Simulated nucleic acid substrate, kit comprising same, and amplification method and detection method of target fragment by using same

Abstract

The invention relates to the technical field of biology, in particular to a mimic nucleic acid substrate, a kit comprising the mimic nucleic acid substrate and a target fragment amplification method and detection method using the mimic nucleic acid substrate. The mimic nucleic acid substrate has the following structural units: a first primer fragment, an identification fragment and a second primer fragment, wherein the first primer fragment and the second primer fragment are both in a 5 '-3' direction, the first primer fragment and the second primer fragment are used for combining PCR amplification primers of the same or different target fragments, and the identification fragment does not comprise the target fragment or partial fragments of the target fragment. The mimic nucleic acid substrate is added into a PCR reaction system, can be subjected to PCR reaction with an amplification primer, can complete library construction, effectively consumes the amplification primer, and avoids generation of a primer dimer.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a simulated nucleic acid substrate, a kit comprising the same, and a method for amplifying and detecting a target fragment using the same. Background Art

[0002] Next-generation sequencing (NGS), also known as high-throughput sequencing, can detect tens, hundreds or even more nucleic acid molecules at a time. Targeted sequencing (tNGS) based on the second-generation sequencing platform is a new detection technology that combines the principle of multiplex PCR or probe enrichment with the second-generation sequencing. It has the characteristics of stronger targeting, low detection cost, fast detection speed, and high detection sensitivity. Compared with the currently commonly used molecular detection technology (such as multiplex PCR), it has the characteristics of wide detection range and high accuracy, and plays an important role in many fields such as microbial detection and gene detection.

[0003] The multiple microbial joint detection technology based on the targeted sequencing (tNGS) technology of multiple PCR, that is, the preparation of the targeted sequencing library by PCR amplification to detect the microorganisms contained in the sample. The current conventional experimental process requires two rounds of PCR, such as CN107904298A, which records a kit for analyzing intestinal microorganisms and its application, specifically relates to the construction of a library by two rounds of PCR, the kit for analyzing and classifying intestinal microorganisms and its application, and the combination adopts a two-step method to amplify and obtain the sequencing library. The experimenter amplifies the target area through the first round of multiple PCR technology, and after purification, uses the library to carry specific label information (specific labels to distinguish samples) to amplify the library to obtain the library required for sequencing and sequence it. After analyzing the sequencing results, the amplification area information in the sample can be obtained, so as to determine the type of microorganisms in the sample and the number of detection sequences. This detection technology can be used for environmental microbial detection, detection of microorganisms that cause animal diseases, and detection of microorganisms that cause plant diseases.

[0004] There are still some difficulties in the practical application of traditional multiplex PCR targeted sequencing technology, including: in order to cover the target fragments to be detected as much as possible, the number of targets is generally above 500x, and in order to improve the specificity of target detection, primers need to be selected, so primers need to be balanced in terms of quantity, sequence, GC content, primer length, combination of primer pools, etc., and finally produce a primer panel. In practical applications, multiple primers work in the same reaction system, and many primers have no target sequence, so primer dimers are easily generated, resulting in competitive waste of reaction reagents.

[0005] In summary, how to effectively avoid the generation of primer dimers in the reaction system is a difficult problem that needs to be solved urgently in the targeted sequencing technology of multiplex PCR. Summary of the invention

[0006] Based on this, one or more embodiments of the present application provide a simulated nucleic acid substrate, a kit including the same, and a method for detecting a target fragment using the same. The following technical solutions are included:

[0007] One or more embodiments of the present application provide a simulated nucleic acid substrate, wherein the simulated nucleic acid substrate has a structural unit as shown below:

[0008] First primer fragment-identification fragment-second primer fragment;

[0009] in,

[0010] The first primer fragment and the second primer fragment are both in a 5'-3' direction,

[0011] The first primer fragment and the second primer fragment are used to bind to PCR amplification primers of the same or different target fragments.

[0012] The identified segment does not include the target segment or a partial segment of the target segment.

[0013] In some embodiments of the present application, the different target fragments include target fragments from different species.

[0014] In some embodiments of the present application, the identification fragment and the target fragment have different species origins, and the sequence of the identification fragment is a conserved sequence;

[0015] Optionally, the species source of the identification fragment is an animal or a plant; further optionally, the species source of the identification fragment includes Arabidopsis thaliana;

[0016] Optionally, the target fragment originates from bacteria, fungi or viruses.

