A fluorescence detection method for Pg bacteria based on dual-temperature zone cyclic amplification

Abstract

This invention relates to the field of microbial detection technology, specifically providing a fluorescence detection method for Pg bacteria based on dual-temperature zone cyclic amplification, including S1, initial fluorescence detection, S2, dual-temperature zone cyclic amplification, S3, endpoint fluorescence detection, and S4, result analysis. It also includes a detection system for implementing the dual-temperature zone cyclic amplification-based fluorescence detection method for Pg bacteria, comprising a housing, an aluminum rod assembly, a high-temperature isothermal module, a low-temperature isothermal module, a light source detection module, a moving mechanism, a lifting mechanism, and a control and processing unit for controlling the moving mechanism, acquiring fluorescence data, and performing result analysis. This invention simplifies the three steps of denaturation, annealing, and extension in traditional qPCR to two steps: high-temperature denaturation and low-temperature annealing and extension, by setting independent high-temperature and low-temperature isothermal modules. This eliminates the time-consuming heating and cooling processes in traditional PCR instruments, significantly shortening the detection cycle and meeting the needs of rapid on-site diagnosis.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to a fluorescence detection method for Pg bacteria based on dual-temperature zone cyclic amplification. Background Technology

[0002] Porphyromonas gingivalis (Pg) is a key pathogen of periodontal disease, especially chronic and aggressive periodontitis. Its detection rate is directly related to the severity of periodontal lesions. Therefore, rapid and accurate detection of Pg is of significant clinical importance for early diagnosis, treatment planning, and efficacy evaluation of periodontal disease. Currently, polymerase chain reaction (PCR) technology has become the "gold standard" for Pg nucleic acid detection due to its high sensitivity and specificity, with real-time quantitative PCR (qPCR) being the mainstream technique.

[0003] However, existing methods for detecting Pg bacteria based on traditional qPCR typically employ a single-temperature-zone module. Through programmed temperature control, these modules cycle through three steps: denaturation (90-98℃, causing DNA double-strand unwinding), annealing (50-65℃, allowing primers to bind to single-stranded templates), and extension (usually 72℃, allowing DNA polymerase to synthesize new strands). This approach has some inherent limitations: 1. Since the three steps of denaturation, annealing, and extension need to be completed within the same module, each cycle requires a process of heating-holding-cooling. In particular, the cooling process mainly relies on the cooling element, and a single temperature conversion can take 30-60 seconds, resulting in an overall testing cycle of 1-2 hours. In the diagnosis of periodontal disease, clinicians need to obtain test results quickly when patients visit in order to formulate immediate treatment plans. The long time consumption of traditional methods seriously limits their on-site application value.

[0004] 2. In order to achieve rapid temperature cycling, the temperature control module needs to be designed with powerful heating and cooling capabilities, which greatly increases the complexity and manufacturing cost of the instrument, and also leads to higher power consumption.

[0005] Therefore, there is an urgent need in this field for a method for detecting Pg bacteria that can shorten detection time, reduce equipment complexity, and ensure detection accuracy. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a fluorescence detection method for Pg bacteria based on dual-temperature zone cyclic amplification, which solves the problems of long detection time and high power consumption in the prior art that uses a single temperature zone module to cycle denaturation, annealing and extension steps, as well as the problems of increased instrument complexity and manufacturing cost.

[0007] To achieve the above and other related objectives, this invention provides a method for detecting Pg bacteria fluorescence based on dual-temperature zone cyclic amplification, comprising the following steps: S1. Initial fluorescence detection: Place the detection reagent containing the sample to be tested, primers, fluorescent dye or probe in the reaction container, move the reaction container under the light source detection module, and perform a fluorescence signal measurement on the detection reagent to obtain the initial fluorescence value F0. S2, Dual-temperature zone cyclic amplification: The reaction vessel is cyclically moved between the high-temperature isothermal module and the low-temperature isothermal module to complete N amplification cycles. Each amplification cycle includes first moving the reaction vessel to the high-temperature isothermal module to denature the detection reagent, and then moving the reaction vessel to the low-temperature isothermal module to anneal and extend the detection reagent. S3. Endpoint fluorescence detection: After completing the cyclic amplification in step S2, the reaction vessel is moved to the light source detection module again to perform a second fluorescence signal measurement on the detection reagent and obtain the endpoint fluorescence value F1. S4. Result Analysis: Calculate the change in fluorescence signal ΔF, where ΔF = F1 - F0. Determine the presence or content of Pg bacteria in the sample based on ΔF.

[0008] In one embodiment of the present invention, in step S2, the high-temperature isothermal module is constantly controlled at a first target temperature of 85°C-100°C, causing the double-stranded DNA of Pg bacteria to denature and break down into single strands.

