CN114410755A

CN114410755A - Method for realizing multi-step reaction in same reaction tube through isolation

Info

Publication number: CN114410755A
Application number: CN202210151919.5A
Authority: CN
Inventors: 吴鸿菲; 谭若颖; 张奉武
Original assignee: BIOVUE TECHNOLOGY Ltd
Current assignee: BIOVUE TECHNOLOGY Ltd
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2022-04-29
Anticipated expiration: 2042-02-18
Also published as: CN114410755B

Abstract

The present invention provides a method for carrying out a multi-step reaction by isolation in the same reaction tube. The method enables multi-step reactions to be carried out in a closed tube by isolating multi-step reaction components that may interfere with each other from each other using a hot melt isolation layer. The method has high isolation rate, good repeatability and simple preparation, and can be applied to RT-PCR, RT-qPCR, multiple fluorescence high resolution dissolution curve (HRM) analysis, Taqman protein expression level detection, hybridization reaction, chemiluminescence detection and the like.

Description

Method for realizing multi-step reaction in same reaction tube through isolation

Technical Field

The invention relates to the fields of biomedicine, genetic engineering and detection, in particular to a method for realizing multi-step reaction in the same reaction tube by isolation, which can be applied to RT-PCR, RT-qPCR, multiple fluorescence high resolution melting curve (HRM) analysis, Taqman protein expression amount detection, hybridization reaction, chemiluminescence detection and the like.

Background

With the development and popularization of in vitro diagnostic tests, the demand for closed-tube reaction of a plurality of steps in the same reaction tube is higher and higher. The closed tube reaction of a plurality of steps in the detection process in the same reaction tube can greatly reduce the possibility of mutual pollution among detection samples and reduce the probability of the pollution of laboratory instruments and environment. The closed tube in one tube is used for carrying out reaction in multiple steps, so that the operation amount and the operation time of detection personnel can be reduced, and the labor cost is greatly reduced.

However, when a plurality of reactions of different steps are carried out in the same reaction tube, the reaction of the plurality of different steps carried out in the same tube can be applied to a narrow range due to the difference in the demands of the different reaction steps for various substances in the reaction system. At present, the application of reactions with multiple different steps in the same reaction tube is limited to RT-qPCR detection of mRNA and pre-amplification-qPCR detection of DNA. And the two applications have high requirements on primer design of two reactions, and have strict requirements on amplifiable fragments, particularly the length of the fragments, so that the application range of the method is limited.

With the development of in vitro diagnosis, a plurality of specific markers are successively found for various diseases, and particularly some non-coding RNAs, especially small RNA (MicroRNA), are excellent disease markers due to large relevance and good stability with diseases.

MicroRNAs (small RNAs) are a newly discovered class of non-coding RNAs that regulate transcription and stability of one or some target mRNAs by sequence-specific binding to the 3 'non-coding region (3's UTR) of the mRNA complementary to it in antisense. Small RNAs play an extremely important role in numerous biological activities such as growth, division, differentiation, development, apoptosis, and the development of diseases. To date, over 1800 small RNAs have been found in human cells (http:// www.mirbase.org /), and these small RNAs are involved in regulating at least 60% of human genes. Small RNAs are important biological markers for tumor classification, disease diagnosis, prediction, and assessment of prognosis. Small tumor-associated RNA in serum or plasma has been used as a biological marker for tumor diagnosis. The detection and quantification of small RNAs are important tools for the discovery of certain target genes and pathways, the study of disease mechanisms, the assessment of drug safety and efficacy, and the diagnosis and prognosis of diseases. With the discovery of more and more non-coding RNAs and the mechanism of the RNAs for regulation and control and disease occurrence, small RNA precursors are not easily degraded under the protection of binding proteins thereof, and are better disease or tumor markers compared with mRNA and the like. Therefore, it is important to be able to determine the number of various small RNAs in a particular cell.

A variety of quantitative analysis methods for small RNAs are available today, including SYBR-Green I-based quantitative reverse transcription PCR (Raymond, C.K., Roberts, B.S., Garrett-Engele, P., Lim, L.P., and Johnson, J.M, (2005) Simple, quantitative primer-extension PCR for direct detection of microRNAs and short-interfering RNAs.RNA 11), stem-loop-based Taq method (Chen C, Ridzon DA, Bromer AJ, Zhou Z, Lee DH, Nguyen JT, CN 102676637B BarbisnM, Xu NL, Mahuavakar VR, Andersen MR, Lao KQ, Livak J, Gueger J2005, Gorgle J-Gorgol, Gorgol-Miq, and Wu-1-fluorescent PCR (Wu-20) PCR), quantitative PCR assay methods based on fluorescent probe-tail DNA, Tanbook-3, PCR assay methods (Tanbuo, Tanbuo-3, PCR).

However, in the prior art, since the length of the cleaved mature small RNA is only about 20bp, almost all detection methods are to perform the next detection by first performing tailing extension RNA and then reverse transcription, then opening the reaction tube, taking a part of the reaction product, adding a specific forward primer and a probe, and a general or specific reverse primer. Even if the small RNA is subjected to tailing extension, the reverse transcription primer and the forward primer, the reverse primer and the probe are inevitably overlapped with each other, and severe non-specific amplification is caused. In addition, the mutual competition between two primers results in poor efficiency of specific amplification, so that no method for detecting small RNA by using a single-tube multi-step reaction method is reported at present.

In addition, the reaction tube needs to be opened after the first-step reaction is finished, and the reaction tube is diluted and subpackaged in a second-time reaction system, so that great pollution risks are brought to detection places and used instruments and equipment, and the clinical detection laboratory or mechanism needing strict pollution control can hardly risk the introduction of the detection.

It can be seen that the existing small RNA quantitative analysis method has the defects, which are mainly shown in the following: low sensitivity, low specificity and easy pollution.

Therefore, there is an urgent need in the art to develop a quantitative analysis method for detecting small RNA that has high sensitivity, high specificity, and can prevent cross-contamination.

Disclosure of Invention

The invention aims to provide a quantitative analysis method for detecting small RNA, which has high sensitivity and specificity and can prevent cross contamination. The method can conveniently and effectively realize the multi-step reaction in the same reaction tube through isolation.

In a first aspect of the invention, there is provided a method of carrying out a multi-step reaction by isolation in the same reaction vessel, said method comprising the steps of:

(a) providing all components required for the multi-step reaction and dividing said all components into component a and component B;

(b) enclosing component a in a reaction vessel using a barrier layer;

(c) adding component B to a reaction vessel;

(d) carrying out a first-step reaction at a temperature below the melting point of the barrier material while the reaction vessel is closed, thereby obtaining a first-step reaction product;

(e) heating the reaction tube to a temperature higher than the melting point of the isolating layer material under the condition that the reaction container is closed, so as to melt the isolating layer material, and mixing the first-step reaction product with other components to obtain a mixture;

(f) subjecting the mixture to a second-step reaction, thereby obtaining a second-step reaction product.

In another preferred embodiment, all the components consist of the components required for the first reaction and the components required for the second reaction.

In another preferred example, in the step (b), the component a is firstly added into the reaction tube, and then the isolating layer substance is added to form the isolating layer, so that the component a is sealed at the bottom of the reaction tube; or

Component a is first encapsulated with a barrier material and then the encapsulated component a is added to the reaction tube, thereby enclosing component a in the reaction tube to form a vesicle form containing component a.

