WO2004101733A1

WO2004101733A1 - A pcr amplification device used for multi-steps reaction and a wash-free pcr amplification tube used for direct gene detection

Info

Publication number: WO2004101733A1
Application number: PCT/CN2004/000438
Authority: WO
Inventors: Zuhong Lu; Quanjun Liu; Yunfei Bai; Qinyu Ge; Tian Wen; Xiao Xie; Jing Tu
Original assignee: Southeast University
Priority date: 2003-05-01
Filing date: 2004-04-30
Publication date: 2004-11-25

Abstract

The present invention provides a PCR amplification tube used for multi-steps reaction. In the PCR amplification tube, another reaction poor or reservoir is added or made in a lump. In a closed environment, the multi-steps reaction can be done in the same PCR amplification tube. The said PCR amplification tube used for multi-steps reaction includes GroupWare’s. The amplification tube GroupWare’s are made up of tube cap and amplification tube. The amplification tube GroupWare’s have up opening solution poor and launder, and the solution poor is positioned in the amplification tube. The present invention also provides a wash-free PCR amplification tube used for direct gene detection, which includes a reaction tube, the tube is made up of tube cap and tube body. The molecule signal is immobilized on the reaction tube used for PCR. There is a transparent window in the region where the molecule signal is immobilized on the reaction tube used for PCR. The molecule signal is immobilized on the inboard of the transparent window.

Description

PGR Amplifier for Multi-Step Reactions and Wash-Free PGR Amplification Tubes for Direct Gene Detection

I. Technical Field

The present invention relates to a PCR amplifier which can be used to perform multi-step inversion, and is used for gene amplification and related gene detection.

The invention also relates to a PCR amplification tube used for gene amplification and direct detection (a PCR amplification tube is a technical term well-known to those skilled in the art), particularly to a flush-free, which can directly detect amplification products. PCR amplification tube. 2. Background Technology

At present, polymerase chain reaction (PCR) technology is indispensable in molecular biology research and medical clinical diagnosis. PCR is usually performed in a closed lumen, which can amplify the target gene being detected by tens to millions of times. However, after gene amplification, it is generally necessary to detect the amplified product by methods such as electrophoresis. Due to the extremely high sensitivity of the PCR method, its amplification products will inevitably enter the air during the detection process. When the next test is carried out, these floating products may be "added" again, which is why the PCR test is easy to obtain so-called "false positives". For this reason, some international biotechnology companies have developed closed genes Amplification and detection technologies, such as fluorescent quantitative PCR technology. However, this technology requires very complicated dynamic optical detection devices, and the detection equipment is very complicated, which is difficult to promote in China. At the same time, this technology can only detect one gene per test. Information cannot meet the requirements of people in the era of bioinformatics for the detection of a large amount of genetic information.

Gene chip technology provides a powerful tool for people to detect a large amount of genetic information at the same time. Gene chip immobilizes many different nucleic acid probes (single-stranded oligonucleotides) on glass On the chip, the amplified product of the gene in the sample is labeled with fluorescence, and hybridized with the nucleic acid probe on the chip, and detected by a fluorescence scanner. Although the amount of information obtained by gene chips is large, most gene chips are currently mainly used for laboratory research applications such as detection of mRNA expression in cells. In terms of detection of important pathogenic microorganisms, there are few mature application examples and excellent products to date. The main reason is that the sample processing, amplification labeling, hybridization, and detection operations are performed separately, which makes the operation of the entire detection process cumbersome and complicated, and therefore increases the chance of contamination and reduces the results obtained. Reliability. Although the gene amplification technology and the chip detection method have been integrated into one, its characteristic is that the entire gene amplification process is performed in a micro-reaction cell. Therefore, it is necessary to repeatedly increase and decrease the temperature of the same part of the device. Results were dynamically tracked and quantitatively analyzed in real time. For this reason, some researchers at home and abroad have proposed to combine gene chips with microfluidics technology to achieve the integration of all operating processes. The applicant has also studied this, and has proposed a gene amplification microarray probe cycle detection type biochip and the like. However, because such a chip is difficult to prepare and a special reactor needs to be developed to support it, its cost is very high. At the same time, the gene chip requires that the amplification product of the target gene is labeled with fluorescence, and the hybridization state of the nucleic acid probe is detected by a fluorescent signal. This requires fluorescence to be incorporated during sample processing, which not only increases the cost of sample processing, but also increases difficulty and uncertainty. Moreover, the reliability of detection is affected by the problem of labeling efficiency in this process. At the same time, due to the increased complexity of the entire operation process, such as the control of sample processing conditions and repeated washing of non-specific probes after hybridization, it is not conducive to integrated detection. Therefore, the development of low-cost biochip technology that can perform unlabeled detection of the detected gene sequence is the third key to achieve a large number of practical applications of biochips in fields such as medicine and life sciences.

