CN112795473A - Neisseria gonorrhoeae integrated nucleic acid detection card box - Google Patents
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Abstract
The invention discloses a neisseria gonorrhoeae integrated nucleic acid detection card box, which comprises a box body with a box cover at the top, wherein a cracking cavity, a first cleaning cavity and a second cleaning cavity are sequentially arranged in the box body from top to bottom, the cracking cavity is communicated with the box cover, and the second cleaning cavity is communicated with a reaction cavity arranged at the bottom of the outer side of the box body; plungers with plunger hole directions perpendicular to the connection direction are arranged in the connection regions of the cracking cavity, the first cleaning cavity, the second cleaning cavity and the reaction cavity, and hydrophobic liquid sealing materials are arranged in plunger holes of the plungers; amplification reaction liquid is arranged in the reaction cavity, a meltable material layer is arranged in the reaction cavity, and primer probe mixed liquid of a PorA pseudogene of Neisseria gonorrhoeae is embedded in the soluble material layer. The invention can rapidly and integrally carry out PCR amplification and detection on the Neisseria gonorrhoeae under the condition of relatively low cost, and solves the problems of poor repeatability, time-consuming detection method, complex operation and the like of the prior art.
Description
Technical Field
The invention relates to a neisseria gonorrhoeae integrated nucleic acid detection card box, and belongs to the technical field of medical detection.
Background
Neisseria Gonorrhoeae (NG) is short for Neisseria gonorrhoeae, is pathogenic bacteria of gonorrhoea, has strict human parasitism, and has strong adaptability and invasiveness to human bodies. Of the neisserial species, only neisseria meningitidis and neisseria gonorrhoeae are pathogenic to humans, and other neisseria species can cause opportunistic infections in humans under certain conditions. Epidemiological investigations have shown that gonorrhea is always the most common and first-ranked disease of sexually transmitted diseases. Gonorrhea is distributed globally, but mainly in developing countries with dense populations. Gonococcal infections are related to age, sex, race, education, sexual behavior pattern and immunity, and are also influenced by social, cultural and economic factors.
Gonorrhea is one of the infectious diseases with the highest incidence in developing countries and is also the venereal disease with the highest incidence in China at present. At the initial stage of NG infection, a human body has no clinical symptoms, but serious genitourinary tract diseases can be caused if the human body cannot be diagnosed and treated in time, and particularly, pelvic inflammation or secondary infertility is often caused by female patients. Therefore, timely and accurate diagnosis of NG infection has become a key to the treatment of gonorrhea.
At present, the detection methods of neisseria gonorrhoeae mainly comprise the following methods:
(1) isolation culture method
The only method recommended by the world health organization at present for screening gonorrhea is a culture method, which has the advantages of high accuracy, sensitivity to female and male patients with light or no symptoms, time consumption, high cost, susceptibility to various factors such as pollution, medication condition, sampling and the like, low detection rate and easy missed diagnosis. NG drug resistance also increases year by year, and the emergence of multiple drug-resistant bacteria makes NG culture increasingly difficult.
(2) Microscopic observation method
The most commonly used method is the gram staining of secretion smears and observation of gonococcal cell morphology under a light microscope. The gram staining method has the advantages of simple operation, easy material taking, low cost, easy popularization and the like, and simultaneously, the pollution of pathogens is greatly reduced after the specimen is stained. The method has the advantages of high sensitivity, simple and convenient operation, rapidness and easy judgment of results, but the false positive rate of the method is higher. Meanwhile, the gram staining method is easy to be confused with other gram-negative cocci, so the positive detection rate is not high, and even if some samples find the gonorrhoea diplococcus, only the gonorrhoea is suspected, the gonorrhoea must be further diagnosed by other laboratory tests.
(3) Nucleic acid molecule detection method
The PCR method has the positive detection rate of the gonococcus higher than that of the gram staining method, has the characteristics of rapidness and sensitivity, is widely used for diagnosing gonorrhea in a laboratory, and has wide application prospect. The infection can be confirmed by detecting gonococcal nucleic acid in the peer-to-peer sample by real-time fluorescence PCR. However, the operation time of the conventional fluorescence PCR integral detection needs 3-4 hours, the process is complicated, a PCR laboratory and professionals are needed, and the restriction conditions are more.