In another preferred embodiment, the first step reaction in step (d) can be performed above the isolation layer, below the isolation layer, or both above and below the isolation layer.

In another preferred embodiment, the isolation layer in step (b) is one isolation layer or multiple isolation layers.

In another preferred embodiment, the multiple isolation layers have the same melting point or different melting points.

In another preferred embodiment, the substance used for the isolation layer can be hydrophilic or hydrophobic.

In another preferred embodiment, the substance used in the isolation layer may or may not be chemically active.

In another preferred embodiment, the density of the substance used in the isolation layer may be greater than or less than the density of the reaction solution.

In another preferred example, the isolation layer is a single-layer paraffin isolation layer or a multi-layer paraffin isolation layer.

In another preferred example, the isolation layer is a double-layer or three-layer paraffin wax isolation layer.

In another preferred embodiment, the melting point of the isolation layer is 50-52 ℃, 52-54 ℃, 54-55 ℃, 56-58 ℃ or 58-60 ℃.

In another preferred embodiment, the multi-layer isolation layer is a combination of a plurality of isolation layers with different melting points.

In another preferred embodiment, the melting points of the respective layers of the multi-layer barrier layer are each independently selected from: 50-52 deg.C, 52-54 deg.C, 54-55 deg.C, 56-58 deg.C, and 58-60 deg.C.

In another preferred embodiment, the melting point combinations of the layers of the multi-layer isolation layer are selected from the group consisting of: 50-52 ℃ and 52-54 ℃, 50-52 ℃ and 54-56 ℃, 50-52 ℃ and 56-58 ℃, 50-52 ℃ and 58-60 ℃, 52-54 ℃ and 56-58 ℃, 52-54 ℃ and 58-60 ℃, 54-56 ℃ and 58-60 ℃, 56-58 ℃ and 58-60 ℃, 52-54 ℃ and 54-56 ℃ and 58-60 ℃.

In another preferred embodiment, the multi-layer isolation layer is a double-layer isolation layer and has melting points of 52-54 ℃ and 58-60 ℃ or 50-52 ℃ and 58-60 ℃ respectively.

In another preferred embodiment, the melting point of the first (lower) layer is 58-60 deg.C, and the melting point of the second (upper) layer is 50-52 deg.C or 52-54 deg.C.

In another preferred embodiment, the isolation layer is a single-layer isolation layer, and the volume of the isolation layer is selected from 5 μ L, 7.5 μ L, 10 μ L, 12.5 μ L and 15 μ L; or a plurality of isolation layers, and the volume of each layer is selected from 5 muL, 7.5 muL, 10 muL, 12.5 muL and 15 muL respectively and independently.

In another preferred embodiment, the multi-layer isolation layer is a combination of a plurality of isolation layers with different volumes.

In another preferred embodiment, the volume combination of each layer of the multi-layer isolation layer is selected from: 5 μ L in combination with 5 μ L, 5 μ L in combination with 7.5 μ L, 5 μ L in combination with 10 μ L, 7.5 μ L in combination with 7.5 μ L, 7.5 μ L in combination with 10 μ L, 7.5 μ L in combination with 12.5 μ L, 7.5 μ L in combination with 15 μ L, 10 μ L in combination with 10 μ L, 5 μ L in combination with 7.5 μ L in combination with 10 μ L.

In another preferred embodiment, the volume of the first layer (lower layer) is 7.5 μ L, and the volume of the second layer (upper layer) is 10 μ L or 12.5 μ L.

In another preferred embodiment, in the step (b), the isolation layer is prepared by a method selected from the group consisting of:

(1) adding the melted isolating layer material into a reaction container with the temperature higher than the melting point of the isolating layer material, enabling the isolating material to naturally flow into the reaction container, and cooling and solidifying to form an isolating layer;

(2) adding the melted isolating layer material into a reaction container at room temperature to solidify the isolating material on the tube wall, heating the reaction container to the melting point of the isolating layer material, naturally flowing the isolating layer material into the reaction container after melting, and cooling and solidifying again to form an isolating layer;

(3) preparing the isolating layer substance into particles, adding the particles into a reaction container at room temperature, heating the reaction container to a temperature higher than the melting point of the isolating layer substance to melt the isolating layer substance, and then cooling again to form an isolating layer; and

(4) the reaction container is maintained at the temperature of 10-15 ℃ lower than the melting point of the isolating layer material, the melted isolating layer material with the melting point higher than 5 ℃ is dripped from the center of the reaction container under the condition of not contacting the pipe wall, and the isolating layer is formed by cooling and solidifying after the formed liquid drops contact the pipe wall.

In another preferred embodiment, the isolation layer is a multi-layer isolation layer, and the preparation method of each isolation layer is independently selected from the group consisting of (1), (2), (3) and (4).

In another preferred embodiment, the isolation layer is a double-layer isolation layer, and the preparation method of the isolation layer comprises the following steps:

(S1) directly melting the lower layer of isolating layer, adding the lower layer of isolating layer into a reaction container with the temperature higher than the melting point of the lower layer of isolating layer, enabling the isolating material to naturally flow into the reaction container, and cooling and solidifying the isolating layer; and

(S2) preparing the upper layer separation layer material into particles, adding the particles into a reaction vessel at room temperature, heating the reaction vessel to a temperature higher than the melting point of the separation layer material to melt the separation layer material, and cooling again to form the separation layer.

In another preferred embodiment, one of the components A and B comprises all the components with the following characteristics:

C1) is a substance required only in the second reaction step;

C2) is interfering with the first reaction step; and

C3) high temperature resistance, wherein the high temperature is greater than or equal to the melting point of the isolating layer material;

and the other component is a substance other than the above-mentioned components in the whole components.

In another preferred embodiment, one of the component a and the component B further comprises all the components having the following characteristics:

C1) is a substance required only in the second reaction step;

C2) is non-interfering with the first reaction step; and

C3) and (3) resisting high temperature, wherein the high temperature is greater than or equal to the melting point of the isolating layer material.

In another preferred embodiment, the components required for the first reaction step and the components required for the second reaction step are interfering.

In another preferred embodiment, the expression "in the presence of an interference" indicates that at least one of the components required for the first reaction step interferes with or adversely affects the second reaction step; and/or at least one of the components required for the second reaction step interferes with or adversely affects the first reaction step.

In another preferred example, the first reaction in step (d) is a reverse transcription reaction, an antigen-antibody reaction, an enzyme digestion reaction, an enzyme ligation reaction, a low-temperature PCR reaction, a low-temperature quantitative PCR reaction, a hybridization reaction, or a chemiluminescence reaction.

In another preferred example, the second reaction in step (f) is a PCR reaction, a quantitative PCR reaction, a multiple fluorescence high resolution melting curve (HRM) analysis, a Taqman protein expression level detection, a hybridization reaction or a chemiluminescence reaction.

In another preferred embodiment, the first reaction step is a reverse transcription reaction.

In another preferred embodiment, the second reaction is a PCR reaction.

In another preferred embodiment, the components required for the first reaction step are selected from: primer, enzyme, catalyst and sample to be tested.

In another preferred embodiment, the sample to be tested is selected from the group consisting of: body fluids, dairy products, vegetables, meats, meat products, or water.

In another preferred embodiment, the components required for the second reaction step are selected from: primers, probes, salts, surfactants, reaction enhancers, reaction inhibitors, catalysts, enzymes, proteins, antigens, antibodies, or combinations thereof.

