CN218202839U

CN218202839U - Portable real-time fluorescence quantitative nucleic acid diagnostic apparatus

Info

Publication number: CN218202839U
Application number: CN202221231282.2U
Authority: CN
Inventors: 郭确; 陈熹; 杨磊
Original assignee: Maplelab Scientific Shenzhen Technology Co ltd
Current assignee: Maplelab Scientific Shenzhen Technology Co ltd
Priority date: 2022-05-23
Filing date: 2022-05-23
Publication date: 2023-01-03
Anticipated expiration: 2032-05-23

Abstract

The utility model discloses a portable real-time fluorescence quantitative nucleic acid diagnostic apparatus, which comprises a shell, a PCR amplification device, an automatic start-stop device, an NFC receiver, a Bluetooth and WIFI transmitting receiver, a light path system consisting of a fluorescence excitation device and a fluorescence detection device, a control circuit and a display, wherein the PCR amplification device, the automatic start-stop device, the NFC receiver, the Bluetooth and WIFI transmitting receiver are fixedly connected on the shell; the PCR amplification device comprises an amplifier; the fluorescent detection device is characterized in that a sample reaction chamber is arranged on the amplifier, the fluorescence excitation device emits light to irradiate the sample reaction chamber through a side hole formed in the amplifier, and the fluorescence emitted by the fluorescence detection device after the sample in the sample reaction chamber is amplified is excited by the light, so that the real-time fluorescent quantitative detection is realized. The utility model discloses a diagnostic apparatus, but both off-line measuring, through the display result promptly, but also the networking detects, with diagnostic data upload to the database, both can supply non professional to operate, still can regard as the single module of the nucleic acid diagnostic system of extensive networking operation to use, have characteristics such as small, low price, easy operation.

Description

Portable real-time fluorescence quantitative nucleic acid diagnostic apparatus

Technical Field

The utility model relates to a medical treatment detects technical field, specifically, relates to a portable real-time fluorescence quantitative nucleic acid diagnostic apparatus.

Background

The main methods for detecting virus infection are nucleic acid and antigen detection, but antigen detection can only be known after human body is infected to generate antibody, and nucleic acid detection can be detected in the early stage of infection without symptoms, so nucleic acid detection is the most effective and direct diagnostic method at present. Nucleic acid detection using different amplification systems and polymerases typically involves different nucleic acid diagnostic methods formed by isothermal amplification, such as Reverse Transcription (RT) -isothermal amplification, loop-mediated isothermal amplification (LAMP), PCR three-stage amplification, and nested PCR reaction modes. The traditional specialized real-time fluorescence quantitative nucleic acid instrument can be used for diagnosing all the nucleic acid amplification modes, but is expensive, large in size, complex to operate and can be operated by specialized personnel with considerable time training.

The real-time fluorescent quantitative PCR technology is a method for adding a fluorescent group into a PCR reaction system, monitoring the whole PCR process in real time by using fluorescent signal accumulation, and finally carrying out quantitative analysis on an unknown template through a standard curve. The existing real-time fluorescent quantitative PCR instrument still needs to be trained by professional personnel to be competent. The cost of the reagent for detecting the antibody is more expensive than that of a plurality of current antigen kits and antigen detectors, and the cost of the reagent for detecting the nucleic acid is higher than that of the current nucleic acid diagnostic reagent due to the fact that a special reagent card and a sampling rod are needed, so that the reagent is difficult to popularize and use.

SUMMERY OF THE UTILITY MODEL

An object of the utility model is to provide a portable real-time fluorescence quantitative nucleic acid diagnostic apparatus to solve the problem that proposes among the above-mentioned background art.

In order to achieve the above purpose, the utility model provides a following technical scheme:

a portable real-time fluorescence quantitative nucleic acid diagnostic apparatus comprises a shell, a PCR amplification device, an automatic start-stop device, an NFC receiver, a Bluetooth and WIFI emission receiver, a light path system, a control circuit and a display, wherein the PCR amplification device, the automatic start-stop device, the NFC receiver, the Bluetooth and WIFI emission receiver, the light path system, the control circuit and the display are fixedly connected to the shell; the control circuit is respectively and electrically connected with the PCR amplification device, the automatic start-stop device, the NFC receiver, the Bluetooth and WIFI emission receiver, the fluorescence excitation device and the fluorescence detection device;

the shell comprises an upper shell and a lower shell; the front end of the upper shell is provided with a display opening, the middle end of the upper shell is provided with a sample loading opening, the display comprises an LCD screen, and the LCD screen is fixed at the display opening of the upper shell;

the PCR amplification device comprises an amplifier, a temperature sensor, a heating refrigerator, a radiator and a fan; the amplifier is provided with a plurality of sample reaction chambers for placing reagent containers; the temperature sensor and the amplifier are integrally arranged, the heating refrigerator is arranged below the amplifier, the radiator is arranged below the heating refrigerator, and the fan is arranged below the radiator;

in order to make the temperature more uniform and stable by heat conduction, it is preferable that a graphite heat-conducting sheet is provided between the heating refrigerator and the amplifier.

In order to improve the amplification efficiency and ensure the stability and reliability of the structure, preferably, the amplifier comprises a plate-shaped structure and a ridge-shaped structure which are integrally arranged, the ridge-shaped structure is arranged in the middle of the plate-shaped structure, and the sample reaction chamber is a cavity arranged on the ridge-shaped structure; the plate-shaped structures of the radiator, the heating refrigerator, the graphite heat-conducting fin, the temperature sensor and the amplifier are fixedly connected with the upper shell, and the ridge-shaped structure on the amplifier extends out of the sample loading opening of the upper shell.

The display, the temperature sensor and the heating cooler are respectively and electrically connected with the control circuit through respective lead wires; the fan is arranged below the radiator and connected with the lower casing, the lower casing is provided with a vent for facilitating heat dissipation, and a power input line of the fan is connected with the control circuit through a plug.

The sample reaction chamber is used for accommodating a reagent container and carrying out detection diagnosis.

In order to facilitate the user to read the information displayed by the display, preferably, the display is a liquid crystal display, and an LCD screen mounting plane of the liquid crystal display forms an angle with a rear end plane of the upper housing, where the angle is preferably 135 degrees.

In order to enable the heating refrigerator to directly adjust the temperature of the reagent tube from the bottom so as to ensure the heating efficiency and uniformity and facilitate the accommodation of the reaction reagent tube, the sample reaction chamber is preferably provided with a cylindrical streamline hole groove matched with the reagent tube.

In order to facilitate sample detection and comparison, preferably, the diagnostic apparatus adopts three-in-one reagent tubes which are a connecting test tube and a sampling reagent bottle specially used for collecting sample reagents, and the connecting test tubes are a sample reagent tube, a positive control reagent tube and a negative control reagent tube respectively; the number of the sample reaction chambers is 3, and the sample reaction chambers are respectively and correspondingly provided with a sample, a positive control reagent tube and a negative control reagent tube; the reagent tube is a transparent cylindrical tube, a corresponding trinity transparent tube cover is arranged on the reagent tube, 1 cylindrical positioning pin is arranged at the outer end of the sample reagent tube of the tube cover, and correspondingly, a positioning hole is arranged on the amplifier. Thus, when the tube cover is covered and placed in the sample reaction chamber, the positioning pin is embedded into the positioning hole at the sample side in the sample reaction chamber of the amplifier, so that the placing position of the sample reagent tube is ensured to be correct, and the diagnosis error caused by the position of the confounded sample and the position of the contrast reagent tube is avoided. The tube caps of the positive control reagent tube and the negative control reagent tube are sealed and covered after the reagents are manufactured, so that the reagents are prevented from overflowing to cause pollution in the transportation process, the tubes and the caps are prevented from being separated, diagnosis errors caused by position confusion during capping are avoided, the sample reagent tubes do not contain any reagents, and the sample reagents are put into the sampling reagent bottles.

For realizing fluorescence excitation better, make the light path act on reagent better, it is preferred, a side opening has been seted up respectively apart from bottom third department in sample reaction chamber one side to the augmenter, light path system's fluorescence excitation device is used for shining the sample and excites fluorescence, including exciting light circuit board and weld three group patch type emitting diode on it, three group patch type emitting diode set up respectively in the position that corresponds with the side opening of sample reaction chamber, and the exciting light that patch type emitting diode sent jets into the sample reaction chamber through the side opening as light path passageway, excites the fluorophore in the sample and sends fluorescence. Because the liquid level in the reagent pipe is usually in the height position 2/3 apart from the sample reaction chamber bottom, can make light shine the middle part position at the reagent liquid through the side opening of light like this, and because sample reaction chamber temperature variation factor lies in the bottom mainly, set up the side opening still can not cause very big influence to rising and falling temperature efficiency and temperature homogeneity like this.

In order to more accurately measure the temperature of the sample reaction chamber and thus the sample reagent tube, preferably, the lower side of the amplifier is provided with sensor grooves corresponding to the positions of the sample reaction chamber, and the temperature sensors are fixedly mounted in the sensor grooves respectively.

