阅读说明：本技术 一种核酸恒温扩增定量检测方法 (Nucleic acid constant-temperature amplification quantitative detection method ) 是由 曹志 单虎 刘盛龙 周士硕 马帅 于 2021-02-05 设计创作，主要内容包括：本发明属于微生物检测技术领域,具体涉及一种核酸恒温扩增定量检测方法。基于环介导等温扩增方法,在反应体系内添加聚丙烯酰胺或海藻糖,以目标基因拷贝数为横坐标、光强度曲线拐点的时间为纵坐标,拟合得到基因拷贝数与曲线拐点的时间的关系曲线,即标准曲线；根据标准曲线和曲线拐点的时间得到定量检测数据。本发明在建立的病毒和细菌的现场快速高灵敏检测技术的基础上,通过对目标基因不同拷贝数检测光强度曲线的研究,确定基因拷贝数与曲线拐点时间关系的定量曲线,并在反应体系内添加一些表面活性剂或提高体系黏度和热稳定性的化学制剂,来分析它们与定量的相关性,优化定量曲线,最终实现病原的快速简便、灵敏特异和定量的检测。(The invention belongs to the technical field of microbial detection, and particularly relates to a nucleic acid constant-temperature amplification quantitative detection method. Based on a loop-mediated isothermal amplification method, adding polyacrylamide or trehalose into a reaction system, and fitting to obtain a relation curve of the copy number of a target gene and the inflection point time of a curve of light intensity by taking the copy number of the gene as an abscissa and the inflection point time of the curve as an ordinate, namely a standard curve; and obtaining quantitative detection data according to the time of the standard curve and the inflection point of the curve. On the basis of the established on-site rapid high-sensitivity detection technology of viruses and bacteria, the quantitative curve of the relationship between the gene copy number and the inflection point time of the curve is determined through the research on the detection light intensity curves of different copy numbers of target genes, and certain surfactants or chemical agents for improving the viscosity and the thermal stability of the system are added into a reaction system to analyze the correlation between the gene copy number and the inflection point time of the curve and optimize the quantitative curve, so that the rapid, simple, sensitive, specific and quantitative detection of pathogens is finally realized.)

1. A nucleic acid isothermal amplification quantitative detection method is characterized in that: based on a loop-mediated isothermal amplification method, adding polyacrylamide or trehalose into a reaction system, and fitting to obtain a relation curve of the copy number of a target gene as a vertical coordinate and the time of the inflection point of a light intensity curve as a horizontal coordinate, namely a standard curve; and obtaining quantitative detection data according to the time of the standard curve and the inflection point of the curve.

2. The method for isothermal amplification quantitative detection of nucleic acid according to claim 1, wherein: the concentration of polyacrylamide was 1.0%.

3. The method for isothermal amplification quantitative detection of nucleic acid according to claim 1, wherein: the trehalose concentration was 15%.

4. The application of polyacrylamide in nucleic acid isothermal amplification quantitative detection.

5. The application of trehalose in nucleic acid isothermal amplification quantitative detection.

The technical field is as follows:

the invention belongs to the technical field of microbial detection, and particularly relates to a nucleic acid constant-temperature amplification quantitative detection method.

Background art:

the Polymerase Chain Reaction (PCR) is the most common method for detecting pathogenic nucleic acid at present, but the method relies on a PCR instrument with high cost, is time-consuming and labor-consuming, and is difficult to meet the requirement of rapid detection and identification of pathogenic microorganisms at present. Nucleic Acid Isothermal Amplification Technology (NAIAT) represents a new trend of future development of Nucleic acid amplification technology due to the advantages of single reaction temperature, simple and rapid detection procedure, high precision instrument and the like. Common NAIATs are: loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA), Rolling Circle Amplification (RCA), Nucleic acid sequence-dependent amplification (NASBA), Strand Displacement Amplification (SDA), Helicase-dependent isothermal amplification (HDA), and the like.

