CN117625753A - Isothermal amplification reagent and application thereof - Google Patents

Abstract

The invention relates to the field of biological detection, in particular to a isothermal amplification reagent and application thereof. Taking influenza virus as an example, the invention develops a reagent capable of improving isothermal amplification efficiency and application thereof; the invention optimizes the traditional isothermal amplification reagent, the enhancement primer is added in an amplification system, the designed position and concentration of the enhancement primer can obviously influence the isothermal amplification efficiency, and sodium alginate is added in the amplification system, and the experimental result shows that the amplification efficiency of isothermal amplification can be obviously improved when the sodium alginate is 0.05 (wt%). The isothermal amplification reagent provided by the invention is used for detecting samples, the amplification efficiency is obviously improved, and the isothermal amplification reagent has wide popularization and application prospects.

Description

Isothermal amplification reagent and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to a isothermal amplification reagent and application thereof.

Background

Real-time fluorescent PCR is an amplification method for quantitatively detecting nucleic acid based on PCR technology, and can monitor amplification reaction in real time by adding fluorescent substances or using molecular beacons. The accumulation of fluorescent signal may reflect the amplification of the target DNA or RNA reverse transcription product. Real-time fluorescence PCR requires precise temperature control, is independent of professional instruments and equipment, and generally requires 1-2 hours in the whole amplification process, and is not suitable for on-site rapid detection.

For this limitation, isothermal amplification technology is increasingly favored by the research and development of extrareceptor diagnostic products or by researchers. The isothermal amplification technology mainly uses the activity of recombinase, does not carry out nucleic acid melting and annealing, and carries out nucleic acid amplification under isothermal conditions. In recent years, with the rapid development of molecular biology technology, a isothermal amplification diagnostic method based on nucleic acid detection has been established in a large number and widely used in laboratory detection of human diseases, and compared with other nucleic acid amplification technologies, isothermal amplification has the advantages of rapidity, high efficiency, specificity, and no need of repeated temperature change, so that once it occurs, it is considered by many students as a detection method that is likely to be comparable to PCR.

However, at present, the amplification efficiency of isothermal amplification is still to be improved, and a isothermal amplification system with higher amplification efficiency and a matched method are required to be developed.

Disclosure of Invention

In view of the above, the present invention aims to provide isothermal amplification reagents and applications thereof.

The invention provides a isothermal amplification reagent, which comprises Buffer premix, isothermal amplification substrate, enzyme premix and isothermal amplification primer;

the isothermal amplification primer comprises: isothermal amplification upstream primers, isothermal amplification downstream primers, amplification primers and/or random primers;

the concentration of the enhancement primer is less than or equal to 0.5 mu M; the 5 'end of the enhanced primer is 0-8 bases away from the 5' end of the isothermal amplification downstream amplification primer, the length of the enhanced primer is obtained by subtracting 9-11 nt from the length of the isothermal amplification downstream primer, and the length of the enhanced primer is 18-22 nt; preferably, the 5 'end of the enhancing primer is about 0 to 5 bases from the 5' end of the isothermal amplification downstream primer;

in particular embodiments, enhancement is best when the 5 'end of the enhancing primer is 0 bases from the 5' end of the downstream amplification primer and the enhancing primer is shorter than 10nt in length than the downstream primer; the enhancement is significantly reduced by the 3' end of the enhancing primer beyond the downstream primer.

Further, the method comprises the steps of,

the concentration of the enhancement primer is 0.1 mu M-0.5 mu M, preferably 0.3 mu M;

in a specific embodiment of the invention, the concentration and location of the enhancing primer are examined; the test result shows that the enhancement primer with the concentration of 0.1 mu M to 0.5 mu M can promote the amplification effect, wherein the effect is best when the addition concentration is 0.3 mu M; in the investigation of the position of the enhanced primer, the enhanced primer has an enhanced effect when the 5 'end of the enhanced primer is 0-8 bases away from the 5' end of the isothermal amplification downstream primer and the length of the enhanced primer is shorter than 9-11 nt of the isothermal amplification downstream primer, and the enhanced primer sequence is completely contained in the isothermal amplification downstream primer sequence and is completely overlapped with a partial sequence of the isothermal amplification downstream primer;

the isothermal amplification primers of the present invention also include probes.

Each of the probes is coupled with a fluorescent group and a fluorescence quenching group;

the fluorescent group is selected from any one of FAM, VIC, ROX or CY-5;

the fluorescence quenching group is selected from any one of BHQ1, MGB or BHQ 2;

specifically, the length of the probe is 46-52 nucleotides, the fluorescent group and the quenching group are marked on thymine, the distance between the fluorescent group and the quenching group is 2-5 bases, a certain base between 2T marked by the fluorescent group and the quenching group is replaced by Tetrahydrofuran (THF), at least 30 nucleotides of the probe are positioned at the 5' end of the THF site, at least 15 nucleotides are positioned at the 3' end of the probe, and the 3' end of the probe is blocked.