In order to make the heat transfer of the heating aluminum plate more uniform, preferably, a layer of graphite heat conducting sheet is arranged below the amplifier. Here, the amplifier was made of a heated aluminum plate. The radiator is arranged below the semiconductor heating and refrigerating sheet, the plate-shaped structures of the radiator, the semiconductor heating and refrigerating sheet, the graphite heat conducting sheet, the temperature sensor and the amplifier are fixedly connected with the upper shell through screws, and the ridge-shaped structure on the amplifier extends out of the sample loading opening of the upper shell. The temperature sensor adopts an NTC sensor.

Preferably, the heating and cooling device is a semiconductor heating and cooling plate, the maximum power of the semiconductor heating and cooling plate is about 40 watts, the input voltage is 12 volts, namely Peltier (Peltier), the temperature rising and falling process is completed by changing the direction of the output direct current through a control circuit, and a relay capable of changing the direction of the direct current is arranged in the control circuit.

Preferably, the automatic start-stop device further comprises a sealing cover and a photosensitive diode; the upper machine shell is provided with a locking port and a photosensitive port, and the photosensitive diode is electrically connected with the control circuit and fixedly arranged at the photosensitive port; the front end of the sealing cover is provided with a hinge with a spring, and the rear end of the sealing cover is provided with a lock catch and a light blocking column; the front end of the sealing cover is hinged with the upper shell through a hinge, the rear end of the sealing cover is in open-close type clamping connection with the locking opening through a lock catch, and the light blocking column shields the photosensitive opening when the sealing cover is covered. Therefore, the opening and closing of the sealing cover can be controlled through the lock catch and the lock opening, when the sealing cover is opened, the sealing cover can be automatically popped out and kept in an open state due to the elasticity of the spring, and the sample reaction chamber is displayed, so that a sample reagent tube can be conveniently placed; the control circuit can automatically sense the opening and closing state of the sealing cover through the matching of the light blocking column, the photosensitive port and the photosensitive diode, so that the diagnostic instrument is automatically started or closed, and the diagnostic instrument is automatically started when the sealing cover is closed; when the sealing cover is opened, the light blocking column is moved away, the photosensitive diode senses that light exists, and the diagnostic instrument is closed.

Furthermore, the automatic start-stop device also comprises a sealing cover and a photosensitive diode; the upper machine shell is provided with a locking port and a photosensitive port, and the photosensitive diode is electrically connected with the control circuit and fixedly arranged at the photosensitive port; the front end of the sealing cover is provided with a hinge with a spring, and the rear end of the sealing cover is provided with a lock catch and a light blocking column; the front end of the sealing cover is hinged with the upper shell through a hinge, the rear end of the sealing cover is in open-close type clamping connection with the locking opening through a lock catch, and the light blocking column shields the photosensitive opening when the sealing cover is covered. Therefore, the function of automatically starting and stopping the diagnostic instrument can be realized under the algorithm programming control of the control circuit.

In order to realize better fluorescence detection effect, the reagent tube is fixed and sealed by the sealing cover, and further, the fluorescence detection device of the optical path system is used for measuring fluorescence intensity and comprises a photoelectric circuit board and 3 groups of photodiodes welded on the outer side of the photoelectric circuit board, wherein the inner side of the photoelectric circuit board is fixedly arranged at the middle section of the sealing cover, and the 3 groups of photodiodes vertically correspond to the upper end opening of the sample reaction chamber one by one and are tightly contacted with the sample reagent tube on the sample reaction chamber to form three independent fluorescence detection optical paths when the sealing cover is covered; a photoelectric circuit board is fixedly arranged on the outer side of the photoelectric circuit board, vertical through holes are respectively formed in the positions of the 3 groups of photodiodes, and when the sealing cover is covered, the outer side of the photoelectric circuit board is hermetically pressed at the upper end of the reagent tube; the upper shell is provided with a wire hole, and the photodiode penetrates through the wire hole through a lead to be electrically connected with the main control circuit. Here, the positions of the 3 groups of patch type exciting light diodes of the fluorescence excitation device are respectively opposite to the side holes of the 3 sample reaction chambers; the light is emitted by the light-emitting diode, irradiates the reagent tube through the corresponding side hole, excites the fluorescent group generated by amplification to generate scattered fluorescence, the upward scattered fluorescence is irradiated on the photodiode through the vertical through hole on the photoelectric through board, the electric signal generated by the photodiode is transmitted to the main control circuit, and the result is obtained through analysis of an algorithm program.

The light path board is preferably a Polytetrafluoroethylene (PTFE) board, has the same length and width as the photoelectric circuit board, and has the functions of isolating heat generated by the sample reaction chamber and forming independent closed light paths of the three sample tubes. The light path detection module forms three paths of non-interfering light paths which are respectively used for detecting a target object, a positive control sample and a negative control sample; each group of light paths are isolated from the outside, so that the interference of an external light source is reduced, the light path paths of exciting light and fluorescence are very short, the efficiency of detecting the fluorescence intensity is higher than that of the fluorescence intensity detected by the prior art, the structure of mirror reflection is higher, the interference of fluorescence detection is reduced, and a filter is not needed; one side of each sample chamber is provided with a corresponding exciting light source; the front end of the sealing cover is hinged with the upper shell through a hinge, the rear end of the sealing cover is in openable clamping with the locking port through a lock catch, the light blocking column shields the photosensitive port when the sealing cover is covered, the middle section of the sealing cover is provided with a fluorescence intensity detection device containing three photodiodes, and the sealing cover is in close contact with a sample reagent tube on the sample chamber to form three independent fluorescence detection light paths when the sealing cover is closed; in the real-time fluorescent quantitative detection, in the amplification process of a sample, exciting light at a side hole of a sample chamber excites fluorescent groups generated in the amplification process to emit fluorescence, and the fluorescence is transmitted to a fluorescent detection system through the top of a reagent tube to carry out real-time quantitative detection on the copy number of nucleic acid; the fluorescence reporter group selects the exciting light wavelength in the near ultraviolet range, the fluorescence emission wavelength is in the visible light wavelength, and the fluorescence spectrum is the emission spectrum and can be detected in the direction perpendicular to the incident light, so that the fluorescence is not interfered by the background of the exciting light; the exciting light adopts three groups of patch type light emitting diodes, which are respectively welded on an exciting light circuit board corresponding to the side hole position of the test tube on the sample reaction chamber, and then are tightly attached to a light path layer of Polytetrafluoroethylene (PTFE) containing a light guide hole and fixed with one side of the sample reaction chamber by screws, and power supply input wires of the three groups of exciting light are connected with a main control board by the circuit board through a plug; because of the totally closed and short distance optical path, the fluorescence detector can adopt a low-cost patch type photodiode instead of an expensive photomultiplier or CCD.

Preferably, the control circuit comprises a main control circuit board provided with a plurality of lead sockets, the main control circuit board is provided with a start-stop module, a display module, a heating and refrigerating module, a temperature transmission module, an AD module, a storage module, a photoelectric module, a fluorescence detection module, a Bluetooth and WIFI module and a CPU, and the display, the heating and refrigerating unit, the temperature sensor, the fan, the NFC sensor, the photoelectric board, the photosensitive sensor and the power supply are respectively and electrically connected with the main control circuit board through leads. Furthermore, the Bluetooth and WIFI module of the main control circuit board is connected with a mobile phone through Bluetooth, connected with the Internet through a corresponding mobile phone application program (APP), and also connected with the Internet through WIFI; SSID and initial password code required by WIFI network connection can be connected with a mobile phone through Bluetooth, then the password of the WIFI network is input from a mobile phone APP, namely a Bluetooth auxiliary distribution network, or the mobile phone APP sends the encrypted WIFI through an AP (access point) mode and an STA (wireless access point) mode and sends the SSID (wireless router name) and the password encrypted through the AES (user datagram protocol), or the mobile phone APP codes the SSID and the password into a UDP (user datagram protocol) message and sends the SSID and the password through a broadcast packet or a multicast message, a WIFI module of an instrument receives the UDP message and then decodes the UDP message to obtain the correct SSID and password, and then the connection is completed by actively connecting a route of the specified SSID, namely a SmartConfig mode; after connection, the instrument can be directly connected with a cloud server or even a server in a networking way through the Internet without a mobile phone and the Internet through the connection of WIFI and the Internet. The nucleic acid diagnostic instrument can be loaded with an opening of the Internet of things, and after the Internet of things card is inserted, the nucleic acid diagnostic instrument can be directly connected with the Internet. Each nucleic acid diagnostic instrument has a unique mac address (media access control address, i.e., the physical address of the nucleic acid diagnostic instrument) that can be used as an identification code for the instrument.

