CN112143780A - Method for confirming sample in gray zone after nucleic acid amplification - Google Patents
Method for confirming sample in gray zone after nucleic acid amplification Download PDFInfo
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Abstract
The invention discloses a method for confirming a sample in a gray area after nucleic acid amplification. Adding a reagent into the detection reagent tube, putting the nucleic acid amplification reaction tube into the nucleic acid amplification reaction tube frame, covering a sealing cover, and forming a closed space by the card box main body and the sealing cover; then pressing the sealing cover downwards to form pre-sealing; then continuously pressing the sealing cover downwards, contacting the blade with the nucleic acid amplification reaction tube, cutting the bottom of the nucleic acid amplification reaction tube, and allowing the liquid in the nucleic acid amplification reaction tube to flow out of the detection reagent tube and then react; confirming and detecting the light source irradiation observation result; the device comprises a card box main body, a nucleic acid amplification reaction tube rack and a sealing cover, wherein the card box main body comprises a shell, a knife rest and a blade. The method does not change any flow of the existing PCR amplification or isothermal nucleic acid amplification operation, can detect the detection sample of the gray zone in a confirmation way, and has the advantages of high confirmation sensitivity and good specificity of gray zone signals, short time of the whole confirmation process, low cost and simple operation.
Description
Technical Field
The invention relates to a detection method for nucleic acid amplification, in particular to a confirmation detection method for a sample in a gray area after nucleic acid amplification, which can avoid repeated detection of the sample in the gray area and prevent aerosol pollution, is suitable for the field of nucleic acid detection, and belongs to the fields of medical detection, analytical chemistry, food detection and the like.
Background
The nucleic acid is a carrier carrying genetic information, and the nucleic acid sequences of different organisms have specificity, so that the substance to be detected can be effectively identified and detected through nucleic acid detection. The current nucleic acid detection technology is widely applied to various aspects such as food safety detection, clinical diagnosis, genotyping, molecular cloning and the like. Because the concentration of the collected nucleic acid in the sample to be detected is low, the nucleic acid sample is required to be amplified frequently. Common nucleic acid amplification techniques can be divided into temperature-variable amplification techniques and constant-temperature amplification techniques depending on whether thermal cycling is required in the amplification reaction. The temperature-variable amplification technique is Polymerase Chain Reaction (PCR). Isothermal amplification techniques include nucleic acid sequence dependent amplification (NASBA), loop-mediated isothermal amplification (LAMP), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), helicase dependent amplification (HAD), and the like. PCR requires a temperature raising and lowering process for the reaction and a precise temperature control instrument. Constant temperature amplification does not need repeated temperature rise and fall processes, but usually needs high temperature pre-denaturation, complex primer design and auxiliary action of other enzymes, and has high requirements on template specificity.
The amplified nucleic acid product needs to be detected to determine whether the product is a target product, and common detection methods can be classified into an end-point detection method and a process detection method. The end-point detection method is to detect the amplified product after the amplification reaction is finished, for example, the length of the amplified nucleic acid is observed by using a gel electrophoresis method, the yield of the nucleic acid is observed by using a turbidity method, and the presence or absence of the target nucleic acid is judged by using a test strip. The end-point method comprises two processes of amplification and detection, the time consumption is long, the detection precision is poor, aerosol pollution is easily formed by uncovering the detection after amplification, and a false positive result of subsequent detection is caused. The real-time detection method is to add DNA fluorescent dye such as SYBR Green, SYTO 9, Evagren, or Taqman probe during nucleic acid amplification. The dye intercalates into the DNA double strand during amplification of the nucleic acid. In the PCR amplification process, polymerase cuts the Taqman probe, and the fluorescent group and the quenching group at two ends of the Taqman probe are separated to generate a fluorescent signal. The real-time detection method has good reproducibility, the detection process is more convenient, the amplification is not needed for detection again, and the possible amplicon pollution caused by uncapping detection after amplification is avoided.
Because the real-time fluorescence PCR has high sensitivity, low detection limit and good reproducibility, the real-time fluorescence PCR is commonly used in the aspects of food safety detection, medical diagnosis and the like, and the fluorescence quantitative PCR result is often used for judging whether a sample is positive according to a Ct value. Taking the detection of the new coronavirus as an example, according to a judgment standard recommended by the national disease control center, if the Ct value is less than 37, the test can be reported as positive, the Ct value is between 37 and 40, repeated experiments are recommended, if the Ct value of a redo result is less than 40, an amplification curve has obvious peaks, and the sample is judged as positive, otherwise, the sample is negative. Therefore, a sample having a Ct value at a certain value in nucleic acid amplification is often referred to as a gray zone sample. Samples with Ct values between 35 and 40 are generally referred to as gray zone samples.
