CN115807128B

CN115807128B - Nucleic acid combination, kit and detection method for detecting respiratory tract pathogens

Info

Publication number: CN115807128B
Application number: CN202211312244.4A
Authority: CN
Inventors: 高方鸣; 张云云; 魏宁
Original assignee: SANGON BIOTECH (SHANGHAI) CO Ltd
Current assignee: SANGON BIOTECH (SHANGHAI) CO Ltd
Priority date: 2022-10-25
Filing date: 2022-10-25
Publication date: 2024-03-01
Anticipated expiration: 2042-10-25
Also published as: CN115807128A

Abstract

The invention discloses a nucleic acid combination, a kit and a detection method for detecting respiratory pathogens, wherein the respiratory pathogens comprise novel coronaviruses, influenza A viruses and influenza B viruses; the nucleic acid combination comprises a primer pair and a probe for amplifying an N gene fragment of the novel coronavirus, an ORF gene fragment of the novel coronavirus, an M1 gene fragment of the influenza A virus and an NS1 gene fragment of the influenza B virus. The primer pair and the probe can detect three viruses simultaneously in the same reaction tube, and the detection specificity and the sensitivity are high because of no cross reaction among various respiratory tract pathogenic microorganisms. Meanwhile, the invention combines the RPA technology and the latex immunochromatography to obtain a rapid detection method for detecting novel coronaviruses, influenza A viruses and influenza B viruses, thereby realizing direct detection from a sample to a result, further simplifying the operation flow and improving the detection efficiency.

Description

Nucleic acid combination, kit and detection method for detecting respiratory tract pathogens

Technical Field

The invention relates to the technical field of biological detection, in particular to a nucleic acid combination, a kit and a detection method for detecting respiratory tract pathogens.

Background

At present, the nucleic acid detection technology mainly comprises Polymerase Chain Reaction (PCR), real-time fluorescence quantitative PCR (q-PCR), isothermal amplification and the like, and has wide application in the fields of food safety, clinical disease diagnosis, animal and plant quarantine and the like. However, the PCR and q-PCR methods have high temperature requirements, and require accurate and rapid temperature rise and reduction, and the amplification time is more than 1 hour, and can be completed only by specific experimenters. The prior art relies on the readout of nucleic acid electrophoresis and the fluorescent signal acquisition by a developing instrument or an amplifying instrument, so that the nucleic acid detection is limited in a laboratory, and the application scene of the nucleic acid detection cannot be generalized, such as port fast food safety detection, family disease detection and the like. The isothermal amplification RPA method can realize rapid amplification within 8-15min, and no kit for simultaneously detecting novel coronavirus, influenza A virus and influenza B virus nucleic acid by directly adopting the multiplex RPA method is currently available on the market.

The latex immunochromatographic test strip can rapidly display a nucleic acid analysis result, after a sample solution is dripped into a sample pad, the sample solution moves to the other side of the test strip due to capillary action, when a component combined with an antibody coated on a T line on an NC film exists in the sample solution, the component is captured, and the result is judged by naked eyes through whether color is developed or not by the color microspheres. The prior art does not have a latex immunochromatographic test strip suitable for reading multiple isothermal amplification products.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a nucleic acid combination, a kit and a detection method for detecting respiratory tract pathogens, which are used for solving the problems that the existing method for detecting respiratory tract pathogens depends on a large instrument and is inconvenient to detect.

The invention is realized in the following way:

in a first aspect, the present invention provides a nucleic acid combination for detecting respiratory pathogens including novel coronaviruses, influenza a viruses and influenza b viruses; the above nucleic acid combination includes a primer pair and a probe;

wherein, the nucleotide sequence of the primer pair for amplifying the N gene fragment of the novel coronavirus is shown as SEQ ID NO:3-4, the nucleotide sequence of the probe is shown as SEQ ID NO: shown in figure 7;

the nucleotide sequence of the primer pair for amplifying the ORF gene of the novel coronavirus is shown as SEQ ID NO:8 and SEQ ID NO:12, the nucleotide sequence of the probe is shown as SEQ ID NO: 14;

the nucleotide sequence of the primer pair for amplifying the M1 gene fragment of the influenza A virus is shown as SEQ ID NO:15 and SEQ ID NO:20, the nucleotide sequence of the probe is shown as SEQ ID NO: 21;

the nucleotide sequence of the primer pair for amplifying the NS1 gene fragment of the influenza B virus is shown as SEQ ID NO:23 and SEQ ID NO:25, the nucleotide sequence of the probe is shown as SEQ ID NO: 28.

