units can be electrical, mechanical, or other forms.
[0045] The units described above as separate components may or may not be physically separate. The components shown as units may or may not be physical units. They may be located in one place or distributed across multiple network units. Some or all of the units may be selected to achieve the purpose of this embodiment according to actual needs.
[0046] In addition, in the various embodiments of the present invention, each functional unit can be integrated into one processing unit, or each unit can be a separate unit, or two or more units can be integrated into one unit; the integrated unit can be implemented in hardware or in the form of hardware plus software functional units.
[0047] Those skilled in the art will understand that all or part of the steps of the above method embodiments can be implemented by hardware related to program instructions. The aforementioned program can be stored in a computer-readable storage medium. When the program is executed, it performs the steps of the above method embodiments. The aforementioned storage medium includes various media capable of storing program code, such as mobile storage devices, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.
[0048] Alternatively, if the integrated units of this invention are implemented as software functional modules and sold or used as independent products, they can also be stored in a computer-readable storage medium. Based on this understanding, the technical solutions of the embodiments of this invention, or the parts that contribute to the prior art, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the methods of the various embodiments of this invention. The aforementioned storage medium includes various media capable of storing program code, such as mobile storage devices, ROM, RAM, magnetic disks, or optical disks.
[0049] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.
Claims
1. A RAA isothermal amplification method for Brucella detection, characterized in that, Includes the following steps: The sample to be tested was co-incubated with a sample pretreatment solution containing cell lysis agent and nucleic acid protectant to obtain crude nucleic acid samples for direct amplification. The crude nucleic acid sample, RAA isothermal amplification lyophilized microspheres, and dual-labeled oligonucleotide probes designed for Brucella specific gene sequences were mixed in a closed system to form a pre-prepared reaction system. The pre-set reaction system is placed in a constant temperature device to trigger the amplification reaction. Under isothermal conditions, the dual-labeled oligonucleotide probe is specifically cleaved during the amplification process to generate a measurable signal molecule. The fluorescence intensity of the measurable signal molecules in the reaction system is monitored in real time or at the endpoint and converted into raw electrical signal data; The raw electrical signal data is analyzed to output a positive result for Brucella.
2. The RAA isothermal amplification method for Brucella detection according to claim 1, characterized in that, The sample to be tested was co-incubated with a sample pretreatment solution containing cell lysis agent and nucleic acid protectant to obtain a crude nucleic acid sample for direct amplification, specifically: The sample to be tested is mixed with the sample pretreatment solution in a predetermined ratio to form a sample pretreatment mixture; The sample pretreatment mixture is incubated at a constant temperature for a predetermined time to allow the cell lysis agent to penetrate and disrupt the cell wall and cell membrane, releasing intracellular nucleic acids. Meanwhile, nucleic acid protectants stabilize nucleic acid molecules by chelating metal ions or inhibiting nuclease activity, forming a stable nucleic acid lysis buffer; The nucleic acid lysis buffer was subjected to solid-liquid separation treatment, and the liquid phase component was collected as a crude nucleic acid sample.
3. The RAA isothermal amplification method for Brucella detection according to claim 1, characterized in that, The crude nucleic acid sample, RAA isothermal amplification lyophilized microspheres, and dual-labeled oligonucleotide probes designed for Brucella-specific gene sequences were mixed in a closed system to form a pre-prepared reaction system, specifically as follows: The crude nucleic acid sample was subjected to volume standardization to obtain a standard volume nucleic acid sample. The standard volume of nucleic acid sample is injected into a sealed reaction container containing the RAA isothermal amplification lyophilized microspheres, so that the RAA isothermal amplification lyophilized microspheres are fully reconstituted to form a homogeneous amplification reaction solution. The dual-labeled oligonucleotide probe is added to the amplification reaction solution, and the probe is uniformly dispersed and integrated into the amplification reaction solution through a closed pulse mixing operation to form a homogeneous pre-set reaction system.
4. The RAA isothermal amplification method for Brucella detection according to claim 1, characterized in that, The pre-set reaction system is placed in a thermostat to trigger the amplification reaction. Under isothermal conditions, the dual-labeled oligonucleotide probe is specifically cleaved during the amplification process to generate a measurable signal molecule, specifically as follows: The pre-set reaction system is placed in a constant temperature device, and the RAA amplification reaction is triggered under preset isothermal conditions to start the enzyme-catalyzed chain displacement polymerization process and generate amplification products. During the amplification process, the dual-labeled oligonucleotide probe specifically hybridizes with the target gene sequence in the amplification product to form a probe-template hybrid; DNA polymerase recognizes the probe-template hybrid during the chain extension stage and specifically cleaves the probe through the 5'→3' exonuclease activity of DNA polymerase to produce cleavage fragments; the cleavage fragments contain isolated fluorescent reporter groups, and their fluorescence intensity increases with the accumulation of cleavage fragments, generating measurable signal molecules; Real-time monitoring of fluorescence intensity changes, and differential calculation between fluorescence intensity values and initial background values to obtain fluorescence intensity increment sequences; When the fluorescence intensity increment exceeds a preset threshold, a probe cleavage event is determined to have occurred, confirming the generation of measurable signal molecules.