[0017] In some embodiments of the present application, the identification fragment is 100 bp-200 bp;

[0018] Optionally, the length of the identification segment is consistent with that of the target segment.

[0019] In some embodiments of the present application, the simulated nucleic acid substrate has a plurality of the structural units, and the identification fragments included in each of the structural units are the same or different;

[0020] Optionally, each of the structural units has a different combination of the first primer fragment and the second primer fragment, and is used for PCR amplification primers that bind to different target fragments.

[0021] In some embodiments of the present application, adjacent structural units are connected by transition segments, and multiple transition segments in the pseudo-nucleic acid substrate are the same or different;

[0022] Optionally, the transition fragment does not exceed 20 bp.

[0023] In some embodiments of the present application, the simulated nucleic acid substrate includes 5-10 different structural units.

[0024] In some embodiments of the present application, the simulated nucleic acid substrate does not exceed 2000 bp.

[0025] One or more embodiments of the present application provide a kit, which includes the simulated nucleic acid substrate.

[0026] In some embodiments of the present application, the kit further comprises a PCR amplification reagent, and the PCR amplification reagent comprises the PCR amplification primer defined above.

[0027] One or more embodiments of the present application provide a method for amplifying a target fragment, the method comprising the following steps:

[0028] The target fragment is amplified using an amplification system including the simulated nucleic acid substrate.

[0029] One or more embodiments of the present application provide a method for detecting a target fragment, the method comprising the following steps:

[0030] amplifying the target fragment using an amplification system comprising the simulated nucleic acid substrate; and,

[0031] Detect the target fragment in the obtained amplified product.

[0032] In some embodiments of the present application, the detection method includes a sequencing method.

[0033] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0035] Figure 1 Schematic diagram of the structure of the simulated nucleic acid substrate designed for the embodiment of the present application;

[0036] Figure 2 Schematic diagram of the simulated nucleic acid substrate and its amplification product designed for the embodiments of the present application;

[0037] Figure 3 A sequence composition logo of a simulated nucleic acid substrate designed for an embodiment of the present application;

[0038] Figure 4 Comparison of multiplex PCR amplification results with and without the addition of the simulated nucleic acid substrate of the present application example;

[0039] Figure 5 This is the target fragment detection flow chart of this application. DETAILED DESCRIPTION

[0040] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0042] the term

[0043] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0044] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0045] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0046] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0047] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0048] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0049] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0050] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0051] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0052] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0053] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0054] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0055] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0056] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0057] In a first aspect of an embodiment of the present application, a simulated nucleic acid substrate is provided, wherein the simulated nucleic acid substrate has a structural unit as shown below:

[0058] First primer fragment-identification fragment-second primer fragment;

[0059] in,

[0060] The first primer fragment and the second primer fragment are both in a 5'-3' direction,

[0061] The first primer fragment and the second primer fragment are used to bind to PCR amplification primers of the same or different target fragments.

[0062] The identified segment does not include the target segment or a partial segment of the target segment.

[0063] The term "nucleic acid substrate" refers to a nucleic acid molecule that is the target of an enzyme in an enzymatic reaction related to nucleic acids. These nucleic acid molecules include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), which can undergo various reactions such as hydrolysis, synthesis, and connection under the action of various enzymes such as nucleases, polymerases, and ligases. In some embodiments of the present application, the terms "nucleic acid substrate" and "nucleic acid template" are used interchangeably herein and refer to nucleic acid molecules that may contain single-stranded or double-stranded DNA or RNA.

[0064] The present application does not specifically limit the species origin of different target fragments, and may be multiple different target fragments from the same species, or different target fragments may be from different species. Species include, for example, bacteria, fungi, viruses, etc., including but not limited to Candida albicans, Aspergillus fumigatus, Pseudomonas aeruginosa, Klebsiella pneumoniae, influenza B virus, etc. In some embodiments of the present application, the different target fragments include target fragments from different species.

[0065] The terms "target fragment", "target nucleic acid fragment", "target molecule", "target nucleic acid molecule" and "target nucleic acid" are used interchangeably to refer to nucleic acid molecules that need to be sequenced, such as in an array. The target nucleic acid can be essentially any nucleic acid of known or unknown sequence. For example, it can be a fragment of genomic DNA or cDNA. Sequencing can result in determining the sequence target molecule of all or part of the sequence. The target can be derived from a randomly fragmented primary nucleic acid sample. In one embodiment, the target can be processed into a template suitable for amplification by placing a universal amplification sequence (e.g., a sequence present in the universal sequence). Adaptor, at the end of each target fragment. The target can also be obtained from a primary RNA sample by reverse transcription into cDNA.

