CN110885907A - Full-automatic sample adding, hybridizing and detecting method for HPV genotyping chip - Google Patents
Full-automatic sample adding, hybridizing and detecting method for HPV genotyping chip Download PDFInfo
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- CN110885907A CN110885907A CN201911289328.9A CN201911289328A CN110885907A CN 110885907 A CN110885907 A CN 110885907A CN 201911289328 A CN201911289328 A CN 201911289328A CN 110885907 A CN110885907 A CN 110885907A
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Abstract
The invention relates to the field of in-vitro diagnostic reagent detection, in particular to a full-automatic sample adding, hybridizing and detecting method of an HPV genotyping chip. The method comprises the following steps: dripping the PCR product into a small test tube of a PCR product middle rotating disc, placing an HPV genotyping chip in a large test tube of a biochemical hybridization large rotating disc, preparing reaction liquid required by hybridization color development and distilled water cleaned by a liquid transfer device, and performing hybridization detection. The invention has the beneficial effects that: the detection method of the invention realizes rapid and convenient absorption and sample application, ensures the continuity of the detection steps, has less workload of manual intervention and improves the detection efficiency.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagent detection, in particular to a full-automatic sample adding, hybridizing and detecting method of an HPV genotyping chip.
Background
At present, the main method for clinically detecting Human Papilloma Virus (HPV) DNA in China is a real-time fluorescent PCR and hybridization capture method, the PCR-membrane hybridization method adopts a gene amplification technology and a diversion hybridization principle, the hybridization result of an amplification product and a membrane coated with a type-specific probe is detected through reverse dot hybridization, and then qualitative detection is carried out by using an alkaline phosphatase system, so that typing detection analysis is carried out on 21 HPV genotypes (6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68 and CP8304), and the method has important clinical application value for preventing cervical carcinoma.
At present, HPV genotyping chips of PCR-reverse dot hybridization method usually adopt the method of sample extraction-PCR amplification-constant temperature hybridization-visible light interpretation, and the workload of manual intervention is large.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a full-automatic sample adding, hybridizing and detecting method of HPV genotyping chip, which can be continuously carried out and reduce the workload of workers.
In order to solve the technical problems, the invention adopts the technical scheme that: the HPV genotyping chip full-automatic sample adding, hybridizing and detecting method is provided, and a detection system is used for detection, and the detection system comprises: a rotary table, a liquid-transferring device, a biochemical hybridization device and an image acquisition device are arranged in the PCR product;
the rotary disc in the PCR product comprises a rotary disc and a small test tube embedded in the circumferential direction of the rotary disc, and the rotary disc rotates clockwise or anticlockwise around a shaft;
the liquid-transfering device comprises a frame rotating around a shaft and a liquid-transfering needle rotating around the shaft and capable of vertically lifting in the vertical direction; a plurality of open containers are arranged in the circumferential direction of the rack, and needle heads of the liquid transferring needles can extend into the open containers;
the biochemical hybridization device comprises a rotatable turntable, wherein a large test tube is embedded on the circumference of the turntable, and the turntable can be used for heating the large test tube;
the image acquisition device comprises a CCD/CMOS imaging camera;
the detection method comprises the following steps:
step 1: placing the HPV genotyping chip in a large test tube of a biochemical hybridization device; the HPV genotyping chip comprises a substrate and HPV genotyping specific detection probes fixed on the substrate;
purifying a sample to be detected, specifically amplifying PCR to form a product to be detected, and then dripping the product to be detected into a small test tube on a rotary disc of the PCR product for storage;
step 2: transferring the product to be tested in the small test tube into a large test tube of a biochemical hybridization device by using a liquid transferring needle;
and step 3: adding a reaction solution required by the hybridization reaction into the large test tube from the open container by using a liquid transfer needle, heating the large test tube according to the reaction requirement, and rotationally stirring the large test tube by using a biochemical hybridization device so as to ensure that the product to be detected and the reaction solution added into the large test tube complete the hybridization reaction with the HPV genotyping specific detection probe on the substrate;
the hybridization reaction process is divided into gene sequence hybridization, washing, marker color development and chip re-cleaning according to the liquid adding steps, reaction solution added in each liquid adding process (each reaction solution and distilled water are respectively arranged in corresponding open containers) needs to submerge the HPV genotyping chip, and corresponding liquid in a large test tube is pumped out by using a liquid transferring needle after the reaction process or the washing process is finished.