In another preferred embodiment, the components required for the second reaction are as follows: primer, probe, PCR intensifier, surfactant, and the combination of two or three of the above substances.

In another preferred embodiment, the components required for the first reaction and the components required for the second reaction are selected from: a liquid, a semi-fluid, a gel, a lyophilized powder, a naturally dried in the shade powder, or a combination thereof.

In another preferred embodiment, the method is a detection method.

In another preferred embodiment, the detection comprises qualitative detection and quantitative detection.

In another preferred embodiment, the sample to be tested is RNA extracted from a sample to be tested.

In another preferred embodiment, the extracted RNA is total RNA extracted from a sample with or without small RNA.

In another preferred embodiment, the extracted RNA is total RNA extracted from a sample with or without mRNA.

In another preferred embodiment, the reaction vessel is a PCR reaction tube.

In another preferred embodiment, the volume of the reaction vessel is less than or equal to 200. mu.L, preferably 200. mu.L.

In another preferred embodiment, the reaction vessel is a commercially available eight-tube or 96-well plate.

In a second aspect of the present invention, there is provided an isolation layer for isolating reaction components in a PCR reaction tube, the isolation layer being composed of ≥ 2 layers of a substance having a heat-fusible property.

In another preferred embodiment, the isolation layer is a plurality of isolation layers with the same melting point or different melting points.

In another preferred example, the isolation layer is a multi-layer paraffin wax isolation layer.

In a third aspect of the present invention, there is provided a method of preparing an isolation layer for isolating reaction components in a PCR reaction tube, the preparation method being selected from the group consisting of:

In a fourth aspect of the present invention, there is provided a reactor tube for carrying out the method of the first aspect of the present invention, comprising:

(Fa) a reaction vessel;

(Fb) a barrier layer according to the second aspect of the invention;

(Fc) component a enclosed by the barrier layer;

wherein the component A is positioned at the bottom of the reaction vessel, and the isolating layer is positioned above the component A and seals the component A at the bottom of the reaction vessel.

In another embodiment, the reaction tube further comprises component B.

In another preferred embodiment, the component B is located above the isolation layer.

In another preferred embodiment, the component A is composed of all the components with the following characteristics:

C1) is a substance required only in the second reaction step;

C2) is interfering with the first reaction step; and

and the component B is other than the above components in the whole components.

C1) is a substance required only in the second reaction step;

C2) is non-interfering with the first reaction step; and

In another preferred embodiment, the reaction vessel is a PCR reaction tube.

It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features described in the following (for example, embodiments) can be combined with each other to form a new or preferred technical solution, which is not described in detail herein.

Drawings

FIG. 1 shows a schematic view of the reaction isolation of the present invention.

FIG. 2 shows the amplification curves for detection of RNA templates using different paraffin-separated conditions.

FIG. 3 shows a dual reaction template concentration gradient amplification curve of miR-151-5p and miR-99a under paraffin partition conditions.

FIG. 4 shows the double reaction under paraffin-partitioned conditions, with linear relationship of small RNA template amount to Ct value.

FIG. 5 shows a linear relationship of detection of different volume templates using a small RNA one-tube multi-step reaction assay array plate.

Detailed Description

The present inventors have made extensive and intensive studies to invent a method for carrying out a multi-step reaction in one reaction tube by means of physical isolation: the two-step reaction substance is separated by hydrophobic substance with proper melting point, and the components required for realizing the second-step reaction are mixed with the first-step reaction product by heating to make the phase change of the separating layer substance, so that the two-step reactions are not influenced mutually, and the multi-step reaction is realized in one tube.

The invention provides an advantageous method, which can realize closed-tube RT-qPCR detection of non-coding RNA, especially small RNA, in a reaction tube, completely solves non-specific reaction initiated by a primer probe of two-step reaction compared with the condition of closed-tube RT-qPCR detection without isolation, and does not influence the sensitivity of template detection.

The inventor of the invention has conducted a large number of experiments, has conducted extensive screening on the number of layers, the melting point and the preparation method of the isolation layer, and has developed a preparation method of the isolation layer capable of effectively isolating two reaction systems. The method can efficiently avoid non-specific interference reaction between the primers, improve the detection sensitivity and avoid pollution risk caused by opening a reaction tube. Experiments prove that the method disclosed by the invention has excellent sensitivity and specificity in quantitative detection of various small RNAs.

The invention provides a method for mixing through physical isolation and heating phase transition, which can be further applied to mRNA, gene expression, HRM quantitative detection and Taqman protein expression detection.

Term(s) for

As used herein, the term "primer" refers to a generic term for an oligonucleotide that, when paired with a template, is capable of synthesizing a DNA strand complementary to the template from its origin by the action of a DNA polymerase. The primer can be natural RNA, DNA, and any form of natural nucleotide. The primers may even be non-natural nucleotides such as LNA or ZNA etc. A primer is "substantially" (or "substantially") complementary to a particular sequence on one strand of the template. The primer must be sufficiently complementary to one strand of the template to begin extension, but the sequence of the primer need not be completely complementary to the sequence of the template. For example, a primer that is complementary to the template at its 3 'end and has a sequence that is not complementary to the template at its 5' end remains substantially complementary to the template. Primers that are not perfectly complementary can also form a primer-template complex with the template, so long as there is sufficient primer binding to the template, allowing amplification to occur.

As used herein, the term "paraffin" refers to a mixture of hydrocarbons having about 18 to about 30 carbon atoms, which may be straight-chain alkanes (about 80% to about 95%) or branched alkanes and monocyclic cycloalkanes with long side chains, and have a melting point of about 47 ℃ to about 64 ℃. The paraffin wax can be divided into different varieties according to melting points, and is generally divided into a brand number every 2 ℃. The type of paraffin wax indicated herein, e.g., "paraffin wax having a melting point of 50-52 ℃" refers to the paraffin wax designation.

The term "tailed," as used herein, refers to an oligonucleotide added at the 3' end of an RNA. Can be poly-A (AMP, adenine nucleotide), poly-C (CMP, cytosine nucleotide), poly-G (GMP, cytosine nucleotide), poly-U (UMP, uracil nucleotide), linear oligonucleotide, or oligonucleotide with stem-loop structure. "tailing" can be added by a single nucleotide polymerase, by reverse transcription with a reverse transcription primer that is not fully complementary, by a PCR reaction with a PCR primer that is not fully complementary, or by RNA or DNA ligase.

Paraffin wax isolation layer and preparation method thereof

The two-step RT and qPCR reactions require very high isolation layers. Firstly, the pipe diameter and the volume of the PCR pipe are both very small, which greatly affects the surface tension of the liquid, and some isolated components float to the surface of the isolation layer with a certain probability to be solidified and contact with an upper reaction system. Meanwhile, the isolation layer with good sealing performance is prepared in the miniature reaction tube, and the requirements on processing precision and other processes are very high. In particular, the phase transition temperature, the addition mode, etc. of the material may have a great influence on the performance of the finally formed isolation layer.

Secondly, the miRNA realizes two-step reaction of RT and qPCR in one tube through isolation, and has high requirements on the tightness of reagent isolation. The leakage of trace amount of primer into the first RT reaction system may cause the NTC to have nonspecific reaction.

Experiments prove that the multilayer paraffin isolation layer can realize the isolation of reactants efficiently even in a PCR tube (for example, a commercially available eight-connecting tube or a 96-well plate) with the volume less than or equal to 200 mu L, and the isolation rate is far higher than that of single-layer paraffin isolation and can reach 100 percent at most.