To better illustrate the present invention, the detection method is now described as follows:

the detection method of the diagnostic apparatus specifically comprises the following steps:

s1, a user samples and then puts the sample into a sampling reagent bottle, the mixed or processed sample is poured into a sample reagent tube, a reagent cover is covered and placed in a sample reaction chamber, and the sealing cover is in an open state;

s2, turning on a power supply, enabling the instrument to enter a self-checking mode, and checking whether the control circuit board, circuit elements connected with the control circuit board, the NFC module, the PCR amplification system and the optical path system work normally or not in the self-checking mode when the instrument is started; if not, prompting error information and stopping the next operation of the instrument; otherwise, through self-checking, after the instrument display prompts a user to input the operating parameters of the instrument, the sample reagent information and the input parameters, the display displays the related sample reagent information and starts the instrument to heat to the set initial temperature of PCR amplification;

s3, when the temperature of the sample reaction chamber reaches a constant value, the instrument reminds a user to cover the sealing cover through the liquid crystal display and press the lock; after the sealing cover is locked, a light source of a photosensitive diode is blocked or a lead passage with a reed is conducted, and an instrument is triggered to enter the detection process of nucleic acid amplification and fluorescence real-time detection; the instrument simultaneously collects fluorescence intensity data of a sample, a positive control and a negative control, wherein the fluorescence intensity of the positive control generates fluorescence intensity higher than a background after an amplification process enters 15-16 cycles, if the positive control does not generate fluorescence after 20 cycles, the result is invalid or false negative, the negative control does not generate fluorescence higher than the background in the whole detection process, if the positive control generates fluorescence, the result is false or false positive, when the sample generates fluorescence intensity higher than the background in the cycle process and generates enhanced or stable fluorescence intensity in two continuous cycles, and the positive control and the negative control do not generate abnormality, the positive control can be diagnosed, the diagnosis is finished, a Ct value (positive value) can be defined as the cycle number when the fluorescence intensity is higher than the background, if the sample does not generate fluorescence higher than the background in the set whole cycle process, the negative control can be defined as negative, and an analysis result is simultaneously stored in a memory and displayed by a liquid crystal display.

Preferably, in the temperature control process, the temperature sensor detects the temperature change of the amplifier to generate an analog signal, the analog signal is converted into a digital signal by the analog-to-digital conversion circuit and is sent to the CPU controller circuit, the digital signal is converted into a power signal through the processing of a PID temperature control program in the CPU controller, the power signal is output by the heating drive circuit, the heating refrigerator works, and the temperature is regulated and controlled through a PID mode in the temperature control process, so that the error range of the temperature is kept within 0.5 ℃;

PID mode accuse temperature comprises proportional unit P, integral unit I and differentiation unit D, wherein:

proportion unit P: the deviation of the system is reflected in proportion, and once the system has the deviation, the proportion adjustment immediately generates an adjusting effect to reduce the deviation; the proportion effect is large, the adjustment can be accelerated, and the error is reduced;

an integration unit I: the system eliminates steady state errors and improves the degree of freedom; when an error exists, integral adjustment is carried out; until there is no difference, the integral regulation is stopped, and the integral regulation outputs a constant value; the strength of the integral action depends on an integral time constant Ti, and the smaller the Ti is, the stronger the integral action is; otherwise, if Ti is large, the integral effect is weak;

a differentiation unit D: the differential action reflects the change rate of the system deviation signal, has foresight and can foresee the trend of the deviation change, thereby generating an advanced control action which is eliminated by the differential regulation action before the deviation is not formed; thereby improving the dynamic performance of the system; under the condition that the selection of the differential time is proper, overshoot can be reduced, and the adjusting time is reduced;

and the PID temperature control program sets parameters of the proportional unit P, the integral unit I and the differential unit D by measuring the temperature change rule of the sample chamber.

Thus, the PID temperature control program controls the temperature of the sensor chamber to a constant value of the set temperature + -0.5 degrees.

Preferably, in the step S2, the parameters of the instrument operation, the sample reagent information and the parameters (including the operation parameters required for operating the reagent and the calibration curve parameters of the batch of reagents required for quantitative detection) are written into the passive IC card, the NFC sensing module is closely attached to the lower part of the upper housing, and data transmission can be performed with the instrument through the corresponding NFC sensing area; the NFC induction module, namely the near field communication wireless communication module is matched with an IC card or other devices containing the NFC induction module, such as a mobile phone and the like, and the batch number and various operation parameters of the reagent are input from the outside and are led into the instrument; meanwhile, the NFC module can reversely write data of the instrument into an IC (integrated circuit) or an RFID (radio frequency identification) card or other devices containing the NFC induction module, such as a mobile phone, and the data are stored in the mobile phone through an application program and then transmitted to a database through the Internet.

Further, reagent information and operating parameters are described as:

1.X, reagent name number; 2.Y, reagent lot number; 3.z, the number of times of using the reagent batch number is written into the IC card reversely by the instrument; ti, initial temperature; t0, pretreatment temperature; t1, a first set temperature; t2, second set temperature; t3, third set temperature; st1, second stage first set temperature; st2, second stage second set temperature; st3, second stage third set temperature; 12.t0, holding time (sec) of pretreatment temperature; t1, hold time (seconds) of the first set temperature of the first and second stages; t2, holding time (seconds) of the second set temperature of the first and second stages; t3, holding time (seconds) of the third set temperature of the first and second stages; c1, number of cycles of the first phase; c2, number of cycles in the second stage; 18.S, number of stages, up to 2.

Description of the parameters: 1. the reagent name number is used for identifying different components and purposes of the reagent 2. The reagent batch number indicates the production batch of the reagent and the standard curve parameters of the batch of products, whether the reagent is invalid (namely the validity period) is confirmed through the batch number 3. The use times of the reagent are used for preventing the use of counterfeit kits. Parameters 4-16 were all used for different nucleic acid amplification regimes. The number of stages (S) is currently designated 1, but in nested (single-tube) amplification, stage S is an integer greater than 1 and less than 2; cycle number (C) is determined primarily by the specified diagnostic index, e.g., ct value, which is 40 for C40; in general, when S =2, if the polymerase used is the same and the denaturation temperature is the same, the second (ST 2) and third set temperatures (ST 2) of the second stage may be the same as the second (T2) and third set temperatures (T2) of the first stage, but in the mixed amplification mode of RT-PCR and three-stage PCR, the first, second, and third set temperatures of the first and second stages may be different, so it is necessary to add the ST1, ST2, ST3 temperature set value parameters of the second stage; in order to reduce the input value of the parameter, the different holding times t1, t2, t3 of the first, second and third setting temperatures of the first and second stages can be shared; before entering the nucleic acid amplification process, a pretreatment stage is set for directly releasing nucleic acid substances in a sample through heating and chemical reagents in the reagents without additional sample extraction and treatment processes, and the method is suitable for samples collected by throat swabs and reduces the sample treatment link; the starting temperature of the diagnostic instrument is typically set as a pre-process temperature value to save temperature equilibration time for the next sample.

The parameters can be increased or decreased as required, and the setting of the parameters is enough to operate the most common PCR amplification reaction at present, such as the traditional three-stage (i.e. denaturation, annealing and extension) amplification reaction, constant temperature amplification, such as RT-PCR (reverse transcription polymerase chain reaction) or LAMP (loop-mediated isothermal amplification), and mixed amplification of PCR, such as RT-PCR and traditional three-stage mixed amplification, and single-tube Nested amplification mode (Nested PCR), and the like, and can be used for the nucleic acid diagnosis of hundreds of different gene segments.

Preferably, the fluorescence detection is not through the nucleic acid amplification process, and in a traditional three-stage (i.e. denaturation, annealing and extension stage), the fluorescence detection only collects data in the annealing stage because the temperature in the denaturation stage is higher (> 85 ℃), although the temperature in the fluorescence detector is lower than that in the denaturation stage due to the thermal insulation layer, the fluorescence detector still has an interference effect on the performance of the photodiode to generate data fluctuation, the fluorescence intensity generated in the extension stage also generates data fluctuation due to the larger variation of the generated nucleic acid amplification product, the temperature in the annealing stage is lower, the nucleic acid amplification product is stable, and the data is most reliable; turning on the fluorescence detection unit (photodiode) throughout the diagnostic process reduces the lifetime of the detection unit, while taking into account the different amplification modes of the nucleic acid diagnostic instrument, which prescribes that data acquisition is started when amplification enters a set value of T1 (S = 1) or ST1 (S = 2), and ended when the temperature enters a set temperature of a different value or after the completion of the entire cycle; detecting data collected at the frequency of 1 time/second, calculating the average value and standard deviation of the stage, and storing the data for comparison, analysis and judgment; the data acquisition system consists of an amplifying circuit and an A/D conversion module, wherein an analog current signal generated by the photodiode is amplified by the amplifying circuit and enters the A/D conversion module, and a corresponding digital signal is output.

Preferably, the nucleic acid diagnostic apparatus can be subjected to online detection after being networked, and the method comprises the following specific steps:

t1, the mobile phone APP guides a user to register identity, including face recognition, the process of sampling and placing a sample and the video of the step S1 of off-line detection are obtained, and the video and information such as an instrument serial number read by a two-dimensional code on a scanning instrument are stored in a memory of the mobile phone;

t2, executing the step S2 of off-line detection, wherein the parameters of the operation of the input instrument and the information of the sample reagent can also be used for scanning the two-dimensional code of the sample reagent through a mobile phone APP, downloading parameters such as a batch number, a production date and a calibration curve of the sample reagent from a database, and inputting the parameters into the instrument through Bluetooth;

and T3, executing the step S3 of the off-line detection, transmitting the obtained diagnosis result into a memory of the mobile phone through Bluetooth, and transmitting the diagnosis result, the stored identity information and the sampling video to a database through the Internet.