[0066] In some embodiments of the present application, the target fragment may be a specific fragment of a species. The PCR amplification primers used to amplify the target fragment include, but are not limited to, the PCR amplification primers shown in SEQ ID NO.1 to SEQ ID NO.2, the PCR amplification primers shown in SEQ ID NO.3 to SEQ ID NO.4, the PCR amplification primers shown in SEQ ID NO.5 to SEQ ID NO.6, the PCR amplification primers shown in SEQ ID NO.7 to SEQ ID NO.8, and the PCR amplification primers shown in SEQ ID NO.9 to SEQ ID NO.10.

[0067] In the present application, the identification fragment is mainly used to distinguish the amplification product of the simulated nucleic acid substrate from the expansion product of the target fragment. Under the condition that this function can be achieved, the present application does not specifically limit the identification fragment. In some embodiments of the present application, the species origin of the identification fragment and the target fragment are different, and the sequence of the identification fragment is a conserved sequence. In some embodiments of the present application, the species origin of the identification fragment is an animal or a plant; further optionally, the species origin of the identification fragment includes Arabidopsis, including but not limited to the identification fragments shown in SEQ ID NO.11 to SEQ ID NO.15.

[0068] The term "animal" is to be understood as relating to mammals. Mammals are humans, non-human primates, domesticated mammals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals, such as zoo animals. It is also to be understood as also relating to non-mammalian vertebrates, such as birds (especially domesticated birds, such as chickens, ducks, geese, turkeys) and fish (especially farmed fish, such as salmon or catfish).

[0069] The term "plant" is to be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants (including but not limited to Arabidopsis thaliana), vegetable crops, including artichokes, Brussels sprouts, rocket, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine lettuce), pakchoi, yellow taro, melons (e.g., melon, watermelon, Crenshaw melon, honeydew melon, cantaloupe), rapeseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, cabbage), cardoon, carrot, cabbage, okra, onion, celery, parsley, chickpea, parsnip, endive, pepper, potato, cucurbits (e.g., zucchini, cucumber, courgette, squash, pumpkin), radish, bulb onion, rutabaga, eggplant (also known as eggplant), salsify, lettuce, shallot, endive, garlic, spinach, green onion, squash, leafy greens, beets (sugar beets and fodder beets), sweet potato, Swiss chard, horseradish, tomato, turnip, and spices; fruit and / or vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, Prunes, cherries, quinces, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pomegranates, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, silver grass, corn / maize (feed corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grains (barley, oats, rye, wheat, etc.) , sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oil plants (rapeseed, mustard, olives, sunflower, coconut, castor oil plants, cocoa beans, peanuts), Arabidopsis, fiber plants (cotton, flax, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, and trees such as forests (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

[0070] In some embodiments of the present application, the species origin of the target fragment is bacteria, fungi or viruses.

[0071] The term "bacteria" refers to a class of single-celled prokaryotes. Prokaryotes are organisms that do not have a nucleus bounded by a nuclear membrane. Bacteria have a relatively simple cell structure and are widely found in various environments in nature, including soil, water, air, and inside and outside the bodies of animals and plants.

[0072] The term "fungus" refers to a class of eukaryotic organisms whose cells have a nucleus and organelles. They are evolutionarily distinct from animals and plants and have unique biological characteristics. There are many types of fungi with different morphologies, including single-celled fungi (such as yeast) and multicellular fungi (such as mold).

[0073] The term "virus" refers to a class of non-cellular microorganisms that have a simple structure and are mainly composed of nucleic acids (DNA or RNA) and a protein shell. Viruses do not have independent metabolic systems and must rely on host cells to carry out life activities, such as reproduction and survival.

[0074] In some embodiments of the present application, the identification segment is 100bp-200bp, including but not limited to 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200bp. Optionally, the identification segment is 150bp-200bp. Further optionally, the identification segment is 175bp-200bp. Further optionally, the identification segment is 185bp-200bp. Optionally, the length of the identification segment is consistent with that of the target segment.