The nucleic acid combination for detecting the respiratory tract pathogenic microorganisms comprises a primer and a probe which are designed by taking a novel coronavirus N gene fragment (total 198 bp) and an ORF gene fragment (total 161 bp) as detection targets, a primer and a probe which are designed by taking an influenza A virus M1 gene fragment (total 180 bp) as detection targets, and an influenza B virus NS1 gene fragment (total 166 bp) as detection targets. When designing primers and probes for detecting novel coronaviruses, influenza A viruses and influenza B viruses, pathogenic gene fragments with high transcription efficiency are required to be found out for design, so that the primers and probes designed in the gene fragments can have a good amplification effect; also, since the amplification primers of the present invention are used for multiplex PCR amplification, it is necessary to amplify a plurality of targets simultaneously in one reaction tube, and thus amplification of one target tends to affect amplification of another target, the amplification efficiency is not simply mixing all primers and templates in the same reaction tube, but by optimizing the primer design and reaction conditions. Therefore, the inventor of the invention screens and obtains the amplification primer pair and the probe after creative labor, and the amplification primer pair and the probe have the characteristics of good specificity and high amplification efficiency.

In some embodiments, the above nucleic acid combinations are used in RT-RPA to amplify nucleic acids of respiratory pathogens.

Recombinase polymerase amplification (Recombinase Polymerase Amplification, RPA) is a novel isothermal amplification technique in which a pair of primers specifically bind to a template homologous region to form a recombinase primer complex, thereby realizing strand displacement and extension under the action of DNA polymerase. The technology can realize amplification of target products under a certain temperature condition so as to achieve the detection purpose, does not need to undergo a thermal cycle process, greatly simplifies the requirements on large-scale instruments, shortens the reaction time, is simple and convenient to operate, and can meet the requirements of site emergency on rapid diagnosis of pathogens.

The primer sequence length of the invention aiming at RT-RPA is 28-32nt, and because the primer is longer, mismatch and secondary structure are easy to generate, thus reducing the amplification efficiency. Therefore, under the condition that the primer of the invention meets the conditions, the primer design and the reaction conditions are optimized, and the inventor of the invention screens after creative labor to obtain the amplification primer pair and the probe which can specifically amplify and avoid the interference of other primers.

In some embodiments, the 5 'end of the probe in the nucleic acid combination is marked with fluorescein or small molecule antigen, the middle position of the probe is marked with tetrahydrofuran, and the 3' end is marked with a middle arm group; the upstream primer in the nucleic acid combination is unlabeled and the downstream primer is labeled with biotin or fluorescein.

In some embodiments, the fluorescein labeled probe includes FAM/FITC, AF488, TAMRA, and Cy5.

In some embodiments, the small molecule antigen comprises Dig, folic acid, glycocholic acid, cholesterol, estradiol, progesterone, penicillin, tetracycline.

In some embodiments, the tetrahydrofuran is labeled at 30bp from the 5' end of the probe, and the Spacer group is C3 Spacer.

The tetrahydrofuran modified site is introduced into the probe, and the tetrahydrofuran modified nucleotide can realize the DNA chain extension with almost 100% efficiency under the action of DNA polymerase.

In some embodiments, the fluorescein that labels the downstream primer is different from the fluorescein that labels the probe.

In a second aspect, the invention provides a kit for detecting nucleic acid of a respiratory pathogenic microorganism comprising a nucleic acid detection test strip and an amplification reagent comprising a nucleic acid combination as described above for detecting a respiratory pathogenic microorganism.

In order to simplify the detection operation flow, the invention designs a latex immunochromatography test strip aiming at the respiratory tract pathogenic microorganisms, and the test strip has high sensitivity, strong stability and clear color development, and can rapidly detect various viruses within 10 minutes.

In some embodiments, a nucleic acid detection test strip includes a base plate, and a sample pad, a label-binding pad, and a chromatographic reaction membrane disposed on the base plate.

In some embodiments, the label-binding pad is adsorbed with a specific complex obtained by coupling the latex microsphere to one or more of SA, chicken IgY, folic acid, glycocholic acid.

In some embodiments, the latex microspheres include red, blue, and violet.