Compared with the prior art, the beneficial effects of the utility model are that: the portable real-time fluorescence quantitative nucleic acid diagnostic instrument of the utility model can perform networking operation on a plurality of diagnostic instruments through the computer terminal; the computer can simultaneously connect a plurality of diagnostic instruments with the distribution network of the wireless router by adopting a SmartConfig mode through corresponding application software to form a terminal computer which is remotely connected with the plurality of diagnostic instruments in the same wireless local area network or through the Internet, and personal information, instrument serial numbers, reagent information, operation parameters and corresponding diagnostic results, which are collected by the mobile phone APP and correspond to the diagnostic instruments, are collected and stored in a memory of the computer or uploaded to a database. Each diagnostic apparatus can operate independently, samples can be sampled instantly, the operation is carried out instantly, data are uploaded instantly, and the method is also suitable for large-scale screening.

To sum up, the nucleic acid diagnostic apparatus of the present invention can be used for diagnosis of all the above-mentioned nucleic acid amplification modes, but has small volume, low price and simple operation, and not only can be used as a point-of-care diagnostic apparatus (POCT) for non-professional operation, but also can be used as a single module of a nucleic acid diagnostic system for large-scale networking operation; the PCR amplification system of the nucleic acid diagnostic instrument also adopts a mode that the conductor heating refrigerating sheet is directly contacted with the heating plate for amplification reaction, and the difference is that the sample reaction chambers on the sample plate are only arranged in a row or a column, thereby facilitating the design of a light path; the exciting light module of the nucleic acid diagnostic apparatus is arranged at one side of each sample reaction chamber, the exciting light is directly emitted to the sample liquid surface of the transparent test tube through the side hole of the sample reaction chamber through the closed light path, the generated fluorescence is emitted in the direction of 90 degrees and is emitted to the fluorescence detector-photodiode on the sealing cover through the transparent cover of the sample tube and the closed fluorescence channel to carry out the detection of the fluorescence intensity, and the design scheme of the light path is completely different from that of all the current real-time fluorescence quantitative PCR apparatuses or nucleic acid diagnostic apparatuses; the positions of the emitted light, the fluorescence channel and the sample reagent tube are fixed, and the whole channel is in a closed state, so that the external light source and other light sources adjacent to the sample reagent tube cannot interfere with the fluorescence detection of the sample; because emission, refraction and focusing are not needed, and an emission light path and a detection channel are short, high-intensity emission exciting light and a high-sensitivity photomultiplier are not needed, and cheap surface-mounted light-emitting diodes and surface-mounted photodiodes can be adopted, so that the cost is greatly reduced; according to the fluorescence emission principle, the excitation light and the emission light generated by fluorescence form an angle of 90 degrees, the interference of the excitation light on the generated fluorescence detection is small, the influence is small if a fluorescent group which is deviated from the wavelength of the excitation light of ultraviolet light is adopted, and the interference from fluorescein can be considered by being incorporated into a fluorescence background, so that an optical filter can be omitted, and the manufacturing cost of an instrument can be reduced; because no optical filter is used, internal standards or internal controls cannot be adopted, positive and negative controls must be set, although the detection result cannot distinguish Ct values of a plurality of gene targets from the correct copy number of each gene, the detected mixed Ct value of each gene target is sufficient for the purpose of rapid disease diagnosis; the detection reagent adopted by the nucleic acid diagnostic instrument can use general cheap reagent in the current market, and the reagent can be used for detection only by writing corresponding operation parameters into an IC/ID card and an RFID card when the reagent is manufactured, and offline test can be performed without networking; the instrument connection Internet can be connected with a mobile phone through Bluetooth and can also be directly connected with the Internet through a wireless module (WIFI) or an Internet of Things card arranged in the instrument, and is a genuine Internet of Things Medical instrument (IoMT, internet of Medical details).

Drawings

FIG. 1 is a general structure diagram of an embodiment of the present invention;

FIG. 2 is an exploded view of an embodiment of the present invention;

FIG. 3 is a diagram of a PCR amplification system according to an embodiment of the present invention;

FIG. 4 is a view of the sealing cover structure of the embodiment of the present invention;

FIG. 5 is a diagram of a structure of a sample reagent tube according to an embodiment of the present invention;

fig. 6 is a schematic structural diagram of a main control circuit board module according to an embodiment of the present invention;

FIG. 7 is a diagram illustrating an example of setting information parameters in a conventional three-stage PCR amplification mode according to an embodiment of the present invention;

FIG. 8 is a diagram of an example of setting information parameters for the loop-mediated isothermal PCR amplification mode according to an embodiment of the present invention;

FIG. 9 is a diagram of an example of setting information parameters in the reverse transcription isothermal PCR amplification mode according to an embodiment of the present invention;

FIG. 10 is a diagram of an example of information parameter settings for a Nested single tube PCR amplification mode according to an embodiment of the present invention;

FIG. 11 is a diagram of an example of setting information parameters for the RT-PCR hybrid PCR amplification mode according to an embodiment of the present invention;

fig. 12 is a flow chart of the system operation according to an embodiment of the present invention;

wherein: 101. an upper case, 102, an NFC response region, 103, a display, 104, a sealing cover, 105, a spring hinge, 106, a light detection device on the sealing cover, 107, a sample reagent tube and a cover, 108, a sample reaction chamber, 109, a lock cylinder opening, 110, a photosensitive sensor of a start-stop device, 111, a lower case, 201, a display opening, 202, an LCD screen, 203, a nucleic acid amplification system, 204, a fan hole, 205, a switch, 206, a DC power supply jack, 207, a main control circuit board, 208, a heat dissipation opening, 209, an NFC induction module, 210, a sample reaction chamber opening, 211, a lock cylinder, 212, a screen lead, 213, an electrical wire hole, 214, an NFC induction module lead, 301, a heating plate containing the sample reaction chamber, 302, a graphite sheet, 303, a semiconductor heating refrigerator, 304, a heat sink, 305, a fan, 306, an NTC temperature sensor lead, 307, an excitation light circuit board, 308, an excitation light board lead, 309, a positioning hole, 310, three excitation light paths, 311, a sample chamber hole, 312, a ptfe excitation light path board, 401, a lock cylinder, 402, a light blocking device, 403, a ptfe fluorescence light path board, 404, a sealing cover, 405, a hinge spring, 406, a circuit board containing three sets of photodiodes, 407, a diode circuit board lead, 501, a sampling reagent bottle, 502, a sample reagent tube, 503, a reagent tube cover, 504, a positioning pin, 601, a photosensitive sensor lead socket of a start-stop device, 602, a display socket, 603, three sets of excitation light lead sockets, 604, three sets of photodiode lead sockets, 605, a power socket, 606, an nfc circuit board lead socket, 607, a semiconductor heating and cooling sheet lead socket, 608, a temperature sensor (NTC) lead socket, 609, a fan socket, 1201, a monitoring tube door database, 1202, a database, 1203, mobile phone or computer application software, APP The system comprises a mobile phone with an NFC function 1205, an instrument starting system 1206, an instrument network communication module system 1207, an instrument display system 1208, an instrument starting and stopping system 1209, an instrument PCR amplification system 1210 and an instrument optical data acquisition system.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative efforts all belong to the protection scope of the present invention.

Referring to fig. 1-2, the portable quantitative fluorescent nucleic acid diagnostic apparatus includes a housing, a liquid crystal display 103, a nucleic acid amplification system 203, a sealing cover 104 including a fluorescence emission detection system 106, a spring hinge 105, etc., an automatic start-stop system including a photodiode 110 and a light blocking device, an information and parameter input system including an NFC sensing area 102 and an NFC module 209, a power supply system, and a main control circuit board 207.

The shell is composed of an upper shell 101 and a lower shell 111, wherein the liquid crystal display 103 passes through the display port 201 to be fixed with the upper shell 101, the screen lead 212 is connected with the socket 602 of the main control circuit board 207, and the LCD screen 202 provided with the liquid crystal display 103 on the upper shell 101 forms a 135-degree included angle with the rear end plane of the upper shell, so that a user can read information displayed by the display 103 conveniently, and the sealing cover 104 is opened without shielding the information of the display 103; the sealing cover 104 is fixed on the upper machine shell 101 through a spring hinge 105, and a lead 407 of the photodiode passes through a lead opening 210 of the upper machine shell to be connected with a socket 604 of the main control circuit board 207; a sample reaction chamber 108 on the PCR amplification reaction system 203 penetrates through a sample reaction chamber opening 211 of the upper shell and is fixed with the upper shell 101, an NFC induction area 102 is marked on the upper shell by a circular dotted line and is used as an IC or RFID card machine and a contact area of a mobile phone, a lock cylinder 211 of a sealing cover is fixed on the outer surface of the upper shell 101 through a lock cylinder opening 109, a photosensitive diode 110 and an NFC induction module 209 of a starting device are fixed at corresponding positions inside the upper shell 101, a lead 214 of the NFC induction module 209 is connected with a socket 606 of the main control circuit board 207, and a connecting line of the photosensitive diode is inserted into the socket 601 of the main control circuit board 207; the lower shell 111 comprises a direct current power supply jack 206 and a switch 205, the power adapter base is fixed at the direct current power supply jack 206, a direct current power supply is provided by an external 12V direct current power supply adapter, and a power line of the power adapter base is inserted into a power supply socket 605 of the main control circuit board; the main control circuit board 207 and the fan are both fixed on the lower casing; the upper casing 101 and the lower casing 111 of the assembled parts are fastened by screws.