Technical problem: The present invention provides a multi-step reaction PCR amplification tube. Adding or forming another reaction cell or reservoir in the tube at one time can complete multiple steps in the same PCR amplification tube in a closed state.

The invention also provides a method that can simplify the operation of the integrated operation method of gene amplification, hybridization and detection, eliminates the need to incorporate fluorescence when the target gene is amplified, reduces the cost, and can improve the reliability of the obtained results. Rinse-free direct detection of genes

PCR amplification tube. Technical solution: In one aspect, the present invention relates to a PCR amplifier for multi-step reaction, which includes an amplification tube assembly. The amplification tube assembly is composed of a tube cover and an amplification tube, and an opening is provided on the amplification tube assembly. The upward solution cell and solution channel are located in the amplification tube.

In another aspect, the present invention relates to a flush-free PCR amplification tube capable of directly detecting genes, including a reaction tube, and the reaction tube is composed of a tube cover 11 and a tube body 12, and is contained in a reaction tube 1 for PCR. Fixed with molecular beacon 2,. Technical effects:

In one aspect, the present invention relates to a PCR amplifier for a multi-step reaction, which includes an amplification tube assembly, the amplification tube assembly is composed of a tube cover and an amplification tube, and an amplification tube assembly is provided with a solution with an upward opening. The cell and the solution channel are located in the amplification tube.

① Since the storage tank is set in the reaction tube in the present invention, the reaction tube itself can form a relatively closed space, so that the gene amplification and post-amplification operations can be completed in the above relatively closed space: A) The prepared to be expanded The gene-enriched extract and the amplification solution (including the corresponding primers and enzymes) are added to the reaction tube body, and the gene amplification reaction system is added to the storage tank, and the PCR reaction tube is sealed; B) the sealed reaction tube is sealed; Placed in the corresponding gene amplification instrument for amplification; The gene amplification instrument can use various popular instruments, or it can be an improved integrated instrument for hybridization and detection; C) After the amplified solution in the tube is poured, the two liquids are mixed, and the mixed liquid is vigorously shaken to the bottom of the PCR reaction tube; D) After the solutions are mixed, the next step of the gene amplification reaction is performed. Therefore, the present invention can enable multi-step reactions such as gene amplification or related gene detection in the same closed system.

② Because the present invention enables two-step RT-PCR (reverse transcription gene amplification) to be realized in a single PCR tube. The reaction reagent in the PCR tube is subjected to a reverse transcription process to generate D fragments in the solution, and then the reverse transcription system is mixed with the solution in the storage tank as described above, and then the PCR reaction is performed. Two-step RT-PCR in a closed system has the advantages of high reaction yield, less contamination, and high sensitivity.

③ The present invention adopts a closed system. The two reaction systems are stored, and two-step gene amplification reactions can be performed without being disturbed by the outside world. For example, in a nested PCR reaction, the operation steps of the experiment can be greatly reduced, and the repeatability of the experiment can be improved. And precision.

④ In the present invention, two reaction systems are stored in a closed system, one of which is a gene amplification mixed system, and the hybridization buffer is stored in a storage tank, which can be mixed with the hybridization buffer after the PCR reaction is completed. Finally, hybridization is performed with a nucleic acid probe fixed on the inner wall of the PCR tube. As a result of detecting the amplified gene, if there is a clear hybridization signal, it can be confirmed that the detected gene exists before the gene is amplified. In another aspect, the present invention relates to a flush-free PCR amplification tube capable of directly detecting genes, including a reaction tube, and the reaction tube is composed of a tube cover 11 and a tube body 12, and is contained in a reaction tube 1 'for PCR. Fixed with molecular beacon 2,.