In some embodiments, the chromatographic reaction membrane is provided with a detection line containing one or more of an anti-FAM antibody, an anti-DIG antibody, an anti-AF 488 antibody, an anti-CY 5 antibody, an anti-folate antibody, or an anti-glycocholic acid antibody.

In some embodiments, the concentration of antibody in the detection line is 0.25-0.5mg/mL.

In some embodiments, a quality control line is further disposed on the chromatographic reaction membrane, wherein the quality control line contains one or more of an anti-chicken IgY antibody, biotin, or an anti-SA antibody.

In some embodiments, the concentration of antibody in the quality control line is 0.25-0.5mg/mL.

In some embodiments, the amplification reagents contain both a primer pair and probe for the N gene fragment of the novel coronavirus, the ORF gene of the novel coronavirus, the M1 gene fragment of the influenza a virus, and the NS1 gene fragment of the influenza b virus.

In some embodiments, the concentration ratio of the primer pair and probe of the N gene fragment of the novel coronavirus, the primer pair and probe of the ORF gene fragment of the novel coronavirus, the primer pair and probe of the M1 gene fragment of the influenza a virus, and the primer pair and probe of the NS1 gene fragment of the influenza b virus in the amplification reagents is 1:1:1:1 to 1:2:2:2. More preferably, the concentration ratio of the primer pair and the probe of the above gene or gene fragment is 6:9:9:10.

in some embodiments, the molar ratio of the N gene fragment of the novel coronavirus, the ORF gene of the novel coronavirus, the M1 gene fragment of the influenza a virus, and the NS1 gene fragment of the influenza b virus in the amplification reagents is 2:2:0.6-2:2:1.

in some embodiments, the amplification reagents include a buffer and a magnesium acetate solution.

In a third aspect, the present invention also provides a method for detecting nucleic acid of a respiratory pathogenic microorganism, comprising: and adding the mixed solution containing the sample into the amplification reagent to perform amplification reaction, diluting the amplification solution after the reaction is finished, taking out part of diluted solution, adding the diluted solution onto a sample pad of the nucleic acid detection test strip, detecting, and reading a result.

In some embodiments, the time of the amplification reaction is 10-20min and the temperature is 37-42 ℃.

In some embodiments, the dilution factor of the amplification solution is 5-10.

In some embodiments, the volume of the partially diluted solution is 40-80 μl.

In some embodiments, the detection time is 10-20 minutes.

In some embodiments, the 50 μl reaction system of the amplification reaction is: 2.5. Mu.L of magnesium acetate solution, 2. Mu.L of 10. Mu.M upstream and downstream primer, 0.6. Mu.L of probe, 2. Mu.L of template, and buffer to 50. Mu.L.

The invention has the following beneficial effects:

(1) The primer probe composition for detecting or assisting in detecting the novel coronaviruses, the influenza A viruses and the influenza B viruses can detect three viruses simultaneously in the same reaction tube, and has no cross reaction among various respiratory tract pathogenic microorganisms during detection, and the detection specificity and the sensitivity are high;

(2) The latex immunochromatography test strip is optimized, has high sensitivity, strong stability and clear color development, and can rapidly detect various viruses within 10 minutes;

(3) The invention combines RPA technology and latex immunochromatography to obtain a rapid detection method for detecting novel coronaviruses, influenza A viruses and influenza B viruses, realizes direct detection from a sample to a result, further simplifies the operation flow and improves the detection efficiency;

(4) The invention gets rid of the requirements of the amplification experiment on instruments and operators, and widens the application field.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of the operation in example 3;

FIG. 2 is a graph of the detection result in example 3;

FIG. 3 shows the results of the detection of different reaction times of the multiplex RT-RPA amplification system of experimental example 5;

FIG. 4 shows the results of the detection of different reaction temperatures of the multiplex RT-RPA amplification system of Experimental example 5;

FIG. 5 shows the detection results of the detection limit of the N gene and the ORF gene in Experimental example 6;

FIG. 6 shows the detection results of single target detection limit of M1 gene in experimental example 6;

FIG. 7 shows the detection results of single target detection limit of NS1 gene in experimental example 6;

FIG. 8 shows the results of detecting the concentration ratios of the primer probes of the N gene, the ORF gene, the M1 gene and the NS1 gene in Experimental example 5;

FIG. 9 shows the detection results of multiple RPA systems in Experimental example 6 on detection limits of three viruses of New crown, A stream and B stream;

FIG. 10 shows the result of the repeatability test in Experimental example 7;

FIG. 11 shows the result of the specific assay in Experimental example 8.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The pseudovirus standards used for detecting novel coronaviruses, influenza A viruses and influenza B viruses in the following examples are all from biological medicine technologies, inc. of Kao (Suzhou) (product numbers: FNRV2593, FNRV2840 and FNRV2839 in order).