In the invention, the isothermal amplification primer comprises at least one of the following I-III):

i) A isothermal amplification upstream primer of the nucleotide sequence shown as SEQ ID NO. 1, a isothermal amplification downstream primer of the nucleotide sequence shown as SEQ ID NO. 2, an enhanced primer of the nucleotide sequence shown as SEQ ID NO. 7, and a probe of the nucleotide sequence shown as SEQ ID NO. 10;

II), a isothermal amplification upstream primer of the nucleotide sequence shown as SEQ ID NO. 3, a isothermal amplification downstream primer of the nucleotide sequence shown as SEQ ID NO. 4, an enhanced primer of the nucleotide sequence shown as SEQ ID NO. 8, and a probe of the nucleotide sequence shown as SEQ ID NO. 11;

III), a isothermal amplification upstream primer of a nucleotide sequence shown as SEQ ID NO. 5 and a isothermal amplification downstream primer of a nucleotide sequence shown as SEQ ID NO. 6; an enhanced primer of the nucleotide sequence shown as SEQ ID NO. 9, and a probe of the nucleotide sequence shown as SEQ ID NO. 12.

The isothermal amplification reagent also comprises sodium alginate;

the concentration of the sodium alginate is 0.02 (w/v)% -0.08 (w/v)%, preferably 0.03 (w/v)% -0.05 (w/v)%, and most preferably 0.05 (w/v)%.

In the isothermal amplification reagent of the present invention,

the random primer includes a random primer having a length of 6nt and a random primer having a length of 8 nt.

The buffer premix comprises buffer solution, potassium ions, magnesium ions, a reducing agent, a stabilizing agent, betaine and an amplification enhancer;

the isothermal amplification substrate comprises ATP, creatine phosphate and dNTPs;

the enzyme premix comprises reverse transcriptase, polymerase, recombinase protein, single-chain binding protein, recombinase protein auxiliary protein, helicase, creatine kinase, RNase inhibitor and Exoneclease III.

Further, in the buffer premix,

the potassium ion is provided by potassium acetate;

the magnesium ions are provided by magnesium acetate;

the reducing agent comprises at least one of DTT, beta-mercaptoethanol, TCEP, or SDS;

the stabilizer comprises at least one of BSA, glycerol, or sodium azide;

the amplification enhancer comprises at least one of DMSO and/or PEG8000.

The isothermal amplification reagent of the invention;

the Buffer premix comprises 60 mM-100 mM Tris-HCl, 100 mM-120 mM potassium acetate, 12 mM-18 mM magnesium acetate, 3 mM-7 mM DTT, 30 ng/. Mu.L-70 ng/. Mu.L BSA, 280 mM-320 mM betaine and 6 (w/v)% -10 (w/v)% PEG8000;

the isothermal amplification substrate comprises: 3 mM-7 mM ATP, 110 mM-150 mM creatine phosphate, and 0.5 mM-1.5 mM dNTP;

the enzyme premix comprises 10U/mu L-30U/mu L reverse transcriptase, 400 ng/mu L-600 ng/mu L polymerase, 600 ng/mu L-800 ng/mu L recombinant enzyme protein, 700 ng/mu L-1000 ng/mu L single chain binding protein, 100 ng/mu L-300 ng/mu L recombinant enzyme protein auxiliary protein, 400 ng/mu L-600 ng/mu L creatine kinase, 0.5U/mu L-1.5U/mu L RNase inhibitor and 1U/mu L-3U/mu L Exonuclease III;

the isothermal amplification primer comprises: 0.4-0.6 mu M isothermal amplification upstream primer, 0.4-0.6 mu M isothermal amplification downstream primer, 0.2-0.5 mu M enhancement primer, 0.1-0.3 mu M probe and 0.05-0.15 mu M random primer.

In a specific embodiment of the present invention,

the Buffer premix included 80mM Tris-HCl, 110mM potassium acetate, 15mM magnesium acetate, 5mM DTT, 50 ng/. Mu.L BSA, 300mM betaine, and 8.3 (w/v)% PEG8000;

the isothermal amplification substrate comprises: 5mM ATP, 130mM creatine phosphate and 1mM dNTP;

the enzyme premix comprises: 20U/. Mu.L reverse transcriptase, 500 ng/. Mu.L polymerase, 700 ng/. Mu.L recombinant enzyme protein, 885 ng/. Mu.L single-stranded binding protein, 200 ng/. Mu.L recombinant enzyme protein helper protein, 500 ng/. Mu.L creatine kinase, 1U/. Mu.L RNase inhibitor and 2U/. Mu. L Exonuclease III.

The isothermal amplification primer comprises: 0.5. Mu.M isothermal amplification upstream primer, 0.5. Mu.M isothermal amplification downstream primer, 0.3. Mu.M amplification primer, 0.2. Mu.M probe and 0.1. Mu.M random primer.

In the invention, the isothermal amplification reagent is optimized; specifically, the method comprises the optimization of primers and sodium alginate; the enhancement primer is added in the reaction system, the concentration of the enhancement primer and the designed position of the enhancement primer have influence on the amplification efficiency, when the subtype H7N9 of the influenza A virus is detected, the amplification efficiency is improved when the concentration of the enhancement primer is 0.1 mu M-0.5 mu M, and when the concentration reaches 0.3 mu M, the enhancement effect is optimal and is improved by about 31% compared with the signal of a non-added group; in the detection of influenza A virus H7N9 subtype, the amplification efficiency is highest when the enhancement primer is R2 (SEQ ID NO: 7); in the detection system of the influenza A virus H7N9 subtype and the influenza A virus, 0 (w/v)% -0.1 (w/v)% sodium alginate is added, the amplification efficiency shows a trend of increasing firstly and then reducing, the highest amplification efficiency can be obtained when the added volume fraction is 0.05%, and compared with the amplification efficiency which is improved by 20% when sodium alginate is not added.