In practice, the judgment of the gray zone sample can also be adjusted according to the performance of a specific kit. However, in general, the sample in the gray zone cannot be directly judged to be negative or positive, and the PCR amplification detection is often required to be carried out again, which consumes great manpower, material resources and financial resources, so that the development of a sample for effectively detecting the Ct value in the gray zone after the PCR amplification can greatly shorten the time for judging the sample, and reduce the workload of experimenters.
Disclosure of Invention
Aiming at a sample to be detected with a Ct value in a gray area after fluorescent quantitative PCR amplification, the invention provides a method for confirming the sample with the Ct value in the gray area after fluorescent quantitative PCR amplification and isothermal amplification, which can avoid secondary nucleic acid amplification and improve the detection efficiency.
The technical scheme and the situation of the invention are as follows:
the prior art specifically needs a detection reagent and a matched closed mixing device, and the conditions are introduced as follows:
the specific nucleic acid probe corresponding to the nucleic acid amplification product has two main functional regions. The first functional region is generally about 20 bases in length, and the base sequence is complementary to a base sequence in the nucleic acid amplification product. The nucleic acid amplification product is referred to as the target sequence. The length of the second functional region is about 20 bases, and the second functional region is mainly combined with Cas protein in a gene editing CRISPR system. The arrangement of the specific base sequence of the second functional region is determined by the sequence of the corresponding Cas protein. The Cas protein is a protein related to CRISPR recognition in a CRISPR gene editing technology.
Although the invention is described by taking the Cas12a protein as an example, other Cas proteins with similar functions can be used in practice.
During detection, the Cas protein will first recognize the base sequence of the second functional region of the nucleic acid probe, and bind to the nucleic acid probe; then under the guide of the nucleic acid probe, the Cas protein searches a nucleic acid sequence which is complementary to the base sequence of the first functional region of the nucleic acid probe, namely a target sequence, and combines the target sequence; upon binding, the Cas protein initiates a bypass cleavage effect, starting to cleave a single DNA strand of the target sequence.
The length of the DNA single strand is about 5-10 bases, and both ends of the DNA single strand are respectively connected with a fluorescent label and a quencher. Based on the fluorescence resonance transfer effect, when the fluorescent label and the quencher are simultaneously attached to both ends of the single strand, no fluorescence is generated. And when the Cas protein starts the bypass cleavage effect, the fluorescence labeling single chain is cleaved, and the fluorescence labeling at the two ends is separated from the quencher to emit fluorescence.
The specific implementation of the invention adopts the device and combines the gene editing method to carry out specificity detection on the nucleic acid amplification product, can carry out high-sensitivity and high-specificity detection on the nucleic acid amplification product in the gray area, and can confirm the nucleic acid amplification result.
The CRISPR gene editing system is incompatible with a normal PCR amplification system and an isothermal amplification system. The CRISPR detection described above interferes with normal PCR detection if added before PCR or isothermal amplification begins. However, if the CRISPR detection reagent is added after PCR or isothermal amplification, there is an uncapping action after amplification, i.e., the PCR or isothermal amplification reaction tube needs to be opened after amplification, which also easily causes contamination of the amplification product and interferes with subsequent loop detection.
The invention aims to solve the problem and provides a detection device and a detection method with a sealing function matched with the detection device.
Putting the nucleic acid amplification tube containing the nucleic acid amplification solution to be confirmed and the amplicon specific detection reagent into a closed container in advance on the premise of not damaging the nucleic acid amplification tube; then, under the condition that the inside and the outside of the closed container are kept isolated, namely, under the condition that the biological macromolecules in the closed container do not leave the closed container to enter the surrounding environment, the nucleic acid amplification tube is opened by flexibly deforming the local part of the closed container or the local part in the closed container, and is mixed with the specific detection reagent of the amplicon which is put into the closed container in advance to carry out confirmation detection.