The NFC sensing module 209 is disposed under the NFC sensing area 102 disposed on the upper housing 101, and an IC or RFID card 1004, or a mobile phone with NFC communication, etc. can perform data transmission with an instrument by approaching the NFC sensing area 102. Here, the NFC sensing module, i.e., the near field communication wireless communication module, is matched with the IC or RFID card 1004, or other devices including the NFC sensing module, such as a mobile phone, and inputs a sensor lot number and various operating parameters from the outside; meanwhile, the NFC module 209 may reversely output data of the instrument including detection data to the IC or RFID card 1004, or a module containing an NFC device such as a mobile phone or the like.

The main control circuit board 207 comprises a bluetooth and WIFI transceiver 1206, an operation parameter input and output system, a self-checking system of instrument working state, a control system in the PCR amplification process, and a light path system composed of a fluorescence excitation device and a fluorescence detection device. The liquid crystal display 103 is a 2-inch color dot-matrix LCD display screen and is used for displaying various prompts, detection results, error prompts and other information of the instrument operation process.

The PCR amplification system 203 consists of a heating aluminum plate 301 containing the sample reaction chamber 108, a temperature sensor (NTC sensor) 306, a graphite sheet 302, a semiconductor heating and cooling sheet 303, a radiator 304 and a fan 305; the sample reaction chamber 108 of the heating aluminum plate is a cylindrical container capable of containing 3 reaction reagent tubes to ensure that a heating heat source directly heats the bottom of the reagent tube from the bottom to ensure the heating efficiency and uniformity, the sample reaction chamber 108 is a cylindrical container capable of containing 3 reagent tubes which are respectively a sample, a positive control reagent tube and a negative control reagent tube, a side hole 310 is respectively drilled at the center of the 3 cylindrical containers at one side of the cylindrical container, which is one third away from the bottom, and the side holes and the exciting light circuit board 307 and the exciting light circuit board 312 form an exciting light path, a lead 308 of the exciting light circuit board 307 is connected with the main control circuit board 207 through a socket 603, the position of the light path must ensure the center of the reagent liquid level after the reagent tube is added with the reagent, and because the temperature rise and fall of the sample reaction chamber mainly comes from the bottom, the side hole 310 of the sample reaction chamber 311 has no great influence on the temperature rise and fall efficiency and temperature uniformity; a NTC temperature sensor 306 is arranged in a sensor groove with the width of about 2 mm below the heating aluminum plate corresponding to the position of the sample reaction chamber so as to accurately measure the temperature of the sample reaction chamber and the sample reagent tube; a layer of graphite heat conducting sheet 302 is arranged below the heating aluminum plate 301 and the NTC temperature sensor 306, so that the heat conduction of the heating aluminum plate 301 is more uniform; the semiconductor heating and refrigerating sheet 303 (Peltier) is arranged below the graphite heat conducting sheet 302, the maximum power is about 40 watts, and the input voltage is 12 volts; the radiator 304 is arranged below the semiconductor heating and refrigerating sheet 303, the radiator 304, the semiconductor heating and refrigerating sheet 303, the graphite heat-conducting sheet 302, the temperature sensor 306 and the heating aluminum plate 301 are connected with the upper case 101 through screws, the sample reaction chamber 108 on the heating aluminum plate 301 penetrates through the sample port 210 of the upper case to be connected with the outside, and power and signal wires of the temperature sensor 306 and the heating and refrigerating device 303 are respectively inserted into a temperature sensor (NTC) lead socket 608 and a semiconductor heating and refrigerating sheet lead socket 607 of the main control circuit board 207; the fan 305 is disposed under the heat sink 304 and connected to the lower housing 111, the lower housing 111 has a heat dissipating vent 208 and a fan opening 204 for dissipating heat, and a plug of a power input line of the fan is inserted into a fan receptacle 609 of the control circuit board 207.

The heating and cooling of the PCR amplification system 203 is performed by the semiconductor heating and cooling plate 303 (Peltier), and the heating and cooling process is performed by changing the direction of the output dc current through the main control circuit board 207, and the direction of the dc current is changed by the circuit system including the relay of the control board 207. In the temperature control process, the NTC temperature sensor 306 detects the temperature change of the heating sample plate 301 to generate an analog signal, the analog signal is converted into a digital signal by the analog-to-digital conversion circuit and is sent to the CPU controller circuit, the digital signal is converted into a power signal which is output by the heating driving circuit after being processed by a PID temperature control program in the CPU controller, the conductor heating and cooling sheet 303 (Peltier) works, and temperature regulation and control are carried out in a PID mode in the temperature control process, so that the error range of the temperature is kept within 0.5 ℃.

The method in the PID temperature control program consists of a proportional unit P, an integral unit I and a differential unit D, wherein:

the function of the proportional unit P is to proportionally reflect the deviations of the system, and as soon as a deviation occurs in the system, the proportional adjustment immediately produces an adjustment function to reduce the deviation. The proportion effect is large, the adjustment can be accelerated, and the error is reduced;

the function of the integration unit I: the system eliminates steady state error and improves the degree of freedom. Because of the error, the integral adjustment is carried out until no difference exists, the integral adjustment is stopped, and the integral adjustment outputs a constant value. The strength of the integration depends on the integration time constant Ti, and the smaller Ti is, the stronger is the integration. Otherwise, if Ti is large, the integral effect is weak;

the function of the differentiating unit D: the derivative effect reflects the rate of change of the system deviation signal, has predictability, and can predict the trend of deviation change, so that the control effect can be generated in advance, and before the deviation is formed, the control effect is eliminated by the derivative regulation effect. Thus, the dynamic performance of the system can be improved. Under the condition that the selection of the differential time is proper, the overshoot and the adjusting time can be reduced.

The PID temperature control program sets parameters of the proportional unit P, the integral unit I and the differential unit D by measuring the temperature change rule of the sample chamber.

The PID temperature control program controls the temperature of the sensor chamber to a constant value of the set temperature plus or minus 0.5 degrees.

The relevant reagent information of the PCR amplification system 203, the operation parameters required when the reagent is operated are written into the passive IC card or other RFID card 1004, the NFC module 209 is disposed under the NFC sensing area 103 disposed on the panel, the NFC sensing module 209 is tightly attached to the lower side of the upper housing 101, and data transmission can be performed with the instrument through the corresponding NFC sensing area 209. The NFC module 209, namely a near field communication wireless communication module, is matched with an IC card or other RFID cards 1004 matched with the NFC module, or other devices containing the NFC induction module, such as a mobile phone and the like, and a batch number of a reagent and various operation parameters are input from the outside and are led into an instrument; meanwhile, the NFC module 201 may reversely write data of the instrument into the IC or RFID card 1004, or other devices including the NFC sensing module, such as a mobile phone, to the mobile phone through an application program, and then transmit the data to the database through the internet.

The reagent information and operating parameters are described as:

1.X, reagent name number; 2.y, reagent lot number; 3.Z, the number of times of using reagent batch number, and reversely writing the reagent batch number into the IC card by an instrument to prevent using counterfeit reagents; ti, initial temperature; 5.T0, pretreatment temperature; t1, a first set temperature; t2, the second set temperature; t3, third set temperature; ST1, second stage first set temperature; 10.t0, holding time (sec) of pretreatment temperature; t1, hold time (seconds) of the first set temperature of the first and second stages; t2, holding time (seconds) of the second set temperature; 13.t3, hold time (sec) of third set temperature; c1, number of cycles of first phase; c2, number of cycles in the second stage; 16.S, number of stages, maximum 2.

Description of the parameters: 1. the reagent name number is used for identifying different components and purposes of the reagent 2. The reagent batch number indicates the production batch of the reagent and the standard curve parameters of the batch of products, whether the reagent is invalid (namely the validity period) is confirmed through the batch number 3. The use times of the reagent are used for preventing the use of counterfeit kits. Parameters 4-16 were all used for different nucleic acid amplification regimes. The number of stages (S) is currently designated 1, but in nested (single-tube) amplification, stage S is an integer greater than 1 and less than 2; cycle number (C) is determined primarily by the specified diagnostic index, e.g., ct value, which is 40 for C40; in general, when S =2, if the polymerase used is the same and the denaturation temperature is the same, the second (ST 2) and third set temperatures (ST 2) of the second stage may be the same as the second (T2) and third set temperatures (T2) of the first stage, but in the mixed amplification mode of RT-PCR and three-stage PCR, the first, second, and third set temperatures of the first and second stages may be different, so it is necessary to add the ST1, ST2, ST3 temperature set value parameters of the second stage; in order to reduce the input value of the parameter, the corresponding different holding times t1, t2, t3 of the first, second and third setting temperatures of the first and second stage can be used in common; before entering the nucleic acid amplification process, a pretreatment stage is set for directly releasing nucleic acid substances in a sample through heating and chemical reagents in the reagents without additional sample extraction and treatment processes, and the method is suitable for samples collected by a pharyngeal swab and reduces the link of sample treatment; the starting temperature of the diagnostic instrument is typically set to a pre-process temperature value, saving the temperature equilibration time for the next sample.

