CN102199531A - Microfluidic chip for multiple loop-mediated isothermal amplification (LAMP) detection and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of nucleic acid isothermal amplification, and particularly relates to a microfluidic chip for multiple loop-mediated isothermal amplification (LAMP) detection and a preparation method thereof. The chip is made of a high polymer, and is prepared by a micro-electromechanical system (MEMS) method. The chip mainly comprises amplification pools which are spatially and sequentially arranged, capillary channels, and connecting pipelines, wherein the amplification pools realize the effective discrimination of multiple LAMP signals through the spatial discrimination of the signals; the capillary channels are used for preventing the intersection and mixing of LAMP primers, amplification products, byproducts and the like among different amplification pools; and the amplification pools and the capillary channels are communicated through the connecting pipelines, so that fluid steadily and uniformly flows into the amplification pools from the capillary channels. The chip is easy to manufacture and operate; and an effective scheme is provided for the realization of the synchronous clinical detection of various pathogens by an LAMP method.

Description

A microfluidic chip for multiple LAMP detection and its preparation method

technical field

The invention belongs to the technical field of nucleic acid isothermal amplification, and in particular relates to a microfluidic chip for multiple LAMP detection and its preparation method and application.

technical background

Isothermal nucleic acid amplification is relative to polymerase chain reaction (Polymerase chain reaction, PCR) variable temperature nucleic acid amplification. This technology can amplify a large amount of nucleic acid at a constant temperature without relying on expensive instruments such as a temperature cycler. In recent years, it has been highly valued by academia and industry, and various types of isothermal nucleic acid amplification have been developed one after another. Loop-mediated isothermal nucleic acid amplification (Loop-mediated isothermal amplification, LAMP) was first invented by Japanese scientists. This technology has the advantages of high sensitivity, good specificity, fast response speed, and the reaction results can be directly judged by naked eyes. After nearly 10 years of research Academic research and clinical verification have obtained nearly 400 related patents and entered the market in the form of various detection kits. However, the "isothermal mode" of the LAMP reaction and its complex nucleic acid probes make it impossible to realize the simultaneous detection of multiple targets in a sample, that is, multiple detection (ie, the simultaneous detection of multiple targets in a sample). The development of microfluidic chip technology provides us with a very effective solution. With the support of relevant projects, the present invention successfully overcomes the multiplicity problem of LAMP detection by utilizing the microfluidic chip related technology.

Contents of the invention

The purpose of the present invention is to provide a microfluidic chip that can be used for multiple LAMP detection and its preparation method and application.

The microfluidic chip for multiple LAMP detection provided by the present invention mainly includes three structural parts: an amplification pool, a connecting pipeline and a capillary channel. See attached drawing 1. The amplification pool is coated with specific probes for the target. The amplification pools can be arranged spatially in various forms, such as arrays or divergences. Its number can be designed according to the needs of target detection, such as three, five or ten, etc., without limitation. The spatially ordered series of amplification pools realizes the effective discrimination of multiple LAMP signals through the spatial differentiation of signals. The capillary channel mainly uses its physical confinement (low mass transfer coefficient) characteristics to limit the cross-contamination of specific nucleic acid probes and reaction products in each amplification pool in different pools, that is, the small-scale structure of the capillary makes the fluid in it With a small diffusion coefficient, it can effectively prevent LAMP primers, amplification products and by-products (such as magnesium pyrophosphate, etc.) from cross-mixing between different amplification pools. At the same time, the pulling force of the capillary is also used to facilitate the injection of samples and reaction solutions. The amplification pool and the capillary are connected by connecting pipes, so that the fluid flows from the capillary channel into the amplification pool smoothly and evenly, and avoids the formation of bubbles during the reaction.

The present invention also provides a method for preparing the above-mentioned microfluidic chip, the specific steps are as follows:

1) Fabrication of the chip template: use the traditional MEMS method to make the chip template.

2) Chip casting, degassing, curing: After mixing dimethyl siloxane and curing agent evenly, pour it on the chip template, vacuum degassing, and curing at high temperature.

3) Chip punching and sealing: use a syringe needle to punch a hole at the sample injection site of the chip, and after plasma treatment, it is permanently bonded to the glass slide.

4) Functionalized chip: the probe corresponding to the nucleic acid target to be detected is coated in the amplification pool corresponding to the chip by the injection-evaporation method to complete the functionalized chip.

The method of the present invention can be made into polymer multiple LAMP detection chips of various types (including chip size, arrangement and number of amplification pools, etc.). Moreover, various functional chips can be easily completed through the injection-evaporation method without cross-contamination.