Firstly, adding an amplicon specificity detection reagent into a detection reagent tube, putting a nucleic acid amplification reaction tube with a nucleic acid amplification reaction liquid needing gray area sample confirmation into a nucleic acid amplification reaction tube frame, covering a sealing cover, and forming a closed space by a card box main body and the sealing cover;
then, the sealing cover is pressed downwards, so that the flexible sealing material on the sealing cover is tightly contacted with the upper port of the shell in a sealing manner to play a role of pre-sealing;
continuously pressing the sealing cover downwards, compressing the flexible sealing material, transmitting the convex pressure on the sealing cover to the elastic material of the nucleic acid amplification reaction tube frame through the supporting plate to compress the elastic material, moving the whole nucleic acid amplification reaction tube frame downwards, contacting the blade with the nucleic acid amplification reaction tube in the nucleic acid amplification reaction tube frame, cutting the bottom of the nucleic acid amplification reaction tube, and allowing the liquid in the nucleic acid amplification reaction tube to flow out of the detection reagent tube and then react;
finally, the housing 1 is illuminated from the entrance window with a light source of the desired wavelength, and the result is observed at the observation window: if fluorescence is generated, the sample is a positive sample; otherwise, the sample is a negative sample, and the confirmation detection of the gray area sample is completed.
The method adopts the following confirmation device, which comprises a card box main body, a nucleic acid amplification reaction tube frame and a sealing cover; the card box main body comprises a shell, a knife rest and a blade, a detection reagent tube is fixedly arranged in the bottom of the shell, the blade is arranged above the detection reagent tube, and the blade is fixed in the shell through the knife rest; a nucleic acid amplification reaction tube frame is arranged in the shell above the blade and comprises a supporting plate, an elastic material and a through hole, the supporting plate covers the elastic material, the through hole is formed in the middle of the supporting plate and the elastic material, and a nucleic acid amplification reaction tube is arranged in the through hole; an upper port of the shell is arranged above the nucleic acid amplification reaction pipe frame, and a sealing cover is arranged at the upper port of the shell and comprises a bulge, a cover plate and a flexible sealing material; a bulge is fixed in the middle of the bottom surface of the cover plate, a circle of cover plate bottom surface around the bulge is covered with a flexible sealing material, the bulge is arranged in an upper port of the shell, and the flexible sealing material is sealed and covered on the end surface outside the upper port of the shell.
The bulges and the cover plate are made of hard materials; the flexible sealing material is made of flexible materials and has thickness, so that the nucleic acid amplification reaction tube frame can be driven to move downwards by pressing the flexible sealing material, and the nucleic acid amplification reaction tube is cut by the blade.
The shell material is made of transparent material.
The shell material comprises a light-tight material, two side faces of the bottom of the shell are provided with a transparent and closed incident window and an observation window, the incident window and the observation window are both right opposite to the detection reagent tube, the incident window is externally connected with an excitation light source, and the observation window is used for observing whether fluorescence is generated in the detection reagent tube.
The shell is provided with a ventilation sealing hole, and an elastic film, a filter element or a silica gel column is embedded in the ventilation sealing hole.
The device of the invention provides a closed space, and avoids the problem of amplification product pollution which is possibly generated when the specific detection reagent of the amplification product is mixed with the amplification product.
The device of the invention is mainly characterized by having the function of pre-sealing, wherein the pre-sealing is firstly carried out, and then the nucleic acid amplification reaction tube is pressed down again in a closed environment to damage and flow into the detection reagent tube of the detection reagent. Thus, in the device, the nucleic acid amplification reaction tube and the detection reagent are in a closed space before being mixed, the tightness of the device cannot be damaged in all subsequent operations, and an amplification product can be prevented from entering an environmental space outside the device, so that the pollution of a PCR amplicon is avoided.
The invention has the technical advantages that:
1) has high fusion degree with the existing nucleic acid amplification detection method
The method does not change any flow of the existing PCR amplification or isothermal nucleic acid amplification operation, and only further confirms the detection samples with Ct larger than 35 and the like in the gray area.
2) The gray zone signal has high confirmation sensitivity and good specificity
The probe contained in the reagent does not participate in nucleic acid amplification, and no dimer or amplification side reaction is generated. The combined sequence is certainly from a detection sample and has high specificity.
Meanwhile, the gene editing functional enzyme has a signal amplification effect and also has good confirmation capability on low-concentration amplification products.
3) The samples in the gray zone were directly confirmed, and the entire confirmation process was less than 5 minutes.