5. The RAA isothermal amplification method for Brucella detection according to claim 1, characterized in that, The fluorescence intensity of the measurable signal molecules in the reaction system is monitored in real time or at the endpoint and converted into raw electrical signal data, specifically as follows: The time-domain fluorescence pulse train generated by measurable signal molecules under specific excitation light is collected, and the time-domain fluorescence pulse train is converted into a primary current signal; The primary current signal is processed, and the target fluorescence signal component synchronized with the excitation light frequency is extracted by mixing and phase-sensitive detection, while suppressing broadband background noise. The target fluorescence signal component is input into an adaptive sliding window. The adaptive sliding window adjusts its width according to the change of the first derivative of the signal to perform mean filtering during the signal stationary period and retain the original sampling rate during the signal rising period, thereby outputting a denoised fluorescence trajectory. Based on the denoised fluorescence trajectory in the early stage of the reaction, the background fluorescence quenching factor is calculated by nonlinear fitting, and the background fluorescence quenching factor is used to perform background correction on the entire subsequent denoised fluorescence trajectory to generate an apparent fluorescence intensity sequence. The apparent fluorescence intensity sequence is aligned and encapsulated with the timestamp generated by the timer to generate structured raw electrical signal data.
6. The RAA isothermal amplification method for Brucella detection according to claim 5, characterized in that, The primary current signal is processed by using frequency mixing and phase-sensitive detection to extract the target fluorescence signal component synchronized with the excitation light frequency, while suppressing broadband background noise. Specifically: The primary current signal is converted into a primary voltage signal by a transimpedance amplifier; The primary voltage signal is synchronously mixed with the quadrature local oscillator signal pair generated by the excitation light modulation frequency. The quadrature local oscillator signal pair includes an in-phase component and a quadrature component. The in-phase modulation signal and the quadrature modulation signal are generated by multiplication operation, respectively. The in-phase modulation signal and the quadrature modulation signal are respectively input into a pair of matched low-pass filters for integral smoothing to filter out high-frequency noise, thereby outputting the DC in-phase component and the DC quadrature component. The sum of squares of the DC in-phase component and the DC quadrature component is calculated to determine their combined vector amplitude, thereby generating the target fluorescence signal component. The target fluorescence signal component is compared with a preset discrimination threshold. When the amplitude of the signal component continuously exceeds the preset discrimination threshold, it is determined that the effective fluorescence signal is extracted, and the final optimized target fluorescence signal component is output.
7. The RAA isothermal amplification method for Brucella detection according to claim 5, characterized in that, The target fluorescence signal component is input into an adaptive sliding window. The adaptive sliding window adjusts its width according to the change of the first derivative of the signal to perform mean filtering during the signal stationary period and retain the original sampling rate during the signal rising period, thereby outputting a denoised fluorescence trajectory. Specifically: The target fluorescence signal component is input into an adaptive sliding window, and the continuous first derivative sequence of the target fluorescence signal component is calculated in real time. The absolute value of the first derivative sequence is compared with a preset derivative threshold, and the width of the adaptive sliding window is adjusted according to the comparison result: when the absolute value of the first derivative is less than the preset derivative threshold, the signal is determined to be in a stable period, and the window width is expanded to a preset maximum width value. At the same time, an arithmetic mean operation is performed on the data points contained in the window to generate a mean-filtered output sequence; when the absolute value of the first derivative is greater than or equal to the preset derivative threshold, the signal is determined to be in an rising period, and the window width is shrunk to a unit width, directly retaining the data points at the original sampling rate as the original output sequence. The mean-filtered output sequence and the original output sequence are seamlessly spliced and integrated in chronological order to form a continuous denoised fluorescence trajectory.
8. The RAA isothermal amplification method for Brucella detection according to claim 5, characterized in that, Based on the denoised fluorescence trajectory in the initial stage of the reaction, the background fluorescence quenching factor is calculated through nonlinear fitting. This background fluorescence quenching factor is then used to perform background correction on the subsequent entire denoised fluorescence trajectory, generating an apparent fluorescence intensity sequence, specifically: Based on the denoised fluorescence trajectory, the initial reaction period is defined as the time interval from the start of the reaction until the absolute value of the first derivative of the denoised fluorescence trajectory first exceeds the preset stability threshold. Data points within this time interval are extracted to form the initial trajectory segment. Nonlinear least squares fitting is performed on the initial trajectory segment to generate a fitting curve and a corresponding set of fitting parameters. The background fluorescence quenching factor is extracted from the set of fitting parameters as a key correction parameter; The background fluorescence quenching factor is convolved with the time axis of the entire denoised fluorescence trajectory to generate a dynamic background fluorescence estimation sequence. The denoised fluorescence trajectory is subjected to point-by-point difference operation with the dynamic background fluorescence estimation sequence to obtain the background-corrected fluorescence intensity value. When the background-corrected fluorescence intensity value is lower than the preset minimum fluorescence threshold, it is clamped to the minimum fluorescence threshold; All processed fluorescence intensity values were integrated in chronological order to generate an apparent fluorescence intensity sequence.
9. The RAA isothermal amplification method for Brucella detection according to claim 1, characterized in that, The raw electrical signal data is analyzed to output a positive Brucella detection conclusion, specifically: The apparent fluorescence intensity sequence is parsed from the original electrical signal data; the apparent fluorescence intensity sequence is differentiated to generate a fluorescence change rate sequence; Local extrema detection is performed based on the fluorescence rate change sequence to identify amplification inflection points; Using the amplification inflection point as the boundary, the apparent fluorescence intensity sequence is divided into a baseline phase and a growth phase; The cumulative increase in fluorescence intensity during the growth period is calculated as the signal amplification. The signal amplification is compared with a preset positive threshold. If the signal amplification exceeds the preset positive threshold, a positive Brucella detection conclusion is output.
10. A RAA isothermal amplification system for Brucella detection, characterized in that, The device includes a memory and a processor. The memory stores a RAA isothermal amplification method program for Brucella detection. When the RAA isothermal amplification method program for Brucella detection is executed by the processor, the steps of the RAA isothermal amplification method for Brucella detection as described in any one of claims 1 to 9 are implemented.