And 4, step 4: after the hybridization reaction in the large test tube is finished, shooting an HPV genotyping chip in the large test tube by using a CCD/CMOS imaging camera to obtain a result image;
and 5: and comparing, analyzing and judging the result image to obtain a detection result.
The invention has the beneficial effects that: in the full-automatic sample adding, hybridizing and detecting method of the HPV genotyping chip, the PCR product is dripped into a small test tube of a rotary disk in the PCR product, the HPV genotyping chip is placed in a large test tube of a biochemical hybridization large rotary disk, and reaction liquid required by hybridization color development and distilled water cleaned by a liquid transfer device are prepared at the same time.
Drawings
FIG. 1 is a schematic structural diagram of a detection system in the full-automatic sample application, hybridization and detection method of an HPV genotyping chip according to an embodiment of the present invention;
description of reference numerals:
1. a PCR product middle rotating disc; 11. a small test tube; 2. a pipetting device; 21. an open container; 22. a liquid transferring needle; 3. a biochemical hybridization device; 31. a large test tube; 4. an image acquisition device; 5. a preheater.
Detailed Description
In order to explain the technical content, the objects and the effects of the present invention in detail, the following description will be given with reference to the embodiments.
The HPV genotyping chip of the invention adopts the hybridization working principle of nucleic acid reverse dot hybridization used in the full-automatic sample adding, hybridization and detection method:
the target sequence is amplified by using a primer marked by biotin and the like, and the product is hybridized with a specific probe fixed on a matrix (a nylon membrane, a nitrocellulose membrane). Firstly, respectively spotting the probes to be used on a nitrocellulose membrane or a nylon membrane, wherein each probe is one spot and numbered, hybridizing the PCR specific amplification product of the sample to be detected with the probes, combining the sample to be detected with the probes with homologous sequences, and then washing to remove the PCR product which is not combined. Because the sample to be detected has the biotin-like marker, the hybridization signal can be displayed through corresponding color reaction by combining the biotin-like marker on the probe point of the sample to be detected, and most or all alleles of a certain gene locus can be judged at one time.
Referring to fig. 1, the present invention provides a full-automatic sample adding, hybridization and detection method for HPV genotyping chip, which uses a detection system for detection, wherein the detection system comprises: a rotary disc 1, a liquid-transfering device 2, a biochemical hybridization device 3 and an image acquisition device 4 in the PCR product;
the rotary disc 1 in the PCR product comprises a rotary disc and a small test tube 11 embedded in the circumferential direction of the rotary disc, and the rotary disc rotates clockwise or anticlockwise around a shaft;
the liquid-transfering device 2 comprises a frame rotating around a shaft and a liquid-transfering needle 22 rotating around the shaft and capable of vertically lifting and descending; a plurality of open containers 21 are arranged in the circumferential direction of the rack, and the needle heads of the liquid transferring needles 22 can extend into the open containers;
the biochemical hybridization device 3 comprises a rotatable turntable, the circumference of the turntable is embedded with a large test tube 31, and the turntable can be used for heating the large test tube;
the image acquisition device comprises a CCD/CMOS imaging camera;
the detection method comprises the following steps:
step 1: placing the HPV genotyping chip in a large test tube of a biochemical hybridization device; the HPV genotyping chip comprises a substrate and HPV genotyping specific detection probes fixed on the substrate;
purifying a sample to be detected, specifically amplifying PCR to form a product to be detected, and then dripping the product to be detected into a small test tube on a rotary disc of the PCR product for storage;
step 2: transferring the product to be tested in the small test tube into a large test tube of a biochemical hybridization device by using a liquid transferring needle;
and step 3: adding a reaction solution required by the hybridization reaction into the large test tube from the open container by using a liquid transfer device, heating the large test tube according to the reaction requirement, and rotationally stirring the large test tube by using a biochemical hybridization device so as to ensure that the product to be detected and the reaction solution added into the large test tube complete the hybridization reaction with the HPV genotyping specific detection probe on the substrate;
the hybridization reaction process is divided into gene sequence hybridization, washing, marker color development and chip re-cleaning according to the liquid adding steps, reaction solution added in each liquid adding process (each reaction solution and distilled water are respectively arranged in corresponding open containers) needs to submerge the HPV genotyping chip, and corresponding liquid in a large test tube is pumped out by using a liquid transferring needle after the reaction process or the washing process is finished.