Method for carrying out multi-step reactions by isolation in the same reaction tube

As used herein, the terms "process of the invention" or "process of the invention for effecting a multi-step reaction by isolation in the same reaction tube" are used interchangeably.

The method of the invention comprises the following steps:

(b) enclosing component a in a reaction vessel using a barrier layer;

(c) adding component B to the reaction vessel;

Typically, a preferred embodiment of the method of the invention comprises the following steps:

1) placing all or part of components required by the second step reaction at the bottom of the reaction tube;

2) adding substances with proper melting point and density, cooling and solidifying to form an isolating layer, and sealing the components mentioned in the step 1) at the bottom of the tube;

3) adding the components required by the first step of reaction, and reacting at the temperature lower than the melting point of the isolating layer material;

4) and heating the reaction tube to the melting point of the substances of the isolation layer after the first-step reaction is finished, and mixing the components required by the second-step reaction with the first-step reaction product above the isolation layer after the isolation layer is melted to perform the second-step reaction.

Applications of

The method can be applied to RT-PCR, RT-qPCR, multiple fluorescence high resolution dissolution curve (HRM) analysis, Taqman protein expression amount detection, hybridization reaction, chemiluminescence detection and the like.

Taking non-coding RNA as an example, because the length of the small RNA is only about 20bp, when the small RNA is detected, the reverse transcription primer, the forward primer, the reverse primer and the probe inevitably overlap with each other in the prior art to initiate severe non-specific amplification, and the two primers compete with each other to cause poor specific amplification efficiency.

When the method is adopted, the components which can cause non-specific reaction in the two-step reaction are separated, so that the problem of poor amplification specificity can be well solved on the premise of not influencing the sensitivity.

The isolation yield of the method and/or the isolation layer is more than or equal to 90%, preferably more than or equal to 95%, preferably more than or equal to 98%, more preferably more than or equal to 99%, and further preferably more than 100%, wherein the yield refers to the ratio of the number of holes (N1) with the Ct value of more than or equal to 37 to the total number of detection holes (N0) (namely N1/N0) in PCR detection without substrate control.

The main advantage of the invention is that

(a) Under the condition that substances required by the two-step reaction are interfered with each other, the closed-tube reaction in one tube is realized, and the isolation efficiency is high.

(b) The method has wide application range, and can be adopted as long as the two-step reaction can adapt to the temperature required by the phase change of the isolating layer material.

(c) The isolation layer and the reaction tube can be prepared in a common laboratory, and special consumables and operating equipment are not needed.

(d) The repeatability is good.

(e) The experimental steps can be simplified, and the labor is saved.

(f) And the pollution risk of laboratories and equipment is reduced.

(g) The detection of the small RNA can avoid non-specific reaction among reverse transcription primers, PCR primers and PCR probes.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1

Isolation effect test under different conditions

In this example, the tests were performed using the primers and probes described in Table 1 under different isolation and test conditions as shown in Table 2.

TABLE 1 Small RNAs and primer sequences

Specifically, the inventor carries out double fluorescence RT-qPCR reaction with mixed solution with hsa-miR-151-5p (SEQ ID No: 5) and hsa-miR-99a (SEQ ID No: 6) respectively being 4000 molecules of template amount and template amount of 0(NTC, NTC being No template substrate contrast) molecules, wherein the detection signal of hsa-miR-151-5p is Fam, and the detection signal of hsa-miR-99a is Vic.

The 21 test conditions were as follows:

condition 1 (reverse transcription primers (SEQ ID Nos: 11-12) of miR-151-5p and miR99a, forward primers (SEQ ID Nos: 17-18) of miR-151-5p and miR99a, reverse primers (SEQ ID Nos: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) and RT-qPCR reaction liquid are subjected to RT-qPCR reaction);

condition 2(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated by using 5 muL of paraffin with the melting point of 50-52 ℃ and reacted at the bottom of a reaction tube, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above the paraffin isolation layer, and the reverse transcription primers and the RT-qPCR reaction liquid are subjected to RT-qPCR reaction);

condition 3(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated by using 5 muL of paraffin with the melting point of 52-54 ℃ and reacted at the bottom of the reaction tube, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above the paraffin isolation layer, and the reverse transcription primers and the RT-qPCR reaction liquid are subjected to RT-qPCR reaction);

condition 4(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated by using 5 muL of paraffin with the melting point of 54-56 ℃ and reacted at the bottom of the reaction tube, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above the paraffin isolation layer, and the reverse transcription primers and the RT-qPCR reaction liquid are subjected to RT-qPCR reaction);

condition 5(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID Nos: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated by using 5 microliter of paraffin with the melting point of 56-58 ℃ and reacted at the bottom of the reaction tube, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above the paraffin isolation layer, and the reverse transcription primers and the RT-qPCR reaction liquid are subjected to RT-qPCR reaction);

condition 6(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated by using 5 microliter of paraffin with the melting point of 58-60 ℃ and reacted at the bottom of the reaction tube, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above the paraffin isolation layer, and the reverse transcription primers and the RT-qPCR reaction liquid are subjected to RT-qPCR reaction);

condition 7(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated at the bottom of a reaction tube by using 5 muL of two layers of paraffin with melting points of 50-52 ℃ and 5 muL of 56-58 ℃, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above a paraffin isolation layer, and RT-qPCR reaction is carried out together with RT-qPCR reaction liquid);

condition 8(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated by using 5 muL of two layers of paraffin with melting points of 50-52 ℃ and 5 muL of 58-60 ℃ to react to the bottom of the reaction tube, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above a paraffin isolation layer, and RT-qPCR reaction is carried out together with RT-qPCR reaction liquid);

condition 9(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated at the bottom of a reaction tube by using 5 muL of two layers of paraffin with melting points of 52-54 ℃ and 5 muL of 56-58 ℃, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above a paraffin isolation layer, and RT-qPCR reaction is carried out together with RT-qPCR reaction liquid);

the method comprises the following steps of (1) carrying out condition 10(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID Nos: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) by using 5 mu L of two layers of paraffin with melting points of 52-54 ℃ and 5 mu L of 58-60 ℃ to isolate and react at the bottom of a reaction tube), adding miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) above a paraffin isolation layer, and carrying out RT-qPCR reaction together with an RT-qPCR reaction solution);

condition 11(miR-151-5p and miR99a forward primer (SEQ ID No: 17-18), reverse primer (SEQ ID No: 25), miR-151-5p and miR99a probe (SEQ ID No: 23-24) are isolated at the bottom of a reaction tube by using 5 muL of two layers of paraffin with melting points of 54-56 ℃ and 5 muL of 58-60 ℃, miR-151-5p and miR99a reverse transcription primer (SEQ ID No: 11-12) are added above a paraffin isolation layer, and RT-qPCR reaction is carried out together with RT-qPCR reaction liquid);

condition 12(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated at the bottom of a reaction tube by using 5 muL of two layers of paraffin with melting points of 52-54 ℃ and 10 muL of 58-60 ℃, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above a paraffin isolation layer, and RT-qPCR reaction is carried out together with RT-qPCR reaction liquid);

condition 13(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated at the bottom of a reaction tube by using 7.5 muL of two layers of paraffin with melting points of 52-54 ℃ and 7.5 muL of 58-60 ℃, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above the paraffin isolation layer, and RT-qPCR reaction is carried out together with RT-qPCR reaction liquid);