[0009] In one embodiment of the present invention, in step S2, the cryogenic module is constantly controlled at a second target temperature of 50°C-70°C, so that the primers specifically bind to the single-stranded DNA template and are extended under the action of DNA polymerase to synthesize a new DNA strand.

[0010] In one embodiment of the present invention, step S4, determining the presence or content of Pg bacteria in the sample based on ΔF, specifically includes: Compare ΔF with the preset threshold 0. If ΔF is greater than 0, the test reagent is determined to be positive for Pg bacteria, that is, the test reagent contains Pg bacteria. If ΔF is less than 0, the test reagent is considered negative for Pg bacteria, meaning the test reagent does not contain Pg bacteria or the Pg bacteria content is below the detection range.

[0011] In one embodiment of the present invention, in step S4, determining the presence or content of Pg bacteria in the sample based on ΔF specifically includes: comparing ΔF with a preset standard curve or concentration range table for Pg bacteria content to determine the concentration range of Pg bacteria.

[0012] In one embodiment of the present invention, in step S2, the time spent in the high-temperature isothermal module during the dual-temperature zone cyclic amplification is 10-30 seconds, and the time spent in the low-temperature isothermal module is 20-50 seconds.

[0013] In one embodiment of the present invention, in step S2, the number of cycles N for the N amplification cycles is 30-45.

[0014] The present invention also provides a detection system for implementing the Pg bacteria fluorescence detection method based on dual-temperature zone cyclic amplification. The detection system includes a housing, an aluminum rod assembly disposed in the housing, a high-temperature constant temperature module, a low-temperature constant temperature module, a light source detection module, a moving mechanism for switching the moving reaction vessel between the three modules, a lifting mechanism for driving the current application module to rise and fall, and a control and processing unit for controlling the moving mechanism, collecting fluorescence data and performing result analysis. The high-temperature constant temperature module and the low-temperature constant temperature module have the same structure, both including: a temperature control block and a heating plate assembly installed inside the temperature control block. The heating plate assembly consists of a heating plate, a heating plate and a sensing coil connected between the heating plate and the heating plate. The light source detection module uses a blue LED as the excitation light source and a light sensor as a fluorescence signal detector. The moving mechanism includes a turntable and a moving drive motor. The turntable is mounted on the upper end of the aluminum rod assembly and has a reaction station on the turntable. The reaction container is placed on the reaction station. The moving motor is mounted on the housing. The moving drive motor and the turntable are connected by a set of meshing gears. The lifting mechanism includes a bracket and a lifting drive motor. The high-temperature constant temperature module, the low-temperature constant temperature module, and the light source detection module are sequentially mounted on the upper end of the bracket in a clockwise direction. The bracket is movably sleeved on the outside of the aluminum rod assembly. The lifting drive motor is mounted on the housing, and the lifting drive motor and the bracket are connected by a set of meshing gear and rack assembly.

[0015] In one embodiment of the present invention, fans are installed on the outer side of the temperature control block of the high-temperature constant temperature module and the low-temperature constant temperature module, as well as on the side of the casing opposite to the high-temperature constant temperature module and the low-temperature constant temperature module.

[0016] In one embodiment of the present invention, the first target temperature applied to the high-temperature constant temperature module is 85°C-100°C, and the second target temperature applied to the low-temperature constant temperature module is 50°C-70°C.

[0017] As described above, the Pg bacteria fluorescence detection method based on dual-temperature zone cyclic amplification of the present invention has the following beneficial effects: 1. This invention simplifies the traditional three-step denaturation, annealing, and extension process of qPCR into two steps: high-temperature denaturation and low-temperature annealing and extension. By setting up independent high-temperature and low-temperature isothermal modules, and using a moving and lifting mechanism to rapidly switch between the two temperature zones, the reaction vessel simplifies the process to just two steps: high-temperature denaturation and low-temperature annealing and extension. Utilizing the characteristic that DNA polymerase maintains high activity at 50-65℃, the annealing and extension steps can be completed in the same low-temperature zone, eliminating the time-consuming heating and cooling processes in traditional PCR instruments. This reduces the cycle time to tens of seconds, significantly shortening the detection cycle and meeting the needs of rapid on-site diagnosis. Furthermore, due to the high temperature... The constant temperature module and the low temperature constant temperature module are set independently. The high temperature constant temperature module can heat at full power and maintain a high temperature stability through a fan, while the low temperature constant temperature module can heat at low power and maintain a low temperature through a fan, without interfering with each other. The control system applies a precise current to the heating device, and heats the temperature control block to the preset target temperature through the electrothermal effect. Since the high temperature constant temperature module and the low temperature constant temperature module only need to maintain a constant temperature, there is no need for a complex programmed heating and cooling control system. Therefore, the requirements for temperature control components are lower, the instrument structure is simpler, the manufacturing cost and operating power consumption are significantly reduced, and it is easier to develop into a portable device.