Example 1

This example is a nucleic acid combination for detecting respiratory pathogenic microorganisms

Downloading genome sequences of novel coronaviruses SARS-CoV-2, influenza A virus FluA and influenza B virus FluB in a genbank database, carrying out sequence comparison by DNAMAN software, and selecting a conserved region of the viruses for primer design. The RPA primer probe sequences designed in this example are shown in Table 1.

TABLE 1

None of the upstream primers in the above sequence was labeled, none of the downstream primers was labeled 5 'with Biotin, the N gene probe was labeled 5' with FAM, the ORF gene probe was labeled 5 'with AF488, the M1 gene probe was labeled 5' with Dig, the NS1 gene probe was labeled 5 'with Cy5, the 31 st position of all probes was modified with Tetrahydrofuran (THF), and the 3' was blocked with a blocking group C3 to block probe extension.

Example 2

The embodiment provides a kit for detecting nucleic acid of respiratory pathogenic microorganisms, which comprises a nucleic acid detection test strip and an amplification reagent.

The amplification reagent includes a nucleic acid combination for detecting a respiratory tract pathogenic microorganism in example 1, a buffer solution, and a magnesium acetate solution.

Wherein, the concentration ratio of the primer pair and the probe of the N gene fragment of the novel coronavirus, the primer pair and the probe of the ORF gene fragment of the novel coronavirus, the primer pair and the probe of the M1 gene fragment of the influenza A virus and the primer pair and the probe of the NS1 gene fragment of the influenza B virus in the amplification reagent is 6:9:9:10; the molar ratio of the N gene fragment of the novel coronavirus, the ORF gene of the novel coronavirus, the M1 gene fragment of the influenza A virus and the NS1 gene fragment of the influenza B virus in the amplification reagent is 10:10:3.

the nucleic acid detection test strip comprises a bottom plate, and a sample pad, a marker binding pad, a chromatographic reaction membrane and absorbent paper are arranged on the bottom plate. Wherein, the label binding pad is adsorbed with a specific compound which is mainly obtained by coupling latex microspheres with SA, chicken IgY, folic acid, glycocholic acid and the like. The preparation method of the detection test strip comprises the following steps:

1. activation of latex microspheres and labeling of couplets

(1) Preparation of label activation buffer (1L): 10-50mM GOODs buffer, ph=5.5-6.5.

(2) Preparation of label coupling buffer (1L): 10-50mM GOODs buffer, ph=5.5-6.5.

(3) Preparation of label-preserving buffer (1L): 10-50mM boric acid-borax buffer solution, 5-20g sucrose, 1-10g casein, 1-10g PVPk40, 1mL Proclin-300, and pH=7.0-8.0.

(4) To the activation vessel, 0.9mL of purified water was added and labeled, indicating the product name, lot number, throughput, date of manufacture.

(5) 100. Mu.L of microspheres were added to the activation vessel, mixed well, ultracentrifuged at 20000rpm/min for 10 minutes on an ultracentrifuge, and the supernatant was discarded.

(6) 1mL of the labeled activation buffer was added to the precipitate, dispersed by sonication, and stirred until completely mixed.

(7) Adding 0.1-1.0mg NHS into the activation container, and stirring to completely mix. Immediately adding 0.1-0.5mg EDC, and stirring to completely mix. The reaction was carried out for 10-20 minutes while stirring, and after the completion of the reaction, the reaction was ultracentrifuged for 10 minutes at 20000rpm/min in an ultracentrifuge, and the supernatant was discarded.

(8) 1mL of the labeled coupling buffer was added to the precipitate, dispersed by sonication, and stirred until completely mixed.

(9) Adding 0.1-0.5mg of the marker into the activated microsphere solution, stirring until the mixture is completely mixed, and performing ultrasonic dispersion for 1-2 minutes. Then the reaction is carried out for 1 to 3 hours while stirring.