The traditional detection method is limited by the storage and transportation conditions of the specimen and the growth condition of bacteria in a culture medium, so that false negative results often occur. The culture of pathogenic bacteria is the 'gold standard' for clinical examination and diagnosis of NG infection, the culture and detection period of the pathogenic bacteria is long, NG drug resistance is increased year by year, and the NG culture is increasingly difficult due to the appearance of multi-drug resistant bacteria. The sensitivity of related non-culture methods such as direct immunofluorescence assay and enzyme immunoassay is also low. The nucleic acid detection is continuously applied to the diagnosis of gonococcal infection at present, and has the characteristics of early diagnosis, high sensitivity and specificity and the like.
Although the method can provide better value clinically, the method cannot be used for two purposes, saves time and labor and cannot ensure the accuracy of results; under such conditions, the sample can only be taken to qualified laboratories, and optimal treatment time can be missed, which is a difficult problem for both doctors and patients in the diagnosis period. The integrated rapid nucleic acid detection card box for neisseria gonorrhoeae, which is time-saving and labor-saving, is developed for the detection, so that various urgent problems are undoubtedly solved for a basic detection mechanism, the threshold problem of nucleic acid detection is avoided, only a manual sample adding process is needed, the nucleic acid extraction and detection are completed by a machine, on one hand, the long-term contact of experimenters with high-risk viruses is reduced, and on the other hand, the reliability of detection results is greatly improved due to the automation of the machine. Provides convenience for medical workers and simultaneously strives for the optimal treatment time for patients.
The conventional nucleic acid detection adopts a real-time fluorescent quantitative PCR method, has strict requirement on operation skills, needs professional training, carries out PCR on-duty certificate on duty and must be carried out in a PCR certification laboratory. Gonococcal nucleic acid detection generally takes 30 to 120 minutes according to different nucleic acid extraction modes, nucleic acid amplification time is within 2 hours, and time for manual operations such as reagent preparation, sample adding, result analysis and the like is generally 4 to 5 hours for one experiment, and if the circulation time of a sample in a hospital is considered, the time from sampling of a patient to taking of a detection result is generally 6 to 8 hours. Most primary hospitals do not have the professional personnel and equipment facilities, and only the sample is transported to qualified laboratories, but in most cases, the qualified laboratories cannot complete the sample in their own hospitals, and the difficulty of nucleic acid diagnosis is conceivable.
Disclosure of Invention
The invention aims to provide an integrated nucleic acid detection card box for Neisseria gonorrhoeae. The method can be used for quickly and integrally carrying out PCR amplification and detection on the neisseria gonorrhoeae at relatively low cost, and solves the problems of poor repeatability, time-consuming detection method, complex operation and the like of the existing technology.
The technical scheme of the invention is as follows: the utility model provides a neisseria gonorrhoeae integration nucleic acid detection card box which characterized in that: the device comprises a box body with a box cover at the top, wherein a cracking cavity with cracking liquid, a first cleaning cavity with first cleaning liquid and a second cleaning cavity with second cleaning liquid are sequentially arranged in the box body from top to bottom, the cracking cavity is communicated with the box cover, and the second cleaning cavity is communicated with a reaction cavity arranged at the bottom of the outer side of the box body; plungers with plunger hole directions perpendicular to the connection direction are arranged in the connection regions of the cracking cavity, the first cleaning cavity, the second cleaning cavity and the reaction cavity, and hydrophobic liquid sealing materials are arranged in plunger holes of the plungers; amplification reaction liquid is arranged in the reaction cavity; a meltable material layer is arranged in the reaction cavity, and primer probe mixed liquor of a Neisseria gonorrhoeae PorA pseudogene is embedded in the soluble material layer; and magnetic beads which can pass through a plunger hole of the plunger are arranged in the lysis cavity (the magnetic beads can be added into the lysis cavity when in use). Through promoting the plunger, can make the plunger hole aim at the intercommunication interval of two adjacent cavitys, two cavity spares rely on the downthehole hydrophobic liquid sealing material of attached to plunger to keep apart this moment, then the magnetic bead (utilize outside magnet steel to drag) alright pass the liquid sealing material of hydrophobic in plunger hole, transfer another cavity from a cavity. The primer probe mixed liquid required by the amplification reaction is in the meltable material, so that the reaction liquid of the reaction liquid system is separated from the primer probe mixed liquid, the PCR reaction is prevented from being carried out in advance, and the validity period of the card box is prolonged. The meltable material is any one or combination of more of paraffin substances such as paraffin, dodecane, tetradecane, hexadecane and the like, the melting point range is 20-95 ℃, paraffin is preferred, and paraffin oil which is melted into liquid state in use can prevent PCR products forming aerosol from polluting the space between rings; the paraffin oil has weak thermal conductivity, and the temperature condition of PCR is effectively ensured; the low-temperature solidification characteristic of the paraffin can fix the position of the magnetic beads after the temperature of the magnetic bead back dragging is reduced. The paraffin is meltable so that the inclusion allows the reaction solution to be combined from a separate state.