Not only quickens the detection process, but also avoids the additional problems caused by sample nonuniformity and the like which may exist when the nucleic acid amplification is carried out again.
4) Low cost and simple operation. The cost of the confirmation reagent is equivalent to that of the normal nucleic acid reagent.
Drawings
FIG. 1 is an overall external view of an apparatus employed in the present invention;
wherein: 1-pipe wall, 2 a-entrance window, 2 b-observation window, 3-ventilation sealing hole, 12-cover plate and 13-flexible sealing material.
FIG. 2 is a view showing the internal construction of an apparatus employed in the present invention;
wherein: 1-tube wall, 2 a-incidence window, 2 b-observation window, 3-ventilation sealing hole, 4a,4 b-knife rest, 5-blade, 6-detection reagent tube, 7-support plate, 8-elastic material, 9, 10-through hole, 11-bulge, 12-cover plate, 13-flexible sealing material and 14-nucleic acid amplification reaction tube.
FIG. 3 is a structural view of a main body portion of the apparatus cartridge;
wherein: 1-tube wall, 2 a-incidence window, 2 b-observation window, 3-ventilation sealing hole, 4a,4 b-knife rest, 5-blade and 6-detection reagent tube.
FIG. 4 is a schematic diagram of a nucleic acid amplification reaction tube holder part of the apparatus;
wherein: 7-support plate, 8-elastic material and 9, 10-through hole.
FIG. 5 is a block diagram of the sealing cap portion of the device;
wherein: 11-bump, 12-cover plate, 13-flexible sealing material.
Detailed Description
The invention is further explained below with reference to specific embodiments and the drawing. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the practice of the present invention.
As shown in FIGS. 1 and 2, the device comprises a cartridge body, a nucleic acid amplification reaction tube holder, and a sealing cover.
As shown in fig. 3, the cartridge main body comprises a shell 1, knife rests 4a,4b and a blade 5, a detection reagent tube 6 is fixedly arranged in the bottom of the shell 1, the blade 5 is arranged above the detection reagent tube 6, and the blade 5 is fixed in the shell 1 through the knife rests 4a,4b on the two sides; a nucleic acid amplification reaction tube frame is arranged in the shell 1 above the blade 5, as shown in figure 4, the nucleic acid amplification reaction tube frame comprises a support plate 7, an elastic material 8 and through holes 9 and 10, the support plate 7 covers the elastic material 8, the through holes 9 and 10 are arranged in the middle of the support plate 7 and the elastic material 8, and nucleic acid amplification reaction tubes 14 are arranged in the through holes 9 and 10; an upper port of the shell 1 is arranged above the nucleic acid amplification reaction tube frame, and a sealing cover is arranged at the upper port of the shell 1, and comprises a bulge 11, a cover plate 12 and a flexible sealing material 13 as shown in FIG. 5; a bulge 11 is fixed in the middle of the bottom surface of the cover plate 12, a circle of the bottom surface of the cover plate 12 around the bulge 11 is covered with a flexible sealing material 13, the bulge 11 is arranged in the upper port of the shell 1, and the flexible sealing material 13 covers the end surface outside the upper port of the shell 1.
The lower part of the sealing cover plate 12 is provided with a bulge 11, the flexible sealing material 13 is made of sponge and the like, and the thickness of the flexible sealing material can be adjusted according to the selected flexible sealing material and elastic material.
In specific implementation, the blades 5 are mounted on the upper frames of the tool holders 4a and 4b, the edges of the knife backs of the blades 5 are sharpened, the edges of the knife backs of the blades 5 close to the edges have a certain thickness, and after the PCR reaction tube is cut, the PCR reaction tube is unfolded into two parts as far as possible, so that the nucleic acid amplification reaction tube 14 can be cut, and liquid in the tube can flow out smoothly. A detection reagent tube 6 is arranged right below the blade 5, and the detection reagent tube 6 contains a detection reagent. The detection reagent tube 6 is supported by a fixed support in the cartridge without obstructing the irradiation of the exciting light to the detection reagent tube 6 and the observation of the result.
The knife seat in the card box body plays a role of fixing the blade, the blade can be further fixed by glue and the like, and a small groove on the knife seat can be blocked after the blade is inserted into the knife seat, so that the blade is completely fixed. The blade edge portion is as thin as possible and is preferably hard, for example, a tungsten steel blade may be used.