① Since the molecular beacon is set in the reaction tube in the present invention, the reaction tube itself can form a relatively closed space, and the operation method for integrating gene amplification, hybridization and detection can be completed in the above relatively closed space according to the following steps: A ) Add the prepared gene extract to be amplified and the amplification solution (including the corresponding primers and enzymes, etc.) to the reaction tube body, and fasten the cap to the reaction tube; B) Close the closed reaction The tube is placed in the corresponding gene amplification instrument for amplification; the gene amplification instrument can use various popular instruments, or it can be an improved integrated instrument for amplification hybridization and detection; C) after the tube is amplified The solution flows down the fixed molecular beacon area, interacts with the molecular beacon on the inner surface of the tube, and controls a certain temperature for specific hybridization; D) the optical detection method is used to read out the fluorescence of the molecular beacon on the reaction tube Signal to obtain gene hybridization information. And this method is precisely because of the present invention, it is possible to achieve continuous gene amplification, hybridization, and detection operations through some simple operations, and it is not necessary to add markers such as fluorescent groups during the target gene amplification process to avoid Opening the amplification tube after PCR amplification to clean target genes for non-specific hybridization, and adding hybridization solution, etc. not only simplifies the operation steps, but more importantly, completely avoids the PCR amplification product being positive for the detection community and making the The method can use existing equipment or slightly improved equipment, which reduces costs. Molecular beacons are different from ordinary nucleic acid probes (single-stranded nucleic acid probes). The molecular beacon is composed of a fluorescent group, a fluorescent quenching group, a stem region of a double-stranded nucleic acid, and a single-stranded circular nucleic acid region. The single-stranded circular nucleic acid region is a region recognized by hybridization of a molecular beacon with a target gene to be detected. However, after the molecular beacon is combined with the rake gene, the stem of the double-stranded nucleic acid is opened, so that the distance between the fluorescent group and the fluorescence quenching group is changed, which causes the fluorescent luminescence property of the molecular beacon to be changed. The DNA strand of a common nucleic acid is detected by hybridizing with a target gene labeled with a fluorescent molecule. Molecular beacons typically detect target genes directly in solution, but this can only detect one target gene. The present invention uses a molecular beacon method to detect a target gene, which eliminates the need for special manipulations such as incorporation of fluorescence into the target gene, and does not require cleaning of non-specific adsorption on the chip after hybridization to reduce the fluorescent background. In the invention, the molecular beacon is directly fixed in the PCR amplification tube, and the integration of gene amplification and hybridization detection is realized in the PRC amplification tube. The fluorescent group and the fluorescence quenching group in the molecular beacon do not need to be fluorescently labeled for the nucleic acid fragment used for hybridization with the molecular beacon during hybridization detection. After hybridization, the nucleic acid fragment is combined with the molecular beacon. The fluorescent group and the fluorescence quenching group inside the molecular beacon are separated in space, and the signal of the fluorescent group can be detected. At the same time, the fluorescent group and the fluorescence quenching group in the molecular beacon section that has not undergone hybridization can be detected. The cluster maintains a small distance in space, and the fluorescent signal cannot be detected, so no flushing is needed, reducing the number of operating steps and reducing the difficulty of detection. In summary, the present invention can simplify the operation of the integrated operation method of gene amplification, hybridization, and detection, reduce the cost, improve the reliability of the obtained results, and achieve flush-free.

② Since the present invention enables the operation method to perform multi-pass amplification, hybridization and detection, the present invention enables the method to achieve dynamic tracking detection and obtain quantitative results.

③ The present invention adopts the connection of "connecting a molecular beacon to a transparent window through a chemically active group", which has the advantages of strong binding, not easy to detach, and can be used repeatedly and repeatedly. At the same time, fewer detection samples are required, and the molecular probe high density.

④ The technical measures of the present invention "fixing molecular beacons in a polymer gel medium and then fixing them on a transparent window" increase the density of the fixed molecular beacons, enhance the hybrid signal, and further improve the reliability of the obtained results.

⑤ The method of "dividing the transparent window into several regions, and assembling molecular beacons of nucleic acid sequence with different or the same base arrangement on different regions respectively" of the present invention can be performed in a single PCR experiment on several or even tens of thousands Hybridization detection of gene fragments enables high-throughput detection.

4. Description of the Drawings

FIG. 1 is a schematic structural diagram of Embodiment 1 of the present invention.

FIG. 2 is a schematic structural diagram of Embodiment 2 of the present invention.

FIG. 3 is a schematic structural diagram of Embodiment 3 of the present invention.

FIG. 4 is a schematic structural diagram of Embodiment 4 of the present invention. In Figs. 1-4, 1: the tube cover, 2: the ordinary PCR tube, 3: the liquid channel, 4: the solution pool. Fig. 5 is a schematic structural diagram of a flush-free PCR amplification tube that can directly detect genes in the present invention.

