CN104862216A - High-throughput visual totally-enclosed split-type LAMP-LFD detection chip device - Google Patents

Abstract

A high-throughput visual fully-sealed split-type LAMP-LFD detection chip device is characterized in that it is composed of a mixing pool, a chip reaction box, and an LFD reaction box. The interface connected to the distribution port is equipped with a reaction amplification pool, and the reaction amplification pool is equipped with probes corresponding to the target to be detected, and is sealed by the rear LFD sealing cover; the LFD reaction box includes two cavities and double chambers. Pipeline needles, LFD nucleic acid test strips are built in the first chamber, buffer solution and one-way valve are installed in the second chamber, the second needle tube of the double-pipeline needle is connected to the one-way valve, the first needle tube is connected to the LFD nucleic acid test strips Connected, when the double-pipeline needle pierces the LFD sealing cover, the one-way valve is opened, and the buffer solution flows into the reaction amplification pool along the way, and is sucked into the LFD nucleic acid test strip by the first needle tube to realize the rapid visual reaction of LAMP-LFD. The invention has the characteristics of simple and reasonable structure, full airtightness, high throughput, multiplicity, multi-target, split type, flexible, convenient and fast operation.

Description

High-throughput visualized fully-sealed split-type LAMP-LFD detection chip device

technical field

The invention relates to the technical field of biological detection, and belongs to the technical field of multi-technology integrated biochips of nucleic acid isothermal amplification technology and lateral analysis technology, in particular to a high-throughput visualized fully-sealed split-type LAMP-LFD detection chip device. .

Background technique

In recent years, with the development of society, the government, society, and people have higher and higher requirements for their own and environmental safety. , the number of samples for various biological tests is increasing at an alarming rate every year. At the same time, the demand for on-site quick inspection is becoming more and more urgent.

LAMP technology was first established by the Japanese scientist Notomi et al. (2000). By designing 4 specific primers for 6 regions of the target gene, using BstDNA polymerase with strand displacement activity, it can be highly efficient under constant temperature conditions (60-65°C). (0.5-1h) Amplify the target DNA. Compared with the currently popular PCR nucleic acid amplification technology, this technology has the advantages of not relying on expensive instruments such as temperature cyclers (PCR), and has high sensitivity, good specificity, and fast response speed. Therefore, as an emerging technology, it has been used in clinical disease diagnosis, on-site rapid detection of epidemic bacteria or viruses, animal embryo sex identification and gene chip development and other fields. In the field of virus detection, methods for rapid diagnosis and detection of various epidemic viruses including hepatitis B virus, influenza virus, SARS coronavirus, and herpes simplex virus have been developed based on LAMP technology. In the field of bacterial detection, this technology is also widely used in the rapid detection of Mycobacterium tuberculosis, Escherichia coli, Streptococcus pneumoniae and Shigella dysenteriae. Based on the difference in specific sequences on the Y chromosome, Hirayama et al. used LAMP technology to quickly detect the sex of early bovine embryos, and applied LAMP technology to a new field. In recent years, some scientists have tried to combine it with nucleic acid lateral flow dipstick (LFD) detection technology (LAMP-LFD) to realize the visualization of LAMP on-site detection. In the whole detection process, the operator does not need to rely on special instruments and equipment, and only needs to immerse the LAMP amplification product and the test strip into the buffer to realize the detection of the target product. The whole operation process is simple and safe. Once the LAMP-LFD technology came out, it was favored by many scientists. Especially in the field of aquaculture disease detection, it has been successfully used in Tarura syndrome virus (TSV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), etc. Detection of viral pathogens. With the increasing development of this technology, two bottlenecks hindering its application and promotion at the grassroots level have gradually emerged. The first is the problem of aerosol pollution caused by the sensitivity of LAMP. Since the LAMP method is sensitive to the expansion of the target sequence and has a large amount of amplification, and the process of LFD detection often involves the process of uncapping and sampling, it is very easy to cause aerosol pollution in the actual application process, which in turn leads to false positive test results. product. To deal with this problem, the actual application of LAMP technology generally requires "strict partitioning and standardized operation". That is, according to the LAMP operation process, the experimental area is divided into "sample processing area, solution preparation area, template addition area, and detection area". concentration to operate. For such strict and demanding requirements, it is difficult to meet the actual grass-roots use process, which makes it difficult to promote this technology in grass-roots on-site inspections. Second is the high-throughput, multi-target problem. Current technical methods can only detect samples one by one and single target. In actual use, users often face the simultaneous detection of dozens or even hundreds of samples of different targets. Existing LAMP technology appears powerless. Therefore, how to reasonably avoid aerosol pollution in the actual detection process, and at the same time solve the high-throughput and multi-target problems of LAMP technology, becomes whether to promote the rapid development of LAMP technology in the detection field and finally truly serve the grassroots A problem that cannot be circumvented or avoided by the testing unit.