The protrusions 11 and the cover plate 12 are made of a hard material such as polymethyl methacrylate; the flexible sealing material 13 is made of flexible material and has a thickness such that pressing the flexible sealing material 13 can drive the nucleic acid amplification reaction tube rack to move downwards to cut the nucleic acid amplification reaction tube by the blade 5. The height of the bulge can be optimized according to the flexible sealing material at the edge of the sealing cover and the compressibility degree of the elastic material on the PCR test tube nucleic acid amplification reaction tube frame. The thickness of the flexible sealing material can be adjusted according to the selected material, and the sealing effect is ensured.
A housing 1 is made of a hard material. The housing 1 material is composed of a transparent material to facilitate observation of the fluorescent signal.
Another material of the shell 1 is made of a light-tight material, as shown in fig. 1-3, two sides of the bottom of the shell 1 are provided with a transparent and closed entrance window 2a and an observation window 2b, both the entrance window 2a and the observation window 2b are opposite to the detection reagent tube 6, the entrance window 2a is externally connected with an excitation light source, and the observation window 2b is used for observing whether fluorescence is generated in the detection reagent tube 6. The incident window and the observation window on the card box main body are both right opposite to the tube body of the detection reagent tube, so that incident light can be ensured to irradiate on liquid in the detection reagent tube, and the fluorescence of the detection reagent tube can be observed.
The shell 1 is provided with a ventilation sealing hole 3, an elastic film, a filter core or a silica gel column is embedded in the ventilation sealing hole 3, and the inner space and the outer space of the card box are isolated through the elastic film, the filter core or the silica gel column under the condition of keeping the air pressure balance inside and outside the card box main body, so that aerosol pollution is prevented.
The embodied support plate 7 consists of a hard material, such as polymethyl methacrylate. An elastic material 8, such as a sponge, is attached to the underside of the support plate 7. The nucleic acid amplification reaction tube is made of rubber materials, and the detection reagent tube 6 is provided with an amplicon specificity detection reagent edited by CRISPR genes.
The support plate 7 and the elastic material 8 at the center of the nucleic acid amplification reaction tube support are provided with through holes 9 and 10 which are penetrated through, the outward protruding part at the edge of the upper end of the nucleic acid amplification reaction tube is just clamped on the through hole 9 of the support plate 7, the tube body at the lower part of the nucleic acid amplification reaction tube is completely placed into the through holes 9 and 10 and penetrates through the through hole channel in whole, the elastic material at the bottom of the nucleic acid amplification reaction tube support can play a role in fixing the PCR reaction tube, so that the center of the PCR reaction tube is just opposite to a blade, and the cutting degree of the PCR reaction tube is ensured to be. The whole nucleic acid amplification reaction tube frame can be just placed in the main body of the cartridge.
If the Ct value of the sample after PCR amplification is in a gray area and cannot be judged to be a positive sample or a negative sample, the device is started.
As shown in fig. 2, the operation process of the method of the present invention is:
firstly, adding an amplicon specificity detection reagent into a detection reagent tube 6, putting a nucleic acid amplification reaction tube with nucleic acid amplification reaction liquid needing gray zone sample confirmation into a nucleic acid amplification reaction tube frame, putting the whole nucleic acid amplification reaction tube frame into a card box, covering a sealing cover, and forming a closed space by a card box main body and the sealing cover;
then, the sealing cover is pressed downwards, so that the flexible sealing material 13 on the sealing cover is tightly contacted with the upper port of the shell 1 in a sealing manner to play a role of pre-sealing; at the moment, according to the arrangement of the ventilation sealing hole 3, the elastic film and the filter element are embedded, the balance of the air pressure inside and outside the card box is kept, the downward pressing operation is facilitated, meanwhile, aerosol in the card box is prevented from entering the surrounding environment, and therefore the pollution of the amplicon aerosol can be avoided.
Then, the sealing cover is continuously pressed downwards, the flexible sealing material 13 is compressed, the pressure of the bulge 11 on the sealing cover is transmitted to the elastic material 8 of the nucleic acid amplification reaction tube frame through the supporting plate 7, so that the elastic material 8 is compressed, the whole nucleic acid amplification reaction tube frame moves downwards, the blade 5 is contacted with the nucleic acid amplification reaction tube in the nucleic acid amplification reaction tube frame and cuts the bottom of the nucleic acid amplification reaction tube, and the liquid in the nucleic acid amplification reaction tube flows out to the detection reagent tube 6 and then reacts;
finally, the housing 1 is illuminated with a light source of the desired wavelength from the entrance window 2a, and the result is observed at the observation window 2 b: if fluorescence is generated, the sample is a positive sample; otherwise, the sample is a negative sample, and the confirmation detection of the gray area sample is completed.