And 4, step 4: after the hybridization reaction in the large test tube is finished, shooting an HPV genotyping chip in the large test tube by using a CCD/CMOS imaging camera to obtain a result image;
and 5: and comparing, analyzing and judging the result image to obtain a detection result.
Preferably, in the above full-automatic sample application, hybridization and detection method for an HPV genotyping chip, the matrix is a nylon membrane or a nitrocellulose membrane.
Preferably, in the above full-automatic sample adding, hybridizing and detecting method for an HPV genotyping chip, a preheater 23 is further disposed in the circumferential direction of the rack, and the preheater is used for preheating the liquid in the open container.
Preferably, in the above-mentioned method for fully automatically loading, hybridizing and detecting an HPV genotyping chip, one of the plurality of open containers is provided with distilled water for washing the pipetting needle.
As can be seen from the above description, in the fully automatic sample adding, hybridization and detection method of the HPV genotyping chip of the invention, distilled water, hybridization reagents and color reagents are respectively arranged in a plurality of open containers. Liquid-transfering device can be pivoted to vertical lift liquid-transfering needle about can to absorb or release definite solution, in addition, liquid-transfering device possesses the self-cleaning function, can add different liquid of sample in succession.
The invention has the beneficial effects that: in the full-automatic sample adding, hybridizing and detecting method of the HPV genotyping chip, as long as the membrane and the PCR product are added on the detection device, other series processes of hybridizing, washing, developing and the like are automatically completed by an instrument, so that the method is automatic and rapid, and avoids the influence of human factors on the hybridizing process. And after the hybridization process is finished, scanning pictures, and analyzing and outputting a signal value and a result by adopting analysis and identification software.
Example 1
A detection system of the full-automatic sample adding, hybridization and detection method of the HPV genotyping chip comprises the following steps: a rotary table, a liquid-transferring device, a biochemical hybridization device and an image acquisition device are arranged in the PCR product;
the turntable in the PCR product can rotate clockwise or anticlockwise around the shaft, and small test tubes are embedded in the circumference of the turntable;
the liquid transferring device can rotate around a shaft and vertically lift the liquid transferring needle up and down so as to absorb or release determined solution, and in addition, the liquid transferring device has an automatic cleaning function and can continuously sample different liquids;
the biochemical hybridization device is also a turntable device which can rotate clockwise and anticlockwise, a large test tube is embedded on the circumference of the turntable, and the large biochemical hybridization turntable has the function of temperature rise and fall;
and finally, an image acquisition device of the detection system is provided with a CCD/CMOS imaging camera, and can shoot the HPV genotyping chip in the large test tube through the side windowing position of the large rotary disc for biochemical hybridization so as to acquire the image of the surface of the chip after reaction.
A full-automatic sample adding, hybridization and detection method of an HPV genotyping chip comprises the following steps:
1. in the detection process, firstly, the test strip (HPV genotyping chip) is placed in a large test tube of a biochemical hybridization turntable, and then a to-be-detected product formed by purifying and PCR amplifying a to-be-detected sample submitted by a hospital is dropped in a small test tube in a PCR product turntable for storage; when the test is started, the liquid-transfering device firstly transfers a certain amount of PCR product solution in the small test tube into the large test tube corresponding to the biochemical hybridization system.