(10) After the coupling reaction time is over, 10-100 mu L of 10% BSA solution is added into the solution, fully mixed and sonicated for 1 minute, and then the reaction is carried out for more than 2 hours under stirring.

(11) After the completion of the blocking reaction time, the reaction mixture was ultracentrifuged at 20000rpm/min for 10 minutes in an ultracentrifuge, and the supernatant was discarded. Then 1mL of the marked preservation buffer is added, dispersed by ultrasonic, and stirred until the mixture is completely mixed. Placing in 2-8deg.C environment for preservation.

2. Preparation of sample pad

Sample pad treatment fluid composition included (1 l, ph=7.0-8.0): 10-50mM phosphate buffer, 10-50g sucrose, 10-50g sodium chloride, 0.1-2g casein, 1-10mL Tween 20, 1-10mL triton-100, 1mL Proclin-300. The sample pad is cut into 1.5cm multiplied by 30cm, soaked in sample pad treating liquid for 1min, taken out and spread on a drying net, and dried in a constant temperature drying oven for more than 12 h (45 ℃).

3. Preparation of bond pads

The composition of the conjugate pad treatment fluid included (1 l, ph=7.0-8.0): 10-50mM phosphate buffer, 10-50g sucrose, 0.1-2g casein, 0.1-2g PVPk40, 0.1-2g BSA, 1mL Proclin-300. Cutting the blank bonding pad into strips of 30cm×1.1cm, soaking in bonding pad treatment solution for 1min, spreading on a drying net, and drying in a constant temperature drying oven for more than 12 hr (45deg.C).

4. Assembling and cutting test paper strip

Tearing the film on the backlight plate, and pasting NC film, bonding pad, sample pad, and absorbent paper, wherein the adjacent parts are overlapped by 1-2 mm. The strips were cut with a cutter and the test strips were 3mm wide.

Example 3

The embodiment provides a method for detecting nucleic acid of respiratory pathogenic microorganism, which has the following operation steps:

(1) After the throat swab is sampled, placing the cotton swab into a sample treatment liquid, and uniformly shaking for 1min;

(2) Adding 50 mu L of sample treatment solution into an amplification reagent tube, and heating at a constant temperature of 40 ℃ for 15min;

(3) The amplified solution was diluted 10 times, 50. Mu.L of the diluted solution was applied to a sample pad of a latex immunochromatographic test strip, and the sample was left to stand for 10 minutes to read the result.

Wherein, the reaction system in the step (2) is as follows: 2.5. Mu.L of magnesium acetate solution, 2. Mu.L of upstream and downstream primer concentration (10. Mu.M), 0.6. Mu.L of probe (10. Mu.M), 2. Mu.L of template (100 copies/. Mu.L) and 50. Mu.L of buffer were added.

The results are shown in FIG. 2. In fig. 2, the detection results of negative influenza a virus, influenza b virus, novel coronavirus n+orf1ab, influenza a virus+influenza b virus+novel coronavirus n+novel coronavirus orf1ab are shown in order from left to right.

Comparative example

Different primers were designed for the target sequences selected in example 1, the corresponding primer sequences are shown in table 2:

table 2 primer sequences for comparison

None of the upstream primers in the above sequences was labeled, and 5' of the downstream primers was labeled with Biotin.

Experimental example 1

The 3 upstream primers and the 3 downstream primers of the novel coronavirus N gene in the example 1 and the comparative example are respectively combined to obtain 9 pairs of primer pairs, the primer pairs are combined with probes, an RPA amplification reagent is utilized, a pseudoviral nucleic acid standard of the novel coronavirus is used as a template, negative reaction of each primer probe combination is free of false positive, and positive reaction strip depth is used as a judgment basis. The experimental results are shown in table 3.

Wherein, the reaction system of the reaction is as follows: 2.5. Mu.L of magnesium acetate solution, 2. Mu.L of upstream and downstream primer concentration (10. Mu.M), 0.6. Mu.L of probe (10. Mu.M), 2. Mu.L of template (100 copies/. Mu.L), and reaction buffer supplemented to 50. Mu.L, the reaction temperature was 40℃and the reaction time was 15min. After the reaction, the amplified product was diluted 10 times, 50. Mu.L of the diluted product was applied to a sample pad of a latex immunochromatographic test strip, and the reaction was left for 10 minutes to observe the presence or absence of a band and the color depth of the band.