The gray zone of the present invention is between negative and positive. That is, the Ct value of nucleic acid amplification is between negative and positive.
In the design operation, as required, after the sealing cover is pressed down, the relative position between the sealing cover and the card box body is fixed by using a device such as a buckle, a screw and the like, and the sealing performance of the whole device is further ensured.
The corresponding fluorescence detector can be configured to read and analyze the fluorescence signal.
The temperature of the detection reagent tube can be controlled according to the requirement so as to promote the reaction speed and improve the detection efficiency.
The materials used in the devices of the present invention, as well as the shapes and configurations of the devices, are set forth in the description and should not be construed as limiting the scope of the invention. As long as the device has the function of pre-sealing, the nucleic acid amplification reaction tube and the detection reagent are in a sealed space before being mixed, and the tightness of the device is not damaged by all subsequent operations, and the device is within the protection scope of the present invention.
The actual nucleic acid detection procedure using the present invention is as follows:
1) and (3) carrying out normal fluorescence PCR amplification detection or isothermal amplification detection on the actual detection sample.
2) And (3) judging the obtained Ct value to be positive if the Ct value is less than 35 (or adjusting a corresponding positive judgment basis according to actual needs), and finishing the detection. For isothermal nucleic acid amplification, corresponding reaction time values can be set as the criterion according to the actual conditions of the kit.
3) If the Ct value is greater than 35 or the detection result is in the gray area according to other standards, the detection method developed by the invention is started.
a) The corresponding specific nucleic acid detection reagent is added into the closed device.
b) And taking out the nucleic acid amplification instrument in the nucleic acid amplification reaction tube corresponding to the grey area result, directly putting the nucleic acid amplification instrument into a closed device added with the reagent, starting the closing and mixing process, and mixing the nucleic acid amplification product and the detection reagent in the closed device.
c) And (4) putting the sealing device into a corresponding fluorescence detector, and reading a fluorescence result after 1-5 minutes to finish the confirmation.
The fluorescence detector required in the method only needs an excitation light source matched with the wavelength of the fluorescent probe and an observation and recording function matched with the emission wavelength. To facilitate the speed of the confirmatory detection, the fluorescence detector device may also have a heating function. The temperature can be controlled to 37 degrees centigrade.
The embodiment of the method of the invention is as follows:
example 1:
the specific steps are as follows.
1) According to the recommendation of China CDC, ORF or N gene fragment can be selected as the gene locus for detecting the new crown. PCR detection of ORF gene recommended by CDC is exemplified.
If the Ct value of the sample is in the gray zone after the PCR amplification is finished and the sample is difficult to judge as a positive sample or a negative sample, the device is started. Firstly, preparing a specific detection reagent for an amplification product, wherein the specific detection reagent comprises 0.2 micromole per liter of specific nucleic acid probe corresponding to a PCR amplification product, 0.2 micromole per liter of Cas protein and 2.5 micromole per liter of fluorescence-labeled single-stranded probe, and in order to ensure that the reaction is better carried out, the system also comprises 1 XNEB buffer solution and 1U RNA inhibitor per microliter. After preparation, 25 microliters of amplification product specific detection reagent is added into the detection reagent tube.
2) And (3) placing the PCR sample tube with the Ct value in the ash area after the PCR detection of the new coronavirus into a PCR test tube nucleic acid amplification reaction tube frame, so that the edge of the tube opening of the PCR sample tube is just clamped on the support plate, and the whole tube body is accommodated in the through hole. The whole PCR test tube nucleic acid amplification reaction tube frame is placed into the main body of the card box.
3) The sealing cover is covered, and the pressure is pressed downwards to the maximum limit. If necessary, the cartridge can be centrifuged or shaken after the pressing is finished to ensure that the liquid in the PCR sample tube flows completely into the detection reagent tube, and if the reaction speed is accelerated, the whole device can be placed in a 37 ℃ incubator.