condition 14(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID Nos: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated at the bottom of a reaction tube by using 7.5 muL of two layers of paraffin with melting points of 52-54 ℃ and 10 muL of 58-60 ℃, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above a paraffin isolation layer, and RT-qPCR reaction is carried out together with RT-qPCR reaction liquid);

the method comprises the following steps of (1) carrying out condition 15 (carrying out RT-qPCR reaction on miR-151-5p and miR99a forward primers (SEQ ID Nos. 17-18), reverse primers (SEQ ID Nos. 25), miR-151-5p and miR99a probes (SEQ ID Nos. 23-24) by using 7.5 muL of two layers of paraffin with melting points of 52-54 ℃ and 12.5 muL of 58-60 ℃ to isolate the bottom of a reaction tube, adding miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos. 11-12) above the paraffin isolation layer and carrying out RT-qPCR reaction together with an RT-qPCR reaction solution);

condition 16(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) are isolated by using 7.5 muL of two layers of paraffin with melting points of 52-54 ℃ and 15 muL of 58-60 ℃ to react to the bottom of the reaction tube, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above the paraffin isolation layer, and RT-qPCR reaction is carried out together with RT-qPCR reaction liquid);

condition 17(miR-151-5p and miR99a forward primer (SEQ ID No: 17-18), reverse primer (SEQ ID No: 25), miR-151-5p and miR99a probe (SEQ ID No: 23-24) are isolated at the bottom of a reaction tube by using 10 muL of two layers of paraffin with melting points of 52-54 ℃ and 10 muL of 58-60 ℃, miR-151-5p and miR99a reverse transcription primer (SEQ ID No: 11-12) are added above a paraffin isolation layer, and RT-qPCR reaction is carried out together with RT-qPCR reaction liquid);

condition 18(miR-151-5p and miR99a forward primers (SEQ ID No: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID No: 23-24), DMSO is isolated at the bottom of the reaction tube by using 7.5 muL of two layers of paraffin with melting points of 52-54 ℃ and 10 muL of 58-60 ℃, miR-151-5p and miR99a reverse transcription primers (SEQ ID No: 11-12) are added above the paraffin isolation layer, and RT-qPCR reaction is carried out together with RT-qPCR reaction liquid);

condition 19(miR-151-5p and miR99a forward primers (SEQ ID Nos. 17-18), reverse primers (SEQ ID No. 25), miR-151-5p and miR99a probes (SEQ ID Nos. 23-24), Tween20 is isolated at the bottom of the reaction tube by using 7.5 muL of two layers of paraffin with melting points of 52-54 ℃ and 10 muL of 58-60 ℃, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos. 11-12) are added above the paraffin isolation layer, and RT-qPCR reaction is carried out together with the RT-qPCR reaction solution);

the method comprises the following steps of (20) carrying out RT-qPCR reaction on a condition (miR-151-5p and miR99a forward primer (SEQ ID No: 17-18), a reverse primer (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID No: 23-24) and magnesium chloride by using 7.5 muL of two layers of paraffin with melting points of 52-54 ℃ and 10 muL of 58-60 ℃ for isolation reaction at the bottom of a reaction tube, adding miR-151-5p and miR99a reverse transcription primers (SEQ ID No: 11-12) above a paraffin isolation layer and an RT-qPCR reaction liquid);

the condition 21(miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24), magnesium chloride is isolated at the bottom of a reaction tube by using 5 muL of three-layer paraffin with a melting point of 52-54 ℃, 7.5 muL of a melting point of 56-58 ℃ and 10 muL of a melting point of 58-60 ℃, miR-151-5p and miR99a reverse transcription primers (SEQ ID Nos: 11-12) are added above the paraffin isolation layer, and RT-qPCR reaction is carried out together with an RT-qPCR reaction solution).

As a result: as shown in table 2.

Table 2: 21 isolation condition detection results

For the detection specificity, RT-qPCR reaction was performed in one tube without paraffin partition (condition 1), Ct values of 28.3 and 26.2 were respectively detected for template amount 0(NTC, no template control), and nonspecific reaction between RT primer and qPCR primer probe was severe. And isolation conditions (condition 2-condition 21) in which one or more layers of paraffin wax with different melting points are used, the Ct value of the template-free control is greater than 37. Condition 16-condition 20 test the isolated material with the addition of PCR reaction enhancer, surfactant, and the Ct value of the no template control is still greater than 37.

For detection sensitivity, the Ct values of the detected RNA are substantially consistent under either the isolation conditions or the non-isolation conditions.

FIG. 2 shows the amplification curves for RNA detection under various isolation conditions, wherein the amplification curves are shown to be smooth for all conditions in Table 2 except condition 1.

The results show that in the reverse transcription-real-time fluorescent quantitative PCR (RT-qPCR) reaction, the paraffin wax isolating layer can be used for avoiding the non-specific reaction among the reverse transcription primer, the qPCR primer and the qPCR probe and improving the specificity of the detection result.

Example 2 specificity and sensitivity assays

In this example, the specificity and sensitivity of the reaction method of the present invention for detecting different target genes were further determined. The method comprises the following steps:

in the test isolation condition experiment, a forward primer, a reverse primer and a probe (the sequence is shown as SEQ ID No: 13-25 or table 1) are isolated at the bottom of a reaction tube by using 7.5 mu L of two layers of paraffin with the melting point of 52-54 ℃ and 10 mu L of 58-60 ℃, and a reverse transcription primer (the sequence is shown as SEQ ID No: 7-12 or table 1) is added above a paraffin isolation layer to carry out RT-qPCR reaction together with RT-qPCR reaction liquid.

Using 2X 10⁴ Human 6 kinds of small RNA (sequence shown as SEQ ID No: 1-5 or table 1) with several copies are used as template, RT-qPCR reaction is carried out, and the specificity and sensitivity of each small RNA to PCR reaction of different PCR primers are detected.

The Ct value detected is converted into the cross-reactivity according to the following rule: the reaction rate of the specific primer reaction was recorded as 100%, and if the Ct value of the specific primer reaction was n Ct values lower than that of the non-specific primer reaction, the non-specific primer reaction corresponded to 1/2 in the specific primer reactionⁿI.e. having a cross-reactivity of 1/2ⁿ(i.e., if the Ct value of the specific primer reaction is 1 Ct value lower than that of the non-specific primer reaction, the non-specific primer reaction corresponds to 50% of that of the specific primer reaction, i.e., the cross reaction rate is 50%; if the Ct value of the specific primer reaction is 2 Ct values lower than that of the non-specific primer reaction, the non-specific primer reaction corresponds to 25% of that of the specific primer reaction, i.e., the cross reaction rate is 25%; if the Ct value of the specific primer reaction is 3 Ct values lower than that of the non-specific primer reaction, the non-specific primer reaction corresponds to 12.5% of that of the specific primer reaction, i.e., the cross reaction rate is 12.5%, and so on).

In practical detection experiments, cross reaction of less than 5 percent can be generally accepted.

As a result: table 3 shows the results of the one-tube process with the isolation layer. Table 4 shows the results of one tube reaction (control) without isolation layer.