[0018] 2. The high-temperature constant temperature module and the low-temperature constant temperature module of this invention can control the temperature independently. A single module can ensure a constant module temperature through closed-loop control of heating plate heating, real-time temperature monitoring by sensing coil, and fan-assisted heat dissipation, avoiding temperature overshoot and drift during the heating and cooling process of traditional single modules. The constant temperature environment of the dual temperature zones makes the denaturation, annealing and extension process of Pg bacterial DNA more stable, and the amplification efficiency is consistently improved. It avoids non-specific amplification or insufficient amplification efficiency caused by temperature fluctuations in traditional equipment, significantly reduces false positive and false negative rates, and avoids detection errors caused by temperature fluctuations.

[0019] 3. This invention introduces a difference analysis mode that combines initial fluorescence detection F0 with endpoint fluorescence detection F1. The result is determined by calculating ΔF = F1 - F0. F0 can subtract the inherent background fluorescence of reagents, reaction containers, and sample matrix in the detection system. ΔF only reflects the newly added specific fluorescence during the amplification process, namely the fluorescence generated by the binding of Pg bacterial DNA amplification products and fluorescent dyes. This effectively avoids the missed detection of low-concentration samples caused by background fluorescence interference in traditional qPCR, and improves the detection accuracy of low-concentration samples. At the same time, ΔF has a good linear relationship with the concentration of Pg bacteria, and accurate quantification can be achieved through a preset standard curve, providing a quantitative basis for assessing the severity of periodontal disease.

[0020] 4. This invention employs a detection scheme combining dual-temperature zone cyclic amplification and fluorescence difference analysis. Specific detection of Pg bacteria is achieved through specific primers and fluorescent dyes or probes. Therefore, by changing the specific primers and probes for different pathogenic microorganisms, such as Helicobacter pylori, influenza virus, and COVID-19, or for gene mutations, such as periodontal disease-related gene polymorphisms, it can be adapted to corresponding detection needs. Furthermore, without changing the hardware structure of the equipment, only the detection reagents need to be replaced to expand to fields such as digestive system infections, respiratory tract infections, and genetic disease gene detection, achieving multi-purpose functionality, reducing equipment investment costs, improving equipment utilization, and possessing broad industrial application prospects. Attached Figure Description

[0021] Figure 1 The flowchart shown is a process for detecting Pg bacteria fluorescence based on dual-temperature zone cyclic amplification disclosed in this invention.

[0022] Figure 2 The diagram shown is a schematic representation of the external structure of the detection system disclosed in this invention.

[0023] Figure 3 The diagram shown is an exploded view of the detection system disclosed in this invention.

[0024] Figure 4 The diagram shown is an enlarged structural schematic of the moving mechanism in the detection system disclosed in this invention.

[0025] Figure 5 Displayed as Figure 4 A structural diagram from another perspective.

[0026] Figure 6 The diagram shows the structure of the moving mechanism in the detection system disclosed in this invention, in conjunction with the high-temperature constant temperature module, the low-temperature constant temperature module, and the light source detection module.

[0027] Figure 7 Displayed as Figure 6 A schematic diagram of the structure of local decomposition.

[0028] Component designation explanation 1. Housing; 2. High temperature constant temperature module; 3. Low temperature constant temperature module; 4. Light source detection module; 5. Moving mechanism including turntable 51; 511 reaction station; 52. Moving drive motor; 53. Gear assembly; 6. Lifting mechanism; 61. Support; 62. Lifting drive motor; 63. Gear and rack assembly; 7. Temperature control block; 8. Heating plate assembly; 81. Heating plate; 82. Sensing coil; 83. Aluminum rod assembly; 9. Fan; 10. Detailed Implementation

[0029] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0030] Please see Figures 1 to 7 It should be understood that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes to aid those skilled in the art and to facilitate understanding. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to size, provided they do not affect the effectiveness or purpose of the invention, should fall within the scope of the disclosed technical content. Furthermore, the terms "upper," "lower," "left," "right," "middle," and "one" used in this specification are merely for clarity and not intended to limit the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention's implementation.

[0031] Example 1, please refer to Figures 1-3 This embodiment provides a fluorescence detection method for Pg bacteria based on dual-temperature zone cyclic amplification, including the following steps: S1. Initial fluorescence detection: Place the detection reagent containing the sample to be tested, primers, fluorescent dyes or probes into the reaction container, move the reaction container to the light source detection module 4, and perform a fluorescence signal measurement on the detection reagent to obtain the initial fluorescence value F0. S2. Dual-Temperature Cyclic Amplification: The reaction vessel is cyclically moved between the high-temperature isothermal module 2 and the low-temperature isothermal module 3 to complete N amplification cycles. Each amplification cycle includes first moving the reaction vessel to the high-temperature isothermal module 2 to denature the detection reagent. The high-temperature isothermal module 2 is constantly controlled at a first target temperature of 85℃-100℃, causing the double-stranded DNA of Pg bacteria to denature and break down into single strands. Then, the reaction vessel is moved to the low-temperature isothermal module 3 to anneal and extend the detection reagent. The low-temperature isothermal module 3 is constantly controlled at a second target temperature of 50℃-70℃, allowing the primers to specifically bind to the single-stranded DNA template and anneal, and then extending the DNA under the action of DNA polymerase to synthesize a new DNA strand. During the dual-temperature cyclic amplification, the single residence time in the high-temperature isothermal module 2 is 10-30 seconds, and the single residence time in the low-temperature isothermal module 3 is 20-50 seconds. The number of cycles N for the N amplification cycles is 30-45.