TABLE 3 detection results of primer probe combinations for N genes

Note that: "-" indicates no band, "+" indicates a band of weaker color, "++" indicates that the color of the strip is moderate, "+++" indicates a strip the color is very dark.

As can be seen from the detection results of Table 3, the detection specificity and sensitivity of the upper and lower primers used in the present invention are better than those of the other upper and lower primer combinations.

Experimental example 2

The 3 upstream primers and 3 downstream primers of the novel coronavirus ORF genes in example 1 and comparative example were combined to obtain 9 pairs of primer pairs, and the combination of the primer pairs and the probe was detected by the method in experimental example 1. The experimental results are shown in table 4.

TABLE 4 detection results of ORF Gene primer probe combinations

Primer probe combination	Negative of	Positive and negative
			O-1F1R	-	+
O-1F2R (example 1)	-	+++
			O-1F3R	-	+
O-2F1R	+	+++
			O-2F2R	+	+++
O-2F3R	+	+++
			O-3F1R	+	+++
O-3F2R	+	+++
			O-3F3R	+	+++

As can be seen from the detection results of Table 4, the detection specificity and sensitivity of the upper and lower primers used in the present invention are better than those of the other upper and lower primer combinations.

Experimental example 3

The 3 upstream primers and 3 downstream primers of the M1 gene of the first stream in example 1 and comparative example were combined to obtain 9 pairs of primer pairs, and the combination of the primer pairs and the probe was detected by the method in experimental example 1. The experimental results are shown in table 5.

TABLE 5M1 Gene primer probe combination detection results

Primer probe combination	Negative of	Positive and negative
			M-1F1R	+	+++
M-1F2R	-	++
			M-1F3R (example 1)	-	+++
M-2F1R	+	+++
			M-2F2R	+	+++
M-2F3R	+	+++
			M-3F1R	+	+++
M-3F2R	+	+++
			M-3F3R	+	+++

As can be seen from the detection results of Table 5, the detection specificity and sensitivity of the upper and lower primers used in the present invention are better than those of the other upper and lower primer combinations.

Experimental example 4

The 3 upstream primers and 3 downstream primers of the B-stream NS1 gene of example 1 and comparative example were combined to obtain 9 pairs of primer pairs, and the combination of the primer pairs and the probe was detected by the method of experimental example 1. The experimental results are shown in table 6.

TABLE 6NS1 Gene primer probe combination detection results

As can be seen from the detection results in Table 6, the detection specificity and sensitivity of the upper and lower primers used in the present invention are better than those of the other upper and lower primer combinations.

Experimental example 5

The nucleic acid combination in example 1 was used to compare the amplification effects of a multiplex RT-RPA amplification system under different reaction conditions using novel corona, A-stream, B-stream pseudoviral RNA (concentrations 100 copies/. Mu.L) as templates.

(1) The concentration ratio of the primers is different

Amplification was performed at the primer concentration ratios shown in Table 7, and the amplification method was the same as in example 3.

TABLE 7 different concentration ratios of multiplex RPA primers

Detection result: amplification was performed using a primer probe at a 1:1:1:1 concentration (combination 1), and the test strip detection found that the bands of the N gene were brightest, and the bands of the other 3 genes were weaker (as shown in FIG. 8). When the concentration ratio of combination 2 is adopted, no N gene band exists, other 3 gene bands are clearly visible, but the NS1 gene band is the weakest. Comparison of combinations 3, 4, and 5 revealed that combination 3 (i.e., N: ORF: M1: NS1 primer probe ratio 6:9:9:10, concentration ratio employed in example 3) was the optimal concentration ratio for the quadruple RPA amplification reaction.

(2) Different reaction times

To verify the effect on the quadruple RPA amplification reaction at reaction times of 5, 10, 15 and 20min, the assay method of example 3 was used. The detection results are shown in FIG. 3.

As can be seen from FIG. 3, the RPA amplification product bands were not visible or were darker and the amount of product was smaller when the reaction time was 5 and 10 min; the band becomes deeper gradually with the extension of the reaction time, and the band becomes significantly deeper when the reaction time is 15min, and the color of the band is slightly enhanced when the reaction time is 20min, preferably the reaction time is 15min.

(3) Different reaction temperatures

To verify the effect on the quadruple RPA amplification reaction at reaction temperatures of 37, 38, 39, 40, 41 and 42 ℃, the assay method of example 3 was used. The detection results are shown in FIG. 4.