4) And (3) transmitting incident light through an incident window by using a corresponding fluorescence detection instrument, and reading and analyzing a fluorescence signal in an observation window.
Example 2:
the specific embodiment is described by taking the detection of transgenic promoter CaMV335 gene in transgenic soybean as an example.
1) And carrying out fluorescent quantitative PCR detection on the transgenic soybean promoter CaMV 335.
If the Ct value of the sample is in the gray zone after the PCR amplification is finished and whether the sample is the transgenic soybean is difficult to judge, the device is started. Firstly, preparing a specific detection reagent for an amplification product. Cas protein, fluorescently-labeled single-stranded probe, NEB buffer and RNA inhibitor are used as in example 1. The concentration of specific nucleic acid probe corresponding to the PCR amplified product of transgenic soybean was the same as in example 1.
2) And (3) placing the PCR sample tube with the Ct value in the ash area after the PCR detection of the transgenic soybean into a PCR test tube nucleic acid amplification reaction tube frame, so that the edge of the tube opening of the PCR sample tube is just clamped on the supporting plate, and the whole tube body is accommodated in the through hole. The whole PCR test tube nucleic acid amplification reaction tube frame is placed into the main body of the card box.
The rest of the procedure was the same as in example 1.
Claims (7)
1. A method for confirming that a sample is in a gray zone after nucleic acid amplification, comprising: putting the nucleic acid amplification tube containing the nucleic acid amplification solution to be confirmed and the amplicon specific detection reagent into a closed container in advance on the premise of not damaging the nucleic acid amplification tube; then, in the case where the inside and outside of the closed vessel are kept isolated, that is, in the case where the inside of the closed vessel has no way for the biological macromolecules to leave the closed vessel and enter the ambient environment, the nucleic acid amplification tube is opened and mixed with the amplicon-specific detection reagent that has been put into the closed vessel in advance to carry out confirmatory detection.
2. The method of claim 1, wherein the method comprises the steps of:
firstly, adding an amplicon specific detection reagent into a detection reagent tube (6), putting a nucleic acid amplification reaction tube with a nucleic acid amplification reaction liquid needing gray zone sample confirmation into a nucleic acid amplification reaction tube frame, covering a sealing cover, and forming a closed space by a card box main body and the sealing cover;
then, the sealing cover is pressed downwards, so that the flexible sealing material (13) on the sealing cover is tightly contacted with the upper port of the shell (1) in a sealing manner to play a role of pre-sealing;
then, the sealing cover is continuously pressed downwards, the flexible sealing material (13) is compressed, the pressure of the bulge (11) on the sealing cover is transmitted to the elastic material (8) of the nucleic acid amplification reaction tube frame through the supporting plate (7), so that the elastic material (8) is compressed, the whole nucleic acid amplification reaction tube frame moves downwards, the blade (5) is contacted with the nucleic acid amplification reaction tube in the nucleic acid amplification reaction tube frame and cuts the bottom of the nucleic acid amplification reaction tube, and the liquid in the nucleic acid amplification reaction tube flows out to the detection reagent tube (6) and then reacts;
finally, the inside of the housing (1) is illuminated with a light source of the desired wavelength from the entrance window (2a), and the result is observed at the observation window (2 b): if fluorescence is generated, the sample is a positive sample; otherwise, the sample is a negative sample, and the confirmation detection of the gray area sample is completed.
3. The method of claim 2, wherein the method comprises the steps of: the method adopts the following confirmation device, which comprises a card box main body, a nucleic acid amplification reaction tube rack and a sealing cover;
the card box main body comprises a shell (1), tool rests (4a, 4b) and a blade (5), a detection reagent tube (6) is fixedly placed in the bottom of the shell (1), the blade (5) is arranged above the detection reagent tube (6), and the blade (5) is fixed in the shell (1) through the tool rests (4a, 4 b);
a nucleic acid amplification reaction tube frame is arranged in the shell (1) above the blade (5), the nucleic acid amplification reaction tube frame comprises a support plate (7), an elastic material (8) and through holes (9, 10), the support plate (7) covers the elastic material (8), the through holes (9, 10) are formed in the middle parts of the support plate (7) and the elastic material (8), and a nucleic acid amplification reaction tube (14) is arranged in the through holes (9, 10);
an upper port of the shell (1) is arranged above the nucleic acid amplification reaction tube frame, a sealing cover is arranged at the upper port of the shell (1), and the sealing cover comprises a bulge (11), a cover plate (12) and a flexible sealing material (13); the middle of the bottom surface of the cover plate (12) is fixed with a bulge (11), the bottom surface of the cover plate (12) around the bulge (11) is covered with a flexible sealing material (13), the bulge (11) is arranged in an upper port of the shell (1), and the flexible sealing material (13) is covered on the end surface outside the upper port of the shell (1).