Contents of the invention

The technical problem to be solved by the present invention is to provide a high-throughput visualized fully-sealed split-type LAMP-LFD detection chip device, which has the characteristics of simple and reasonable structure, high throughput, multiplicity, multiple targets, and flexible, convenient and quick operation.

The technical solution adopted by the present invention to solve the above technical problems is: a high-throughput visualized fully-sealed split-type LAMP-LFD detection chip device, which is characterized in that: the detection chip system device is connected with a centrifuge to achieve uniform material mixing It is composed of three parts: the mixing pool, the detachable chip reaction box and the visualized LFD reaction box.

The mixing tank is disc-shaped, and the upper end surface is provided with the first capillary pipe for adding samples. The mixing tank is equipped with multi-layer mixing tanks, and the outer circumference of the mixing tank is distributed with multiple Dispensing port for connecting chip reaction box;

The front end of the chip reaction box is provided with an interface connected to the distribution port of the mixing pool, and the chip reaction box is sequentially provided with a second capillary and a reaction amplification pool connected to the interface, and the reaction amplification pool is provided with a The probe corresponding to the object to be inspected is sealed through the LFD sealing cover at the rear end;

The LFD reaction box consists of a hollow shell. A double-pipe needle for piercing into the LFD sealing cover is built in the front of the shell. The rear part of the shell is divided into two upper and lower closed cavities. There are LFD nucleic acid test strips used for nucleic acid visualization test verification after LAMP reaction. The buffer reagent is sealed in the second cavity, and the front of the second cavity is equipped with a one-way valve. The one-way valve of the second cavity is connected, and the first needle tube is connected with the LFD nucleic acid test strip of the first cavity. When the needle of the double-pipe needle pierces the LFD sealing cover of the reaction amplification pool, the one-way valve is opened. , the buffer reagent flows into the reaction amplification pool along the second needle tube, and is sucked into the LFD nucleic acid test strip by the first needle tube using the siphon principle to realize the rapid visual reaction of LAMP-LFD.

As an improvement, the mixing pool is formed by combining a disc-shaped upper cover and a disc-shaped base, the first capillary is arranged on the upper cover, and one end of the first capillary is connected to the uniform mixing tank The other end is connected to the sampling device, and the uniform mixing tank is set on the base. The uniform mixing tank is composed of several layers of concentric segmented rings, and there is a gap for the feed liquid to flow from the inner tank to the outer tank on the segmented rings. , the dispensing ports are evenly arranged radially on the outermost dividing ring.

As an improvement, the split ring has three layers, and the gaps are 3 to 4 arc-shaped gaps. The arc-shaped gaps are curved in the same direction and arranged on the split ring at even intervals. The outermost split ring inside the distribution port A parabola-shaped storage tank is provided on the inner wall of the storage tank, and fine holes are provided at the bottom of the storage tank, and the fine holes communicate with the interface of the chip reaction box through the distribution port.

As an improvement, the chip reaction box includes a box body with openings at both ends, and one end of the box body is detachably sealed with a connecting tube with a second capillary inside, the interface is arranged at the end of the connecting tube, and the reaction amplification pool The detachable sealing sleeve is set on the other end of the box body. The front end of the reaction amplification pool is provided with a capillary interface connected to the second capillary tube. Buffer relief tank for air return.

Further improvement, the reaction amplification pool includes a cylindrical shell, the bottom of the cylindrical shell is recessed inwardly to form a cylindrical cavity, the capillary pipe interface is arranged at the front end of the shell to communicate with the cylindrical cavity, and the probe is arranged in the cylindrical cavity. In the chamber, the LFD sealing cover is made of airtight and thin material, and the upper end of the LFD sealing cover is provided with a slot for sealingly connecting the double-pipe needles of the LFD reaction box after being inserted.

As a further improvement, the lower end of the buffer and pressure relief pool is connected to the capillary interface of the reaction amplification pool, and the upper end of the buffer and pressure relief pool is provided with an elastic sealing cover, which is in a concave state during non-airtight sampling.

As a further improvement, a one-way valve switch is provided on the casing of the LFD reaction box corresponding to the upper part or the side of the second cavity.