Table 3: cross-reactivity ratio of one-tube reaction under isolated conditions

Table 4: cross-reactivity ratio of one-tube reaction under non-isolated conditions

As shown in Table 3, the cross-reactivity rate of all non-specific small RNA primers was much lower than 5% when the reaction tube with the isolation layer of the present invention was used. In contrast, as shown in table 4, in a one-tube detection reaction without using a separation layer, the cross-reactivity rate was very high, even reaching 272569.59% in some groups (such as miR141), suggesting that there was a large amount of non-specific reaction therein, causing serious interference to the detection results.

In addition, in the one-tube reaction without using an isolation layer, more than half of the groups showed a phenomenon that the nonspecific reaction rate was > specific reaction rate (e.g., miR125b, miR141, miR143#, miR151-5p, miR-99a, and all nonspecific reaction rates were > 100%). This suggests that for some specific reactions, the efficiency of specific detection may be worse than for non-specific reactions due to the competition between the reverse transcription primers of the first step and the PCR primers of the second step.

The results show that when the method of the invention is used for detecting various target genes, the nonspecific reaction can be effectively reduced, the effective proceeding of the specific reaction is ensured, and the specificity and the sensitivity of the detection are improved.

Example 3

Detection sensitivity and linearity under different template quantity conditions and dynamic linear range of template

In this example, under the preferred isolation conditions (example 1, condition 19) obtained by the test, the miR-151-5p and miR99a forward primers (SEQ ID Nos: 17-18), reverse primers (SEQ ID No: 25), miR-151-5p and miR99a probes (SEQ ID Nos: 23-24) were isolated from the bottom of the reaction tube using 7.5. mu.L of two-layer paraffin having melting points of 52-54 ℃ and 10. mu.L of 58-60 ℃, and the reverse transcription primers (SEQ ID Nos: 11-12) were added above the paraffin isolation layer to perform RT-qPCR reaction together with the RT-qPCR reaction solution.

The inventor carries out gradient dilution on the mixed solution of hsa-miR-151-5p and hsa-miR-99a and tests 6 multiplied by 10⁵，6×10⁴，6×10³，6×10²60 and 0(NTC) (NTC is no substrate control) molecular template amount of mixed liquid.

As a result: FIG. 3 shows the detection sensitivity, linearity and dynamic linear range of the template. In the isolation conditions tested,

6X

10⁵，6×10⁴，6×10³，6×10²The double reactions with 60 molecular templates all have smooth amplification curves, and 0(NTC) molecular templates have no amplification.

FIG. 4 shows that the small RNA template amount and Ct value in the double reaction are in good linear relationship under paraffin isolation conditions (R)²Value greater than 0.99, R²The closer the value is to 1, the better the linear relationship).

FIG. 5 shows a linear relationship of detection of different volume templates using a small RNA one-tube multi-step reaction assay array plate. In the tested isolation conditions, at least 5 gradients of dynamic linear range can be detected, and the linear correlation is good.

The detection results of different template amounts show that the method of the invention has high sensitivity.

Example 4

Comparison of different isolation methods

In this example, the present inventors tested different methods of preparing the isolation layer and compared them. The method comprises the following steps: the method comprises the steps of using a plurality of isolation layer preparation methods for miR-151-5p and miR99a forward primers (SEQ ID Nos. 17-18), reverse primers (SEQ ID Nos. 25), miR-151-5p and miR99a probes (SEQ ID Nos. 23-24), preparing a plurality of multiple pores by each isolation preparation method, selecting 1 pore to detect 4000 copies of a mixed solution of hsa-miR-151-5p and hsa-miR-99a (positive control), and detecting 0 molecules (NTC) in the rest multiple pores (NTC is substrate-free control), so as to test the isolation effect of different isolation layer preparation methods (finished product standard: No template control detects that Ct value is more than or equal to 37).

The volume of the eight-tube used in this example was 200. mu.L, and the volume of the reaction tube for the 96-well PCR plate was 200. mu.L.

The specific isolation method is as follows:

the isolation method 1: placing 2ul of primer probe mixture liquid at the bottom of the eight-connection pipe, placing the eight-connection pipe on a constant temperature heater at 70 ℃, adding 10 mu L of paraffin which is melted into liquid and has a melting point of 58-60 ℃, naturally flowing into the bottom of the eight-connection pipe, and placing the eight-connection pipe at room temperature for cooling until the paraffin is solidified.

An isolation method 2: 2ul of primer probe mixture liquid is placed at the bottom of the 96-hole PCR plate, 10 mu L of paraffin which is melted into liquid and has a melting point of 58-60 ℃ is added on the 96-hole PCR plate which is placed on a constant temperature heater at 70 ℃, and the paraffin naturally flows into the bottom of the 96-hole PCR plate and then the 96-hole PCR plate is placed at room temperature to be cooled until the paraffin is solidified.

An isolation method 3: 2ul of primer probe mixture liquid is placed at the bottom of the eight-way tube, and the eight-way tube is placed at room temperature to naturally volatilize and dry the liquid. And placing the eight-connection pipe on a constant temperature heater at 70 ℃, adding 10 mu L of paraffin which is melted into liquid and has a melting point of 58-60 ℃, naturally flowing into the bottom of the eight-connection pipe, and placing the eight-connection pipe at room temperature for cooling until the paraffin is solidified.

The isolation method 4: 2ul of primer probe mixture liquid is placed at the bottom of the 96-well PCR plate, and the plate is placed at room temperature to naturally volatilize and dry the liquid. And (3) placing the 96-hole PCR plate on a constant temperature heater at 70 ℃, adding 10 mu L of paraffin which is melted into liquid and has a melting point of 58-60 ℃, naturally flowing the paraffin into the bottom of the tube, and then placing the 96-hole PCR plate at room temperature for cooling until the paraffin is solidified.

An isolation method 5: 2ul of primer probe mixture liquid is placed at the bottom of the 96-well PCR plate, and the plate is placed at room temperature to naturally volatilize and dry the liquid. And (3) placing the 96-hole PCR plate on a constant temperature heater at 70 ℃, adding 7.5 mu L of melted paraffin with the melting point of 58-60 ℃ into the constant temperature heater, naturally flowing the paraffin into the bottom of the tube, and then placing the 96-hole PCR plate at room temperature for cooling until the paraffin is solidified to form a first isolation layer. And (3) placing the 96-hole PCR plate on a constant temperature heater at 55 ℃, melting paraffin with the melting point of 50-52 ℃ with 70 ℃, adding 12.5ul of paraffin into the 96-hole PCR plate, naturally flowing the paraffin into the bottom of the tube, and then placing the 96-hole PCR plate at room temperature for cooling until the paraffin is solidified.

An isolation method 6: 2ul of primer probe mixture liquid is placed at the bottom of the 96-well PCR plate, and the plate is placed at room temperature to naturally volatilize and dry the liquid. And (3) placing the 96-hole PCR plate on a constant temperature heater at 70 ℃, adding 7.5 mu L of melted paraffin with the melting point of 58-60 ℃ into the constant temperature heater, naturally flowing the paraffin into the bottom of the tube, and then placing the 96-hole PCR plate at room temperature for cooling until the paraffin is solidified to form a first isolation layer. And (2) placing the 96-hole PCR plate at room temperature, melting paraffin with the melting point of 50-52 ℃ and 95 ℃, adding 12.5ul of paraffin to the wall of the 96-hole PCR plate, solidifying the paraffin on the wall of the 96-hole PCR plate, placing the 96-hole PCR plate on a constant temperature heater at 55 ℃, re-melting the paraffin on the wall of the 96-hole PCR plate, naturally flowing the paraffin into the bottom of the tube, and then placing the 96-hole PCR plate at room temperature for cooling until the paraffin is solidified.