Verification of the microfluidic chip of the present invention:

The present invention uses a LAMP amplification system to verify the validity of the chip. In the ten-channel microfluidic chip, specific probes (Probe 1, Probe 2, Probe 3, Probe 4) for four nucleic acid targets are coated in the 1, 2, 3, and 4 amplification pools, Nucleic acid probes are not coated in the fifth amplification pool. Pass through the sample (containing four kinds of target nucleic acids), and then pass through the LAMP reaction solution, and react with LAMP at a constant temperature for 30 min-60 min. The experimental results are shown in Figure 2. LAMP signal (c) white precipitate, (d) green fluorescence appeared in the amplification pool coated with the target probe; a characteristic LAMP ladder also appeared in the agarose gel electrophoresis Amplified band (e). However, no LAMP signal appears in the amplification pool not coated with nucleic acid probes, and no amplified band appears in electrophoresis analysis.

The basic operation steps of using the microfluidic chip of the present invention are as follows:

1) Sample injection: Use a pipette gun to add the sample to be tested into the sample pool of the chip, and then add a certain amount of LAMP amplification solution. Under the pulling force of the capillary, the sample and amplification solution are quickly and evenly distributed into the amplification detection pool.

2) Constant temperature amplification: Seal the chip with uncured polydimethylsiloxane, and perform constant temperature reaction in a water bath for about 30 min-60 min.

3) Judgment of results: The test result can be judged directly according to whether the white precipitate in the amplification pool is produced or not, or it can be judged according to whether fluorescence is generated under the ultraviolet light.

Compared with prior art, the present invention has following advantage:

1) The present invention realizes the effective distinction of multiple LAMP signals through the different spatial positions of the amplification pools on the chip. The result is intuitive and the effect is obvious without the need for other expensive instruments, which is very conducive to the promotion and application of the LAMP method at the grassroots level.

2) Through the MEMS chip fabrication method, any spatial arrangement of the amplification pool on the chip can be realized as required.

3) The chip structure model has unlimited scalability in theory, and can complete any multiple effective amplification of LAMP according to actual needs, so as to meet the effective and rapid diagnosis of any variety of pathogens in clinical practice.

4) It takes less than 1 hour from sample addition to result judgment, and the operation is very simple and convenient, and non-professionals can successfully operate through simple demonstration.

Description of drawings

Fig. 1 Microfluidic chip structure of multiple LAMPs.

Figure 2 Validation of the effect of the microfluidic chip used for multiple LAMPs.

Figure 3 Multiplex LAMP chip rapid typing detection of three influenza viruses.

Figure 4 Multiplex LAMP chip typing detection of eight important swine disease pathogens.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the specific examples do not limit the present invention in any way.

Example 1 Rapid typing and detection of influenza A virus (Flu A virus), seasonal influenza A virus (Seasonal flu A virus) and 2009 swine-derived influenza A virus (Pandemic flu A virus).

The specific design size of the chip: the size of the amplification pool is (length: 10 mm, width: 0.6 mm, depth: 0.8 mm); the size of the capillary channel is (length: 2 mm, width: 0.1 mm, depth: 0.1 mm).

The specific LAMP probes of the three influenza viruses were respectively coated on the 1st-4th amplification pool 1 (1'), 2 (2'), 3 (3'); the amplification pool 4 (4') package A positive control LAMP probe was applied; the fifth amplification pool 5 (5') was not coated with a probe, and served as a blank control. We use this functional chip to detect and type three important influenza viruses.

Take 4 μL of the three virus standard solutions and clinical reference materials respectively, add them to the sample wells of the chip, then add 46 μL of LAMP amplification solution, seal the chip, and place it in a 65 ^o C water bath for 1 hour.

The results are shown in Figure 3 and Attached Table 1:

For the three standard products, LAMP signals (white precipitate, a-f, or green fluorescence a’-f’) appear in the corresponding amplification pools, and no signals appear in other amplification pools.

For the three clinical reference products, the obtained results are also in line with the expected, influenza A virus control 2 (2'), 4 (4') amplification pool signal, seasonal influenza A virus control 1 (1'), 2 (2' ) and 4 (4') amplification pool signals, porcine influenza A virus control 2 (2'), 3 (3') and 4 (4') amplification pool signals. The results were in line with expectations, and the amplification signal in the 4 (4') amplification pool indicated that the sample contained the nucleic acid sequence of the human genome.