In the neisseria gonorrhoeae integrated nucleic acid detecting cassette, the amplification reaction solution contains Taq DNA polymerase.
In the neisseria gonorrhoeae integrated nucleic acid detection card box, the primer-probe mixed solution of the PorA pseudogene of neisseria gonorrhoeae comprises: the PorA pseudogene upstream primer and the PorA pseudogene downstream primer of the neisseria gonorrhoeae, the internal standard upstream primer and the internal standard downstream primer, and the PorA pseudogene probe and the internal standard probe of the neisseria gonorrhoeae, in particular to
In the neisseria gonorrhoeae integrated nucleic acid detecting cartridge, the lysis solution contains 1-1000mM acetic acid-sodium acetate, 0.1-20% polyethylene glycol, 0.1-10M guanidine isothiocyanate, the pH value is 2.0-7.0, and the solvent is ddH2And O. The above percentages are mass percentages. Guanidine isothiocyanate denatures proteins to destroy cells and organelles, releasing nucleic acids. High concentrations of guanidine isocyanate and PEG combined action to make DNA adsorption to magnetic beads (in this process acidic pH than neutral or alkaline pH better effect).
In the neisseria gonorrhoeae integrated nucleic acid detecting cartridge, the first cleaning solution comprises 1-1000mM Tris-HCl, and 0.1-10% PEG; the second wash solution comprises 1-1000mM Tris-HCl, 0.1-10% PEG and 0.1-2% non-protein blocking agent. The solvent is ddH2And O. The above percentages are mass percentages. The non-Protein Blocking agent may be Pierce TM Protein-free (TBS) Blocking Buffer (Thermo Scientific, cat # 37570). The acetic acid-sodium acetate solution provides a low pH environment for DNA adsorption to the magnetic beads. The nucleic acid purification reagent (cleaning solution) and the lysis solution do not contain protease K, so that the product does not need to be stored at the temperature of-20 ℃. Alcohol is not needed in the cleaning solution, and PEG is used for replacing ethanol. The non-protein blocking agent in the second cleaning solution is used for blocking the surface groups of the magnetic beads, so that reaction failure caused by DNA adsorption during elution in the PCR reaction solution is avoided, and no protein exists, the non-protein blocking agent is not a biological source, and no biohazard risk exists. Non-protein blocking agents are not of biological origin and do not risk biohazards.
In the neisseria gonorrhoeae integrated nucleic acid detection card box, the diameter of a plunger hole of the plunger is 3-5mm, the diameter of a magnetic bead is 0.1-100 mu m, and the density of the hydrophobic liquid sealing material is 0.1-1.5g/cm3The viscosity was 2000-. This setting can ensure that the liquid sealing material of hydrophobicity can keep apart liquid, can let the magnetic bead pass again.
In the neisseria gonorrhoeae integrated nucleic acid detecting card box, the hydrophobic liquid-state sealing material is silicone oil.