And the amplification reagent is matched with the enzyme premix, the Exonuclease III is combined with the probe for use, and is specifically complementary matched with a detection target, and under the condition of a specific amplification product, the probe is cut by the Exonuclease III to release a fluorescent group, so that the instrument can capture a fluorescent signal.

The invention provides a kit which comprises the isothermal amplification reagent and a quality control product.

The invention provides application of the isothermal amplification reagent or the kit in influenza virus detection or preparation of influenza detection products.

The invention provides a method for detecting influenza virus, which comprises the step of detecting a sample by using the isothermal amplification reagent.

Taking influenza virus as an example, the invention develops a reagent and a method isothermal amplification reagent capable of improving isothermal amplification efficiency and application thereof; the invention optimizes the traditional isothermal amplification reagent, the enhancement primer is added in an amplification system, the designed position and concentration of the enhancement primer can obviously influence the isothermal amplification efficiency, and sodium alginate is added in the amplification system, and the experimental result shows that the sodium alginate is 0.05 (w/v)% and can obviously improve the isothermal amplification efficiency. The isothermal amplification reagent provided by the invention is used for detecting samples, the amplification efficiency is obviously improved, and the isothermal amplification reagent has wide popularization and application prospects.

Drawings

FIG. 1 is the effect of isothermal amplification enhancement primer concentrations for influenza A subtype H7N9 nucleic acid and influenza B nucleic acid;

FIG. 2 is a graph showing the effect of the presence or absence of an enhancer primer on influenza B nucleic acid amplification;

FIG. 3 is a graph showing the effect of the presence or absence of an enhancement primer on influenza A (non-typing) nucleic acid amplification;

FIG. 4 is a map of isothermal amplification enhancement primer positions for influenza A subtype H7N9 nucleic acid;

FIG. 5 is a graph of the effect of isothermal amplification of influenza A subtype H7N9 nucleic acid and influenza A (non-typing) nucleic acid on the positions of the enhancing primers;

FIG. 6 is a graph showing the effect of sodium alginate on detection of influenza A subtype H7N9 nucleic acid;

FIG. 7 is a graph showing the effect of sodium alginate on influenza B nucleic acid detection.

Detailed Description

The invention provides isothermal amplification reagents and applications thereof, and one skilled in the art can properly improve the process parameters by referring to the disclosure herein. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

Influenza a virus H7N9 subtype nucleic acid fragment in influenza a virus H7N9 subtype virus lentivirus quality control:

gtttcagatggagggccaaacctgtacaatatccggaacctccacattccagaggtctgcttgaaatgggaattgatggatgaagactaccaaggcaggttgtgtaatcctatgaacccgtttgtcagtcataaggaaattgattcagtcaacaatgctgtggtgatgccagctcatggcccagccaaaagcatggagtatgatgccgttgcaaccacacattcatggattcctaagaggaatcgctccattctcaacaccagccaaagggggattcttgaggacgaacagatgtaccagaagtgctgcaacctattcgaaaagttcttccccagcagttcgtacaggaggccagttggaatttccagcatggtggaggccatggtgtctagggcccgaattgatgcacgaattgacttcgaatctggaaggattaagaaagaagagtttgctgagatcatgaagatctgttccaccattgaagagctcagacggcaaaaatagtgaatttagcttgtccttcatgatgaatccaaatcagaagattctatgcacttcagccactgctatcataataggcgcaatcgcagtactcattggaatggcaaacctaggattgaacataggactgcatctaaaaccgggctgcaattgctcacactcacaacctgaaacaaccaacacaagccaaacaataataaacaactattataatgaaacaaacatcaccaacatccaaatggaagagagaacaagcaggaatttcaataacttaactaaagggctctgtactataaattcatggcacatatatgggaaagacaatgcagtaagaattggagagagctcggatgttttagtcacaagagaaccctatgtttcatgcgacccagatgaatgcaggttctatgctctcagccaaggaacaacaatcagagggaaacactcaaacggaacaatacacgataggtcccagtatcgcgccctgataagctggccactatcatcaccgcccacagtgtacaacagcagggtggaatgcattgggtggtcaagtactagttgccatgatggcaaatccaggatgtcaatatgtatatcaggaccaaacaacaatgcatctgcagtagtatggtacaacagaaggcctgttgcagaaattaacacatgggcccgaaacatactaagaacacaggaatctgaatgtgtatgccacaacggcgtatgcccagtagtgttcaccgatgggtctgccactggacctgcagacacaagaatatactattttaaagaggggaaaatattgaaatgggagtctctgactggaactgctaagcatattgaagaatgctcatgttacggggaacgaacaggaattacctgcacatgcagggacaattggcagggctcaaatagaccagtgattcagatagacccagtagcaatgacacacactagtcaatatatatgcagtcctgttcttacagacaatccccgaccgaatgacccaaatataggtaagtgtaatgacccttatccaggtaataataacaatggagtcaagggattctcatacctggatggggctaacacttggctagggaggacaataagcacagcctcgaggtctggatacgagatgttaaaagtgccaaatgcattgacagatgatagatcaaagcccattcaaggtcagacaattgtattaaacgctgactggagtggttacagtggatctttcatggactattgggctgaaggggactgctatcgagcgtgtttttatgtggagttgatacgtggaagacccaaggaggataaagtgtggtggaccagcaatagtatagtatcgatgtgttccagtacagaattcctgggacaatggaactggcctgatggggctaaaatagagtacttcctctaa(SEQ ID NO:18)。