FIGS. 7-11 show different modes of nucleic acid amplification resulting from different parameter inputs, wherein FIG. 7 shows a conventional three-stage PCR amplification mode, where the temperatures T1, T2, and T3 represent the set values of the three-stage temperatures, respectively, T1 is an annealing temperature, generally set at 52 degrees, T2 is an elongation temperature, i.e., an optimum temperature for enzymatic reaction of nucleic acid polymerase, generally set at 72 degrees, and T3 is a denaturation temperature, generally set at 95 degrees; ti is the starting temperature, i.e. the temperature setting value at the starting of the instrument starting and the end of the diagnosis, generally equal to T1 or T0, T1, T2, T3 are the holding time (seconds) when the temperature is set at T1, T2, T3, generally 30 seconds at the same time, and different time can be set according to different target nucleic acids; t0 is a pretreatment temperature setting value which is mainly determined according to the temperature required during the extraction of the nucleic acid sample, T0 is the time (seconds) required for the pretreatment of the sample, T0 is set to be 60 degrees for the throat swab sample, and T0 is set to be 60 seconds; c1 is the cycle number of the temperature rise and drop process and is mainly determined by a specified diagnostic index, such as a Ct value, when the Ct value is 40, C1 is 40; the traditional three-stage PCR amplification mode is a cycle of denaturation, annealing and extension in sequence, namely, a double-stranded DNA template is denatured into single-stranded DNA, the single-stranded DNA is combined with a primer through annealing, and then single corresponding nucleotide is linked through nucleic acid polymerase, and the process is repeated in cycles to complete the amplification of the target DNA; during PCR amplification, taq enzyme with polymerization and 5'-3' exonuclease activity cuts and degrades a probe, so that a report fluorescent group and a quenching group are separated, a fluorescence monitoring system can receive a fluorescent signal, namely, one fluorescent molecule is formed when each DNA chain is amplified, the fluorescent signal accumulation and the PCR product formation are completely synchronous, and during PCR amplification, taq enzyme with polymerization and 5'-3' exonuclease activity cuts and degrades the probe, so that the report fluorescent group and the quenching group are separated, so that the fluorescence monitoring system can receive the fluorescent signal, namely, one fluorescent molecule is formed when each DNA chain is amplified, and the fluorescent signal accumulation and the PCR product formation are completely synchronous; before the diagnosis starts, the instrument can estimate the diagnosis time, and the diagnosis time of the nucleic acid amplification mode = T0+ the time required to heat up from T0 to T3+ 40 × (T3 + the time required to cool down from T3 to T1+ the time required to heat up from T1 to T2+ the time required to heat up from T2 to T3) + the time required to cool down from T3 to Ti, the temperature increase and decrease times are determined by the temperature increase and decrease rate of the instrument, the temperature increase rate is 3 degrees/sec, the temperature decrease rate is 1.5 degrees/sec, so that the diagnosis time =60+ (95-60)/3 +40 (30 + (95-52)/1.5 +30+ (72-52)/3 +30+ (95-72)/3) =5391.7 sec =89.9 min; the calculated estimated diagnosis time is displayed on a display screen, and the diagnosis progress is prompted in a countdown manner.

FIG. 8 is a diagram showing a loop-mediated isothermal amplification (LAMP) mode in which amplification is performed by strand displacement DNA polymerase (Bst DNApolymerase), the T1 value is set to 65 ℃ which is the optimum temperature for amplification, the pretreatment temperature setting T0 is the same as T1, and the initiation temperature Ti = T0= T1; the detection of the Lamp amplification product is mainly characterized in that the turbidity change formed by the reaction of pyrophosphoric acid generated in the amplification process and manganese ions of a sample solution is detected, or a fluorescent aminocarboxylic acid complexing agent (calcein-Mn) is added into the sample solution, the pyrophosphoric acid generated by the amplification reaction reacts with the manganese ions to generate manganese pyrophosphate, and meanwhile, the calcein-Mn which does not have the fluorescence reaction is converted into calcein-Mg which generates fluorescence, so that the amplification product is quantitatively detected; because the constant temperature amplification mode has no multi-cycle process, the Ct value cannot be used as a diagnostic index, but the earliest time node for generating fluorescence or the copy number of the target object calculated by a standard curve is used as a judgment basis, and the earlier the time for generating the fluorescence value is, the higher the concentration or the copy number of the target object is; the diagnosis time of this nucleic acid amplification pattern = t0+ t1, assuming that t0=60 seconds =1 minute, t1=45 minutes, and the calculated estimated diagnosis time =46 minutes, is also displayed on the display screen, and the progress of the diagnosis is prompted in a countdown manner.

Fig. 9 shows a reverse transcription (RT-PCR) isothermal amplification mode, in which amplification is performed by reverse Transcriptase (Reversed transcription), a T1 value is set to 37 degrees, an optimal amplification temperature is set to 65 degrees, a pretreatment temperature setting value T0 is set to 65 degrees, and an initial temperature Ti = T0=65 degrees; the content of the double-stranded product can be directly detected by a fluorescent reporter group of the probe or by a SYBR dye method; because the constant temperature amplification mode has no multi-cycle process, the Ct value cannot be used as a diagnostic index, but the earliest time node for generating fluorescence or the copy number of the target object calculated by a standard curve is used as a judgment basis, and the earlier the time for generating the fluorescence value is, the higher the concentration or the copy number of the target object is; the diagnosis time of this nucleic acid amplification pattern = T0+ the time required for decreasing the temperature from T0 to T1+ the time required for increasing the temperature from T1 to Ti, assuming that T0=60 seconds =1 minute and T1=45 minutes, the calculated estimated diagnosis time =60+ (65-37)/1.5 +2700+ (65-37)/3 =2788 seconds =46.46 minutes, which is also displayed on the display screen, and prompts the progress of the diagnosis in a countdown manner.

FIG. 10 is a single tube Nested (Nested) PCR amplification mode, wherein the Nested PCR amplification mode is two rounds of PCR amplification using two sets of PCR primers, the second round of amplification product is the target gene fragment, the use of the two sets of primers improves the specificity of amplification because the target sequence complementary to both sets of primers is very small, and if the first amplification generates a wrong fragment, the probability of pairing and amplifying the wrong fragment with the inner primer is very low, thus improving the specificity and sensitivity of the PCR amplification reaction; the single tube nest is characterized in that two pairs of PCR primers are specially designed on the basis of the traditional nest PCR, the two primers on the outer side of the nest are 25bp, and the annealing temperature is higher (68 ℃); the two primers at the inner side of the nest are 17bp, and the annealing temperature is lower (46 ℃). The outer primer is amplified first by controlling the annealing temperature (68 ℃), and after 20-30 cycles (the first round of PCR is finished), the inner primer is amplified in a nested manner by taking the first PCR product as a template by reducing the annealing temperature (46 ℃). The single-tube nested PCR and two-round PCR reactions are carried out in one PCR tube, so that the possibility of cross contamination is reduced; a variable parameter of the introduction stage of the diagnostic instrument, S; s has a maximum value of 2, i.e., the diagnostic device performs two PCR amplifications at the maximum, and the nucleic acid amplification scheme of fig. 7-9 performs only one PCR amplification, thus S =1, corresponding to a cycle number of C1; when S =2, the amplification temperatures T1, T2, T3 represent the annealing temperature, the elongation temperature, and the denaturation temperature, respectively, of the first round (C1); the amplification temperatures ST1, ST2, ST3 represent the initiation temperature, elongation temperature, and denaturation temperature, respectively, of the second round (C2); t1, T2, T3 are the holding times (seconds) when the temperatures are set at T1, T2, T3, ST1, ST2, ST3, respectively; in fig. 10, S =2, ti = T0= T1=68 degrees, T1= T2= T3=30 seconds, T0=60 seconds, ST1=46 degrees, T2, = ST2=72 degrees, T3= ST3=95 degrees, C1= C2=20, the temperature increase rate is 3 degrees/second, the temperature decrease rate is 1.5 degrees/second, the diagnostic time of this nucleic acid amplification pattern = T0+ the time required for raising the temperature from T0 to T3+ 20 × (T3 + the time required for lowering the temperature from T3 to T1+ the time required for raising the temperature from T1 to T2+ the time required for raising the temperature from T2 to T3+ T2) +20 × (T3 + the time required for lowering the temperature from ST3 to ST 1+ T1+ the time required for raising the temperature from ST1 to ST 1+ T1+ the time required to ST 2+ T2+ the time required to heat from ST2 to ST 3) + the time required to cool from T3 to Ti =60+ (95-68)/3 +20 + (30 + (95-68)/1.5 +30+ (72-68)/3 +30+ (95-72)/3) +20 + (30 + (95-46)/1.5 +30+ (72-46)/3 + (95-72)/3) + (95-68)/3 =5198 seconds =86.6 minutes; the calculated estimated diagnosis time is displayed on a display screen, and the diagnosis progress is prompted in a countdown manner.