[0075] In some embodiments of the present application, the simulated nucleic acid substrate has a plurality of the structural units, and the identification fragments included in each of the structural units are the same or different; optionally, each of the structural units has a different combination of the first primer fragment and the second primer fragment, which is used to bind to PCR amplification primers of different target fragments. Optionally, each of the structural units has a different combination of the first primer fragment and the second primer fragment, which is used to bind to PCR amplification primers of target fragments of bacteria (including but not limited to Pseudomonas aeruginosa, Klebsiella pneumoniae, etc.), fungi (including but not limited to Candida albicans and Aspergillus fumigatus) and viruses (including but not limited to influenza B virus).

[0076] In some embodiments of the present application, the adjacent structural units are connected by transition segments, and the multiple transition segments in the pseudo-nucleic acid substrate are the same or different. In the present application, the transition segment and the identification segment can provide some spatial sequences to avoid the amplification preference of the two primers at the corresponding binding positions due to steric hindrance. In the case of satisfying this effect, the present application does not specifically limit the transition segment. Optionally, the transition segment does not exceed 20bp, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20bp, further optionally 15bp-20bp, and further optionally 15bp-17bp.

[0077] In some embodiments of the present application, the simulated nucleic acid substrate includes 5-10 different structural units, for example, 5, 6, 7, 8, 9, or 10 different structural units.

[0078] In some embodiments of the present application, the simulated nucleic acid substrate is no more than 2000bp, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 200bp. Alternatively, the simulated nucleic acid substrate is 1000bp-2000bp. Further alternatively, the simulated nucleic acid substrate is 1100bp-1500bp. Further alternatively, the simulated nucleic acid substrate is 1300bp-1400bp.

[0079] In some embodiments of the present application, the simulated nucleic acid substrate is shown as SEQ ID NO.21.

[0080] A second aspect of an embodiment of the present application provides a kit, which includes the simulated nucleic acid substrate.

[0081] In some embodiments of the present application, the kit is also used for PCR amplification reagents, and the PCR amplification reagents include the PCR amplification primers defined above, including but not limited to the PCR amplification primers shown in SEQ ID NO.1 to SEQ ID NO.2, the PCR amplification primers shown in SEQ ID NO.3 to SEQ ID NO.4, the PCR amplification primers shown in SEQ ID NO.5 to SEQ ID NO.6, the PCR amplification primers shown in SEQ ID NO.7 to SEQ ID NO.8, and the PCR amplification primers shown in SEQ ID NO.9 to SEQ ID NO.10.

[0082] It is understandable that the kit of the present application may also include other required detection reagents, such as nucleic acid lysis reagents, purification reagents, etc.

[0083] According to a third aspect of the embodiments of the present application, a method for amplifying a target fragment is provided, the method comprising the following steps:

[0084] The target fragment is amplified using an amplification system including the simulated nucleic acid substrate.

[0085] The term "amplification" or "amplification" refers to the production of multiple copies of polynucleotides or parts of polynucleotides when they occur together in the context of the term "nucleic acid", usually starting from a small amount of polynucleotides (e.g., as few as a single polynucleotide molecule), wherein the amplification product or amplicon is usually detectable. The amplification of polynucleotides includes a variety of chemical and enzymatic methods. The production of multiple DNA copies from one or several copies of a target DNA or template DNA molecule during polymerase chain reaction (PCR), rolling circle amplification (RCA) or ligase chain reaction (LCR) is a form of amplification. Amplification is not limited to the strict replication of the starting molecule. For example, the use of reverse transcription RT-PCR to produce multiple cDNA molecules from a limited amount of RNA in a sample is a form of amplification. In addition, the production of multiple RNA molecules from a single DNA molecule during the transcription process is also a form of amplification.

[0086] A fourth aspect of an embodiment of the present application provides a method for detecting a target fragment, the method comprising the following steps:

[0087] amplifying the target fragment using an amplification system comprising the simulated nucleic acid substrate; and,

[0088] Detect the target fragment in the obtained amplified product.