In the neisseria gonorrhoeae integrated nucleic acid detection card box, the reaction cavity is a conical tube arranged outside the box body, one side of the conical tube is provided with a plane, external magnetic steel is convenient to pull a magnetic bead back into a meltable material above the conical tube (the magnetic bead is solidified after being cooled), and then interference on optical detection cannot be generated.
Compared with the prior art, the neisseria gonorrhoeae integrated nucleic acid detection card box can be used for quickly and accurately detecting neisseria gonorrhoeae. By combining the primer and the probe screened by repeated research and test, the invention is superior to other molecular diagnostic kits in the aspects of detection timeliness, specificity, sensitivity, convenience, technical parameters and the like.
The neisseria gonorrhoeae integrated nucleic acid rapid detection card box has the following advantages:
1) and (3) totally-enclosed design: 1 AIGS device, and a small amount of laboratory basic small equipment can complete complex nucleic acid detection experiments. The method integrates the processes of nucleic acid extraction, amplification, detection and the like into a totally-enclosed cassette, thereby structurally avoiding aerosol pollution, protecting the safety of detection personnel and avoiding a PCR laboratory. Greatly reducing the requirements of nucleic acid detection on the laboratory construction standard. The joint defense deployment and control of the area with limited conditions are facilitated;
2) the method comprises the following steps: the manual operation time is 2-5 minutes, the machine is operated for 30-90 minutes to obtain results, the nucleic acid extraction and amplification integrated technology design and the automatic judgment software really realize the full automation from sample processing to result reporting, the requirement on the professional skill of a tester is greatly reduced, and the method is suitable for rapid on-site bedside diagnosis;
3) and (4) refrigerating and preserving: the product is stored at 2-8 ℃ in a refrigeration way, and the conventional-20 ℃ freezing storage is not needed, so that the cold chain transportation and storage of the product are facilitated.
In addition, the invention uses the plunger to combine the mode of hydrophobic liquid sealing material to isolate a plurality of functional cavities, so that the liquid between each cavity can not overflow, the biological sample can be respectively cracked, cleaned, purified and amplified in each cavity, and the magnetic beads can be used for bringing the nucleic acid into each cavity in turn in an attachment mode, wherein, the hydrophobic liquid sealing material can have a dispersion effect on the magnetic beads, can allow the magnetic beads and the nucleic acid on the magnetic beads to pass through and block other impurities, when the magnetic beads pass through, the impurities such as some nonpolar large particles attached to the magnetic beads can be infiltrated into the hydrophobic liquid sealing material and blocked, and the influence of the impurities on the detection result can be reduced.
FIG. 1 is a schematic view of the construction of the cartridge of the present invention;
fig. 2 is a side schematic view of fig. 1.
The labels in the figures are: 1-box body, 2-box cover, 3-cracking cavity, 4-first cleaning cavity, 5-second cleaning cavity, 6-reaction cavity, 7-plunger and 8-magnetic bead.
Detailed Description
The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.
Examples are given. An integrated neisseria gonorrhoeae nucleic acid detection cartridge as shown in figure 1: the device comprises a box body 1 with a box cover 2 at the top, wherein a cracking cavity 3 with cracking liquid, a first cleaning cavity 4 with first cleaning liquid and a second cleaning cavity 5 with second cleaning liquid are sequentially arranged in the box body 1 from top to bottom, the cracking cavity 3 is communicated with the box cover 2, and the second cleaning cavity 5 is communicated with a reaction cavity 6 arranged at the bottom of the outer side of the box body 1; plungers 7 with plunger hole directions perpendicular to the connection direction are respectively arranged in the connection areas of the cracking cavity 3, the first cleaning cavity 4, the second cleaning cavity 5 and the reaction cavity 6, and hydrophobic liquid sealing materials are arranged in plunger holes of the plungers 7; an amplification reaction solution is arranged in the reaction cavity 6; a meltable material layer is arranged in the reaction cavity 6, and primer probe mixed liquor of a Neisseria gonorrhoeae PorA pseudogene is embedded in the soluble material layer; and magnetic beads 8 capable of penetrating through a plunger hole of the plunger 7 are arranged in the cracking cavity 3. The amplification reaction solution contains Taq DNA polymerase. The primer probe mixed solution of the neisseria gonorrhoeae PorA pseudogene comprises: the PorA pseudogene upstream primer and the PorA pseudogene downstream primer of the neisseria gonorrhoeae, the internal standard upstream primer and the internal standard downstream primer, and the PorA pseudogene probe and the internal standard probe of the neisseria gonorrhoeae, in particular to
The lysis solution contains 1-1000mM acetic acid-sodium acetate, 0.1-20% polyethylene glycol, 0.1-10M guanidinium isothiocyanate, and has a pH value of 2.0-7.0. The first wash comprises 1-1000mM Tris-HCl, and 0.1-10% PEG; the second wash solution comprises 1-1000mM Tris-HCl, 0.1-10% PEG and 0.1-2% non-protein blocking agent.