2. The biochemical hybridization system is heated and kept at a determined optimal reaction temperature, the liquid transfer device is used for sequentially adding required reaction solution according to a determined biochemical reaction process (the required reaction solution is added every time, the HPV genotyping chip is submerged, the front liquid is pumped away before the reaction solution is added next time, and then new reaction solution is added), and meanwhile, the rotation function of the hybridization device can stir the hybridization solution in the large test tube and simulate the shaking function, so that the PCR product, the hybridization solution and the color development solution added in the large test tube can complete corresponding biochemical hybridization reaction with the detection probe on the HPV genotyping chip as required.
3. When the biochemical hybridization reaction time is up, the biochemical hybridization large loading disc rotates to a detection position, the CCD/CMOS imaging system shoots the HPV genotyping chip in the large test tube through the side windowing position of the biochemical hybridization large rotating disc, if the surface of the HPV genotyping chip is not aligned with the camera at the moment, the corresponding large test tube is rotated through the stepping motor at the detection position, so that the surface of the HPV genotyping chip reaches the camera imaging position, and the shooting of the surface of the HPV genotyping chip is completed.
4. And (3) judging the image on the surface of the HPV genotyping chip by computer processing software to obtain a detection result.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.
Claims (3)
1. A full-automatic sample adding, hybridization and detection method of an HPV genotyping chip is characterized in that a detection system is used for detection, and the detection system comprises: a rotary table, a liquid-transferring device, a biochemical hybridization device and an image acquisition device are arranged in the PCR product;
the rotary disc in the PCR product comprises a rotary disc and a small test tube embedded in the circumferential direction of the rotary disc, and the rotary disc rotates clockwise or anticlockwise around a shaft;
the liquid-transfering device comprises a frame rotating around a shaft and a liquid-transfering needle rotating around the shaft and capable of vertically lifting in the vertical direction; a plurality of open containers are arranged in the circumferential direction of the rack, and needle heads of the liquid transferring needles can extend into the open containers;
the biochemical hybridization device comprises a rotatable turntable, wherein a large test tube is embedded on the circumference of the turntable, and the turntable can be used for heating the large test tube;
the image acquisition device comprises a CCD/CMOS imaging camera;
the detection method comprises the following steps:
step 1: placing the HPV genotyping chip in a large test tube of a biochemical hybridization device; the HPV genotyping chip comprises a substrate and HPV genotyping specific detection probes fixed on the substrate;
purifying a sample to be detected, specifically amplifying PCR to form a product to be detected, and then dripping the product to be detected into a small test tube on a rotary disc of the PCR product for storage;
step 2: transferring the product to be tested in the small test tube into a large test tube of a biochemical hybridization device by using a liquid transferring needle;
and step 3: adding a reaction solution required by the hybridization reaction into the large test tube from the open container by using a liquid transfer needle, heating the large test tube according to the reaction requirement, and rotationally stirring the large test tube by using a biochemical hybridization device so as to ensure that the product to be detected and the reaction solution added into the large test tube complete the hybridization reaction with the HPV genotyping specific detection probe on the substrate;
and 4, step 4: after the hybridization reaction in the large test tube is finished, shooting an HPV genotyping chip in the large test tube by using a CCD/CMOS imaging camera to obtain a result image;
and 5: and comparing, analyzing and judging the result image to obtain a detection result.
2. The HPV genotyping chip of claim 1, wherein the matrix is a nylon membrane or a nitrocellulose membrane.
3. The HPV genotyping chip of claim 1, wherein one of the open containers is filled with distilled water for washing the pipetting needle.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111849750A (en) * | 2020-06-23 | 2020-10-30 | 福建工程学院 | Automatic detection system of genotyping chip |
| CN112774240A (en) * | 2021-01-19 | 2021-05-11 | 胡艳皇 | Bioengineering is with circulated extraction equipment |
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Cited By (2)
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| CN112774240A (en) * | 2021-01-19 | 2021-05-11 | 胡艳皇 | Bioengineering is with circulated extraction equipment |
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