influenza a virus (non-typing) packaging nucleic acid fragment in influenza a virus lentivirus quality control:

tgagtcttctaaccgaggtcgaaacgtacgttctctctatcattccatcaggccccctcaaagccgagatcgcacagagacttgaggatgtttttgcagggaagaacgcagatctcgaggctctcatggagtggataaagacaagaccaatcctgtcacctctgactaaggggattttagggtttgtgttcacgctcaccgtgcccagtgagcgaggactgcagcgtagacggtttgtccaaaacgccctaaatgggaatggagacccaaacaacatggacaaggcggttaaattatacaagaaactgaagagggaaatgacatttcatggagcaaaggaagttgcactcagttactcaactggtgcgcttgccagctgcatgggtctcatatacaacagaatggggactgtgaccgcagaaggggctcttggactagtatgtgccacttgtgagcagattgctgacgcacaacatcggtcccacaggcagatggcgactactactaacccactaattaggcatgagaatagaatggtactagccagcactacggctaaggctatggagcagatggctggatcaagtgaacaggcagcggaagccatggaagttgcaagtcaggctaggcaaatggtgcaggctatgagaacagttgggactcaccctaactccagtacaggtctaaaagatgatcttattgaaaatttgcaggcctaccagaaccggatgggagtgcaactgcagcggttcaagtgagcctctagtcgttgcagctaacattattgggatattgcacttgatattgtggattcttgatcgtcttttcttcaaatgcatttatcgtcgttttaaatacggtttgaaaagagggccttctacggaaggaatgcctgagtctatgagggaagaatatcggcaggaacagcagaatgctgtggatgttgacgatggtcattttgtcaacatagagctgaagtaaaaa(SEQ ID NO:19)。

influenza b lentiviral nucleic acid fragment in influenza b lentiviral quality control:

tgctaccttcaactatacaaacgttaaccctatttctcacatcagggggagtattattatcactatatgtgtcagcttcattatcatacttactatattcggatatattgctaaaattctcaccaacagaaataactgcaccaacaatgccattggattgtgcaaacgcatcaaatgttcaggctgtgaaccgttctgcaacaaaaggggtgacacttcttctcccagaaccggagtggacatacccgcgtttatcttgcccgggctcaacctttcagaaagcactcctaattagccctcatagattcggagaaaccaaaggaaactcagctcccttgataataagggaaccttttgttgcttgtggaccaaatgaatgcaaacactttgctttaacccattatgcagcccaaccagggggatactacaatggaacaagaggagacagaaacaagctgaggcatctaatttcagtcaaattgggcaaaatcccaacagtagagaactccattttccacatggcagcatggagcgggtccgcgtgccatgatggtaaggaatggacatatatcggagttgatggccctgacaataatgcattgctcaaagtaaaatatggagaagcatatactgacacataccattcctatgcaaacaacatcctaagaacacaagaaagtgcctgcaattgcatcgggggaaattgttatctaatgataactgatggctcagcttcaggtgttagtgaatgcagatttcttaagattcgagagggccgaataataaaagaaatatttccaacaggaagagtaaaacacactgaggaatgcacatgcggatttgccagcaataaaaccatagaatgtgcctgtagagacaacaggtacacagcaaaaagaccttttgtcaaattaaacgtggagactgatacagcagaaataaggttgatgtgcacagatacttatttggacacccccagaccaaatgatggaagcataacaggcccttgtgaatctgatggggacaaagggagtggaggcatcaagggaggatttgttcatcaaagaatgaaatccaagattggaaggtggtactctcgaacgatgtctaaaactgaaaggatggggatgggactgtatgtcaagtatggtggagacccatgggctgacagtgatgccctagcttttagtggagtaatggtttcaatgaaagaacctggttggtattcctttggcttcgaaataaaagataagaaatgcgatgtcccctgtattgggatagagatggtacatgatggtggaaaagagacttggcactcagcagcaacagccatttactgtttaatgggctcaggacagctgctgtgggacactgtcacaggtgttgacatggctctgtaa(SEQ ID NO:20)。

the reagent consumable adopted by the invention is a common commercial product and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1 isothermal amplification reagent and use thereof

1. Experimental protocol

1. Reagent(s)

The isothermal amplification (Isothermal Amplification, hereinafter abbreviated as IA) in the present invention includes reverse transcription (Reverse Transcription, hereinafter abbreviated as RT) and isothermal amplification.

RT-IA is divided into 2 components, namely Buffer premix and enzyme premix.

The Buffer premix contains 80mM Tris-HCl (pH 8.0), 110mM potassium acetate, 5mM DTT, 5mM ATP, 130mM creatine phosphate, 1mM dNTP, 50 ng/. Mu.L BSA, 8.3 (w/v)% PEG8000, 300mM betaine, 0.about.0.1 (w/v)% sodium alginate, 0.5. Mu.M isothermal amplification primer, 0.about.0.6. Mu.M enhancement primer, 0.2. Mu.M probe, 0.1. Mu.M random primer (6 nt), 0.1. Mu.M random primer (8 nt), 15mM magnesium acetate, specific primer sequences are detailed in Table 1.