The diameter of a plunger hole of the plunger 7 is 3-5mm, the diameter of the magnetic bead 8 is 0.01-100 mu m, and the density of the hydrophobic liquid sealing material is 0.1-1.5g/cm3The viscosity was 2000-. The hydrophobic liquid blocking material is preferably silicone oil. The reaction chamber 6 is a conical tube arranged outside the box body 1, and a plane is arranged on one side of the conical tube, as shown in part A of figure 2.
Firstly, establishing a neisseria gonorrhoeae integrated nucleic acid detection card box. 30ul of PCR amplification solution and 5ul of DNA polymerase solution were added to each PCR tube. And 5ul of template DNA was added.
1 subpackaging PCR buffer solution: 30ul of PCR buffer solution is dispensed to the bottom of the amplification area; the composition of the PCR buffer was: 50mmol/L Tris-HCl (pH8.0), 20mmol/L (NH)4)SO4，30mmol/L KCl，4.0mmol/L MgCl20.2mmol/L dNTPs, 5U Taq DNA polymerase and 5ul template DNA.
2 embedding primer probe mixed solution: 5ul of primer probe mixture was embedded in the upper layer of PCR buffer with meltable material: the use concentration of 5ul of primer probe for detecting the probe of the PorA pseudogene is 500nM, the use concentration of probe for detecting the internal standard is 200nM, the use concentration of upstream and downstream primers for detecting the PorA pseudogene is 400nM, and the use concentration of upstream and downstream primers for detecting the internal standard is 250 nM.
3, sealing: sealing the reaction solution with a proper amount of silicone oil;
4, subpackaging a second cleaning solution: subpackaging 140ul of second cleaning solution, 100mM acetic acid-sodium acetate, 1% PEG and 3.5M GTC into the second cleaning cavity;
5, sealing: sealing the second cleaning solution by using a proper amount of silicone oil;
6, subpackaging a first cleaning solution: 140ul of first cleaning solution is dispensed into the first cleaning cavity: 100mM Tris-HCl, 2% PEG;
7, sealing: sealing the first cleaning solution with a proper amount of silicone oil;
8, split charging of lysate: the lysate area was filled with 800ul of lysate. Lysis solution: 10mM Tris-HCl, 2% PEG, 1.8% protein-free blocking solution.
Second, using method and rapid identification.
Collection, preservation and transportation of the sample.
1.1 sample type: a swab;
1.2 sample collection:
the recommended samples are according to the national standard: GB 15975 + 1995 gonorrhea diagnosis standard and treatment principle and industry standard: standard for diagnosis of gonorrhea in the sanitary industry of the people's republic of China (WS268-2019)
The urethra swab is used for male patients, the urethral orifice is cleaned by physiological saline, the male material-taking swab is inserted into the urethra for 2cm to 3cm, the male material-taking swab is rotated slightly and is taken out after being reserved for 5s to 10 s.
Female genital tract swab: dilating vagina with a vaginal dilator, and wiping off cervical secretion with a sterile cotton ball; taking materials from deep part of vagina or fornix behind vagina, cervical orifice, etc. and taking out the swab to the sampling tube.
The use of the test cartridge.
The lid of the cartridge was opened, the sealing membrane was opened again, and 10. mu.L of magnetic beads were added (thoroughly suspended by shaking before use). A further 200. mu.L of sample was added, the reagent was pipetted and pipetted (at least 15 strokes are recommended) and the cap was closed.