The isolation method 7: 2ul of primer probe mixture liquid is placed at the bottom of the 96-well PCR plate, and the plate is placed at room temperature to naturally volatilize and dry the liquid. And (3) placing the 96-hole PCR plate on a constant temperature heater at 70 ℃, adding 7.5 mu L of melted paraffin with the melting point of 58-60 ℃ into the constant temperature heater, naturally flowing the paraffin into the bottom of the tube, and then placing the 96-hole PCR plate at room temperature for cooling until the paraffin is solidified to form a first isolation layer. Melting paraffin with melting point of 50-52 deg.C with 95 deg.C, taking 12.5ul to prepare paraffin particles, placing the particles into the 96-hole PCR plate, placing the 96-hole plate on a constant temperature heater with 55 deg.C, melting the paraffin particles again, naturally flowing into the bottom of the tube, and placing the 96-hole PCR plate at room temperature for cooling until the paraffin is solidified.

The isolation method 8: 2ul of primer probe mixture liquid is placed at the bottom of the 96-well PCR plate, and the plate is placed at room temperature to naturally volatilize and dry the liquid. And (3) placing the 96-hole PCR plate on a constant temperature heater at 70 ℃, adding 5 mu L of melted paraffin with the melting point of 58-60 ℃ into the constant temperature heater, naturally flowing the paraffin into the bottom of the tube, and then placing the 96-hole PCR plate at room temperature for cooling until the paraffin is solidified to form a first isolation layer. And (3) placing the plate on a constant temperature heater at 55 ℃, melting the paraffin with the melting point of 54-56 ℃ and 95 ℃, adding 7.5ul of paraffin into the plate hole, naturally flowing the paraffin into the bottom of the tube, and placing the PCR plate with 96 holes at room temperature for cooling until the paraffin is solidified to form a second isolation layer. And (3) placing the plate on a constant temperature heater at 53 ℃, melting paraffin with the melting point of 50-52 ℃ and 95 ℃, adding 12.5ul of paraffin into the plate hole, naturally flowing the paraffin into the bottom of the tube, and placing the PCR plate with 96 holes at room temperature for cooling until the paraffin is solidified to form a third layer of isolation layer.

As a result: as shown in table 5.

Table 5: yield of 8 paraffin wax isolation layer preparation methods

Of the 8 tested isolation layer preparation methods, the yield of method six and method seven was the highest (100%), and the other preparation methods also had higher yields.

This shows that it is advantageous to have 2 or 3 isolating layers. Furthermore, the direct addition of wax to the nth layer (n.gtoreq.2 positive integer) is not a preferred way to form the barrier layer. Particularly advantageous ways of forming the isolation layer include: (a) after the wall built-up is solidified, heating to about 55 +/-2 ℃ to ensure that the wax is melted again and flows into the bottom of the pipe; (b) the wax particles were placed in the tube and the wax melted again (about 55. + -. 2 ℃ C.) to flow into the bottom of the tube.

Example 5

Quantitative detection of small RNA for clinical samples

In this example, total RNA extracted from urine was used as a template for quantitative detection of small RNA. The method comprises the following steps:

in the experiment of testing isolation conditions, a forward primer, a reverse primer and a probe (the sequence is shown as SEQ ID No: 13,19 and 25) of miR125 b; a forward primer, a reverse primer and a probe of miR141 (the sequence is shown as SEQ ID No: 15,21 and 25); a forward primer, a reverse primer and a probe of miR151-5p (the sequence is shown as SEQ ID No: 17,23 and 25); a forward primer, a reverse primer and a probe of miR99a (the sequence is shown as SEQ ID No: 18,24 and 25); 7.5 mu L of two layers of paraffin wax with melting point of 52-54 ℃ and 10 mu L of 58-60 ℃ are used for isolating the bottom of the reaction tube, reverse transcription primers (with the sequence shown as SEQ ID No: 7-12) are added above the paraffin wax isolating layer, and the reverse transcription primers and RT-qPCR reaction liquid are prepared into a related small RNA one-tube multi-step reaction method to detect the whole array plate. The inventor uses total RNA extracted from human urine as a template, adds 4 ul/hole, 2 ul/hole and 1 ul/hole urine extracted total RNA in a reaction alignment plate respectively, and carries out RT-qPCR reaction by using a group without adding urine extracted RNA as a non-template control.

As a result: as shown in table 6 and fig. 5

Table 6: detection of Ct values for templates of different volumes using small RNA one-tube multistep reaction method for detection of entire array of plates

Table 6 shows the results of the prepared small RNA one-tube multi-step reaction method for detecting the small RNA extracted from the whole array of plate detection samples, the Ct values of the NTC groups are all larger than 37, the linear relation of the detection of the templates with different volumes is shown in figure 5, and the results have good linearity and can be used for detecting the RNA extracted from the samples.

EXAMPLE 6 reaction tube preparation method

In this example, a preferred method of manufacturing the reaction tube of the present invention was conducted. The reaction tube structure is shown in fig. 1A, wherein,

(a) the components required for the first reaction include: reverse transcriptase, reverse transcription primer, dNTP, magnesium ions and Tris hydrochloric acid;

(b) the components required for the second reaction include: a forward primer, a reverse primer, a probe, Taq enzyme, dNTP, enzyme ions and Tris hydrochloric acid;

(c) the isolating layer is two layers of paraffin with different melting points.

The preparation method comprises the following steps:

1) adding a primer and probe mixed solution required by the second reaction into the bottom of the tube, and naturally drying the tube at room temperature;

2) placing the 96-well PCR plate containing the primers and probes required by the second reaction on a constant temperature heater at 70 ℃, adding 7.5 mu L of melted paraffin with the melting point of 58-60 ℃ and naturally flowing the paraffin into the bottom of the tube;

3) placing the 96-hole PCR plate at room temperature, cooling, and solidifying paraffin to form a first isolation layer;

4) melting paraffin with melting point of 50-52 deg.C at 95 deg.C, and making into granules at 12.5 ul;

5) placing the particles into the holes of the 96-hole PCR plate added with the first isolation layer, and placing the 96-hole plate on a constant temperature heater at 55 ℃ to ensure that the paraffin particles are melted again and naturally flow into the bottom of the tube;

6) and (3) cooling the 96-hole PCR plate at room temperature to solidify paraffin, thereby preparing the small RNA one-tube RT-qPCR reaction tube.

The prepared reaction tube is used for RT-qPCR reaction, and the operation steps are as follows:

1) adding reverse transcriptase and reverse transcription primer required by the first reaction, excessive dNTP, magnesium ions and Tris hydrochloric acid and Taq enzyme required by the second reaction at room temperature, and sealing the reaction tube;

2) heating the reaction tube under the condition that the reaction tube is closed, and carrying out the first-step reaction at the temperature of less than 50 ℃;

3) after the first step reaction is completed, the reaction tube is heated to a temperature higher than 60 ℃ to melt the isolation layer and carry out the second step reaction.

In this example, dNTP, magnesium ion and Tris-HCl are required for the reaction in two steps, and these 3 substances are added in excess for the first reaction step, and after the first reaction is completed, the remaining substances can be reacted further in the second reaction. The Taq enzyme is a component required by the second reaction, does not affect the first reaction, and is added on the upper layer of the isolation layer in order to avoid the inactivation of the Taq enzyme affected by high temperature when the isolation layer is prepared.