[0089] The detection method of nucleic acid may or may not rely on amplification of nucleic acid. As used herein, the term "detection method of nucleic acid" refers to any method for determining the nucleotide composition of a target nucleic acid, including but not limited to DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assays, (Hologic, Inc.) and described in, e.g., U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al. , PNAS, USA, 97:8272 (2000), and US2009 / 0253142, each of which is incorporated herein by reference in its entirety for all purposes); enzymatic mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, which are incorporated herein by reference in their entirety); polymerase chain reaction (PCR) as described above; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246 and 5,6 24,802, which is incorporated herein by reference in its entirety); rolling circle replication (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960, and 6,235,502, which are incorporated herein by reference in their entirety); NASBA (e.g., U.S. Pat. No. 5,409,818, which is incorporated herein by reference in its entirety); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, which is incorporated herein by reference in its entirety); E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,17 0 and 6,063,573, which are incorporated herein by reference in their entirety); circular probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769 and 5,660,988, which are incorporated herein by reference in their entirety); Dade Behring signal amplification method (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867 and 5,792,614, which are incorporated herein by reference in their entirety); ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridization method (e.g., U.S. Pat. No. 5,288,609, which is incorporated herein by reference in its entirety). In some embodiments of the present application, the detection method comprises a sequencing method.

[0090] In some embodiments of the present application, the detection method is for non-diagnostic purposes.

[0091] In some embodiments of the present application, the detection method is for diagnosis purposes.

[0092] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0093] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0094] The present application designs a nucleic acid with a unique sequence structure, which is essentially a simulated nucleic acid substrate (or called a primer consumption fragment, a consumption primer) to solve the problem of primer dimer generation in the process of multiple PCR library construction. This simulated nucleic acid substrate is added to the PCR reaction system, and can react with the amplification primer, and can complete the construction of the library, effectively consume the amplification primer, and avoid the generation of primer dimers; the amplified fragment generated by amplifying this simulated nucleic acid substrate can be effectively distinguished from the amplified product of the target fragment based on the identification fragment it contains (for example, Arabidopsis nucleic acid fragment, of course, it can also be a fragment of other species that can be distinguished). In some examples, 5-10 pairs of primers can be accommodated on a simulated nucleic acid substrate of the present application, and these primer pairs can be selected according to the actual application situation. Primers that are easy to generate primer dimers. The present application does not specifically limit the species source of the target fragment targeted by the primer pair in the simulated nucleic acid substrate, for example, one end is a primer for species A, and the other end is a primer for species B, or both can be used for the same species. In samples that do not contain the target pathogen, because of the presence of this nucleic acid, the amplification primers can use this nucleic acid to complete PCR and produce products. In this process, primer dimers can be reduced while negative samples can also be sequenced, thereby achieving quality control of blank samples. Adding this simulated nucleic acid substrate to the blank sample can also complete the construction of the library.

[0095] Combination Figure 1 The simulated nucleic acid substrates in the embodiments of the present application are explained as follows:

[0096] 1. Sequence structure such as Figure 1As shown, primer sequences 1 and 2 are a pair of primers (as mentioned above, they can be the same species or a combination of two different species), and the primer directions are all 5'-3'. This primer direction is relatively novel. Generally, it is a combination of 5'-3' and 3'-5'. This design can complete the library construction and reduce primer dimers. Both primers are occupied, but only one effective amplification fragment is generated, reducing the complexity of the target fragment.

[0097] 2. The middle of the sequence is an Arabidopsis sequence. Primer sequences 1 and 2 and the Arabidopsis sequence form a synthetic unit of a primer consumption sequence. 5-10 pairs of synthetic units can be connected in series to form the final sequence.

[0098] 4. Since this sequence can complete library construction, it can also be successfully completed in blank samples. After delivery, its usage will be evaluated. Use blank samples, set the gradient of total amount added, conduct gradient experiments, and evaluate the optimal addition amount.

[0099] 5. The purpose of setting up Arabidopsis is to distinguish it from the normally occurring amplified sequence. The sequence length needs to be consistent with the normal amplified sequence length, which is about 200 bp, or related to the length of the sequencing. For example, if the sequencing is 75 nt, the length can be greater than 75 nt.

[0100] 6. The design length of the transition sequence is 16 bp, with no other rules. Its function is to distinguish the two primer amplification sequences and add spatial positions without causing primer dimers.

[0101] 5. Five pairs of primers were synthesized this time. The total length of a single sequence is 1304 bp. Theoretically, more fragments of different lengths can be generated, and some of the fragments can be as long as the above Figure 2 As shown in , large fragments are filtered out in the magnetic bead fragment selection.