Fig. 6 is a direct chemical connection diagram of molecular beacons used in the no-wash PCR amplification tube of the present invention.

FIG. 7 is a schematic diagram of a partial structure of an embodiment of a flush-free PCR amplification tube of the present invention. V. Specific implementation plan

Example 1

A PCR amplifier that can be used to complete a multi-step reaction, using the method:

1. Preparation of the chip

According to the sequence of each target gene to be detected, design a 20-50 base oligonucleotide probe that can effectively hybridize with the negative strand (that is, the same as the positive strand). One end of the probe is labeled with an amino group. These probes are fixed to the inner surface of the processed tube cap according to a certain array to prepare a tube cap chip.

2. Primer design for amplification

According to each target gene sequence to be detected, a pair of amplification primers of about 20 bases are designed at both ends, and one of the primers 5 is complementary to the positive strand, and a fluorescent molecule (such as FAM, CY3, CY5, etc.) is labeled at the end. ).

3. Due to amplification

Add a mixed PCR or RT-reaction system, primers, and processed DNA or RNA templates to a common PCR amplification tube in advance. Then plug it into the solution pool and add 30ul of hybridization buffer to the solution pool. Finally, close the prepared tube cap. Perform the amplification reaction on the PCR instrument according to a certain procedure. 4.Hybrid detection

After the PCR amplification is completed, invert the amplification tube, and vigorously mix the PCR reaction solution and the hybridization solution and shake to the end of the tube cover, and place at 37 ° C for 1 hour for hybridization.

5. Chip detection

Place the tube cover chip under a fluorescent scanning microscope to detect the hybridization results.

Example 2

A PCR amplification tube for multi-step reaction includes a reaction tube and the reaction tube is composed of a PCR tube and a liquid storage tank in the tube. Specific primers for detection of atypical pneumonia-associated coronaviruses (see WHO published primers), reverse transcription system in PCR tube, gene amplification system in reservoir, and appropriate amount of SYBGREEN . Using the gene amplification conditions recommended by the WHO, after setting on the PCR instrument, after reverse transcription, invert the PCR tube to mix the two solutions, and then perform PCR amplification. Before and after amplification, you can directly observe under UV light. If fluorescence appears, the gene is amplified, and the test system contains the atypical pneumonia-associated coronavirus.

Example 3

1. Preparation of the chip

According to the sequence of each target gene to be detected, a 20-50 base oligonucleotide probe that can effectively hybridize with the negative strand (that is, the same as the positive strand) is designed, and the software is used to analyze the target with Beacon des igner 2.1 software. The probe is designed into a molecular beacon, and then these probe solutions are adhered to the inner surface of the treated tube cap according to a certain array by spotting. Prepare the tube cap chip.

2. Primer design for amplification

Design a pair of 20 bases at each end based on each target gene sequence to be detected Left and right amplification primers.

3.PCR amplification, hybridization and detection

PCR amplification, hybridization and detection are the same as protocol 1

Example 4

A PCR amplifier for multi-step reaction includes an amplification tube assembly. The amplification tube assembly is composed of a tube cover 1 and an amplification tube 2. The amplification tube assembly is provided with a solution pool and a solution flow opening upward. In lane 3, the solution cell is located in the amplification tube 2. The solution cell is suspended on the tube cover 1. The outer bottom surface of the solution cell is an inclined bottom surface.

Example 5

A PCR amplifier for multi-step reaction includes an amplification tube assembly. The amplification tube assembly is composed of a tube cover 1 and an amplification tube 2. The amplification tube assembly is provided with a solution pool and a solution flow opening upward. Lane 3, the solution pool is located in the amplification tube 2, the solution pool is a circular solution 4, the solution flow channel 3 is located in the inner peripheral wall of the annular solution pool 41, the solution pool is located in the amplification tube 2, the outer wall of the solution pool and the amplification The inner walls of the tubes coincide.

Example 6

The solution cell is a round-shaped solution cell 42, and its cross section is round-shaped. The solution channel 3 is the gap between the round-shaped solution cell 42 and the inner wall of the amplification tube 2. The cross-section of the round-shaped solution cell 42 is A circle shape with a central angle greater than 180 ° is provided in the amplification tube 2. The outer wall of the circle shape solution pool 42 coincides with the inner wall of the amplification tube 2. The top surface of the circle shape solution pool 42 is an inclined surface.