[0032] This invention simplifies the traditional three-step denaturation, annealing, and extension process of qPCR into two steps: high-temperature denaturation and low-temperature annealing and extension. By setting up independent high-temperature and low-temperature isothermal modules 2 and 3, and using a moving mechanism 5 and a lifting mechanism 6 to rapidly switch between the two temperature zones, the reaction vessel simplifies the process to two steps: high-temperature denaturation and low-temperature annealing and extension. Utilizing the characteristic that DNA polymerase maintains high activity at 50-65℃, the annealing and extension steps can be completed in the same low-temperature zone, eliminating the time-consuming heating and cooling processes in traditional PCR instruments. This reduces the cycle time to tens of seconds, significantly shortening the detection cycle and meeting the needs of rapid on-site diagnosis. Furthermore, since the high-temperature and low-temperature isothermal modules 2 and 3 are independently set up, they only need to maintain a constant temperature, eliminating the need for a complex programmed heating and cooling control system. Therefore, the requirements for temperature control components are lower, the instrument structure is simpler, and manufacturing costs and power consumption are significantly reduced, making it easier to develop into a portable device.

[0033] S3. Endpoint Fluorescence Detection: After completing the cyclic amplification in step S2, move the reaction vessel back to the light source detection module 4 to perform a second fluorescence signal measurement on the detection reagent and obtain the endpoint fluorescence value F. 1。 S4. Result Analysis: Calculate the change in fluorescence signal ΔF, ΔF = F1 - F0. Determine the presence or content of Pg bacteria in the sample based on ΔF. Specifically, determining the presence or content of Pg bacteria in the sample based on ΔF includes: comparing ΔF with a preset threshold of 0. If ΔF is greater than 0, the test reagent is determined to be positive for Pg bacteria, i.e., the test reagent contains Pg bacteria; if ΔF is less than 0, the test reagent is determined to be negative for Pg bacteria, i.e., the test reagent does not contain Pg bacteria or the Pg bacteria content is below the detection range. Alternatively, determining the presence or content of Pg bacteria in the sample based on ΔF includes: comparing ΔF with a preset Pg bacteria content standard curve or concentration range comparison table to determine the concentration range of Pg bacteria.

[0034] This invention introduces a difference analysis mode that combines initial fluorescence detection F0 with endpoint fluorescence detection F1. The result is determined by calculating ΔF = F1 - F0. F0 can subtract the inherent background fluorescence of reagents, reaction containers, and sample matrix in the detection system. ΔF only reflects the newly added specific fluorescence during the amplification process, namely the fluorescence generated by the binding of Pg bacterial DNA amplification products and fluorescent dyes. This effectively avoids the missed detection of low-concentration samples caused by background fluorescence interference in traditional qPCR, and improves the detection accuracy of low-concentration samples. At the same time, ΔF has a good linear relationship with the concentration of Pg bacteria, and accurate quantification can be achieved through a preset standard curve, providing reference data for the assessment of the severity of periodontal disease.

[0035] Example 2, please refer to Figures 2-7This embodiment provides a detection system for implementing the Pg bacteria fluorescence detection method based on dual-temperature zone cyclic amplification described in Embodiment 1. The detection system includes a housing 1, an aluminum rod assembly 9 disposed in the housing 1, a high-temperature isothermal module 2, a low-temperature isothermal module 3, a light source detection module 4, a moving mechanism 5 for switching the reaction vessel between the three modules, a lifting mechanism 6 for driving the current application module to rise and fall, and a control and processing unit for controlling the moving mechanism, collecting fluorescence data, and performing result analysis. Fans 10 are installed on the outside of the temperature control blocks 7 of the high-temperature isothermal module 2 and the low-temperature isothermal module 3, and on the side of the housing 1 opposite to the high-temperature isothermal module 2 and the low-temperature isothermal module 3. The first target temperature applied to the high-temperature isothermal module 2 is 85℃-100℃, and the second target temperature applied to the low-temperature isothermal module 3 is 50℃-70℃.