As a further improvement, there are 12 distribution ports evenly spaced, and there are 12 corresponding chip reaction boxes.

Compared with the prior art, the present invention has the advantages of utilizing the different spatial positions of the amplification pools on the chip to realize high-throughput multiplexed multi-target visualization, fully-sealed detachable split-type micro-volume LAMP-LFD detection chip system device discrimination signal The effective distinction, the result is intuitive, the effect is obvious, no need to use other expensive instruments, the production is all industrialized, and the supporting equipment, reagent consumables, all use the existing conventional experimental equipment and consumables, easy to apply, suitable for all kinds of reagents The detection reaction is especially suitable for the supporting needs of on-site and field detection. The micro-dose reagent detection technology provides an effective guarantee for the low-cost operation of detection products, which is very conducive to the promotion and application of the LAMP-LFD method at the grassroots level; it can be expanded as needed The pools are arranged in any space on the chip; the structural model has theoretically unlimited scalability, and any multiple LAMP-LFD effective amplification can be completed according to actual needs, so as to meet the effective and rapid diagnosis of various nucleic acid molecular targets; It takes less than 1 hour to determine the result from the sample, and the operation is very simple and convenient. Non-professionals can operate it through a simple demonstration; due to the use of LFD technology, the content of the sample can be found as low as only a small amount of nucleic acid molecules, preventing missed detection. , which is conducive to primary screening; due to the use of bioinformatics principles of biochip technology, the collected samples do not need to be specially separated, and can be directly added to the high-throughput visualized fully-sealed split LAMP-LFD detection chip device after extraction, and the specific probe It is beneficial to rapid detection on site and in the field, and adapts to the promotion and application at the grassroots level.

Description of drawings

Fig. 1 is the structural representation of detection chip device of the present invention;

Fig. 2 is a schematic structural view of the mixing pool of the present invention, wherein a is a schematic structural view of the upper cover, and b is a schematic structural view of the base;

Fig. 3 is a schematic structural view of the split detachable chip reaction box of the present invention;

Fig. 4 is a schematic structural view of the visualized LFD reaction box of the present invention;

Fig. 5 is a graph showing the experimental results of the rapid analysis and detection of ten important food-borne pathogenic bacteria in the embodiment of the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

As shown in Figures 1-4, the high-throughput visualized fully-sealed split-type LAMP-LFD detection chip device of this embodiment, the detection chip system device is a mixing and receiving pool 1 that is connected to a centrifuge to achieve uniform mixing. 1, a detachable chip reaction box 2 and a visualized LFD reaction box 3 are composed of three parts. The mixer 1 is disc-shaped, which is formed by combining a disc-shaped upper cover 11 and a disc-shaped base 12. In this way, the upper cover 11 is provided with internal threads, the base 12 is provided with corresponding external threads, the upper cover 11 and the base 12 are fixed by threaded connection, and a sealing ring is lined between the upper cover 11 and the base 12;

The upper cover 11 is provided with a first capillary channel 111 for adding samples. One end of the first capillary channel 111 communicates with the uniform mixing tank 122, and the other end is connected with the sample adding device for adding reagents such as nucleic acids and enzymes and buffers. liquid; the base 12 is provided with three layers of concentric segmented rings 123, and the base 12 forms three mixed segmented grooves 122 by the segmented rings 123, and 12 holes for connecting the chip reaction box 2 are distributed on the outer peripheral surface of the base 12 Dispensing port 121, distributing port 121 is radially arranged on the outermost dividing ring 123 of base 12 at even intervals, distributing port 121 is provided with through fine hole 125, the outermost dividing circle of distributing port 121 inner side The inner wall of the ring 123 is provided with a parabola-shaped receiving groove 126, the thin hole 125 is arranged at the bottom of the receiving groove 126, and there are 3 to 4 arc-shaped gaps 124 on the inner and middle dividing rings 123, the arc-shaped gaps 124 Curved in the same direction and evenly spaced on the split ring 123, so that the material and liquid flow from the inner tank to the outer tank after being mixed; the chip reaction box 2 includes a box body with two ends open, and one end of the box body is detachably sealed and sleeved. There is a connecting pipe 26 with a second capillary 21 inside, the other end of the box body is provided with a reaction amplification pool 22, the end of the connecting pipe 26 is provided with an interface 27 connected to the distribution port 121, and the reaction amplification pool 22 is detachable The ground seal is set on the other end of the box body, and the front end of the reaction amplification pool 22 is provided with a capillary interface 25 connected to the second capillary tube 21, and a connection with the reaction amplification pool 22 is also provided in the box body. The buffer and pressure relief pool 23 for excess air backflow, the second capillary tube 21 is composed of a tube with an inner diameter of 0.1-0.9mm, and presents a multi-circuit S-shaped, and the reaction amplification pool 22 includes a cylindrical shell, and the bottom of the cylindrical shell is Arc-shaped spherical surface, the spherical surface and the cylindrical surface are in a smooth transition, the bottom of the cylindrical shell is sunken inward to form a cylindrical cavity, and the capillary pipe interface 25 is arranged at the front end of the cylindrical shell to communicate with the cylindrical cavity. There are probes corresponding to the target to be detected inside. The probes are coated in the reaction amplification pool by the injection-evaporation method through the biochip spotter, and sealed by the LFD sealing cover 24 at the rear end. The LFD sealing cover 24 For airtight thin materials, the upper end of the LFD sealing cover 24 is provided with a card slot 28 that can be inserted into the LFD reaction box 3 and then sealed and connected. The upper end of the pressure relief pool 23 is provided with an elastic sealing cover, which is in a concave state when adding samples in a non-airtight manner. Pressurize and ensure that excess air is not leaked outward;