Example 7

A PCR amplifier for multi-step reaction includes an amplification tube assembly. The amplification tube assembly is composed of a tube cover 1 and an amplification tube 2. The amplification tube assembly is provided with a solution pool and a solution flow opening upward. In lane 3, the solution pool is located in the amplification tube 2. The solution pool is fixed in the amplification tube 2 by the bracket 5. Embodiment 8 A flush-free PCR amplification tube capable of directly detecting genes, including a reaction tube, and the reaction tube is composed of a tube cover 11 and a tube body 12, and a molecular beacon is fixed in a reaction tube Γ for PCR 2, There is a transparent window 3 on the area where the molecular beacon is fixed in the PCR reaction tube, Molecular beacon 2, Fixed transparent window 3, Inside, Molecular beacon 2, Connected to the transparent window 3, through a chemically active group R, In this embodiment, the following specific connection methods can be adopted to fix the molecular beacon connection on the transparent window:

1. On the surface of transparent plastic windows such as polystyrene and glass, plasma activation, ultraviolet radiation activation, and chemical activation are used to connect chemically active groups, such as amino, aldehyde, cyano, The mercapto group and the like use the above chemically active group to react with the chemical group on the molecular beacon to connect the molecular beacon to the transparent window.

2. There are many methods for modifying transparent plastic windows and glass surfaces, such as glutaraldehyde modification method, polylysine modification method, thiol modification method, polysaccharide dream decoration method, BSA-NHS modification method, hydrogel modification method, etc. ,

Chemical treatment of transparent glass window: Put it in a solution consisting of 1/3 hydrogen peroxide (30%) and 2/3 sulfuric acid (18M), and soak it for 1 hour ⁽²¹⁾ . Rinse 3 times with deionized distilled water; put in deionized distilled water and boil for 10 minutes; dry under argon flow, save in a dry place for future use.

Aminosilylation treatment: The cleaned glass slide is immersed in a 95% acetone / water solution containing 1% 3-aminopropyl tr iethoxys i lane for 10 minutes. After taking out, the transparent plastic window, the glass slide with acetone and Rinse with deionized water, dry at 120 ° C for 45 minutes, and store in a dry place. Place the silanized glass slide at 3 ° / ° C. Soak in ~ 4% glutaraldehyde in PBS at room temperature for 2 hours, remove, wash, blow dry with nitrogen, store at 4 ° C for future use.

Preparation of BSA-NHS modified slides: Put transparent plastic windows and slides that have been silanized into 1.76g Ν-Ν'-di succinimidyl carbonate, 1. 2 ml NN '-di i sopropylethylamine, and 68.8 ml of NN' -dimethylformamide (DMF) solution A. After reaction at room temperature for 3 hours, remove it, immerse it in a PBS solution containing 1% BSA, leave it at room temperature for 12 hours, and then remove it into solution A. The reaction was continued at room temperature for 3 hours, then taken out, washed and dried for later use.

Agarose modification treatment: Agarose plus silent distilled water is formulated into a 1% agarose aqueous solution, and thoroughly mixed and boiled for 3 minutes. Pour 2 ml of agarose water on each piece of transparent plastic window that has been silanized, and wait for 37 minutes after solidification. Dry overnight at C and store at room temperature. Before use, activate with 20 mM NaI0 ₄ aqueous solution and wash with double distilled water.

Thiol modification treatment: Prepare a 1% toluene solution containing mercaptosilane, put the plastic window into the solution, and

Warm overnight. Take out the slide glass, wash it with organic solvents such as chloroform and acetone, and blow dry.

Polylysine modification treatment: Put the cleaned transparent plastic window in 3% polylysine in PBS solution for 2 hours, wash, blow dry, and store at 4 ° C.

Embodiment 9 A flush-free PCR amplification tube capable of directly detecting genes, including a reaction tube, and the reaction tube is composed of a tube cover 11 and a tube body 12, and a plurality of molecular signals are fixed in a reaction tube Γ for PCR. Marker 2, and are used to detect specific gene fragments of atypical pneumonia-associated coronavirus, and specific gene fragments of type VII and influenza B virus, respectively. There is a transparent window 3 in the area where the molecular beacon is fixed in the PCR reaction tube. Beacon 2, fixed transparent window 3, inside. In this embodiment, the transparent window is divided into several regions, and nucleic acid sequence molecular beacons with different or identical base arrangements are assembled on different regions, respectively. This embodiment can also be The molecular beacon 2 is fixed in the polymer gel medium 4 (such as polyacrylamide, agarose, polyvinyl alcohol, etc.), and then fixed on the transparent window 3 ,. After the virus is lysed, the test sample is placed in a wash-free PCR amplification tube, and the reverse transcription PCR (RT-PCT) reagent and the corona are added at the same time. Amplification primers for viruses and influenza A and B viruses are closed in the amplification tube and then subjected to an amplification reaction on a PCR reaction device. After the amplification is completed, the amplification product is introduced into the window area; if the detected virus is present in the detection sample, a fluorescent signal can be detected in the corresponding molecular beacon fixed area.