TABLE 1 primer sequences

The enzyme premix contains 20U/. Mu.L reverse transcriptase, 500 ng/. Mu.L polymerase, 700 ng/. Mu.L recombinant protein, 885 ng/. Mu.L single-stranded binding protein, 200 ng/. Mu.L recombinant protein helper protein, 500 ng/. Mu.L creatine kinase, 1U/. Mu.L RNase inhibitor, 2U/. Mu. L Exonuclease III.

2. The specific detection flow is as follows

(1) Slow virus quality control materials packed with a part of nucleic acid fragments of influenza A (SEQ ID NO: 19) or B (SEQ ID NO: 20) are diluted to a concentration of 100 copies/. Mu.L in a 10-fold gradient by using a sample releasing agent (purchased from St. Hunan, cat. No. S1014), and the slow virus quality control materials are synthetically packaged and quantified by the complex hundred Australian biological medicine technology Co., ltd., and placed at room temperature (24-30 ℃) for 5 minutes to release nucleic acids, so that a lysate is obtained;

(2) mu.L of Buffer premix, 10. Mu.L of the lysate from step (1) and 10. Mu.L of enzyme premix were mixed in the same clean centrifuge tube or eight-tube with a final volume of 50. Mu.L using a pipette, and after mixing, fluorescence signals were collected using ABI7500, procedure as in Table 2:

TABLE 2 amplification procedure

Temperature (temperature)	Time	Cycle number	Collecting fluorescence
				37℃	1min	20	√

2. Condition exploration

1. Enhancement of primer influence

1.1 Effect of enhancing primer concentration

(1) Using sample releasing agent (purchased from Sanxiang organism, product number S1014) to dilute slow virus quality control product packed with influenza A virus H7N9 subtype partial nucleic acid fragment (SEQ ID NO: 18) or influenza B virus partial nucleic acid fragment (SEQ ID NO: 20) to a concentration of 100 copies/. Mu.L in a 10-fold gradient, taking diluted enzyme-free water as negative control, synthesizing and packaging the slow virus quality control product by complex hundred Australian biological medicine science and technology Co., ltd, quantifying, and standing for 5 minutes at room temperature (24-30 ℃) to release nucleic acid, thus obtaining a lysate;

(2) mu.L of Buffer premix containing 80mM Tris-HCl (pH 8.0), 110mM potassium acetate, 5mM DTT, 5mM ATP, 130mM creatine phosphate, 1mM dNTP, 50 ng/. Mu.L BSA, 8.3 (w/v)% PEG8000, 300mM betaine, 0.5. Mu.L isothermal amplification primer, 0 to 0.6. Mu.L of influenza A virus H7N9 subtype nucleic acid isothermal amplification enhancement primer R2 (or 0 to 0.6. Mu.M influenza B virus nucleic acid isothermal amplification enhancement primer), 0.2. Mu.M probe, 0.1. Mu.M random primer (6 nt), 0.1. Mu.M random primer (8 nt), 15mM magnesium acetate was used with a pipette tip, 10. Mu.L of the lysate of step (1) and 10. Mu.L of enzyme premix were mixed in the same clean 8-well with a final volume of 50. Mu.L, and fluorescent signals were collected using ABI7500 following the procedure.

TABLE 3 influence of enhanced primer concentration

As shown in Table 3 and FIG. 1, the results show that the amplification product signal of the RNA fragment of influenza virus increases with increasing concentration of the enhancement primer, and the enhancement effect is optimal when the concentration of the enhancement primer in the H7N9 subtype detection system reaches 0.3. Mu.M, and the enhancement effect is about 31% compared with the signal of the non-added group, and the enhancement effect is about 42% compared with the signal of the non-added group, and the detection signal is significantly improved (P < 0.05). When the concentration is higher than 0.5. Mu.M, the amplification efficiency is affected, possibly by magnesium ions in the competitive system of the reverse transcription process, which affects the isothermal amplification efficiency. Thus, the concentration of the enhanced primer for RNA isothermal amplification detection of influenza virus is in the range of 0 to 0.5. Mu.M, and most preferably 0.2 to 0.3. Mu.M.

1.2 Effect of the presence or absence of the enhancement primer on influenza B Virus detection

(1) The slow virus quality control product packed with the influenza B virus partial nucleic acid fragment (SEQ ID NO: 20) is diluted to a concentration of 10 copies/. Mu.L by a 10-fold gradient by using a sample releasing agent (purchased from St. Hunan organism, product number S1014), and the slow virus quality control product is synthesized and packed and quantified by Fubai biological medicine technology Co., ltd., and placed at room temperature (24-30 ℃) for 5 minutes to release nucleic acid, so as to obtain a lysate;

(2) 30. Mu.L of Buffer premix containing 80mM Tris-HCl (pH 8.0), 110mM potassium acetate, 5mM DTT, 5mM ATP, 130mM creatine phosphate, 1mM dNTP, 50 ng/. Mu.LBSA, 8.3 (w/v)% PEG8000, 300mM betaine, 0.05 (w/v)% sodium alginate, 0.5. Mu.M isothermal amplification primer, 0.3. Mu.M enhancement primer, 0.2. Mu.M probe, 0.1. Mu.M random primer (6 nt), 0.1. Mu.M random primer (8 nt), 15mM magnesium acetate was removed using a pipette gun, 10. Mu.L of the lysate of step (1) and 10. Mu.L enzyme premix were mixed in the same clean 8-piece tube to a final volume of 50. Mu.L, and after mixing, fluorescent signals were collected using ABI7500, the same procedure was used to verify if the enhancement primer had the same effect on isothermal amplification of other RNA viruses.

