CN105349530B - A kind of nucleic acid detection method and detection pipe - Google Patents

Abstract

The invention discloses a kind of nucleic acid detection method and detection pipe,By setting multiple separate layers in detection pipe by the lysate in detection pipe,Cleaning fluid and reaction solution are isolated,Pass through the control of external heat source simultaneously,Separate layer is set to be respectively melted in the different phase of detection to control the sequential of each detection-phase,And using being controlled the evenly mixing device being magnetized of motion to be mixed to adding the sample in detection pipe and realize nucleic acid extraction and the purifying of sample by external magnetic body in lysate,Then the nanometer magnetic bead in detection pipe is driven to enter reaction solution after cleaning fluid using external magnetic body,Nucleic acid entrained by nanometer magnetic bead elutes in reaction solution after purification in cleaning fluid,Amplified reaction occurs in reaction solution for nucleic acid,Last external equipment realizes the detection to sample nucleic by optical detection,Nucleic acid extraction and purifying are carried out in same detection pipe so as to realize,Cleaning,Elution and the multiple steps of amplified reaction.

Description

A nucleic acid detection method and detection tube

technical field

The invention relates to a nucleic acid detection method and a detection tube, in particular to a nucleic acid detection method and a detection tube mainly used in nucleic acid extraction and amplification technologies.

Background technique

Laboratory testing is an important means of screening and diagnosing bacterial or viral infections, mainly including microscopy, culture, immunological testing and nucleic acid testing. Microscopic examination is low in cost, simple, and convenient, but the positive rate is low and it is easy to miss; the results of culture method are highly reliable, but the cycle is long and the cost is high; immunological detection is easy to operate, but there is a window period, so it is easy to miss The nucleic acid detection cycle is short, and the sensitivity and specificity are comparable to those of the culture method, but there are the following problems: ①The operation process of sample preparation (nucleic acid extraction), nucleic acid amplification and amplified product detection is carried out separately, which requires the operation of professional operators There are many and cumbersome steps, which increases operational errors; ②Excessive manual operations can easily cause cross-contamination of samples and false positive test results; ③Most nucleic acid amplification reagents need cold chain transportation and cryopreservation, and the cost is relatively high. high.

Contents of the invention

The object of the present invention is to provide a nucleic acid detection method and a detection tube. It can realize the steps of sample processing, nucleic acid extraction, cleaning and amplification reaction in the same detection tube, so that the nucleic acid of the sample can be detected efficiently, and the detection process is not easy to introduce pollution, and there will be no cross-contamination, and the detection accuracy is high. higher.

The technical solution of the present invention: a nucleic acid detection method, which is characterized in that the lysate, cleaning solution and reaction solution in the detection tube are isolated by setting a plurality of separation layers in the detection tube, and at the same time, the separation layer is controlled by an external heat source. Melt at different stages of the detection to control the timing of each detection stage, and use a magnetizable mixing device controlled by an external magnetic body in the lysate (all or part of it can be magnetized and attracted by an external magnetic material) Mix the sample added into the detection tube and realize the nucleic acid extraction and purification of the sample, and then use an external magnetic body to drive the nano-magnetic beads (carrying the extracted nucleic acid) in the detection tube to pass through the cleaning solution and then enter the reaction solution. The nano-magnetic beads carried After the nucleic acid is purified in the cleaning solution, it is eluted in the reaction solution, and at the same time, the magnetizable carrier (all or part of it can be magnetized and is attracted by the external magnetic material) is moved into the reaction solution through the control of the movement of the external magnetic body and the control of the external heat source. The reaction solution, and the reaction components on the carrier are dissolved and mixed in the reaction solution, the nucleic acid is amplified in the reaction solution, and finally the external device realizes the detection of the sample nucleic acid through optical detection, so as to realize the detection in the same Multiple steps of nucleic acid extraction and purification, washing, elution and amplification reactions are carried out in the tube.

In the above nucleic acid detection method, a plurality of branch tubes are arranged at the lower part of the detection tube, and under the action of an external magnetic body, multiple clusters of nano-magnetic beads carry nucleic acids to perform independent cleaning and reaction in multiple branch tubes, thereby realizing Nucleic acid from one sample is detected under multiple different reaction systems at the same time.

