WO2023155403A1 - Nucleic acid test kit and test method - Google Patents

Abstract

Provided are a nucleic acid test kit and a test method. The kit, taking a microfluidic chip as a carrier, comprises a sample injection region, a microchannel, and a waste liquid region; a plurality of T lines and a C line are arranged in the microchannel; different T lines are coated with nucleic acid single strands containing different target fragments; the C line is coated with a single strand as an internal reference. Nucleic acids in a test sample are amplified and denatured into nucleic acid single strands, and the nucleic acid single strands are mixed with a nucleic acid dye and combined, in an ice bath, with the corresponding nucleic acid single strands with which the T lines are coated to form nucleic acid double strands dyed with the nucleic acid dye, so that fluorescence is generated, and detection of multiple target fragments is achieved. The method is simple to operate and is convenient and efficient.

Description

A nucleic acid detection kit and detection method technical field

The invention belongs to the technical field of biotechnology and medical detection, and in particular relates to a nucleic acid detection kit and a detection method.

Background technique

Nucleic acid detection has been widely used in many fields, including criminal investigation, detection of pathogenic microorganisms, and disease diagnosis. Under normal circumstances, the target nucleic acid content in environmental samples or physiological samples is very small. In order to make the results more accurate, a small amount of target fragments are often amplified, and then the amplified products are subjected to constant voltage electrophoresis. After the electrophoresis, the gel is taken out and placed Observe in the ultraviolet imager, take pictures and record the test results, and compare the size of the target fragment with the standard reference object, so as to obtain a negative or positive test result. This analysis method is mainly concentrated in traditional laboratories, which requires high professionalism of operators, and the operation is cumbersome and time-consuming, so it is not suitable for rapid on-site detection.

For this reason, nucleic acid detection methods based on microfluidic chips emerged at the historic moment. At present, nucleic acid detection methods based on microfluidic chips mainly rely on polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP), but These techniques can only detect one target fragment. When multiple target fragments appear in a mixed sample of multi-gene fragments, they cannot be specifically distinguished, and multiple detections are required, which has great limitations in the field of multiple gene detection. .

Contents of the invention

In order to overcome the problems of complex nucleic acid detection process and low detection efficiency, the present invention provides a nucleic acid detection kit and detection method, which can accurately and quantitatively detect multiple nucleic acid target fragments in a relatively short period of time, which is fast, efficient, simple and safe.

In order to achieve the above object, the present invention proposes a nucleic acid detection kit, using a microfluidic chip as a carrier, the microfluidic chip includes a microchannel and a sampling area and a waste liquid area respectively connected to both ends of the microchannel, The waste liquid area is communicated with the outside atmosphere, and the waste liquid area is provided with a water-absorbing material that can move along the length direction of the microchannel. There are detection areas with multiple detection lines (T lines) and quality control areas with quality control lines (C lines). The T-lines of the T-lines are respectively coated with nucleic acid single-strands paired with different target fragments, the C-lines are coated with internal reference single-strands, and nucleic acid dyes are immobilized between the sample application area and the T-lines and can be paired with the internal reference single-strands on the C-line The combined internal reference single chain (hereinafter referred to as paired internal reference).

It should be noted that the nucleic acid dye and the paired internal reference can be fixed at the same position or at different positions. In one embodiment, the nucleic acid dye and the paired internal reference are both set in the sample wells of the sample application area.

When the liquid sample enters the microchannel from the sample hole, the nucleic acid dye and the paired internal reference dissolve and the nucleic acid denatured product flows to the T line.

Of course, the nucleic acid dye and the paired internal reference may not be set on the above-mentioned microfluidic chip. When performing nucleic acid detection, the sample to be tested is mixed with the nucleic acid dye and the paired internal reference and then added to the microfluidic chip without affecting the detection results. ; In this embodiment, the nucleic acid dye and the paired internal reference are integrated on the microfluidic chip, which can reduce the detection steps and improve the detection efficiency.

In order to avoid crossover with the target fragment in the sample to be tested, the internal reference single strand uses a non-human nucleic acid product to ensure the specificity of the result.