[0036] The high-temperature constant temperature module 2 and the low-temperature constant temperature module 3 have the same structure, both including: a temperature control block 7 and a heating plate assembly 8 installed inside the temperature control block 7. The heating plate assembly 8 consists of a heating plate 81, a heating plate 82, and a sensing coil 83 connected between the heating plate 81 and the heating plate 82. The high-temperature constant temperature module 2 and the low-temperature constant temperature module 3 of the present invention can independently control the temperature. A single module can ensure a constant module temperature through closed-loop control of heating by the heating plate 81, real-time temperature monitoring by the sensing coil 83, and auxiliary heat dissipation by the fan 10, avoiding temperature overshoot and drift during the heating and cooling process of traditional single modules. The constant temperature environment of the dual temperature zones makes the denaturation, annealing, and extension processes of Pg bacterial DNA more stable, and the amplification efficiency is consistently improved. This avoids non-specific amplification or insufficient amplification efficiency caused by temperature fluctuations in traditional equipment, significantly reduces false positive and false negative rates, and avoids detection errors caused by temperature fluctuations.

[0037] The light source detection module 4 uses a blue LED as the excitation light source and a light sensor as a fluorescence signal detector. The moving mechanism 5 includes a turntable 51 and a moving drive motor 52. The turntable 51 is mounted on the upper end of the aluminum rod assembly 9, and a reaction station 511 is provided on the turntable 51, on which the reaction container is placed. The moving motor 52 is mounted on the housing 1. The moving drive motor 52 and the turntable 51 are connected by a set of meshing gear assemblies 53. The moving drive motor 52 provides the moving driving force, which, together with the set of meshing gear assemblies 53, drives the turntable 51 to rotate, so that the reaction station 511 on which the reaction container is placed corresponds to any one of the high temperature constant temperature module 2, the low temperature constant temperature module 3, and the light source detection module 4. The lifting mechanism 6 includes a bracket 61 and a lifting drive motor 62. The high-temperature constant temperature module 2, the low-temperature constant temperature module 3, and the light source detection module 4 are sequentially mounted clockwise on the upper end of the bracket 61. The bracket 61 is movably sleeved on the outside of the aluminum rod assembly 9. The lifting drive motor 62 is mounted on the housing 1. The lifting drive motor 62 and the bracket 61 are connected by a set of meshing gear and rack assembly 63. After the moving mechanism 5 moves the reaction vessel to the corresponding module, it provides lifting driving force through the lifting drive motor 62, and works with the set of meshing gear and rack assembly 63 to lift and lower the bracket 61, so that the high-temperature constant temperature module 2 or the low-temperature constant temperature module 3 comes into contact with the reaction vessel. Springs are provided at the lower ends of the reaction station 511, the high-temperature constant temperature module 2, and the low-temperature constant temperature module 3. When the temperature control block 7 rises to the point where the reaction container is fully inserted, the lifting mechanism 6 continues to rise slightly, compressing the springs and generating a continuous elastic force that tightly presses the inner surface of the interface of the temperature control block 7 against the outer surface of the reaction container, eliminating air gaps and achieving efficient heat conduction over the maximum area.

[0038] This invention simplifies the traditional three-step qPCR process of denaturation, annealing, and extension into two steps: high-temperature denaturation and low-temperature annealing / extension. By setting up independent high-temperature and low-temperature isothermal modules 2 and 3, and using a moving mechanism 5 and a lifting mechanism 6 to rapidly switch between the two temperature zones, the reaction vessel simplifies the process to two steps: high-temperature denaturation and low-temperature annealing / extension. Utilizing the characteristic that DNA polymerase maintains high activity at 50-65℃, the annealing and extension steps can be completed in the same low-temperature zone, eliminating the time-consuming heating and cooling processes in traditional PCR instruments. This reduces the cycle time to tens of seconds, significantly shortening the detection cycle and meeting the needs of rapid on-site diagnosis. Furthermore, because the high-temperature and low-temperature isothermal modules 2 and 3 are independently set up, the high-temperature isothermal module 2 can be heated at full power and continuously heated. The high-temperature constant temperature module 2 and the low-temperature constant temperature module 3 can be heated with low power and maintained at a low temperature through the fan 10, without interfering with each other. The control system applies a precise current to the heating device, and the temperature control block is heated to the preset target temperature through the electrothermal effect. Since the high-temperature constant temperature module 2 and the low-temperature constant temperature module 3 only need to maintain a constant temperature, there is no need for a complex programmed temperature rise and fall control system. Therefore, the requirements for temperature control components are lower, the instrument structure is simpler, the manufacturing cost and operating power consumption are significantly reduced, and it is easier to develop into a portable device. Of course, this invention adopts a dual-temperature zone, but the actual implementation is not limited to a dual-temperature zone. The inventors can also use three or more multi-temperature zones. Therefore, multi-temperature zones are also within the protection scope of this invention.