In recent years, NAIAT is widely applied to the fields of detection of epidemic disease pathogens, food safety monitoring, detection of environmental microorganisms and the like, and particularly, the application of LAMP technology is the most obvious. The selection of the PubMed database at the National Center for Biotechnology Information (NCBI) in the united states, which is used as reference when "Loop-mediated isothermal amplification, LAMP" is entered to best match, can be found in 2421 (11/30 days by 2020), and LAMP is the most suitable isothermal amplification method for nucleic acids for low cost detection in the field as seen from the relevant literature data analysis. The subject group is dedicated to solving various problems of LAMP in practical application, for example, the problem of aerosol pollution is effectively solved by using a paraffin segmentation system; the TaqMan probe is introduced into the LAMP technology, so that the direct detection of the amplification product is realized, and the problem of non-specific amplification is solved fundamentally. At present, the LAMP method has the biggest problem that whether absolute quantification can be realized or not, some clinical virus detection requires absolute quantification, and the LAMP method is required to be applied to clinical application in the future, because the LAMP amplification process always presents 'disordered' amplification, product fragments are different in size, a complete mathematical model does not exist so far, a mathematical derivation formula does not exist so as to support quantitative application of LAMP, and the fact that the LAMP reproducibility is very good for some templates with certain concentrations and irregular for other templates with certain concentrations is discovered in the experiment. How to realize quantitative detection is a key scientific problem needing to be solved in the LAMP-based detection at present.

The invention content is as follows:

the technical problems to be solved by the invention are that the LAMP amplification process often presents 'disordered' amplification, the product fragments are different in size, and a complete mathematical model is not available so far, and a mathematical derivation formula is not available to support quantitative application of LAMP, so that the problem of urgent need to be solved if quantitative detection can be realized based on LAMP detection.

In order to solve the problems, the invention provides a nucleic acid constant-temperature amplification quantitative detection method, which is characterized in that on the basis of the established pathogen on-site rapid high-sensitivity detection technology, a quantitative curve of the relationship between the gene copy number and the inflection point time of the curve is determined through the research on the detection light intensity curve of different copy numbers of a target gene, and certain surfactants or chemical preparations for improving the viscosity and the thermal stability of the system are added into a reaction system to analyze the correlation between the gene copy number and the curve and optimize the quantitative curve, so that the rapid, simple, sensitive, specific and quantitative detection of the pathogen is finally realized.

In order to achieve the purpose, the invention is realized by the following technical scheme: a nucleic acid constant temperature amplification quantitative detection method is based on a loop-mediated isothermal amplification method, polyacrylamide or trehalose with a certain concentration is added into a reaction system, a relationship curve of the copy number of a target gene and the time of the inflection point of a curve of light intensity is obtained by fitting by taking the copy number of the gene as a vertical coordinate and the time of the inflection point of the curve as a horizontal coordinate, namely a standard curve; and obtaining quantitative detection data according to the time of the standard curve and the inflection point of the curve.

Among them, Polyacrylamide (PAM) is a commonly used nonionic polymeric flocculant, and the amide group in PAM molecule has very high activity, including various properties such as thickening, flocculation and resistance reduction, so that it has wide application in industries such as oil exploitation, water treatment, textile printing and dyeing, papermaking, coal washing, medicine, sugar manufacturing, agriculture and the like, and is called "all-purpose assistant" and "all-purpose product". After PAM is dissolved in a reaction system, the surface tension or the interfacial tension of the solution can be obviously reduced, the solubilizing and dispersing capacity of the solution can be improved, the detection repeatability of the reaction system is obviously improved, and the ordered amplification is further realized.

Furthermore, the quantitative detection of the target gene has good correlation by adding 1.0 percent of PAM into the basic reaction body, and the standard curve is that Y is-0.2377X +11.303, R²＝0.9907。

Trehalose is the most stable of the natural disaccharides. Because of the non-reducibility, the product has very good stability to heat and acid and alkali. Trehalose also has low moisture absorption characteristics and a high glass transition temperature, which combine with the process stability of trehalose, making it a high protein protectant and an ideal spray-dried flavor retention agent. Secondly, trehalose has a non-specific protective effect on biological macromolecules and organisms, and the protective mechanism is generally considered that a part of organisms containing trehalose strongly binds water molecules, and the trehalose and membrane lipids share the function of binding water or replacing membrane binding water, so that the denaturation of biological membranes and membrane proteins and the like are prevented. Based on the functions of trehalose, the trehalose is added into a reaction system, so that the stability and activity of heat-sensitive enzyme are increased at high temperature, the detection repeatability is further improved, and the ordered amplification is facilitated to be realized.