The nucleic acid dye refers to a dye that can only bind non-specifically to a nucleic acid double strand and not bind to a nucleic acid single strand. In one embodiment, the nucleic acid dye is selected from SYBR Green I, and SYBR Green I is free in liquid. There is almost no fluorescence display in the state, but the fluorescence intensity emitted by non-specific binding to the nucleic acid double-stranded structure can be enhanced by 1000 times, and the design is simple (no pre-binding of signal probes is required), and the cost is low.

The water-absorbing material can be conventional water-absorbing material such as water-absorbing cotton, which is embedded in the strip-shaped through-hole provided in the waste liquid area, and one end of the strip-shaped through-hole is close to the outlet end of the microchannel.

The present invention also includes a method for detecting nucleic acid using the above kit, comprising the following steps:

(a) Nucleic acid amplification: the nucleic acid double-stranded product containing the target fragment is amplified by PAP method, RAP or PCR and other methods that can amplify the target fragment;

(b) Nucleic acid denaturation: denature the double-stranded nucleic acid in the double-stranded nucleic acid product into a single-stranded nucleic acid, thereby obtaining a denatured nucleic acid product;

(c) Detection of target nucleic acid: the denatured nucleic acid product enters the microfluidic chip from the sampling hole in the sampling area, and then flows through the nucleic acid dye and paired internal reference on the microfluidic chip, so that the nucleic acid dye and the paired internal reference dissolve and flow through the microchannel together The T line and C line set inside, after the nucleic acid denaturation product fully reacts with the T line and C line, move the water-absorbing material away from the microchannel to contact with the outlet end of the microchannel, and the excess unbound by the T line and C line The liquid is absorbed and the reaction is terminated.

(d) Result analysis: qualitatively or quantitatively detect the T-line and C-line signal values with a fluorometer.

There may be nucleic acids of non-target fragments in the sample to be detected. In order to further reduce the interference of non-target fragment nucleic acids, the above detection method also includes the following steps: after the nucleic acid detection reaction is terminated, add buffer to the sample hole to wash away the microchannel The remaining non-target fragment nucleic acid stained by the nucleic acid dye, after the water-absorbent material completely absorbs the buffer, then analyze the results, which improves the accuracy of the test results.

There are many specific operation methods for denaturing nucleic acid double strands into nucleic acid single strands. In one embodiment, the nucleic acid double strands are directly heated to 80-100°C to denature the nucleic acid double strands into nucleic acid single strands; then the steps In (c), it is necessary to cool down the denatured nucleic acid product to react with the T line. There are also many ways to cool down, such as air cooling, water cooling, natural cooling, etc. In one embodiment, the microfluidic chip is directly Place in an ice bath.

There are other ways to denature nucleic acid double strands into nucleic acid single strands, such as adding DNA helicase to nucleic acid double strands to obtain nucleic acid single strands, and then heating at high temperature to inactivate DNA helicase to obtain nucleic acid denatured products, avoiding nucleic acid denatured products and During the T-line reaction, DNA helicase affects the formation of nucleic acid double strands; similarly, it is necessary to cool down the denatured products of nucleic acid after high temperature heating.

Different T lines are respectively coated with nucleic acid single strands paired with different target fragments. If there is a corresponding nucleic acid single strand in the nucleic acid denaturation product, when the nucleic acid denaturation product is reduced to 55°C-70°C (annealing temperature), the nucleic acid denaturation product The nucleic acid single strand in the dye will pair with the nucleic acid single strand coated on the T line to form a nucleic acid double strand structure, and the nucleic acid dye will immediately combine with the nucleic acid double strand structure formed by the T line to produce fluorescence. No fluorescence is produced. The higher the fluorescence signal value, the higher the concentration of the target fragment, thereby realizing the quantitative detection of multiple nucleic acid amplification products.

At the same time, the paired internal reference will combine with the internal reference single strand on the C line to form an internal reference double-stranded structure, which will then be stained by nucleic acid dyes to generate fluorescence, which verifies the effectiveness of the reaction process.