[0039] In this invention, each temperature controller, whether used for high or low temperatures, is an independent and precise temperature control unit. Each temperature controller contains a heater directly mounted on a temperature control block with excellent thermal conductivity. The control system applies a precise current to the heater, heating the temperature control block 7 to a preset target temperature, such as 90°C for high-temperature denaturation, through the electrothermal effect. Each temperature controller is independently equipped with a fan 10, which removes excess heat and environmental influences generated during heating through forced convection, stabilizing the temperature at the preset level. The heaters and fans 10 of the high-temperature constant temperature module 2 and the low-temperature constant temperature module 3 are independently driven by the control system. One module can heat at full power and maintain high-temperature stability through the fan, while the other low-temperature constant temperature module can heat at low power and maintain low temperature through the fan, without interfering with each other.

[0040] Example 3, based on Examples 1 and 2, specifically implements a method for detecting dental plaque fluorescence based on dual-temperature zone cyclic amplification, including: 1) Implementation conditions and equipment: High-temperature constant temperature module 2: adopts a constant temperature metal bath, which is constantly set at 90℃.

[0041] Low-temperature constant temperature module 3: adopts a constant temperature metal bath, which is constantly set at 54℃.

[0042] Light source detection module 4: Uses blue LED as excitation light source and light sensor as fluorescence signal detector.

[0043] Reaction vessel: specially designed transparent detection tube.

[0044] Detection reagents: contain primer pairs targeting the specific gene sequence of Pg bacteria, SYBR Green I fluorescent dye, DNA polymerase, dNTPs, and a buffer system.

[0045] 2) Testing steps: ① Sample preparation and loading: Use a sampling tube to take 1 μL of the dental plaque sample to be tested, insert it into the test tube which already contains 4 μL of test reagent, and tighten the cap.

[0046] ② Initial fluorescence value F0 measurement: The detection tube is moved to the detection position of the light source detection module 4 by a motor. The blue LED is turned on for excitation, and the fluorescence intensity emitted by the reagent at this time is detected and recorded as the initial fluorescence value F0.

[0047] ③ Cyclic amplification: The number of loops is set to N=30 for this implementation.

[0048] First denaturation: Move the detection tube into the 85-100℃ high temperature constant temperature module 2 and hold for 10-30 seconds.

[0049] The process is repeated 30-45 times: a. Annealing and extension: Move the test tube into the 50-70℃ low temperature constant temperature module 3 and hold for 20-50 seconds.

[0050] b. Denaturation: Move the detection tube back to the 85-100°C high-temperature constant temperature module 2 and hold for 10-30 seconds.

[0051] ④ Measurement of endpoint fluorescence value F1: After the cycle is completed, the detection tube is moved back to the light source detection module 4 by the robotic arm, and the endpoint fluorescence value F1 is measured and recorded under the same conditions.

[0052] ⑤ Results Analysis: Calculate the fluorescence increment: ΔF = F1 - F0.

[0053] The preset judgment threshold is 0, which was determined after testing a large number of negative samples and low-concentration positive samples.

[0054] 3) Result determination: If ΔF ≥ 0, report a positive result for Pg bacteria; If ΔF < 0, then report a negative result for Pg bacteria.

[0055] Alternatively, three different display levels can be used: the relationship between ΔF and concentration is established in advance using standard samples, and the results are divided into multiple levels such as "negative (-)", "0-20 concentration (+)", "20-100 concentration (+)", and "100-1000 concentration (+)".

[0056] I. The specific reagent and consumable specifications for this embodiment are as follows: II. Testing Steps Sample preparation: manual operation beforehand, taking approximately 10 minutes; Sample collection: Use a sterile interdental brush to scrape approximately 1 mg of subgingival plaque from the subject and place it in a sampling tube containing 110 μL of enzyme-free pure water. Shake the tube up and down at least 10 times to suspend the bacterial solution. Add sample: Add 4 μL of test reagent to the special transparent test tube, then add 1 μL of test sample or standard / negative control, tighten the cap and centrifuge at 3000 rpm for 10 seconds, then place it in the reaction station 511 of the moving mechanism 5.

[0057] 2. Initial fluorescence value F0 measurement The control and processing unit drives the moving mechanism 5 to move the detection tube directly above the light source detection module 4; the lifting mechanism 6 lifts the light source detection module 4, so that the detection tube is in the detection optical path; the blue LED excitation light source is turned on with a power of 10mW, and the light sensor collects the fluorescence signal. After the fluorescence signal stabilizes, it is collected and recorded as the initial fluorescence value F0.

[0058] 3. Dual-temperature zone cyclic amplification Loop parameter settings: Number of loops N = 30 times, and the single loop process is as follows: High-temperature denaturation: The moving mechanism 5 moves the detection tube directly above the high-temperature module, and the lifting mechanism 6 drives the high-temperature module to rise and fit the detection tube, staying for 25 seconds to completely destrand the Pg bacterial DNA double strand; Low-temperature annealing / extension: The moving mechanism 5 moves the PCR tube directly above the low-temperature module, and the lifting mechanism 6 drives the low-temperature module to rise and fit the detection tube. It stays for 45 seconds to allow the primers to bind to the single-stranded DNA and complete the extension under the action of Taq enzyme. Cyclic monitoring: The control and processing unit records the number of module switching times and the temperature of each module in real time (an alarm is triggered when the error exceeds ±0.5℃) to ensure the stability of the amplification process.