TABLE 4 Effect of enhancing primers on influenza B virus detection

Lentivirus (copy/. Mu.L)	Non-enhanced primer	0.3. Mu.M enhancement primer
			1000	281483	321589
100	165483	195680
			10	62159	82014
NTC	20147	23549

The results are shown in Table 4 and FIG. 2, and the results show that the detection signal added with 0.3 mu M enhancement primer is improved remarkably (P < 0.05), the detection signal of the low-copy target (10 copies/. Mu.L) is improved by about 32% compared with the detection result without adding the enhancement primer, and the enhancement primer is beneficial to the improvement of the isothermal amplification detection signal and the sensitivity of the low-concentration influenza B virus nucleic acid.

1.3 Effect of the presence or absence of the enhancer primer on influenza A Virus (non-typing) nucleic acid detection

(1) Downloading influenza A virus nucleic acid sequences on NCBI according to different subtypes, hosts, regions and the like, using DNAMAN sequence checking software to perform sequence comparison, designing isothermal amplification primers according to the conserved region sequences, and designing enhanced primers according to the downstream primer sequences, wherein the primer sequences are shown in Table 1;

(2) The slow virus quality control product packed with the non-parting influenza A virus partial conserved region nucleic acid fragment (SEQ ID NO: 19) is diluted to the concentration of 10 copies/. Mu.L by 10 times gradient by using a sample releasing agent (purchased from St. Hunan organism, product number S1014), the slow virus quality control product is synthesized and packed and quantified by complex hundred Australian biological medicine technology Co., ltd, and the slow virus quality control product is placed for 5 minutes at room temperature (24-30 ℃) to release nucleic acid, thus obtaining a lysate;

(3) 30. Mu.L of Buffer premix containing 80mM Tris-HCl (pH 8.0), 110mM potassium acetate, 5mM DTT, 5mM ATP, 130mM creatine phosphate, 1mM dNTP, 50 ng/. Mu.LBSA, 8.3 (w/v)% PEG8000, 300mM betaine, 0.05 (w/v)% sodium alginate, 0.5. Mu.M isothermal amplification primer, 0.3. Mu.M enhancement primer, 0.2. Mu.M probe, 0.1. Mu.M random primer (6 nt), 0.1. Mu.M random primer (8 nt), 15mM magnesium acetate was removed using a pipette, 10. Mu.L of the lysate of step (2) and 10. Mu.L enzyme premix were mixed in the same clean 8-piece tube, and after mixing, fluorescent signals were collected using ABI7500, the same procedure as in Table 2, to verify whether the enhancement primer for influenza A virus (non-typing) nucleic acid amplification had the same promoting effect.

TABLE 5 Effect of enhancing primers on influenza A Virus (non-typing) nucleic acid detection

Lentivirus (copy/. Mu.L)	Non-enhanced primer	0.3. Mu.M enhancement primer
			1000	215248	235602
100	132698	160083
			10	39874	46517
NTC	23689	20057

The results are shown in Table 5 and FIG. 3, the influenza A virus non-typing detection results show a slight increase in detection signal with the addition of 0.3. Mu.M enhancement primer compared to the detection results without the addition of enhancement primer, but the statistical analysis results were insignificant (P > 0.05), with about 16% increase in detection signal for the low copy target (10 copies/. Mu.L) and about 9% increase in detection signal for the high copy target (1000 copies/. Mu.L). The influenza A virus nucleic acid sequence has a plurality of mutation sites, a limited conserved region and more severe isothermal amplification primer design and amplification conditions, so that the design and screening of amplification primers and enhancement primers can be limited, but the enhancement primers can improve the detection signal of isothermal amplification of RNA virus nucleic acid by improving the reverse transcription efficiency, so that the detection signal or sensitivity of RNA virus with low concentration abundance can be improved by designing and adding the enhancement primers.

1.4 Effect of enhancing primer position

The low-temperature isothermal amplification of nucleic acid is a complex reaction process, isothermal amplification primers are longer, the design requirement is higher, the amplification efficiency is limited, and the amplification efficiency can be improved by enhancing the design and addition of the primers. The enhancement primer has the same partial sequence as the isothermal amplification downstream primer, but has a shorter sequence length than the amplification primer, does not participate in cDNA amplification, and does not compete with the isothermal amplification primer. The effect of the enhanced primer at different positions is different, and the test steps are as follows:

1.4.1 Effect of the enhanced primer position on isothermal amplification of influenza A subtype H7N9 and non-typed influenza A viruses

(1) The slow virus quality control product packed with influenza A virus H7N9 subtype partial nucleic acid fragment (SEQ ID NO: 18) is diluted to a concentration of 100 copies/. Mu.L by a 10-fold gradient by using a sample releasing agent (purchased from St. Hunan organisms and product number S1014), and the slow virus quality control product is synthesized and packed and quantified by Fubai biological medicine technology Co., ltd, and is placed at room temperature (24-30 ℃) for 5 minutes to release nucleic acid, so that a lysate is obtained;