In the aforementioned nucleic acid detection method, the separation layer can be a combination of a hydrophilic substance and a hydrophobic substance, a combination of a hydrophobic substance or a plurality of hydrophobic substances (hydrophobic substances such as paraffin, stearic acid, grease, resin, etc.; combinations such as paraffin and combination of fat or agar and hydrophobic substances); when the nucleic acid extraction is completed, the separation layer above the cleaning solution is heated and melted by an external device, and the nano-magnetic beads can enter the cleaning solution; the separation layer above the reaction solution is wrapped with Magnetizable carrier, with reaction components on the carrier, when the nucleic acid carried by the nano-magnetic beads is purified in the cleaning solution, the separation layer above the reaction solution is melted, so that the nano-magnetic beads can enter the reaction solution, and in When nano magnetic beads enter the reaction solution, the magnetizable carrier and reaction components are also brought into the reaction solution.

A nucleic acid detection tube for realizing the aforementioned method: including a main pipe and a branch pipe below the main pipe, the branch pipe is provided with a first separation layer and a second separation layer, the first separation layer is located at the junction of the pipe and the branch pipe, and the second separation layer is located at the first separation layer. Below the layer; the main pipe is provided with a lysate, and a mixing device that can be magnetized is provided in the lysate; a cleaning liquid is provided between the first separation layer and the second separation layer, and a reaction liquid is provided below the second separation layer .

In the aforementioned nucleic acid detection tube, the main tube is provided with nano-magnetic beads, the second separation layer is wrapped with a magnetizable carrier, and the carrier is loaded with reaction components.

In the aforementioned nucleic acid detection tube, the number of branch tubes is 2 or more.

In the aforementioned nucleic acid detection tube, the main pipe is provided with an internal control object (nucleic acid or microorganism) for the internal control system.

In the aforementioned nucleic acid detection tube, the upper end of the main pipe is an opening, and the opening is provided with a stop ring, the stop ring is sealed with a sealing film, the sealing film is provided with a pre-pressed seam, and an upper cover is provided outside the opening.

In the aforementioned nucleic acid detection tube, one or more additional separation layers are provided between the first separation layer and the second separation layer to separate the cleaning liquid into multiple layers, so as to realize multiple cleanings.

In the aforementioned nucleic acid detection tube, the branch tube is a thin tube with a diameter smaller than that of the main tube.

Compared with the prior art, the present invention uses the separation layer in the detection tube to isolate the raw materials required for multiple processing steps, and at the same time sets nano-magnetic beads and a magnetized mixing device in the detection tube, so that the external magnetic body can be used as a The power source performs isolated mixing and transfer processing on the sample in the detection tube, so that multiple processing steps of the sample take place in the same detection tube, which is easy to operate, less prone to pollution and cross-contamination, high sample utilization rate, and short detection time , The cost of supporting instruments and equipment is low, so not only the detection efficiency is higher, but also the detection accuracy is higher.

In addition, the present invention enables different amplification reactions to take place in the same detection tube by arranging multiple branch tubes, and simultaneously performs optical detection under multiple different systems, with higher efficiency.

Description of drawings

Fig. 1 is a schematic structural view of the nucleic acid detection tube of the present invention.

Marks in the drawings: 1-main pipe, 2-branch, 3-first separation layer, 4-second separation layer, 5-lysate, 6-cleaning solution, 7-reaction solution, 8-nano magnetic beads, 9 - internal control substance, 10 - carrier, 11 - stop ring, 12 - parafilm, 13 - upper cover, 14 - mixing device.

detailed description

The present invention will be further described below in conjunction with the examples, but not as a basis for limiting the present invention. Any modifications and/or changes made to the present invention will fall within the protection scope of the present invention.