[0059] 4. Measurement of endpoint fluorescence value F1 After the cyclic amplification is completed, the moving mechanism 5 moves the detection tube back to the light source detection module 4 and collects the fluorescence signal under the same conditions as the F0 measurement. After the fluorescence signal stabilizes, it is collected and recorded as the endpoint fluorescence value F1.

[0060] 5. Results Analysis The control and processing unit automatically calculates the fluorescence change ΔF = F1 - F0 and calls up a preset standard curve, from 20-10 6 The CFU / mL standard test results were plotted, and the nonlinear curve equation was y = 12.8·x. 0.85 -1982, x is the concentration of lg Pg bacteria, CFU / mL, and y is ΔF.

[0061] III. Result Determination 1. Qualitative judgment based on threshold Threshold determination: By testing 20 negative controls, including dental plaque from healthy individuals and enzyme-free pure water, the ΔF value range was calculated to be -1982 to 1294 RFU. The threshold value was taken as "mean ΔF of negative controls + 3 times the standard deviation" - 1638.5 + 3 × 296.47 = -749 RFU. Results criteria: If ΔF ≥ -749 RFU, it is considered Pg bacteria positive, meaning the sample contains Pg bacteria; if ΔF < -749 RFU, it is considered Pg bacteria negative, meaning the sample does not contain Pg bacteria or the concentration is below the detection limit.

[0062] 2. Quantitative judgment based on standard curve Based on the ΔF value, consult the standard curve to determine the concentration range of Pg bacteria. The specific levels are divided as follows: IV. Method Validation Data Fifty clinical dental plaque samples were selected, including 20 samples that were known to be positive for Pg bacteria and 30 samples that were negative. This method was used for detection, with conventional qPCR (gold standard) used as a control to verify the method's performance. This embodiment clearly defines the precise parameters, reagent composition and dosage, and standard gradient of the dual-temperature zone module and the light source detection module 4, ensuring high repeatability and standardization of the detection method and avoiding errors caused by operational differences. The entire process requires only manual sample collection and addition; all other steps are automated by the control unit, reducing errors caused by human intervention, making it suitable for non-professional operation, and lowering the barrier to on-site testing. It innovatively combines the difference analysis of ΔF=F1-F0 with the standard curve y=12.8·x 0.85 The method, developed in 1982, enables both qualitative and quantitative identification of Pg bacteria. Validated with 50 clinical samples, its sensitivity and specificity are consistent with traditional qPCR, achieving an accuracy of 98%. The entire process takes only about 55 minutes, a 54.2% reduction compared to traditional qPCR, meeting the needs for rapid on-site diagnosis. Furthermore, by clarifying precautions such as sample processing, temperature preheating, and avoidance of strong light, the reliability of the detection is further ensured. This effectively promotes the transformation of this detection method from theory to a clinically applicable practical technology, providing accurate and efficient Pg bacteria detection support for periodontal disease diagnosis and treatment.

[0063] In summary, this invention employs a detection scheme combining dual-temperature zone cyclic amplification and fluorescence difference analysis. Specific detection of Pg bacteria is achieved through specific primers and fluorescent dyes or probes. Therefore, by changing the specific primers and probes targeting different pathogens, such as Helicobacter pylori, influenza virus, and SARS-CoV-2, or targeting gene mutations, such as periodontal disease-related gene polymorphisms, it can be adapted to various detection needs. Furthermore, without altering the equipment hardware structure, only the detection reagents need to be replaced, allowing expansion into fields such as digestive system infections, respiratory infections, and genetic disease gene detection. This achieves multi-purpose functionality, reduces equipment investment costs, and improves equipment utilization, demonstrating broad industrial application prospects. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and possesses high industrial application value.

[0064] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A fluorescence detection method for Pg bacteria based on dual-temperature zone cyclic amplification, characterized in that, Includes the following steps: S1. Initial fluorescence detection: Place the detection reagent containing the sample to be tested, primers, fluorescent dye or probe in the reaction container, move the reaction container under the light source detection module, and perform a fluorescence signal measurement on the detection reagent to obtain the initial fluorescence value F0. S2, Dual-temperature zone cyclic amplification: The reaction vessel is cyclically moved between the high-temperature isothermal module and the low-temperature isothermal module to complete N amplification cycles. Each amplification cycle includes first moving the reaction vessel to the high-temperature isothermal module to denature the detection reagent, and then moving the reaction vessel to the low-temperature isothermal module to anneal and extend the detection reagent. S3. Endpoint fluorescence detection: After completing the cyclic amplification in step S2, the reaction vessel is moved to the light source detection module again to perform a second fluorescence signal measurement on the detection reagent and obtain the endpoint fluorescence value F1. S4. Result Analysis: Calculate the change in fluorescence signal ΔF, where ΔF = F1 - F0. Determine the presence or content of Pg bacteria in the sample based on ΔF.