Embodiment 10 A flush-free PCR amplification tube capable of directly detecting genes, including a reaction tube, and the reaction tube is composed of a tube cover 11 and a tube body 12, and a plurality of molecular signals are fixed in a reaction tube Γ for PCR. Marker 2 'is used to detect specific gene fragments of atypical pneumonia-associated coronavirus and specific gene fragments of influenza A and B viruses, respectively. There is a transparent window 3 in the area where the molecular beacon is fixed in the PCR reaction tube. Beacon 2, fixed transparent window 3, inside, in this embodiment, a transparent polymer gel layer (such as polyacrylamide, agarose, polyvinyl alcohol, etc.) is provided on the transparent window 3, and then the molecule The beacon 2 is fixed on the polymer gel medium layer.

Claims

1. A PCR amplifier for multi-step reaction, comprising an amplification tube assembly, the amplification tube assembly is composed of a tube cover (1) and an amplification tube (2), and is characterized in that the amplification tube assembly is provided with There is a solution cell and a solution flow channel (3) with an upward opening, and the solution cell is located in the amplification tube.

(2) Within.

2. The PCR amplifier according to claim 1, characterized in that the solution tank is suspended on the tube cover (1).

3. The PCR amplifier according to claim 1 or 2, characterized in that the outer bottom surface of the solution tank is an inclined bottom surface.

4. The PCR amplifier according to claim 1, characterized in that the solution pool is a circular solution pool (41), and the solution flow channel (3) is located in an inner peripheral wall of the circular solution pool (41); preferably, the solution pool It is set in the amplification tube (2), and the outer wall of the solution pool matches the inner wall of the amplification tube.

5. The PCR amplifier according to claim 1, characterized in that the solution tank is a round-shaped solution tank (42), its cross-section is round-shaped, and the solution flow channel (3) is a round-shaped solution tank ( 42) the gap between the inner wall of the amplification tube (2);

Preferably, the cross-section of the round-shaped solution pool (42) is a round-shaped solution with a central angle greater than 180 °, and is arranged in the amplification tube (2). The outer wall of the round-shaped solution pool (42) and the amplification tube The inner wall of (2) coincides; among them, it is particularly preferred that the top surface of the round-shaped solution tank (42) is an inclined surface.

6. The PCR amplifier according to claim 1, characterized in that the solution pool is fixed in the amplification tube (2) by a bracket (5).

A directly detectable PCR amplification of the gene for Free irrigation tube, comprising a reaction tube and the reaction tube (1;) by a tube cap (11) and body (1 ²⁾ composition, characterized in that with A molecular beacon (2,) is fixed in the reaction tube (Γ;) of the PCR.

8. The flush-free PCR reaction tube capable of directly detecting genes according to claim 7, wherein a transparent window (3,) is fixed on a region where the molecular beacon is fixed in the PCR reaction tube, and the molecular beacon (2,) is fixed and transparent Inside the window (3, :).

The flush-free PCR amplification tube capable of directly detecting genes according to claim 7 or 8, characterized in that the molecular beacon (2,) is connected to the transparent window (3,;) through a chemically active group (R). on.

10. The flush-free PCR amplification tube capable of directly detecting genes according to claim 7 or 8, characterized in that the molecular beacon (2,) is fixed in a polymer gel medium (4,), and then fixed in On the transparent window (3,;). ,

The flush-free PCR amplification tube capable of directly detecting genes according to claim 7 or 8, characterized in that a transparent polymer gel layer is provided on the transparent window (3,), and the molecular beacon ( 2 ') is fixed on the polymer gel medium layer.

12. The flush-free PCR amplification tube capable of directly detecting genes according to claim 8, characterized in that the transparent window is divided into a plurality of regions, and nucleic acid sequences having different or identical base arrangements are assembled on different regions, respectively. Molecular beacon.