[0102] The two sequences that can form primer dimers in different multiple primer pool combinations are very different. The consumed primer sequences are aimed at the two primer sequences that are more likely to form primer dimers. The evaluation standard for the easy formation of primer dimers is that two primers that can be complementary to each other can be easily obtained through bioinformatics analysis. After analysis, the primer sequences with more reads are suitable for elimination. Therefore, in different primer pools, the consumed primer sequences are not exactly the same; in this embodiment, the species name of the primer sequence source in the consumed primer sequence, as well as the specific sequence of the designed primers are as follows:

[0103] Table 1

[0104]

[0105]

[0106] The sequences of the various parts of the consumable primers (simulated nucleic acid substrates) used in this example are shown in the table below:

[0107] Table 2

[0108]

[0109] Consumption primer (simulated nucleic acid substrate, 5'-3', SEQ ID NO.21):

[0110]

[0111]

[0112] In the sequence, the underlined mark is the primer sequence, the box mark is the transition sequence, and the bold italic mark is the Arabidopsis sequence.

[0113] As shown in the above table, the sequence composition of a single depletion primer includes two multiple amplification primers i5 and i7, an Arabidopsis sequence, and a transition sequence. The above sequences are connected in series to obtain a depletion primer sequence.

[0114] Combination Figure 3 , the primer sequence in the sequence of the consumed primer can be amplified with the corresponding sequence in the multiple amplification pool primer after synthesizing the double strand during multiple amplification, and can generate an amplification product sequence containing the Arabidopsis sequence. In this process, the two primers in the multiple amplification pool that should have formed dimers can be amplified and generate recognizable sequences, thereby achieving the reduction of primer dimers. The Arabidopsis sequence and transition sequence in the consumed primer sequence can provide some spatial sequences when the primers bind to the DNA chain, avoiding the amplification preference caused by steric hindrance at the corresponding binding positions of the two primers.

[0115] Example 1

[0116] The concentration of the consumed primers in the detection amplification system was diluted to the corresponding concentration level in the table below. Water samples were used for verification. The specific experimental process is shown in the subsequent detailed detection process, in which the templates were replaced by water. This example is to verify the amount of consumed primers.

[0117] Table 3

[0118] Input concentration Input amount PCR concentration 1 1 10 3.63 2 0.1 1 0.756 3 0.01 0.1 0.117 4 0.001 0.01 0.051 5 0.0001 0.001 low 6 0.00001 0.0001 0.066

[0119] In the water sample test, when the primer input amount is at the level of 10 ng, the library construction concentration is greater than 1 ng / μL and is qualified.

[0120] Example 2

[0121] In order to verify the actual application effect of depletion primers, positive references were prepared using Candida albicans, Aspergillus fumigatus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and influenza B virus, and a gradient of 5000-10 copies of standard samples was set for each species. The commercial kit was used for extraction (magnetic bead pathogenic microorganism DNA / RNA extraction kit NG550). The extracted nucleic acid was constructed according to the following library construction process, in which the multiple primer pool used was 1000, and the experimental group was added with depletion primers, and the control group was not added with depletion primers. 10ng of depletion primers were added to the amplification system. When positive samples and primer depletion sequences exist at the same time, the library construction concentration is qualified.

[0122] Table 4

[0123]

[0124]

[0125] Table 5

[0126] 5000 1000 500 200 100 50 10 No_Dimer_rate 75.51％ 48.10％ 64.49％ 52.34％ 29.17％ 31.14％ 16.64％ Dimer_rate 24.49％ 51.90％ 35.51％ 47.66％ 70.83％ 68.86％ 83.36％ Normal capture reads number 33623 5786 4594 2905 1880 956 157 Number of sequence reads consumed by primers 2113 1567 977 1041 3705 1530 3906

[0127] The number of reads detected for consumed primer sequences was relatively stable and did not dominate.

[0128] Table 6

[0129]

[0130] like Figure 4 As shown. This experiment set up a control group and an experimental group. The experimental group added depletion primers (including 5 pairs of primers, SEQ ID NO.21), and the control group did not add them. The coordinate axis of the bar graph in the figure is on the left, and the coordinate axis of the line graph is on the right. The bar graph represents the number of reads of dimers in the experimental group and the control group, and the line graph represents the ratio of dimers reduced in the experimental group. It can be seen that the addition of depletion primers can effectively reduce primer dimers, and the degree of reduction is reduced at low copy numbers. After adding more depletion primers, better results can be achieved.