FIG. 11 shows a RT-PCR amplification scheme, in which when the target is single-stranded RNA, reverse transcription is performed first, and then the resulting DNA is amplified; at this time, the parameters are as follows: s =2, the first round is isothermal amplification, and since the corresponding values of the maintaining times t1, t2, and t3 in the second stage must be the same, the maintaining time in the first stage must be set to be an integer multiple of t1+ t2+ t3, for example, C1=1, t1= t2= t3=30 seconds, the maintaining time at the isothermal temperature in the first stage is 30+30=90 seconds, for example, C1=10 is changed, and the maintaining time at the isothermal temperature in the first stage is 90x10=900 seconds =15 minutes; in fig. 11, let S =2, ti = T0=65 degrees, T1= T2= T3=30 seconds, T0=60 seconds, T1= T2= T3=37 degrees, ST1=55 degrees, ST2=72 degrees, ST3=95 degrees, C1=10, C2=40, the temperature increase rate is 3 degrees/second, the temperature decrease rate is 1.5 degrees/second, the diagnostic time for this nucleic acid amplification modality = T0+ the time required for a temperature decrease from T0 to T3+ 10 × (T3 + T1+ T2) + the time required for a temperature decrease from T2 to ST 3+40 × (T3 + the time required for a temperature decrease from ST3 to ST 1+ T1+ the time required for a temperature increase from ST1 to ST 2+ the time required for a temperature increase from ST2 to ST 3) + the time required for a temperature decrease from ST3 to Ti =60+ (65-37)/3 +10 +++ 30) + (95-37)/1.5 +40 ++ 30+ (95-55)/1.5 +30+ (72-55)/3 + 0+ (95-72)/3) + (95-65)/3 =6218 seconds =103.63 minutes; the calculated estimated diagnosis time is displayed on a display screen, and the diagnosis progress is prompted in a countdown manner.

One end of the sealing cover 104 is connected with the upper casing 101 through a hinge 105 with a spring structure, the sealing cover 104 is opened due to the elastic force of the spring, the sample reaction chamber 108 is displayed, the sample reagent tube 107 is placed, the other end of the sealing cover 104 comprises a locking device 401 and a starting and stopping device 402 for shielding a light source, after the sealing cover 104 is covered, the light blocking sheet 402 shields the photodiode 110 under the upper casing to change the state of the photodiode 110, so that the trigger instrument is started, after the diagnosis process is finished, the light blocking sheet 402 is opened along with the sealing cover 104 after the cover is opened, the photodiode 110 recovers to the initial state, and the diagnosis is finished. The inner side of the sealing cover 104 is fixedly connected with a light path detection system 406 for measuring the fluorescence intensity, and the position of the light path detection system corresponds to the position of the three sample reaction chambers 108 when the sealing cover is closed; the optical path detection system consists of a photodiode circuit board 406 and an optical path board 403; one surface of the photodiode circuit board contains three welded patch type photodiodes, and the other surface of the photodiode circuit board contains three groups of leads 407 corresponding to the three photodiodes; the optical path board 403 is a Polytetrafluoroethylene (PTFE) board having a certain thickness, the same width as the photodiode circuit board 406, and three sets of holes for accommodating patch type photodiodes, and has functions of isolating heat generated by the reaction chamber 108 and forming independent closed optical paths for the three sample tubes 107; the side of the photodiode circuit board 406 containing the diodes is bonded to the optical circuit board and fixed to the sealing cover, and three sets of leads 407 of the photodiodes are connected to the socket 604 of the main control circuit board 207 through the wire holes 213 of the upper case 101.

The light path system consists of three paths of light paths which do not interfere with each other and are respectively used for detecting a target object, a positive control sample and a negative control sample; each group of light paths are isolated from the outside, so that the interference of an external light source is reduced, the light path paths of exciting light and fluorescence are very short, the efficiency of detecting the fluorescence intensity is higher than that of the fluorescence intensity detected by the prior art, the structure of mirror reflection is higher, the interference of fluorescence detection is reduced, and a filter is not needed; the fluorescence reporter group selects the exciting light wavelength in the near ultraviolet range, the fluorescence emission wavelength is in the visible light wavelength, and the fluorescence spectrum is an emission spectrum and can be detected in the direction at right angle with the incident light, so that the fluorescence is not interfered by the background of the exciting light; the exciting light adopts three groups of patch type light emitting diodes, which are respectively welded on an exciting light circuit board corresponding to the side hole position of the test tube on the sample reaction chamber, and then are tightly attached to a light path system 312 of Polytetrafluoroethylene (PTFE) containing a light guide hole and fixed with one side of the sample reaction chamber by screws, and power input wires of the three groups of exciting light are connected with a socket 603 of the main control board 207 by the circuit board through a plug; because of the totally closed and short distance optical path, the fluorescence detector can adopt a low-cost patch type photodiode instead of an expensive photomultiplier or CCD.

As shown in fig. 7-11, the fluorescence detection is not through the nucleic acid amplification process, and in the conventional three-stage (i.e. denaturation, annealing, and extension stages), the fluorescence detection only collects data in the annealing stage because the temperature in the denaturation stage is higher (> 85 ℃), although the temperature in the fluorescence detector is lower than that in the denaturation stage due to the thermal insulation layer, the fluorescence detector still has an interfering effect on the performance of the photodiode to generate data fluctuation, the fluorescence intensity generated in the extension stage also generates data fluctuation due to the larger variation of the generated nucleic acid amplification product, the temperature in the annealing stage is lower, the nucleic acid amplification product is stable, and the data is most reliable; turning on the fluorescence detection unit (photodiode) throughout the diagnostic process reduces the lifetime of the detection unit, while taking into account the different amplification modes of the nucleic acid diagnostic instrument, which provides that when amplification enters a set value of T1 (S = 1) or ST1 (S = 2), data collection is started, when the temperature enters a set temperature of a different value or after the completion of the entire cycle, data collection is ended, data collection at a frequency of 1/sec is detected, the average and standard deviation at this stage is calculated, and this data is stored for comparison, analysis and judgment; the data acquisition system consists of an amplifying circuit and an A/D conversion module, wherein an analog current signal generated by the photodiode is amplified by the amplifying circuit and enters the A/D conversion module, and a corresponding digital signal is output.

When C1=1 and T1= T2= T3, namely the constant temperature amplification mode, comparing the data acquired along with the time with the data acquired at the previous time, and when the fluorescence value is continuously increased and the change is obvious, judging that the sample contains the target object, wherein the shorter the time of the increase is, the higher the concentration of the target object is; when C1>1, the fluorescence value at each cycle can be compared with the value at the previous cycle, and when the fluorescence value is 10 times the standard deviation of the fluorescence value at the previous cycle, the cycle number at this time is defined as the Ct value.

The main control circuit board 207 of the nucleic acid diagnostic instrument comprises a Bluetooth and wireless connection (WIFI) module 1206, is connected with a mobile phone through Bluetooth, is connected with the Internet through a corresponding mobile phone application program (APP), and can also be connected with the Internet through WIFI; SSID and initial password code required by WIFI network connection can be connected with a mobile phone through Bluetooth, then the password of the WIFI network is input from a mobile phone APP, namely a Bluetooth auxiliary distribution network, or the mobile phone APP sends the encrypted WIFI through an AP (access point) mode and an STA (wireless access point) mode and sends the SSID (wireless router name) and the password encrypted through the AES (user datagram protocol), or the mobile phone APP codes the SSID and the password into a UDP (user datagram protocol) message and sends the SSID and the password through a broadcast packet or a multicast message, a WIFI module of an instrument receives the UDP message and then decodes the UDP message to obtain the correct SSID and password, and then the connection is completed by actively connecting a route of the specified SSID, namely a SmartConfig mode; after connection, the instrument can be directly connected with a cloud server or even a server in a networking way through the Internet without a mobile phone and the Internet through the connection of WIFI and the Internet. The nucleic acid diagnostic instrument can be loaded with an opening of the Internet of things, and after the Internet of things card is inserted, the nucleic acid diagnostic instrument can be directly connected with the Internet. Each nucleic acid diagnostic instrument has a unique mac address (media access control address, i.e., the physical address of the nucleic acid diagnostic instrument) that can be used as an identification code for the instrument.

The reagent tube is a three-in-one connection test tube 502, which is respectively a sample, a positive control tube and a negative control tube, the reagent tube is a transparent cylindrical plastic tube, a corresponding three-in-one transparent plastic tube cover 503 is arranged on the reagent tube, a cylindrical protrusion 504 is arranged at the position 5 mm away from one end of the sample tube cover, when the reagent tube is placed in a sample reaction chamber after being covered on the reagent tube cover, the protrusion is embedded into a small hole 309 on the sample side in the sample reaction chamber 108 and is used as a positioning device for placing the reagent tube to ensure the placing position of the sample reagent tube, so that the diagnosis error caused by the position of the sample and the control reagent tube is avoided, the corresponding tube covers are sealed and covered after the reagent is manufactured by the positive control tube cover and the negative control tube cover, so that the reagent is prevented from overflowing and generating pollution in the transportation process, the tube and the cover are not separated, the diagnosis error caused by the position of the cover is avoided, the sample reagent tube has no reagent, and the sample reagent is placed in the sampling reagent bottle 501.

As shown in fig. 12, the diagnostic apparatus can be used for off-line detection for self-test, or can be used for transmitting the diagnosis result, personal information and comprehensive data monitored in the sampling process as the judgment basis of an authority, so that the diagnostic apparatus becomes an important means for epidemic disease monitoring and tracing, and simultaneously reduces cross infection caused by sampling, sample pollution and adverse factors caused by labor cost.