Example. A nucleic acid detection method: by setting multi-layer partitions in the detection tube to isolate the lysate, cleaning solution and reaction solution in the detection tube, and at the same time, through the control of an external heat source, the separation layer is melted at different stages of the detection to control each detection In the lysate, a magnetized mixing device controlled by an external magnetic body (a plastic component with an iron core in the middle) is used to mix the sample added to the detection tube and realize the nucleic acid extraction of the sample. Then use an external magnetic body to drive the nano-magnetic beads in the detection tube to enter the reaction solution after passing through the cleaning solution, and at the same time seal the magnetizable carrier (may be a substance with an iron core) and the reaction components in the bottom separation layer. (Usually enzymes, primers, probes, dNTP, etc.) are also brought into the reaction solution, the nucleic acid carried by the nano-magnetic beads is washed in the cleaning solution and then eluted in the reaction solution, and reacts with the reaction solution, and finally the external equipment passes through Optical detection realizes the detection of sample nucleic acid, thereby realizing multiple steps of nucleic acid extraction, cleaning and elution reactions in the same detection tube.

A plurality of branch tubes are arranged at the lower part of the detection tube, and under the action of an external magnetic body, a plurality of nano magnetic beads carry nucleic acid to carry out independent cleaning and reaction in the plurality of branch tubes, thereby realizing the nucleic acid in multiple branch tubes at the same time. Optical detection was performed under different reaction systems.

Nano magnetic beads are arranged in the main tube. The separation layer above the reaction solution is wrapped with carriers and reaction components. When the nucleic acid carried by the nucleic acid nano-magnetic beads is cleaned, the separation layer above the reaction solution is melted, so that the nano-magnetic beads can enter the reaction solution, and in the nano When the magnetic beads enter the reaction solution, the carrier and reaction components are also brought into the reaction solution.

Realize the nucleic acid detection tube of above-mentioned method, as shown in Figure 1: comprise main pipe 1 and the branch pipe 2 below main pipe 1, be provided with first separating layer 3 (paraffin) and second separating layer 4 (paraffin) in branch pipe 2, first The separation layer 3 is located at the junction of the pipe 1 and the branch pipe 2, and the second separation layer 4 is located below the first separation layer 3; the main pipe 1 is provided with a lysate 5, and the lysate 5 is provided with a magnetizable mixing device 14 ; a cleaning solution 6 is provided between the first separation layer 3 and the second separation layer 4 , and a reaction solution 7 is provided below the second separation layer 4 . The main pipe 1 is provided with nano magnetic beads 8, and the second separation layer 4 is wrapped with a magnetizable carrier 10 and reaction components. A plurality of branch pipes 2 are arranged below the main pipe 1 . The number of the branch pipe 2 is two, wherein the first separation layer 3 of one branch pipe 2 is wrapped with nano magnetic beads 8, and the first separation layer of the other branch pipe 2 is wrapped with nucleic acid or microorganism 9 used by the internal control system; The second separation layer 4 of each branch pipe 2 is wrapped with a carrier 10 and reaction components. The upper end of the main pipe 1 is an opening, the opening is provided with a stop ring 11, the stop ring 11 is sealed with a sealing film 12, the sealing film 12 is provided with a pre-pressed seam, and an upper cover 13 is provided outside the opening. The branch pipe 2 is a thin pipe with a diameter smaller than that of the main pipe.

Principle of work of the present invention (taking the detection tube of 2 branch pipes as example):

First, the sample to be tested is pierced through the sealing film, and then the magnetized mixing device in the detection tube is controlled by an external magnetic body to move up and down, so that it is fully mixed with the lysate. During this period, the external heat source heats the lysate to crack the sample. Realize nucleic acid extraction. Then the heat source melts the first separation layer, and the external magnetic body drives the nano-magnetic beads, so that the nano-magnetic beads carry nucleic acids into the cleaning solution for cleaning, and then the second separation layer melts, and the nano-magnetic beads carry nucleic acids and magnetizable carriers and reaction groups. After the nucleic acid is eluted in the reaction solution, the nano magnetic beads leave the reaction solution. The external heat source keeps the bottom of the branch pipe at a constant temperature, and the nucleic acid undergoes CPA amplification reaction in the reaction solution. Molecular beacons are introduced into the reaction solution, so that the CPA amplification reaction can be observed in real time through external optical detection (fluorescence quantitative detection), and analyzed. The fluorescence value yields the amount of nucleic acid in the sample. Different reaction solutions (with different specific primers) can be set in the two branch pipes to realize detection under different systems.