A nucleic acid detection kit and detection method obtained through the above technical scheme have the beneficial effects of:

1. In the whole process, only one non-specific nucleic acid dye can be used to detect multiple target fragments. By setting multiple T lines coated with nucleic acid single strands paired with different target fragments, the target to be detected The fragments are denatured from nucleic acid double strands to nucleic acid single strands, and then react with the nucleic acid single strands on the T line to regenerate nucleic acid double strands that can be stained by nucleic acid dyes. It can specifically detect multiple target fragments at one time, with high detection efficiency and easy operation. Simple;

2. The detection process does not require pre-combination of signal probes, which reduces the detection setup and operating costs, and at the same time reduces the need for professional detection personnel, which is simple and safe;

3. Setting internal references on the quality control line can verify the effectiveness of the entire reaction process. The internal references are non-human nucleic acid products, which have no crossover with the samples to be tested, ensuring the specificity and accuracy of the results;

4. The carrier adopts a microfluidic chip, which can realize liquid flow without external force drive. By setting a water-absorbing material that can move along the length of the microchannel, the reaction time can be controlled, so that the nucleic acid dye can fully combine with the nucleic acid double strand , improve the degree of color development, and can absorb excess liquid, reduce the impact of non-target fragment nucleic acid double strands, easy to operate, safe and hygienic;

5. Using buffer to wash out further reduces the influence of non-target fragment nucleic acid double-strand staining and improves the accuracy of structure detection;

6. The nucleic acid dye and the paired internal reference are integrated on the microfluidic chip. After the nucleic acid denaturation product is added, the nucleic acid dye and the paired internal reference dissolve and flow to the detection area along with the nucleic acid denaturation product, which further simplifies the entire detection step.

Description of drawings

Fig. 1 is a graph of the linear relationship between template nucleic acid copy number and signal value (the abscissa is the template nucleic acid copy number, and the ordinate is the fluorescence signal value).

Detailed ways

It should be noted that, in the case of no conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods, unless otherwise specified, are conventional methods.

The purpose of the present invention is to provide a nucleic acid detection kit capable of detecting multiple target fragments, set a plurality of T lines coated with single-stranded nucleic acid of different target fragments, and denature the nucleic acid in the sample to be tested into nucleic acid after amplification Single strand, the nucleic acid single strand is mixed with the nucleic acid dye set on the microfluidic chip and then cooled to make the nucleic acid single strand combine with the corresponding nucleic acid single strand coated on the T line to form a nucleic acid double strand stained by the nucleic acid dye, thereby generating fluorescence , realizing rapid detection of multiple nucleic acid target fragments, the method is simple, convenient and efficient.

In order to make the object, technical solution and effect of the present invention more definite and clear, the present invention will be further described in detail below in conjunction with the embodiments and accompanying drawings.

Embodiment 1: to detect HIV core fragment (207 to 331 residues), HCV core region gene fragment (C70-140), TP core fragment (Fla B1) and HBsAg core fragment (C protein) in the sample to be tested as an example.

1. Materials and instruments

1.1 Microfluidic chip

The composition of the chip card: including a sample loading area, a microchannel and a waste liquid area composed of a cover sheet and a substrate, the microchannel includes a detection area and a quality control area, and the nucleic acid dye SYBR Green I and a paired internal reference are immobilized on the upper end of the sample hole Arabidopsis thaliana nucleic acid single strand, the detection area is sequentially set along the liquid flow direction T1 (coated with HIV core fragment nucleic acid single strand), T2 (coated with HCV core region gene fragment nucleic acid single strand), T3 (coated with TP Nucleic acid single strand of core fragment) and T4 (nucleic acid single strand coated with HBsAg core fragment), C line coated Arabidopsis nucleic acid single strand.