(2) mu.L of Buffer premix containing 80mM Tris-HCl (pH 8.0), 110mM potassium acetate, 5mM DTT, 5mM ATP, 130mM creatine phosphate, 1mM dNTP, 50 ng/. Mu.L BSA, 8.3 (w/v)% PEG8000, 300mM betaine, 0.05 (w/v)% sodium alginate, 0.5. Mu.M isothermal amplification primer, 0.3. Mu.M enhancement primer (R1 to R4 primer positions see FIG. 4), 0.2. Mu.M probe, 0.1. Mu.M random primer (6 nt), 0.1. Mu.M random primer (8 nt), 15mM magnesium acetate was removed using a pipette, 10. Mu.L of the lysate of step (1) and 10. Mu.L of enzyme premix were again mixed in the same clean 8-well tube to a final volume of 50. Mu.L, and fluorescent signals were collected using ABI7500 after mixing, as in Table 2.

TABLE 6 influence of the position design of the enhanced primers on isothermal amplification

The results are shown in Table 6 and FIG. 5, and the position design of the enhanced primer has a certain effect on the improvement of isothermal amplification efficiency, and the result shows that the effect of enhancing the gain of the primer R2 is optimal for isothermal amplification of the H7N9 subtype, compared with the signal improvement of the non-added group by about 21%. The optimal enhancement primer for non-typed influenza a nucleic acid detection is R3, which increases the signal by about 40% compared to the signal of the non-added group. From the trend of the result, it can be deduced that the closer the enhancement primer is to the 3 'end of the downstream primer for isothermal amplification, the more the promotion effect is reduced or the promotion effect is possibly not generated, the sequence beyond the 5' end is not suitable, and the 5 'end of the enhancement primer is about 0-8 bases away from the 5' end of the downstream primer for isothermal amplification and the length is 18-22 nt.

2. Effect of sodium alginate on isothermal amplification

2.1 Effect of influenza A Virus subtype H7N9

(1) Downloading nucleic acid sequences on NCBI according to different hosts of influenza A virus H7N9 subtype, regions and other information, using DNAMAN sequence checking software to perform sequence comparison, designing isothermal amplification primers according to the conserved region sequence, and designing enhanced primers according to the downstream primer sequence, wherein the primer sequences are shown in Table 1;

(2) The slow virus quality control product packed with influenza A virus H7N9 subtype partial nucleic acid fragment (SEQ ID NO: 18) is diluted to a concentration of 100 copies/. Mu.L by a 10-fold gradient by using a sample releasing agent (purchased from St. Hunan organisms and product number S1014), and the slow virus quality control product is synthesized and packed and quantified by Fubai biological medicine technology Co., ltd, and is placed at room temperature (24-30 ℃) for 5 minutes to release nucleic acid, so that a lysate is obtained;

(3) mu.L of Buffer premix containing 80mM Tris-HCl (pH 8.0), 110mM potassium acetate, 5mM DTT, 5mM ATP, 130mM creatine phosphate, 1mM dNTP, 50 ng/. Mu.L BSA, 8.3 (w/V)% PEG8000, 300mM betaine, 0 to 0.1 (w/V)% sodium alginate, 0.5. Mu.M isothermal amplification primer, 0.3. Mu.M enhancement primer R2, 0.2. Mu.M probe, 0.1. Mu.M random primer (6 nt), 0.1. Mu.M random primer (8 nt), 15mM magnesium acetate, specific primer sequences are shown in Table, 10. Mu.L of lysate from step (2) and 10. Mu.L enzyme premix are taken in the same clean 8-way, the final volume is 50. Mu.L, fluorescent signals are collected using ABI7500 after mixing, the procedure as in Table 2, the effect of sodium alginate on influenza A virus H7N9 subtype amplification is shown in Table 7 and FIG. 6.

TABLE 7 Effect of sodium alginate on influenza A Virus subtype H7N9 amplification

Sodium alginate (%)	Fluorescence value
		0.00	357102
0.02	330639
		0.03	378776
0.05	430694
		0.06	418097
0.08	366528
		0.09	183833
0.10	66532

The effect test result of sodium alginate shows that when the concentration of sodium alginate is 0.03 (w/v)% -0.08 (w/v)%, the constant-temperature amplification is promoted to a certain extent, the detection signal (P is less than 0.05), and the optimal value is 0.05 (w/v)%, compared with the signal of a non-added group, the concentration is about 20%, when the concentration exceeds 0.08 (w/v)%, the serious inhibition effect is generated, the amplification reaction efficiency is reduced, and the product yield is greatly reduced.

2.2 Effect of sodium alginate on detection of B-stream Gene

(1) Designing isothermal amplification primers according to the sequence of the conserved region of the influenza B virus nucleic acid, and designing enhanced primers according to the sequence of the downstream primers, wherein the sequences of the primers are shown in Table 1;

(2) The slow virus quality control product packed with the influenza B virus partial nucleic acid fragment (SEQ ID NO: 20) is diluted to a concentration of 100 copies/. Mu.L by a 10-fold gradient by using a sample releasing agent (purchased from St. Hunan organism, product number S1014), and the slow virus quality control product is synthesized and packed and quantified by Fubai biological medicine technology Co., ltd., and placed at room temperature (24-30 ℃) for 5 minutes to release nucleic acid, so as to obtain a lysate;