And (3) real-time fluorescence quantitative PCR amplification and detection.
2.1 sample detection
And (3) analyzing the PCR amplification product by using a full-automatic nucleic acid detection fluorescent quantitative PCR instrument Life Ready K1000 and a full-automatic nucleic acid detection analysis system, and judging the result by integrating an amplification curve and a Tm value. The fluorescence channel setup is as follows.
| Detector | Target |
| FAM | Pora pseudogene |
| ROX | Internal standard |
The amplification program was set up as follows:
2.2 control set up.
The invention analyzes and researches the NG sequence of neisseria gonorrhoeae and designs a primer probe group shown in the following table.
Primer probes of the invention
The detection results of the reaction solution prepared by each group of primer probes are as follows:
| primer probe combination | Pora pseudogene CT efficiency | Internal standard CT efficiency |
| The invention | 30.82 | 26.5 |
| |
29.23 | 26.42 |
| |
31.65 | 27.98 |
The results show that: compared with the control group 1-2, the primer probe of the invention has optimal detection sensitivity and specificity.
2.3 analysis of results.
1. After the operation of the detection program is finished, the detection result is automatically reported by an applicable instrument.
2. The results showed the detection results of PorA pseudogene (FAM) and internal quality control (ROX) of Neisseria Gonorrhoeae (Neisseria Gonorhoeae).
3. The gonococcus (NG) PorA pseudogene (FAM) gene result should present a typical S-type amplification curve (including an S-curve before the plateau), and the Ct is less than or equal to 38, and the gonococcus (Neisseria Gonorhoeae) is judged to be positive.
4. The internal quality control ROX fluorescence channel detection result should present a typical S-shaped amplification curve (including an S-curve before the plateau stage). When the target (FAM) is negative and the internal quality control Ct is less than or equal to 38, the experimental result is valid. Otherwise, it needs to be re-sampled and detected.
2.4 to test the accuracy and effectiveness of the cartridges of the present invention for UU detection, we tested the sensitivity and specificity of the cartridges.
1) Detecting NG quality control products with different concentrations to confirm the detection limit;
2)1.2 testing other pathogens (including ureaplasma urealyticum, Mycoplasma hominis, herpes simplex virus, human papilloma virus, Chlamydia trachomatis, human cytomegalovirus, Mycoplasma pneumoniae, Mycoplasma genitalium, Candida krusei, Staphylococcus epidermidis, Streptococcus faecalis, Candida tropicalis, enterococcus faecalis, enterococcus faecium, Escherichia coli, Staphylococcus aureus subsp. aureofaciens, lactococcus lactis, Streptococcus agalactiae (GBS), etc.) that may be obtained at the same sampling site to verify the specificity.
As a result: we tested UU quality control at different concentrations, and the following table shows the results of PorA pseudogenes.
The detection limit of the detection cartridge of the invention on UU is confirmed to be 250 copies/ml.
3) Other pathogens that may be taken from the same sample site (including ureaplasma urealyticum, mycoplasma hominis, herpes simplex virus, human papilloma virus, neisseria gonorrhoeae, human cytomegalovirus, mycoplasma pneumoniae, mycoplasma genitalium, and candida krusei, staphylococcus epidermidis, streptococcus faecalis, candida tropicalis, enterococcus faecalis, enterococcus faecium, escherichia coli, staphylococcus aureus subspecies aureofaciens, lactococcus lactis, streptococcus agalactiae (GBS), etc.) are not non-specifically amplified.
And thirdly, detecting an example.
Zhejiang Shaofeng hospital verification result.
Sample type: male urethra swab and female genital tract swab
Number of samples: 196 examples in
The results of the clinical sample testing are shown in the following table:
after the 52 positive samples are confirmed by the comparison method, the detection results are consistent: the coincidence rate of 52 positive samples and the coincidence rate of 100% of 144 negative samples in 196 sample detection results.
The above-described embodiments are not intended to limit the present invention, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and scope of the present invention should be construed as equivalents thereof.