1.2 Fluorescence immunoassay analyzer (F10pro)

1.3 20 test samples containing HIV core fragments, HCV core region gene fragments, TP core fragments and HBsAg core fragments (the concentrations of the target fragments contained in different test samples are different);

20 samples to be tested that do not contain HIV core fragments, HCV core region gene fragments, TP core fragments and HBsAg core fragments;

Template nucleic acid samples with copy numbers of 1000copies/μL, 500copies/μL, 100copies/μL and 50copies/μL;

0.01M PBS buffer.

2. Detection method

Place the microfluidic chip in an ice bath, obtain the amplified double-stranded product of the sample to be tested by the PAP method, heat it to a high temperature of 95° to denature it, and add the denatured nucleic acid product dropwise to the microfluidic chip. In the sample well, it flows along the microfluidic channel. During the flow process, the nucleic acid dye and the paired internal reference are dissolved. At this time, the temperature of the nucleic acid is lowered to 55°C-70°C. The denatured nucleic acid products flow through the detection area and the quality control area in turn, and fluorescence occurs. After the reaction, excess samples were washed away with a buffer solution, and then analyzed and detected using a fluorescent immunoassay analyzer.

PAP (Pyrophosphorylation-Activated Polymerization) is a nucleic acid amplification method that uses 3'-end blocking primers to catalyze pyrophosphorylation reactions coupled in series with polymerases.

3. Accuracy test

20 samples to be tested containing HIV core fragments, HCV core region gene fragments, TP core fragments and HBsAg core fragments were detected according to the above-mentioned detection method, and the obtained results are shown in Table 1:

Table 1 Signal values corresponding to different samples containing target fragments

sample name	T1 signal value	T2 signal value	T3 signal value	T4 signal value	C signal value
sample 1	422831	424470	89528	376478	291950
sample 2	123829	400031	208193	481262	258771
sample 3	230877	226111	401049	480935	254329
sample 4	495285	216678	436668	328921	271859
Sample 5	337237	439108	400855	297063	278652
Sample 6	433673	213311	192336	141206	299576
Sample 7	168743	153847	479868	480784	285129
Sample 8	167511	395426	265209	403550	265756
Sample 9	488428	153187	484743	494075	279082
Sample 10	468733	364676	299081	224727	285847
Sample 11	244833	341473	411606	84952	268233
sample 12	255714	243765	438527	498205	251567
Sample 13	359623	175133	189478	124376	281277
sample 14	80148	384363	406954	469896	265583
Sample 15	156333	155785	281844	204086	258777
sample 16	276945	421633	486633	163950	251403
Sample 17	492313	239864	452370	258069	276370
sample 18	376674	242350	79679	107838	280245
sample 19	114375	110808	243542	225586	288850
sample 20	457560	321120	212128	480527	271413

The above results show that 20 samples containing the target fragment were tested and all showed positive results, which met the experimental expectations. The accuracy of the kit was good, and the C lines were all positive, indicating that the experimental process was accurate and reliable.

4. Specificity test

20 samples to be tested that do not contain HIV core fragments, HCV core region gene fragments, TP core fragments and HBsAg core fragments were detected according to the above-mentioned experimental method, and the results obtained are shown in Table 2:

Table 2 Signal values corresponding to samples that do not contain target fragments

sample name	T1 signal value	T2 signal value	T3 signal value	T4 signal value	C signal value
sample 1	633	299	600	506	266910
sample 2	144	155	537	549	295801
sample 3	398	776	221	53	298187
sample 4	680	722	693	451	251251
Sample 5	576	741	436	584	277848
Sample 6	197	377	732	510	274703
Sample 7	498	247	288	524	290270
Sample 8	615	100	377	594	266662
Sample 9	563	693	50	20	292528
sample 10	340	205	89	613	257554
Sample 11	290	551	448	465	268676
sample 12	642	397	338	56	250211
Sample 13	346	492	216	631	254588
sample 14	440	384	292	734	272695
Sample 15	685	317	515	111	285205
sample 16	619	601	96	572	253564
Sample 17	514	543	44	704	279977
sample 18	415	798	757	321	285848
sample 19	106	790	183	592	270985
sample 20	615	118	537	262	261460

The above results show that the 20 samples that do not contain the target fragment were tested, all of which were negative, indicating that the specificity of the kit is good, and the C lines are all positive, indicating that the experimental process is accurate and reliable.