The LFD reaction box 3 comprises a hollow shell, the front portion of the shell is a tapered diameter reduction portion 31, and a double-pipe needle 6 for piercing the LFD sealing cover 24 is built in, and the rear portion of the shell is divided into two upper and lower Airtight cavity, wherein the first cavity 33 is equipped with LFD nucleic acid test strips 5 for nucleic acid visualization test verification after the LAMP reaction, the second cavity 32 is sealed to store buffer reagents, and the front of the second cavity 32 A one-way valve 4 is provided on the top of the housing, and a one-way valve switch 41 is provided on the side of the housing corresponding to the second cavity 32. The second needle tube 62 of the double-pipeline needle 6 is connected to the one-way valve 4 of the second cavity 32. Pass, the first needle tube 61 is connected with the LFD nucleic acid test strip 5 of the first cavity 33, when the needle of the double-pipe needle 6 punctures the LFD sealing cover 24 of the reaction amplification pool 22, open the one-way valve 4, buffer The liquid reagent flows into the reaction amplification pool 22 along the second needle tube 62, and is sucked into the LFD nucleic acid test strip 5 by the first needle tube 61 using the siphon principle to realize the rapid visual reaction of LAMP-LFD.

Next, the detection chip device of the present invention is used to perform rapid analysis and detection of ten important food-borne pathogenic bacteria to verify the effectiveness of the present invention. The basic operation steps are as follows:

1) Embedded probes: pre-embedded probes in the reaction amplification pool of the high-throughput visualized fully-sealed split LAMP-LFD detection chip device. First, in the reaction amplification pools of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, use the crystal core PersonalArrayer 16 personal spotting instrument (model: ADV-I0006ADV) coated on (Vibrio cholerae, Listeria monocytogenes, Vibrio parahaemolyticus, Shigella, Escherichia coli, Vibrio vulnificus, Yersinia, Vibrio alginolyticus, Ha Primers suitable for LAMP amplification of ten kinds of targets of Vibrio vernerii and Salmonella, and labeled with fluorescein for subsequent LFD detection on the ring-adding primer. Standard nucleic acid positive probes are coated in the 11th amplification pool, Nucleic acid probes are not coated in the twelfth amplification pool.

2) Sealed sampling: Use a closed sampling device system to mix and inject the sample to be tested, enzyme, and LAMP reaction buffer into the mixing pool 1, and then centrifuge evenly into each chip reaction box 2 through a centrifuge. In the reaction amplification pool 22, the closed sample loading process of the LAMP reaction system is completed.

3) Constant temperature amplification: place the detection chip system device of the present invention in a constant temperature water bath, and incubate at a constant temperature of 60-65°C for 40-60min.

4) Judgment of results: Pierce the double-pipe needle 6 of the visualized LFD reaction box 3 and insert it into the reaction amplification pool 22 in the chip reaction box 2, and open the one-way valve switch 41 on the buffer solution. The second needle tube 62 of the pipeline needle flows into the reaction amplification pool 22, and the reactant after the LAMP isothermal amplification reaction reacts with the LFD kit, and is sucked into the LFD nucleic acid test strip by the test strip on the first needle tube 61 using the siphon principle 5. For the reaction, the hybridization solution is immersed in the nucleic acid lateral flow test strip (LFD), and the color detection is performed to judge the LAMP amplification result, and complete the LAMP-LFD rapid visual reaction.