(3) mu.L of Buffer premix containing 80mM Tris-HCl (pH 8.0), 110mM potassium acetate, 5mM DTT, 5mM ATP, 130mM creatine phosphate, 1mM dNTP, 50 ne/. Mu.LBSA, 8.3 (w/v)% PEG8000, 300mM betaine, 0 to 0.1% (w/v) sodium alginate, 0.5. Mu.M isothermal amplification primer, 0.3. Mu.M enhancement primer, 0.2. Mu.M probe, 0.1. Mu.M random primer (6 nt), 0.1. Mu.M random primer (8 nt), 15mM magnesium acetate, specific primer sequences were tabulated, 10. Mu.L of the cleavage products of 2 and 10. Mu.L of enzyme premix were mixed in the same clean 8-way tube to a final volume of 50. Mu.L, and after mixing, fluorescent signals were collected using ABI7500, the procedure are as in Table 8 and FIG. 7.

TABLE 8 Effect of sodium alginate on B-stream Gene amplification

Sodium alginate (%)	Fluorescence value
		0.00	183687
0.02	203698
		0.03	215873
0.05	240369
		0.06	235871
0.08	201587
		0.09	165332
0.10	90024

The effect of sodium alginate on the amplification of the B-stream gene shows that when the concentration of sodium alginate is lower than 0.08 (w/v)%, the isothermal amplification efficiency of the B-stream gene can be obviously improved (P is smaller than 0.05), and the optimal value is 0.05 (w/v)%, compared with the signal of a non-added group, the effect of the sodium alginate on the amplification of the B-stream gene is improved by about 30.8%, and when the concentration exceeds 0.08 (w/v)%, the serious inhibition effect can occur, the amplification reaction efficiency is reduced, and the product yield is greatly reduced. Therefore, in the isothermal amplification detection system for influenza virus nucleic acid, when the concentration of sodium alginate in the isothermal amplification system is lower than 0.08 (w/v)%, the amplification efficiency can be improved, the detection performance is improved, and the optimal concentration is 0.05 (w/v)%.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. The isothermal amplification reagent is characterized by comprising Buffer premix, isothermal amplification substrate, enzyme premix and isothermal amplification primers;

the isothermal amplification primer comprises: isothermal amplification upstream primers, isothermal amplification downstream primers, amplification primers and/or random primers;

the concentration of the enhancement primer is less than or equal to 0.5 mu M; the 5 'end of the enhanced primer is 0-8 bases away from the 5' end of the isothermal amplification downstream amplification primer, and the length of the enhanced primer is 9-11 nt subtracted from the length of the isothermal amplification downstream primer.

2. The isothermal amplification reagent according to claim 1, wherein the concentration of the enhancing primer is 0.3 μm.

3. The isothermal amplification reagent according to claim 1 or 2, wherein the isothermal amplification primer further comprises a probe.

4. A isothermal amplification reagent according to any one of claims 1 to 3, wherein the isothermal amplification primers comprise at least one of the following I to III):

i) A isothermal amplification upstream primer of the nucleotide sequence shown as SEQ ID NO. 1, a isothermal amplification downstream primer of the nucleotide sequence shown as SEQ ID NO. 2, an enhanced primer of the nucleotide sequence shown as SEQ ID NO. 7, and a probe of the nucleotide sequence shown as SEQ ID NO. 10;

II), a isothermal amplification upstream primer of the nucleotide sequence shown as SEQ ID NO. 3, a isothermal amplification downstream primer of the nucleotide sequence shown as SEQ ID NO. 4, an enhanced primer of the nucleotide sequence shown as SEQ ID NO. 8, and a probe of the nucleotide sequence shown as SEQ ID NO. 11;

III), a isothermal amplification upstream primer of a nucleotide sequence shown as SEQ ID NO. 5 and a isothermal amplification downstream primer of a nucleotide sequence shown as SEQ ID NO. 6; an enhanced primer of the nucleotide sequence shown as SEQ ID NO. 9, and a probe of the nucleotide sequence shown as SEQ ID NO. 12.

5. The isothermal amplification reagent according to any one of claims 1 to 4, wherein the isothermal amplification reagent further comprises sodium alginate, and the concentration of sodium alginate is 0.02 (w/v)% to 0.08 (w/v)%.

6. The isothermal amplification reagent according to claim 5, wherein the concentration of sodium alginate is 0.05 (w/v)%.

7. The isothermal amplification reagent according to claim 1, wherein the random primer comprises a random primer of 6nt in length and a random primer of 8nt in length.

8. A isothermal amplification reagent according to claim 1, wherein,

the buffer premix comprises buffer solution, potassium ions, magnesium ions, a reducing agent, a stabilizing agent, betaine and an amplification enhancer;

the isothermal amplification substrate comprises ATP, creatine phosphate and dNTPs;

the enzyme premix comprises reverse transcriptase, polymerase, recombinase protein, single-chain binding protein, recombinase protein auxiliary protein, helicase, creatine kinase, RNase inhibitor and Exoneclease III.

9. The isothermal amplification reagent according to claim 1, wherein in the buffer premix,

the potassium ion is provided by potassium acetate;

the magnesium ions are provided by magnesium acetate;

the reducing agent comprises DTT;

the stabilizer comprises BSA;

the amplification enhancer comprises DMSO and/or PEG8000.

10. Use of a isothermal amplification reagent according to any one of claims 1 to 9 for influenza virus detection or for the preparation of an influenza detection product.