5. Sensitivity test

Different concentrations of template nucleic acid samples containing HIV core fragments, HCV core region gene fragments, TP core fragments and HBsAg core fragments were dropped onto the microfluidic chip for detection, and the results obtained are shown in Table 3:

Table 3 Signal values corresponding to different copy number template nucleic acid samples

The results show that when the copy number of the template nucleic acid sample is greater than 100copies/μL, the performance is stable, but when the copy number of the nucleic acid sample is 50copies/μL, the performance becomes unstable (false negatives may occur in individual samples), so the sensitivity of the method is template When the copy number of the nucleic acid sample is greater than 100copies/μL (see Figure 1), and Figure 1 shows that the linear gradient is good when measuring nucleic acid amplification products, the nucleic acid products can be quantitatively detected.

The above-mentioned technical solutions only reflect the preferred technical solutions of the technical solutions of the present invention, and some changes that those skilled in the art may make to certain parts reflect the principles of the present invention and fall within the protection scope of the present invention.

Claims (10)

1. A nucleic acid detection kit, using a microfluidic chip as a carrier, characterized in that the microfluidic chip includes a microchannel and a sample adding area and a waste liquid area respectively connected to both ends of the microchannel, and the waste liquid area is connected to the outside world The atmosphere is connected, and the waste liquid area is provided with a water-absorbing material that can move along the length of the microchannel. The water-absorbing material can contact the outlet end of the microchannel after moving in the direction of the microchannel. The detection area with two T lines and the quality control area with C lines, each T line is coated with nucleic acid single strands paired with different target fragments, the C line is coated with internal reference single strands, the sample loading area and the T line Nucleic acid dyes and paired internal controls are immobilized between the lines.
2. The nucleic acid detection kit according to claim 1, wherein the internal reference single strand is a nucleic acid product of non-human origin.
3. The nucleic acid detection kit according to claim 1, wherein the nucleic acid dye is SYBR Green I.
4. A nucleic acid detection method, characterized in that, the application of any kit in claims 1-3, comprising the following steps:

   (a) Nucleic acid amplification: Nucleic acid amplification to obtain nucleic acid double-stranded products containing various target fragments;

   (b) Nucleic acid denaturation: denature the double-stranded nucleic acid in the double-stranded nucleic acid product into a single-stranded nucleic acid, thereby obtaining a denatured nucleic acid product;

   (c) Detection of target nucleic acid: the denatured nucleic acid product enters the microfluidic chip from the sampling hole in the sampling area, and then flows through the nucleic acid dye and paired internal reference on the microfluidic chip. After the denatured nucleic acid product fully reacts with the T line and the C line , move the water-absorbing material away from the microchannel to contact with the outlet of the microchannel, the excess liquid not combined with the T line and the C line is absorbed, and the reaction is terminated;

   (d) Result analysis: qualitatively or quantitatively detect the T-line and C-line signal values with a fluorometer.
5. The nucleic acid detection method according to claim 4, in step (b), the nucleic acid double-strand product is heated to a high temperature of 80-100° C. to denature the nucleic acid double-strand into a nucleic acid single-strand.
6. The nucleic acid detection method according to claim 4, in step (b), adding DNA helicase to the nucleic acid double-stranded product, and then heating at high temperature to inactivate the DNA helicase.
7. According to the nucleic acid detection method described in claim 5 or 6, in step (c), the nucleic acid denaturation product is cooled.
8. According to the nucleic acid detection method according to claim 7, in step (c), the microfluidic chip is placed in an ice bath environment to cool down the denatured nucleic acid products.
9. The nucleic acid detection method according to claim 4, further comprising the following steps: after the nucleic acid detection reaction is terminated, adding a buffer solution into the sample well, and after the water-absorbent material completely absorbs the buffer solution, the result analysis is performed.
10. According to the nucleic acid detection method according to claim 4, in step (a), one of PAP, RAP or PCR method is used for nucleic acid amplification.

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