The experimental results are as shown in Figure 5, in the reaction amplification pool 22 coated with the target probe, the reactant reacts with the LFD; a characteristic reaction band appears on the LFD detection line, while in the amplification pool 22 not coated with the nucleic acid probe There is no reaction zone in the LFD corresponding to Zengchi.

Claims (8)

1. A high-throughput visual fully-sealed split-type LAMP-LFD detection chip device, characterized in that: the detection chip system device is connected to a centrifuge to achieve uniform mixing and receiving pool, split and detachable The installed chip reaction box and the visualized LFD reaction box are composed of three parts. The mixing tank is disc-shaped, and the upper end surface is provided with the first capillary pipe for adding samples. The mixing tank is equipped with multi-layer mixing tanks, and the outer circumference of the mixing tank is distributed with multiple Dispensing port for connecting chip reaction box; The front end of the chip reaction box is provided with an interface connected to the distribution port of the mixing pool, and the chip reaction box is sequentially provided with a second capillary and a reaction amplification pool connected to the interface, and the reaction amplification pool is provided with a The probe corresponding to the object to be inspected is sealed through the LFD sealing cover at the rear end; The LFD reaction box consists of a hollow shell. A double-pipe needle for piercing into the LFD sealing cover is built in the front of the shell. The rear part of the shell is divided into two upper and lower closed cavities. There are LFD nucleic acid test strips used for nucleic acid visualization test verification after LAMP reaction. The buffer reagent is sealed in the second cavity, and the front of the second cavity is equipped with a one-way valve. The one-way valves of the two chambers are connected, and the first needle tube is connected with the LFD nucleic acid test strip of the first chamber. When the needle of the double-pipe needle pierces the LFD sealing cover of the reaction amplification pool, the one-way valve is opened. The buffer reagent flows into the reaction amplification pool along the second needle tube, and is sucked into the LFD nucleic acid test strip by the first needle tube using the siphon principle to realize the rapid visual reaction of LAMP-LFD. 2. The detection chip device according to claim 1, characterized in that: the mixing pool is formed by combining a disc-shaped upper cover and a disc-shaped base, and the first capillary is arranged on On the upper cover, one end of the first capillary pipe is connected to the uniform mixing tank, and the other end is connected to the sample adding device. The uniform mixing tank is arranged on the base. There is a notch on the ring for the feed liquid to flow from the inner tank to the outer tank, and the distribution ports are arranged radially and evenly on the outermost dividing ring. 3. The detection chip device according to claim 2, characterized in that: the segmented ring has three layers, and the notches are 3 to 4 arc-shaped notches, and the arc-shaped notches are curved in the same direction and are evenly spaced on the segmented circle. On the ring, the inner wall of the outermost dividing ring inside the distribution port is provided with a parabola-shaped storage groove, and the bottom of the storage groove is provided with fine holes, and the fine holes communicate with the interface of the chip reaction box through the distribution port. 4. The detection chip device according to claim 1, characterized in that: the chip reaction box comprises a box body with openings at both ends, and one end of the box body is detachably sealed with a second capillary tube. Take over, the interface is set at the end of the takeover, the reaction amplification pool is detachably sealed and sleeved at the other end of the box, the front end of the reaction amplification pool is provided with a capillary interface connected with the second capillary, inside the box There is also a buffer pressure relief pool for excess air backflow connected with the reaction amplification pool. 5. The detection chip device according to claim 4, characterized in that: the reaction amplification pool comprises a cylindrical shell, the bottom of the cylindrical shell is recessed inwardly to form a cylindrical cavity, and the capillary pipe interface is arranged at the front end of the shell and The cylindrical cavities are connected, the probes are arranged in the cylindrical cavities, the LFD sealing cover is made of airtight thin material, and the upper end of the LFD sealing cover is provided with a card slot that can insert the double-pipe needles of the LFD reaction box and then seal the connection. 6. The detection chip device according to claim 4, characterized in that: the lower end of the buffer pressure relief pool communicates with the capillary interface of the reaction amplification pool, and the upper end of the buffer pressure relief pool is provided with an elastic sealing cover, The sealing cap presents a concave state during non-airtight sample addition. 7 . The detection chip device according to any one of claims 1 to 6 , wherein a one-way valve switch is provided on the shell of the LFD reaction box corresponding to the upper part or the side of the second cavity. 8 . The detection chip device according to any one of claims 1 to 6 , characterized in that: there are 12 distribution ports evenly spaced, and there are 12 corresponding chip reaction boxes.