[0131] Table 7

[0132] LOD 5000 1000 500 200 100 50 10 Candida albicans 10 18584 5495 5267 6764 5354 6888 327 Aspergillus fumigatus 10 4194 1439 578 1061 548 0 307 Pseudomonas aeruginosa 10 326166 180197 254399 181390 41997 52417 2498 Klebsiella pneumoniae 10 390 163 520 66 496 563 81 Epstein-Barr virus 100 2292 556 1157 1459 156 0 0 RSV-B 1000 760 130 0 0 0 0 0 Human herpesvirus type 1 100 2852 1275 3993 1923 548 0 0

[0133] Panel detection was not affected by the relative proportion of consumed primers, and the detection effect was accurate, reaching 10 copies for some species.

[0134] The specific experimental steps of the above examples 1 to 4 are as follows:

[0135] 1. See the test flow chart Figure 5 .

[0136] 2. Experimental Procedure

[0137] 2.1 Sample processing and nucleic acid extraction (template preparation)

[0138] A commercial extraction kit with verified performance (magnetic bead-based pathogenic microorganism DNA / RNA extraction kit NG550) was used. Nucleic acid extraction was performed according to the manufacturer's instructions. Water samples were set as negative controls according to the sample type.

[0139] 2.2 Preparation of reverse transcription reaction system (reagent preparation)

[0140] Table 8

[0141] Reagents Volume added for a single reaction (μL) 5×RT mix 4 Nuclease-Free Water 6 Template (extraction product) 10 Total 20

[0142] 2.3. Reverse transcription reaction (template preparation)

[0143] Place the PCR reaction tube into the sample slot of the thermal cycler and start the PCR reaction. The cycle parameters are set as follows:

[0144] Table 9

[0145] step temperature time Number of cycles 1 25℃ 10min 1 2 55℃ 15min 1 3 85℃ 5min 1 4 4℃ ∞ /

[0146] Place the reaction product cDNA solution on ice for subsequent experiments; or immediately store it at -20°C for later use.

[0147] 2.4 Configuration of multiplex PCR amplification system

[0148] Table 10

[0149] Reagents Volume added for a single reaction (μL) Multiplex PCR amplification mix 10 After nucleic acid extraction 10 Reverse transcription product 5 Multiplex Amplification Primers 4 Primer consumption fragment 1 Total 30

[0150] 2.5 Multiplex Amplification

[0151] Place the reaction tube of the reaction system prepared for one round of PCR into the sample slot of the thermal amplification instrument and start the PCR reaction. The cycle parameters are set as follows:

[0152] Table 11

[0153]

[0154] 6. Multiplex PCR Product Purification

[0155] Preparation: Use commercially available anhydrous ethanol with verified performance. Take the DNA clean beads out of the refrigerator, equilibrate at room temperature for at least 30 minutes, vortex or fully invert the DNA clean beads to fully resuspend, and prepare 80% ethanol.

[0156] (1) Add 15 μL of DNA clean beads to the first round of PCR amplification products; vortex to mix and incubate at room temperature for 5 min.

[0157] (2) Briefly centrifuge the tube and place it on a magnetic rack. After the solution becomes clear (approximately 2 minutes), carefully transfer the supernatant to a clean centrifuge tube.

[0158] (3) Add 15 μL DNA clean beads to the supernatant; vortex to mix and incubate at room temperature for 5 min.

[0159] (4) Briefly centrifuge the tube and place it on a magnetic rack. After the solution becomes clear (approximately 2 minutes), carefully remove the supernatant.

[0160] (5) Add 30uL Nuclease-Free Water to the DNA clean beads, vortex to mix, then add 30uL DNA clean beads to the system again, vortex to mix, and incubate at room temperature for 5 minutes.

[0161] (6) Briefly centrifuge the tube and place it on a magnetic rack. After the solution becomes clear (approximately 2 minutes), carefully remove the supernatant.

[0162] (7) Keep the centrifuge tube in the magnetic rack, add 100 μL of freshly prepared 80% ethanol, and use the magnetic rack to repeatedly adsorb the DNA clean beads back and forth on different sides to fully suspend the DNA clean beads for washing. Carefully remove the supernatant.

[0163] (8) Repeat step (7).

[0164] (9) Keep the centrifuge tube in the magnetic rack and open the lid to dry the DNA clean beads (about 5 minutes). Note: The drying time should not be too long. Excessive drying of the DNA clean beads will affect the purification effect.

[0165] 8. Second round of amplification

[0166] Transfer the prepared 30 μL reaction system to the product analysis area 1, and add it directly to the purified DNA clean beads (combined with one round of PCR products) of the corresponding sample. Resuspend the DNA clean beads reaction solution and centrifuge to the bottom of the tube, then perform the following reaction:

[0167] Table 12

[0168]

[0169] Preparation: Take the DNA clean beads out of the refrigerator, equilibrate at room temperature for at least 30 minutes, vortex or repeatedly invert the DNA clean beads to ensure sufficient resuspending, and prepare 80% ethanol.