Furthermore, the quantitative detection of the target gene has good correlation by adding 15% of trehalose into the reaction body, and the standard curve is that Y is-0.2072X +12.16, R²＝0.9917。

The invention has the beneficial effects that:

(1) the method has the characteristics of simplicity, low cost and quantification, and can be applied to the quantitative detection of various pathogen nucleic acids by adopting isothermal amplification detection.

(2) Provides the application of polyacrylamide and trehalose in the aspect of nucleic acid isothermal amplification quantitative detection.

Drawings

FIG. 1 shows the correlation between the quantitative detection of a target gene by adding 1.0% PAM to a reaction body;

FIG. 2 shows the correlation between the quantitative determination of the target gene by adding 15% trehalose to the reaction.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the specific techniques or conditions are not indicated in the examples, and the techniques or conditions are described in the literature in the field or according to the product specification; the reagents and materials, both of which are analytically pure reagents, are commercially available without specific reference. The adopted solution is prepared by deionized water for sterilizing and inactivating degradation enzyme.

The equipment for detection of target genes, Burley CFX Connect real-time fluorescence quantitative PCR instrument, was purchased from Bio-Rad laboratories, Ltd., California, USA. Reaction conditions are as follows: isothermal amplification at 65 ℃ and continuous reading of fluorescence values for 75min every 30s reading.

The tissues of the litopenaeus vannamei (Acute hepatopancreatic necrosis disease, AHPND) positive and Specific Pathogen Free (SPF) are preserved and provided by the seawater culture disease control key laboratory of the yellow sea aquaculture institute of the Chinese Aquaculture institute.

Basic components of the reaction system: bst 2.0DNA polymerase, 10 XBst buffer, 10mM dNTPs, 5M Betae, 100mM MgSO₄、350mM MnCl₂3.5mM CaClein, pathogen specificity primerMaterials and RNase/DNase free water. Wherein:

Bst 2.0DNA polymerase and matched 10 XBstbuffer and 100mM MgSO₄(# M0538L) from NEB.

5M Betaine (non-hydrochloride salt): purchase of betaine (C)₅H₁₁NO₂Molecular weight 117.15), analytical pure reagent. 117.15g of betaine was weighed, dissolved in 100mL of RNase-free water, adjusted to pH 8.0. + -. 0.2 with 1M HCl on a special pH meter, and added with RNase-free water to a volume of 200 mL. Subpackaging and freezing at-20 ℃.

350mM MnCl₂: purchase of Anhydrous manganese chloride (MnCl)₂MW 125.91) or manganese chloride tetrahydrate (MnCl)₂·4H₂O, MW197.91), analytically pure. 4.407g of anhydrous manganese chloride or 6.927g of tetrahydrate manganese chloride is weighed and dissolved in 80mL of RNase-free water, the volume is determined to be 100mL, the obtained product is subpackaged to 2 mL/piece and frozen at-20 ℃.

3.5mM CaClein: purchase calcein (C)₃₀H₂₆N₂O₁₃MW 622.55) or sodium calcein (Na)₂C₃₀H₂₆N₂O₁₃MW 666.5), analytically pure. 43.58mg of calcein was weighed out and added to 6mL of DMSO, and after dissolution, approximately 14mL of RNase-free water was added to make 20mL, or 46.66mg of sodium calcein was weighed out and dissolved in 20mL of RNase-free water. The prepared solution is subpackaged to 0.5 mL/piece and frozen at-20 ℃.

RNase/DNase free water from TAKARA.

Specific primers for detecting AHPND were synthesized by the generation of Biotechnology engineering (Shanghai) Inc.