At the same time, we applied this chip to the detection of 8 strains of clinical samples, and the detection results are shown in Attached Table 1. The test results of samples 1, 2, 3, and 4 showed that the samples were swine-derived influenza viruses, samples 5 and 6 may contain three influenza viruses at the same time, samples 7 and 8 did not contain the three influenza viruses, and the test results were basically the same as those tested in the hospital match.

For all the tested samples, no signal appeared in the negative amplification pool of the chip, indicating that there was no signal hybridization phenomenon in different amplification pools, and the detection results were reliable.

Embodiment 2 , the typing detection of eight kinds of important swine disease viruses

The specific size of the chip is the same as above, and LAMP multiple chip amplification pools 1-8 are respectively coated for porcine foot-and-mouth disease virus (FMDV), swine fever virus (CSFV), porcine blue ear disease virus (PRRSV), porcine transmissible gastroenteritis Virus (TGEV), porcine pseudorabies virus (PRV), porcine parvovirus (PPV), porcine circovirus (PCV), swine influenza virus (SIV) LAMP-specific nucleic acid probes for eight important porcine disease viruses, forming an eight Functional typing chip of swine disease virus; No. 9 amplified pool was coated with other probes and used as a negative control; No. 10 amplified pool was not coated with probes and used as a blank control.

Add sample 1 (FMDV), sample 2 (CSFV), sample 3 (PRRSV), sample 4 (TGEV), sample 5 (PRV), sample 6 (PPV), sample 7 (PCV), Sample 8 (SIV), Sample 9 (FMDV, CSFV), Sample 10 (FMDV, CSFV, PRRSV), Sample 11 (FMDV, CSFV, PRRSV, TGEV), Sample 12 (FMDV, CSFV, PRRSV, TGEV, PRV), Sample 13 (FMDV, CSFV, PRRSV, TGEV, PRV, PPV), Sample 14 (FMDV, CSFV, PRRSV, TGEV, PRV, PPV, PCV), Sample 15 (FMDV, CSFV, PRRSV, TGEV, PRV, PPV, PCV , SIV), then add LAMP amplification solution, seal the chip, and place it in a 65 ^o C water bath for 1 hour.

The experimental results are shown in Figure 4 (a-o), according to the results, it shows that the LAMP multi-chip can well distinguish eight swine disease viruses, and can also determine the type of the target pathogen in the sample.

Table 1 Detection results of rapid typing of three influenza viruses by multiple LAMP chips

Claims (3)

1. one kind is used for the micro-fluidic chip that multiple LAMP detects, and it is characterized in that being made of three essential parts: amplification pond, capillary channel and connecting tube; Wherein, described amplification pond is that spacial ordering is arranged, and bag is by last specific probe at target compound in the amplification pond, and effective differentiation of multiple LAMP signal is assigned to realize in the amplification pond by the space region of signal; Described capillary channel is used for limiting the specific nucleic acid probe and the crossed contamination of reaction product in different ponds in each amplification pond, also is used for the sample introduction of sample and reaction solution simultaneously; Described connecting tube is used for being communicated with amplification pond and capillary channel, makes fluid steadily flow into the amplification pond from capillary channel uniformly, and avoids forming in the reaction process steam bubble.

2. the preparation method of micro-fluidic chip as claimed in claim 1 is characterized in that concrete steps are:

1) making of chip template: utilize the MEMS method to make the chip template;

2) chip cast, the degassing is solidified: after dimethyl siloxane and solidifying agent are mixed, be poured on the chip template vacuum outgas, hot setting;

3) chip punching, involution: punch at chip sample introduction place with syringe needle, after plasma treatment, with the permanent bonding of slide;

4) functionalization chip: the probe of tested target set nucleic acid thing correspondence is passed through injection-method of evaporation bag quilt in the amplification pond of chip correspondence, finish the functionalization chip.

3. the using method of micro-fluidic chip as claimed in claim 1 is characterized in that concrete steps are:

1) sample injects: with liquid-transfering gun test sample is added to the chip addition pool, adds LAMP amplification liquid then, under pulling force capillaceous, test sample and amplification liquid distribute equably rapidly and enter the amplification pond;

2) constant-temperature amplification: with uncured polydimethylsiloxane with the chip involution, isothermal reaction 30 min-60 min in water-bath;

3) result judges: directly whether produces according to white precipitate in the amplification pond and judge detected result, perhaps whether basis produces fluorescence and comes result of determination under ultraviolet lamp.

Images