Claims (10)

1. A kind of 1. nucleic acid detection method, it is characterised in that：By setting multiple separate layers in detection pipe by splitting in detection pipe Solution liquid, cleaning fluid and reaction solution are isolated, while by the control of external heat source, make separate layer in the different phase point of detection Do not melt to control the sequential of each detection-phase, and being magnetized for motion is controlled using by external magnetic body in lysate Evenly mixing device mixed to adding the sample in detection pipe and realize nucleic acid extraction and the purifying of sample, then utilize outside Magnetic drives the nanometer magnetic bead in detection pipe to enter reaction solution after cleaning fluid, and the nucleic acid entrained by nanometer magnetic bead is cleaning Eluted after purification in reaction solution in liquid, while made by the motion control of external magnetic body and the control of external heat source can be by magnetic The carrier of change moves into reaction solution, and the reactive component on carrier is dissolved in and is mixed in reaction solution, and nucleic acid is sent out in reaction solution Raw amplified reaction, last external equipment realizes the detection to sample nucleic by optical detection, so as to realize in same detection Nucleic acid extraction and purifying, cleaning, elution and the multiple steps of amplified reaction are carried out in pipe.
2. 2. nucleic acid detection method according to claim 1, it is characterised in that：The detection pipe bottom sets multiple branches Pipe, more cluster nanometer magnetic beads in the presence of outside magnetic, carry nucleic acid carried out respectively in multiple branched pipes it is independent clear Wash and react, it is achieved thereby that the nucleic acid from a sample carries out detection of nucleic acids under multiple differential responses systems simultaneously.
3. 3. nucleic acid detection method according to claim 1 or 2, it is characterised in that：After the completion of nucleic acid extraction, on cleaning fluid The separate layer of side is heated by external equipment to be melted, and nanometer magnetic bead can enter cleaning fluid；Wrapped up in separate layer above reaction solution Have a carrier that can be magnetized, with reaction component on carrier, when the nucleic acid entrained by nanometer magnetic bead in cleaning fluid after purification, instead The separate layer above liquid is answered to be melted, so that nanometer magnetic bead can enter reaction solution, and will when nanometer magnetic bead enters reaction solution Carrier and reactive component are also brought into reaction solution.
4. 4. realize the detection of nucleic acids pipe of claims 1 to 3 any claim methods described, it is characterised in that：Including being responsible for (1) With the branch pipe (2) below supervisor (1), branch pipe (2) is interior to be provided with the first separate layer (3) and the second separate layer (4), the first separate layer (3) positioned at the junction of pipe (1) and branch pipe (2), the second separate layer (4) is located at below the first separate layer (3)；The supervisor (1) It is interior to be provided with lysate (5), it is provided with the evenly mixing device (14) that can be magnetized in lysate (5)；First separate layer (3) and second separates Cleaning fluid (6) is provided between layer (4), reaction solution (7) is provided with below the second separate layer (4).
5. 5. detection of nucleic acids pipe according to claim 4, it is characterised in that：Nanometer magnetic bead (8) is provided with the supervisor (1), The carrier (10) that can be magnetized is enclosed with second separate layer (4), carrier carries reactive component on (10).
6. 6. detection of nucleic acids pipe according to claim 4, it is characterised in that：The quantity of the branch pipe (2) be 2 and 2 with On.
7. 7. detection of nucleic acids pipe according to claim 4, it is characterised in that：It is provided with the supervisor (1) and is used for internal control system Nucleic acid internal control thing or microorganism internal control thing.
8. 8. detection of nucleic acids pipe according to claim 4, it is characterised in that：Described supervisor (1) upper end is opening, opening Provided with fluid stopping circle (11), envelope has sealed membrane (12) on fluid stopping circle (11), and sealed membrane (12) is provided with pre- tooled joint, and on the outside of opening Provided with upper lid (13).
9. 9. detection of nucleic acids pipe according to claim 4, it is characterised in that：First separate layer (3) and the second separate layer (4) cleaning fluid (6) is separated into multilayer provided with one or more additional spacer layers between, is cleaned multiple times with realizing.
10. 10. the detection of nucleic acids pipe according to claim 4 to 9 any claim, it is characterised in that：The branch pipe (2) is Diameter is less than the tubule of supervisor.