[0170] (1) Add 24 μL DNA clean beads to the second-round PCR amplification product; vortex to mix and incubate at room temperature for 5 min.

[0171] (2) Briefly centrifuge the tube and place it on a magnetic rack. After the solution becomes clear (approximately 2 minutes), carefully remove the supernatant.

[0172] (3) Keep the centrifuge tube in the magnetic rack, add 100uL of freshly prepared 80% ethanol, and use the magnetic rack to repeatedly adsorb the DNA clean beads on both sides to fully suspend the DNA clean beads for washing. Carefully remove the supernatant.

[0173] (4) Repeat step (3).

[0174] (5) Keep the centrifuge tube in the magnetic rack and open the lid to dry the DNA clean beads (about 5 minutes). Note: The drying time should not be too long. Excessive drying of the DNA clean beads will affect the purification effect.

[0175] (6) Remove the centrifuge tube from the magnetic rack, add 25uL Nuclease-Free Water, vortex to mix, and incubate at room temperature for 2min.

[0176] (7) Briefly centrifuge the tube and place it on a magnetic rack to separate the DNA clean beads and liquid. After the solution is clarified (about 2 minutes), carefully pipette the supernatant into a clean tube to complete the library construction. Label and store at -20±5℃ for short-term use or at -70±5℃ for long-term storage.

[0177] 9. Sequencing

[0178] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0179] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A simulated nucleic acid substrate, characterized in that: The simulated nucleic acid substrate has the following structural units: First primer fragment-identification fragment-second primer fragment; in, The first primer fragment and the second primer fragment are both in a 5'-3' direction, The first primer fragment and the second primer fragment are used to bind to PCR amplification primers of the same or different target fragments. The identified segment does not include the target segment or a partial segment of the target segment.

2. The simulated nucleic acid substrate according to claim 1, characterized in that The different target fragments include target fragments from different species.

3. The simulated nucleic acid substrate according to claim 1, characterized in that The species sources of the identification fragment and the target fragment are different, and the sequence of the identification fragment is a conserved sequence; Optionally, the species source of the identification fragment is an animal or a plant; further optionally, the species source of the identification fragment includes Arabidopsis thaliana; Optionally, the target fragment originates from bacteria, fungi or viruses.

4. The simulated nucleic acid substrate according to claim 1, characterized in that The identification fragment is 100bp-200bp; Optionally, the length of the identification segment is consistent with that of the target segment.

5. The simulated nucleic acid substrate according to any one of claims 1 to 4, characterized in that The simulated nucleic acid substrate has a plurality of the structural units, and the identification fragments included in each of the structural units are the same or different; Optionally, each of the structural units has a different combination of the first primer fragment and the second primer fragment, and is used for PCR amplification primers that bind to different target fragments.

6. The simulated nucleic acid substrate according to claim 5, characterized in that The adjacent structural units are connected by transition segments, and the multiple transition segments in the pseudo-nucleic acid substrate are the same or different; Optionally, the transition fragment does not exceed 20 bp.

7. The simulated nucleic acid substrate according to claim 5, characterized in that The simulated nucleic acid substrate comprises 5-10 different structural units.

8. The simulated nucleic acid substrate according to any one of claims 1 to 4 and 6 to 7, characterized in that The simulated nucleic acid substrate does not exceed 2000 bp.

9. A kit, characterized in that The kit comprises the simulated nucleic acid substrate according to any one of claims 1 to 8.

10. The kit according to claim 9, characterized in that The kit further comprises a PCR amplification reagent, wherein the PCR amplification reagent comprises a PCR amplification primer as defined in any one of claims 1 to 8.

11. A method for amplifying a target fragment, characterized in that: The amplification method comprises the following steps: A target fragment is amplified using an amplification system comprising the simulated nucleic acid substrate according to any one of claims 1 to 8.

12. A method for detecting a target fragment, characterized in that: The detection method comprises the following steps: Amplifying a target fragment using an amplification system comprising the simulated nucleic acid substrate according to any one of claims 1 to 8; and Detect the target fragment in the obtained amplified product.

13. The method for detecting a target fragment according to claim 12, characterized in that: The detection method includes sequencing.

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