2. The method for detecting Pg bacteria fluorescence based on dual-temperature zone cyclic amplification according to claim 1, characterized in that: In step S2, the high-temperature isothermal module is kept constant at a first target temperature of 85℃-100℃, causing the double-stranded DNA of Pg bacteria to denature and break down into single strands.

3. The method for detecting Pg bacteria fluorescence based on dual-temperature zone cyclic amplification according to claim 2, characterized in that: In step S2, the cryostat module is kept constant at a second target temperature of 50°C-70°C, which allows the primers to specifically bind to the single-stranded DNA template and extend under the action of DNA polymerase to synthesize a new DNA strand.

4. The method for detecting Pg bacteria fluorescence based on dual-temperature zone cyclic amplification according to claim 1, characterized in that, In step S4, determining the presence or content of Pg bacteria in the sample based on ΔF specifically includes: Compare ΔF with the preset threshold 0. If ΔF is greater than 0, the test reagent is determined to be positive for Pg bacteria, that is, the test reagent contains Pg bacteria. If ΔF is less than 0, the test reagent is considered negative for Pg bacteria, meaning the test reagent does not contain Pg bacteria or the Pg bacteria content is below the detection range.

5. The method for detecting Pg bacteria fluorescence based on dual-temperature zone cyclic amplification according to claim 1, characterized in that, In step S4, determining the presence or content of Pg bacteria in the sample based on ΔF specifically includes comparing ΔF with a pre-set standard curve or concentration range table for Pg bacteria content to determine the concentration range of Pg bacteria.

6. The method for detecting Pg bacteria fluorescence based on dual-temperature zone cyclic amplification according to claim 1, characterized in that: In step S2, the time spent in the high-temperature isothermal module during the dual-temperature zone cyclic amplification is 10-30 seconds, and the time spent in the low-temperature isothermal module is 20-50 seconds.

7. The method for detecting Pg bacteria fluorescence based on dual-temperature zone cyclic amplification according to claim 6, characterized in that: In step S2, the number of cycles N for the N amplification cycles is 30-45.

8. A detection system for implementing the Pg bacteria fluorescence detection method based on dual-temperature zone cyclic amplification as described in any one of claims 1-7, characterized in that, The detection system includes a housing (1), an aluminum rod assembly (9) housed in the housing (1), a high-temperature constant temperature module (2), a low-temperature constant temperature module (3), a light source detection module (4), a moving mechanism (5) for switching the moving reaction vessel between the three modules, a lifting mechanism (6) for driving the current application module to rise and fall, and a control and processing unit for controlling the moving mechanism, collecting fluorescence data and performing result analysis. The high-temperature constant temperature module (2) and the low-temperature constant temperature module (3) have the same structure, both including: a temperature control block (7) and a heating plate assembly (8) installed inside the temperature control block (7). The heating plate assembly (8) consists of a heating plate (81), a heating plate (82) and a sensing coil (83) connected between the heating plate (81) and the heating plate (82). The light source detection module (4) uses a blue LED as the excitation light source and a light sensor as a fluorescence signal detector. The moving mechanism (5) includes a turntable (51) and a moving drive motor (52). The turntable (51) is mounted on the upper end of the aluminum rod assembly (9), and a reaction station (511) is provided on the turntable (51). The reaction container is placed on the reaction station (511). The moving motor (52) is mounted on the housing (1). The moving drive motor (52) and the turntable (51) are connected by a set of meshing gear assemblies (53). The lifting mechanism (6) includes a bracket (61) and a lifting drive motor (62). The high temperature constant temperature module (2), the low temperature constant temperature module (3), and the light source detection module (4) are installed in a clockwise direction on the upper end of the bracket (61). The bracket (61) is movably sleeved on the outside of the aluminum rod assembly (9). The lifting drive motor (62) is installed on the housing (1). The lifting drive motor (62) and the bracket (61) are connected by a set of meshing gear and rack assembly (63).

9. The detection system according to claim 8, characterized in that: Fans (10) are installed on the outside of the temperature control block (7) of the high temperature constant temperature module (2) and the low temperature constant temperature module (3), as well as on the side of the casing (1) relative to the high temperature constant temperature module (2) and the low temperature constant temperature module (3).

10. The detection system according to claim 8, characterized in that: The first target temperature applied to the high-temperature constant temperature module (2) is 85℃-100℃, and the second target temperature applied to the low-temperature constant temperature module (3) is 50℃-70℃.

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