Example 1: research on quantitative detection of target gene by nucleic acid isothermal amplification technology

The method comprises the steps of taking AHPND as a research object, adding methanol, ethanol, polyvinylpyrrolidone (PVP), Polyacrylamide (PAM), Triton X-100, Tween 20 and trehalose with different concentrations into a basic reaction system, determining the time of the gene copy number and the curve inflection point through research on detection light intensity curves of target genes 'pirA/B' with different copy numbers, fitting to obtain a relation curve of the gene copy number and the time of the curve inflection point by taking the target gene copy number as a vertical coordinate and the time of the curve inflection point of the light intensity as a horizontal coordinate, and analyzing quantitative correlation of the relation curve.

The method specifically comprises the following steps: respectively 10 are provided⁷The target gene of copies/. mu.L was diluted 10-fold in gradient to 10²Taking 1 muL of copies/muL as templates, respectively, researching technical parameters such as precision, accuracy and dynamic range of quantitative detection of genes by adding surfactants or chemical agents for improving system viscosity and thermal stability by adding methanol (2%, 4%, 6%), ethanol (2%, 4%, 6%), PVP (0.2%, 0.5%, 1.0%), PAM (0.2%, 0.5%, 1.0%), Triton X-100 (0.2%, 0.5%, 1.0%), Tween 20 (0.2%, 0.5%, 1.0%) and trehalose (5.0%, 10.0%, 15.0%) with different concentrations into a reaction system respectively, analyzing changes of light intensity curve inflection point time caused by target genes with gradient copy number by a Berle CFX Connect real-time fluorescence quantitative PCR instrument, and examining the dependency relationship between the gene copy number and the changes of the light intensity curve inflection point time.

The results are shown in table 1, which indicates that the quantitative determination of genes by adding 1.0% PAM and 15% trehalose to the reaction body has good correlation, and the standard curves are-0.2377X +11.303, R²0.9907 and Y-0.2072X +12.16, R²0.9917. (details are shown in Table 1)

TABLE 1 quantitative detection of target genes by isothermal nucleic acid amplification

Note: "-" indicates that this concentration significantly inhibited the amplification reaction.

Example 2: application research of nucleic acid isothermal amplification quantitative detection method in clinical sample detection

The established quantitative detection method of the constant temperature amplification instrument is used for detecting a suspected AHPND sample for clinical examination, and a Taq Man probe real-time fluorescent quantitative PCR method for detecting AHPND is adopted for rechecking by the world animal health Organization (OIE) Manual of diagnostic Tests for Aquaticanimalls, the test is repeated for 3 times, and the copy number of the DNA of each microliter of AHPND is calculated according to the standard curves of the two. And (4) respectively carrying out p value analysis on the detection results of the two by using a t detection method.

The results are shown in tables 2 and 3, when 1.0% of PAM or 15% of trehalose is added into the reaction body, the quantitative detection results of the clinical samples are not significantly different (p is more than 0.05) by adopting a Taq Man probe real-time fluorescence quantitative PCR method and a nucleic acid isothermal amplification quantitative detection method respectively, and the nucleic acid isothermal amplification quantitative detection method has excellent repeatability on the quantitative detection of the clinical samples.

TABLE 2 quantitative determination of clinical samples by adding 1.0% PAM to the reaction

Note: the Taq Man probe real-time fluorescent quantitative PCR method is fitted with a standard curve of-0.2938X +12.834, R²0.9991; p > 0.05 means no significant difference, and p < 0.05 means significant difference.

TABLE 3 quantitative determination of clinical samples by adding trehalose 15% to the reaction

Note: the Taq Man probe real-time fluorescent quantitative PCR method is fitted with a standard curve of-0.2938X +12.834, R²0.9991; p > 0.05 means no significant difference, and p < 0.05 means significant difference.

The embodiments show that the invention provides a nucleic acid isothermal amplification quantitative detection method, which has the characteristics of simplicity, low cost and quantification, and can be applied to the quantitative detection of various pathogen nucleic acids by isothermal amplification detection.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.