4. The method of claim 3, wherein the method comprises the steps of: the bulges (11) and the cover plate (12) are made of hard materials; the flexible sealing material (13) is made of flexible materials and has a thickness such that the nucleic acid amplification reaction tube frame can be driven to move downwards by pressing the flexible sealing material (13) to be cut by the blade (5).
5. The method of claim 3, wherein the method comprises the steps of: the shell (1) is made of transparent materials.
6. The method of claim 3, wherein the method comprises the steps of: the material of the shell (1) is composed of a light-tight material, two side faces of the bottom of the shell (1) are provided with a transparent and closed incident window (2a) and an observation window (2b), the incident window (2a) and the observation window (2b) are both right opposite to the detection reagent tube (6), the incident window (2a) is externally connected with an excitation light source, and the observation window (2b) is used for observing whether fluorescence is generated in the detection reagent tube (6).
7. The method of claim 3, wherein the method comprises the steps of: the shell (1) is provided with a ventilation sealing hole (3), and an elastic film, a filter element or a silica gel column is embedded in the ventilation sealing hole (3).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114703048A (en) * | 2022-06-08 | 2022-07-05 | 珠海市尚维高科生物技术有限公司 | Miniaturized nucleic acid amplification detection device |
| WO2022213383A1 (en) * | 2021-04-09 | 2022-10-13 | 广州市达安医疗器械有限公司 | Closed nucleic acid test consumable and test method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888902A (en) * | 2006-08-11 | 2007-01-03 | 杭州优思达生物技术有限公司 | Full closed target nucleic amplifier fast testing device |
| CN102534011A (en) * | 2012-01-18 | 2012-07-04 | 曹利民 | Totally-enclosed fast fluorescence detection method and device for target nucleic acid amplification products |
| CN105199940A (en) * | 2015-09-22 | 2015-12-30 | 广州和实生物技术有限公司 | Anti-pollution portable gene detection method and device |
| CN107557433A (en) * | 2017-08-23 | 2018-01-09 | 浙江大学 | A kind of visible detection method of nucleic acid amplification product |
| CN111334425A (en) * | 2020-04-09 | 2020-06-26 | 杭州奥盛仪器有限公司 | Reaction tube for coupling double systems for nucleic acid amplification and CRISPR detection and double system coupling method |
| CN111548900A (en) * | 2019-02-12 | 2020-08-18 | 浙江大学 | Device and method for amplifying and detecting nucleic acid |
-
2020
- 2020-09-01 CN CN202010903774.0A patent/CN112143780B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888902A (en) * | 2006-08-11 | 2007-01-03 | 杭州优思达生物技术有限公司 | Full closed target nucleic amplifier fast testing device |
| CN102534011A (en) * | 2012-01-18 | 2012-07-04 | 曹利民 | Totally-enclosed fast fluorescence detection method and device for target nucleic acid amplification products |
| CN105199940A (en) * | 2015-09-22 | 2015-12-30 | 广州和实生物技术有限公司 | Anti-pollution portable gene detection method and device |
| CN107557433A (en) * | 2017-08-23 | 2018-01-09 | 浙江大学 | A kind of visible detection method of nucleic acid amplification product |
| CN111548900A (en) * | 2019-02-12 | 2020-08-18 | 浙江大学 | Device and method for amplifying and detecting nucleic acid |
| CN111334425A (en) * | 2020-04-09 | 2020-06-26 | 杭州奥盛仪器有限公司 | Reaction tube for coupling double systems for nucleic acid amplification and CRISPR detection and double system coupling method |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022213383A1 (en) * | 2021-04-09 | 2022-10-13 | 广州市达安医疗器械有限公司 | Closed nucleic acid test consumable and test method |
| CN114703048A (en) * | 2022-06-08 | 2022-07-05 | 珠海市尚维高科生物技术有限公司 | Miniaturized nucleic acid amplification detection device |
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| CN112143780B (en) | 2022-04-08 |
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