SEQUENCE LISTING
<110> Hangzhou Zhangzhen Biotechnology Ltd
<120> Neisseria gonorrhoeae integrated nucleic acid detection card box
<130> 2021012906
<160> 18
<170> PatentIn version 3.5
<210> 1
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<213> Artificial sequence (Artificial sequence)
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<211> 20
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<400> 3
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<213> Artificial sequence (Artificial sequence)
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<213> Artificial sequence (Artificial sequence)
<400> 5
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<400> 6
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<213> Artificial sequence (Artificial sequence)
<400> 7
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<213> Artificial sequence (Artificial sequence)
<400> 8
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<210> 9
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
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<400> 10
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<213> Artificial sequence (Artificial sequence)
<400> 11
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<213> Artificial sequence (Artificial sequence)
<400> 12
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<213> Artificial sequence (Artificial sequence)
<400> 18
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Claims (8)
1. A neisseria gonorrhoeae integrated nucleic acid detecting cartridge, characterized in that: the device comprises a box body (1) with a box cover (2) at the top, wherein a cracking cavity (3) with cracking liquid, a first cleaning cavity (4) with first cleaning liquid and a second cleaning cavity (5) with second cleaning liquid are sequentially arranged in the box body (1) from top to bottom, the cracking cavity (3) is communicated with the box cover (2), and the second cleaning cavity (5) is communicated with a reaction cavity (6) arranged at the bottom of the outer side of the box body (1); plungers (7) with plunger hole directions perpendicular to the connecting direction are arranged in the connecting areas of the cracking cavity (3), the first cleaning cavity (4), the second cleaning cavity (5) and the reaction cavity (6), and hydrophobic liquid sealing materials are arranged in plunger holes of the plungers (7); amplification reaction liquid is arranged in the reaction cavity (6); a meltable material layer is arranged in the reaction cavity (6), and a primer probe mixed solution of a PorA pseudogene of Neisseria gonorrhoeae is embedded in the soluble material layer; and magnetic beads (8) capable of penetrating through the plunger hole of the plunger (7) are arranged in the lysis cavity (3).
2. The Neisseria gonorrhoeae integrated nucleic acid detecting cartridge according to claim 1, wherein: the amplification reaction solution contains Taq DNA polymerase.
3. The Neisseria gonorrhoeae integrated nucleic acid detecting cartridge according to claim 1, wherein: the primer probe mixed solution of the Neisseria gonorrhoeae PorA gene comprises: the PorA pseudogene upstream primer and the PorA pseudogene downstream primer of the neisseria gonorrhoeae, the internal standard upstream primer and the internal standard downstream primer, and the PorA pseudogene probe and the internal standard probe of the neisseria gonorrhoeae, in particular to
4. The Neisseria gonorrhoeae integrated nucleic acid detecting cartridge according to claim 1, wherein: the lysis solution contains 1-1000mM acetic acid-sodium acetate, 0.1-20% polyethylene glycol, 0.1-10M guanidinium isothiocyanate, and has a pH value of 2.0-7.0.
5. The Neisseria gonorrhoeae integrated nucleic acid detecting cartridge according to claim 1, wherein said first washing solution comprises 1 to 1000mM hydrochloric acid buffer, and 0.1 to 10% polyethylene glycol; the second cleaning solution comprises 1-1000mM hydrochloric acid buffer solution, 0.1-10% polyethylene glycol and 0.1-2% non-protein blocking agent.
6. The Neisseria gonorrhoeae integrated nucleic acid detecting cartridge according to claim 1, wherein: the diameter of a plunger hole of the plunger (7) is 3-5mm, the diameter of the magnetic bead (8) is 0.1-100 mu m, and the density of the hydrophobic liquid sealing material is 0.1-1.5g/cm3Viscosity of 2000-200000CSt。
7. The Neisseria gonorrhoeae integrated nucleic acid detecting cartridge according to claim 6, wherein: the hydrophobic liquid sealing material is silicone oil.
8. The Neisseria gonorrhoeae integrated nucleic acid detecting cartridge according to claim 1, wherein: the reaction chamber (6) is a taper pipe arranged on the outer side of the box body (1), and a plane is arranged on one side of the taper pipe.
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