The first step reaction is carried out at the temperature of less than 50 ℃, at the moment, because of the existence of the isolating layer, the qPCR primer and the probe at the lower layer can not influence the reverse transcription reaction system at the upper layer, and the Taq enzyme can not be inactivated at the temperature. In the second reaction, substances above and below the separation layer are mixed with each other by heating, so that the second reaction can be carried out.

Discussion of the related Art

In the prior art, almost all detection methods need to carry out the detection of the next step by firstly carrying out tailing extension on small RNA and then carrying out reverse transcription, then opening a reaction tube, taking a part of reaction products, adding a specific forward primer and a probe, and adding a general or specific reverse primer.

In this process, even if the small RNA is extended by tailing, the reverse transcription primer inevitably overlaps with the forward primer, the reverse primer and the probe, and causes severe non-specific amplification. In addition, the competition between the two primers results in poor efficiency of specific amplification.

In addition, after the first-step reaction is finished, the reaction tube needs to be opened, and the reaction tube is diluted and subpackaged into a second-time reaction system, so that great pollution risk is brought.

Although the prior art provides a paraffin-isolated one-tube closed reaction method, the inventor finds that under the conditions of small volume and small reaction tube diameter, due to the action of electrostatic force and liquid surface tension, paraffin does not perfectly isolate isolated components, and some isolated components float to the surface of an isolation layer to be solidified with a certain probability and still can be in contact with an upper reaction system. This is unacceptable in RT-qPCR reactions where isolation accuracy is very demanding.

The method provided by the invention can realize the detection of non-coding RNA, especially the quantitative detection of small RNA, in one tube without opening the cover. The inventor of the invention has conducted a large number of experiments, has conducted extensive screening on the number of layers, the melting point and the preparation method of the isolation layer, and has developed a preparation method of the isolation layer capable of effectively isolating two reaction systems. The method can efficiently avoid non-specific interference reaction between the primers, improve the detection sensitivity and avoid pollution risk caused by opening a reaction tube. Experiments prove that the method disclosed by the invention has excellent sensitivity and specificity in quantitative detection of various small RNAs.

Experimental results show that the paraffin isolation effect of the double layers and above is better than that of the single layer. In the condition of double-layer isolation, the preferred preparation method is as follows: the second layer adopts paraffin with lower melting point, which is put in a solid form, and the second layer of paraffin particles which are put in are melted at the temperature that the first layer of isolation layer is not melted (the melting point of the paraffin which is adopted by the second layer is lower than that of the first layer of paraffin, the melting temperature of the second layer of paraffin is controlled to be higher than the melting point of the second layer of paraffin and lower than that of the first layer of paraffin), so that the conditions for forming the isolation layer by itself are better.

After the isolated substance is isolated by the paraffin wax of the first layer, even if a small amount of isolated substance is solidified on the surface of the first isolation layer, the isolated substance is fixed, the second layer is put into the step of remelting in a solid form, the temperature control is easy, and as long as the first isolation layer is not melted, the isolated substance is sealed between the first isolation layer and the second isolation layer and cannot be contacted with the reaction system of the first step above. In the fifth method, if the second layer is to be kept in a liquid state and put into a pipe added with the first layer of paraffin, the second layer of paraffin is necessarily kept at a higher temperature, and the surface of the first layer of isolation layer is slightly melted after the second layer of paraffin with the higher temperature is added, so that the isolated substance solidified on the surface of the first layer of isolation layer is released and floats to the surface of the second isolation layer again. For this reason, it can be seen that in the above embodiment, the method eight using three layers of paraffin sealing is adopted, and since the second and third isolation layers are both added in the high-temperature liquid state, the isolation effect is not the same as that of the method six and the method seven.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Xiangqiong Biotechnology Ltd

<120> a method for realizing multi-step reaction by isolation in the same reaction tube

<130> P2021-1426

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<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtcgtatcca gtgcagggtc cgaggtctgg atacgactca caa 43

<210> 8

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtcgtatcca gtgcagggtc cgaggactgg atacgaccag ctgg 44

<210> 9

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gtcgttacca gtgcagggtc cgaggactgg taacgaccca tct 43

<210> 10

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctcgttacca gtgcagggtc cgaggactgg taacgagacc aga 43

<210> 11

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctcgttacca gtgcagggtc cgaggactgg taacgagact agac 44

<210> 12

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtcgtatcca gtgcagggtc cgaggtctgg atacgaccac aag 43

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tccgctccct gagaccctaa 20

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gcgcgtttgg tccccttcaa cc 22

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gcgcgtaaca ctgtctggta aag 23

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ccgcggtgca gtgctgcatc tct 23

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ccgctcgagg agctcacagt c 21

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ccgcaacccg tagatccga 19

<210> 19

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ctggatacga ctcacaagt 19

<210> 20

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ctggatacga ccagctg 17

<210> 21

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ctggtaacga cccatc 16

<210> 22

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ctggtaacga gaccag 16

<210> 23

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ctggtaacga gactagac 18

<210> 24

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ctggatacga ccacaaga 18

<210> 25

<211> 13

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gcagggtccg agg 13

Claims

1. A method for carrying out a multi-step reaction by isolation in the same reaction vessel, said method comprising the steps of:

(b) enclosing component a in a reaction vessel using a barrier layer;

(c) adding component B to the reaction vessel;

2. The method of claim 1, wherein said barrier layer of step (b) is a multilayer barrier layer.

3. The method of claim 2, wherein each melting point of each of the plurality of barrier layers is independently selected from the group consisting of: 50-52 deg.C, 52-54 deg.C, 54-55 deg.C, 56-58 deg.C, and 58-60 deg.C.

4. The method of claim 2, wherein the multi-layer barrier layer is a dual-layer barrier layer and has a melting point of 52-54 ℃ and 58-60 ℃, or 50-52 ℃ and 58-60 ℃, respectively.

5. The method of claim 2, wherein the volumes of each layer of said plurality of barrier layers are each independently selected from the group consisting of: 5 μ L, 7.5 μ L, 10 μ L, 12.5 μ L, and 15 μ L.

6. The method of claim 2, wherein the multi-layer separator layer has a first (lower) layer volume of 7.5 μ L and a second (upper) layer volume of 10 μ L or 12.5 μ L.

7. The method of claim 1, wherein the release layer is a bilayer release layer and the release layer is prepared by a method comprising the steps of:

8. An isolation layer for isolating reaction components in a PCR reaction tube, which is characterized in that the isolation layer is composed of more than or equal to 2 layers of hot-melt substances.

9. A method of preparing an isolation layer for isolating reaction components in a PCR reaction tube, the method being selected from the group consisting of:

(1) after the isolating layer material is melted, adding the isolating layer material into a reaction container with the temperature higher than the melting point of the isolating layer material, enabling the isolating material to naturally flow into the reaction container, and cooling and solidifying to form an isolating layer;

(2) melting the isolating layer material, adding the melted isolating layer material into a reaction container at room temperature to solidify the isolating material on the tube wall, heating the reaction container to the melting point of the isolating layer material, naturally flowing into the reaction container after the melting point of the isolating layer material, and cooling and solidifying again to form an isolating layer;

10. A reactor tube for carrying out the method of claim 1, wherein the reactor tube comprises:

(Fa) a reaction vessel;

(Fb) the isolation layer of claim 2;

(Fc) component a enclosed by the barrier layer;