The off-line detection does not need a mobile phone or a computer device 1203 as a terminal device, instrument information and parameters do not need to be input into an instrument through the mobile phone, and generated data are only displayed on a display 103 in a display system 1207; the diagnosis result is also not transmitted to the database 1202 and the supervision gate database 1201 via the terminal 1203 or directly via the network communication system 1206:

s1, a user samples and then puts the sample into a sampling reagent bottle 501, the mixed or processed sample is poured into a sample reagent tube 502, a reagent cover 503 is covered, the sample reagent tube is correctly placed in 3 sample chamber holes 311 in a sample reaction chamber according to the directions of a positioning pin 504 and a corresponding positioning hole 309, and a sealing cover 104 is in an open state;

s2, turning on a power supply, enabling the instrument 1205 to enter a self-checking mode, and checking whether the control circuit board 207 and circuit elements connected with the control circuit board, the communication module 1206 such as NFC (near field communication), the PCR amplification system 1209 and the optical path system 1210 work normally or not in the self-checking mode when the instrument is started; if the instrument is abnormal, prompting error information and stopping the next operation of the instrument; otherwise, through self-checking, the instrument display prompts the user to place the IC card or RFID 1204 in the NFC sensing region 102 to input the instrument operating parameters and sample reagent information, and after the information and parameters are input, the display system 1207 displays the relevant sample reagent information and starts the instrument to heat to the set starting temperature for PCR amplification;

s3, when the temperature of the sample reaction chamber 108 reaches a constant value, the instrument reminds a user to cover the sealing cover 104 through the liquid crystal display 103 and press and lock the sealing cover; after the sealing cover is locked, the light source of the photosensitive diode 110 is blocked, and the instrument is triggered to enter the detection process of nucleic acid amplification and fluorescence real-time detection; the instrument simultaneously collects fluorescence intensity data of a sample, a positive control and a negative control, wherein the fluorescence intensity of the positive control generates fluorescence intensity higher than a background after an amplification process enters 15-16 cycles, if the positive control does not generate fluorescence after 20 cycles, the result is invalid or false negative, the negative control does not generate fluorescence higher than the background in the whole detection process, if the positive control generates fluorescence, the result is false or false positive, when the sample generates fluorescence intensity higher than the background in the cycle process and generates enhanced or stable fluorescence intensity in two continuous cycles, and the positive control and the negative control do not generate abnormality, the diagnosis can be positive, the diagnosis is finished, a positive value (Ct value) can be defined as the cycle number when the fluorescence intensity is higher than the background, if the sample does not generate fluorescence higher than the background in the whole set cycle process, the positive value can be defined as negative, and an analysis result is simultaneously stored in a memory and displayed by a liquid crystal display 103.

The nucleic acid diagnostic instrument can be automatically networked through a mobile phone, a computer 1203 or a network communication system 1206 for online detection, and the steps are as follows:

t1, the mobile phone APP1203 guides a user to register identity, including face recognition, the process of sampling and placing a sample and the video of the step S1 of off-line detection are obtained, and the video and information such as an instrument serial number read by a two-dimensional code on a scanning instrument are stored in a memory of the mobile phone;

t2, executing the step S2 of off-line detection, wherein the parameters of the operation of the input instrument and the sample reagent information can also be used for scanning the two-dimensional code of the sample reagent through a mobile phone APP, downloading parameters such as the batch number, the production date and the like of the sample reagent from a database, and inputting the parameters into the instrument through Bluetooth;

The portable quantitative fluorescent nucleic acid diagnostic apparatus can perform networking operation on a plurality of diagnostic apparatuses through a computer terminal 1203; the computer can adopt a SmartConfig mode to connect a plurality of diagnostic instruments with a distribution network of a wireless router through a communication module 1206 through corresponding application software, so that a terminal computer and the plurality of diagnostic instruments are connected in the same wireless local area network or remotely through the Internet, personal information collected by mobile phone APP corresponding to each diagnostic instrument, instrument serial numbers, reagent information, operation parameters and corresponding diagnostic results are stored in a memory of the computer or uploaded to a database 1202. Each diagnostic apparatus can operate independently, samples can be sampled instantly, the operation is carried out instantly, data are uploaded instantly, and the method is suitable for large-scale screening.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A portable real-time fluorescence quantitative nucleic acid diagnostic apparatus is characterized by comprising a shell, a PCR amplification device, an automatic start-stop device, an NFC receiver, a Bluetooth and WIFI emission receiver, a light path system consisting of a fluorescence excitation device and a fluorescence detection device, a control circuit and a display, wherein the PCR amplification device, the automatic start-stop device, the NFC receiver, the Bluetooth and WIFI emission receiver are fixedly connected to the shell; the control circuit is respectively and electrically connected with the PCR amplification device, the automatic start-stop device, the NFC receiver, the Bluetooth and WIFI emission receiver, the fluorescence excitation device and the fluorescence detection device;

and a graphite heat-conducting fin is arranged between the heating refrigerator and the amplifier.

2. The diagnostic apparatus according to claim 1, wherein the amplifier comprises a plate-like structure and a ridge-like structure integrally arranged, the ridge-like structure is arranged in the middle of the plate-like structure, and the sample reaction chamber is a cavity opened on the ridge-like structure; the plate-shaped structures of the radiator, the heating refrigerator, the graphite heat-conducting fin, the temperature sensor and the amplifier are fixedly connected with the upper shell, and the ridge-shaped structure on the amplifier extends out of the sample loading opening of the upper shell;

the display, the temperature sensor and the heating cooler are respectively and electrically connected with the control circuit through respective lead wires; the fan is arranged below the radiator and connected with the lower shell, the lower shell is provided with a vent for facilitating heat dissipation, and a power input wire of the fan is connected with the control circuit through a plug; the display is a liquid crystal display, and an LCD screen mounting plane of the display forms a certain included angle with a plane at the rear end of the upper shell.

3. The diagnostic apparatus of claim 2, wherein the sample reaction chamber is configured as a cylindrical streamlined orifice groove that is mated with a reagent tube;

the diagnostic instrument adopts three-in-one connecting test tube and a sampling reagent bottle special for collecting sample reagents, wherein the connecting test tube is a sample reagent tube, a positive control reagent tube and a negative control reagent tube respectively; the number of the sample reaction chambers is 3, and the sample reaction chambers are respectively and correspondingly provided with a sample, a positive control reagent tube and a negative control reagent tube; the reagent tube is a transparent cylindrical tube, a corresponding trinity transparent tube cover is arranged on the reagent tube, 1 cylindrical positioning pin is arranged at the outer end of the sample reagent tube of the tube cover, and correspondingly, a positioning hole is arranged on the amplifier.

4. The diagnostic apparatus as claimed in claim 3, wherein the amplifier has a side hole formed at a position on one side of the sample reaction chamber, which is one third away from the bottom;

the fluorescence excitation device of the light path system is used for irradiating a sample to excite fluorescence, and comprises an excitation light circuit board and three groups of patch type light emitting diodes welded on the excitation light circuit board, wherein 3 groups of light emitting diodes are respectively arranged at positions corresponding to side holes of the sample reaction chamber, and excitation light emitted by the light emitting diodes is emitted into the sample reaction chamber through the side holes serving as light path channels.

5. The diagnostic apparatus as claimed in claim 4, wherein the lower side of the amplifier is provided with sensor grooves corresponding to the positions of the sample reaction chambers, and the temperature sensors are fixedly mounted in the sensor grooves respectively;

a layer of graphite heat conducting sheet is arranged below the amplifier;

the heating refrigerator adopts a semiconductor heating and refrigerating sheet.

6. The diagnostic instrument of claim 5 wherein the automatic start-stop device further comprises a sealing cap, a photodiode; the upper machine shell is provided with a locking port and a photosensitive port, and the photosensitive diode is electrically connected with the control circuit and fixedly arranged at the photosensitive port; the front end of the sealing cover is provided with a hinge with a spring, and the rear end of the sealing cover is provided with a lock catch and a light blocking column; the front end of the sealing cover is hinged with the upper shell through a hinge, the rear end of the sealing cover is in open-close type clamping connection with the locking opening through a lock catch, and the light blocking column shields the photosensitive opening when the sealing cover is covered.

7. The diagnostic apparatus as claimed in claim 6, wherein the fluorescence detection device of the optical path system is used for measuring fluorescence intensity, and comprises a photoelectric circuit board and 3 sets of photodiodes welded to the outer side of the photoelectric circuit board, the inner side of the photoelectric circuit board is fixedly installed at the middle section of the sealing cover, and the 3 sets of photodiodes vertically correspond to the upper end opening of the sample reaction chamber one by one when the sealing cover is covered, and are in close contact with the sample reagent tube on the sample reaction chamber to form three independent fluorescence detection optical paths;

a photoelectric circuit board is fixedly arranged on the outer side of the photoelectric circuit board, vertical through holes are respectively formed in the positions of 3 groups of photodiodes of the photoelectric circuit board, and when the photoelectric circuit board is covered by a sealing cover, the outer side of the photoelectric circuit board is hermetically pressed at the upper end of the reagent tube;

the upper shell is provided with a wire hole, and the photodiode penetrates through the wire hole through a lead to be electrically connected with the main control circuit.

8. The diagnostic apparatus according to any one of claims 1 to 7, wherein the control circuit comprises a main control circuit board provided with a plurality of lead sockets, the main control circuit board is provided with a start-stop module, a display module, a heating and refrigerating module, a temperature transmission module, an AD module, a storage module, a photoelectric module, a fluorescence detection module, a Bluetooth and WIFI module and a CPU, and the display, the heating and refrigerating device, the temperature sensor, the fan, the NFC sensor, the photoelectric board, the photosensitive sensor and the power supply are electrically connected with the main control circuit board through leads respectively.