As can be seen from fig. 4, the RPA amplification products were darker in bands and less in product amounts at the reaction temperatures of 37 and 38 ℃; the bands are deeper and increase with temperature when the reaction temperature is 39, 40, 41 ℃; when the temperature reached 42 ℃, the band was instead weakened, thus determining the reaction temperature to 40 ℃, allowing for an error of 1 ℃ up and down.

Experimental example 6

The kit of example 2 was subjected to a sensitivity test, and the specific procedure was as follows:

(1) Diluting 100 copies/mu L of novel coronavirus RNA to obtain 50, 10, 5, 2, 1 and 0 copies/mu L of RNA diluent, adding 1 mu L of RNA diluent for each reaction, amplifying for 15min at the temperature of 40 ℃ by using the optimal primer probe combination of N genes and ORF genes, and testing the single target detection limit of the N genes. The results are shown in FIG. 5.

(2) 100 copies/. Mu.L of the A-flow pseudovirus RNA is diluted to obtain 50, 10, 5, 2, 1 and 0 copies/. Mu.L of RNA diluent, 1 mu.L of RNA diluent is added to each reaction, the optimal primer probe combination of the M1 gene is used for amplification for 15min at the temperature of 40 ℃, and the single target detection limit of the M1 gene is tested. The results are shown in FIG. 6.

(3) 100 copies/mu L of Eyew virus RNA is diluted to obtain 50, 10, 5, 2, 1 and 0 copies/mu L of RNA diluent, 1 mu L of RNA diluent is added to each reaction, the optimal primer probe combination of the NS1 gene is used for amplification for 15min at the temperature of 40 ℃, and the single target detection limit of the NS1 gene is tested. The results are shown in FIG. 7.

(4) 100 copies/. Mu.L of novel coronavirus RNA, A-stream RNA and B-stream RNA are diluted to obtain 50, 10, 5, 2, 1 and 0 copies/. Mu.L of RNA diluent, 1 mu.L of RNA diluent is added to each reaction, the mixture is amplified for 15 minutes at the temperature of 40 ℃ by using the optimal primer probe combination, and the detection limit is tested. As a result, FIG. 9 shows that the detection limits of the amplification system for the new crown, the first stream and the second stream are 1 copy/reaction, respectively.

Experimental example 7

The kit of example 2 was subjected to a reproducibility test, and the specific procedure is as follows:

the repeatability test is carried out by using the optimal primer probe combination, and each 100copies of new coronal, A-stream and B-stream pseudovirus RNA is reacted at the temperature of 40 ℃ for 15min. The results are shown in FIG. 10, demonstrating that the kit has good reproducibility.

Experimental example 8

The kit of example 2 was subjected to a specificity test. The novel coronavirus is known to have high similarity with HcoV-229E, hcoV-OC43, hcoV-NL63, hcoV-HK-Mm L, SARS-coV and MERS-coV genes, so that detection comparison is performed. In addition, the presence of non-specific bands was detected as compared to influenza a, b and respiratory syncytial virus. The specific operation steps are as follows:

according to a single target system, 100copies of new crown, hcov-229E, hcoV-OC43, hcov-NL63, hcov-HK m L, SARS-coV, MERS-coV, a flow A, a flow B and pseudoviral RNA of respiratory syncytial virus are sequentially added into each reaction, the temperature is 40 ℃, and the amplification is carried out for 15 minutes, and a test strip is used for testing. The detection result is shown in FIG. 11, which shows that the primer probe designed in the kit has specificity.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A nucleic acid composition for detecting respiratory pathogens, wherein the respiratory pathogens are novel coronaviruses, influenza a viruses, and influenza b viruses; the nucleic acid composition comprises a primer pair and a probe;

the nucleotide sequence of the primer pair for amplifying the N gene fragment of the novel coronavirus is shown as SEQ ID NO:3-4, the nucleotide sequence of the probe is shown as SEQ ID NO: shown in figure 7;

the nucleotide sequence of the primer pair for amplifying the NS1 gene fragment of the influenza B virus is shown as SEQ ID NO:23 and SEQ ID NO:25, the nucleotide sequence of the probe is shown as SEQ ID NO: 28; the nucleic acid composition is used in RT-RPA to amplify nucleic acids of the respiratory tract pathogen.

2. The nucleic acid composition for detecting respiratory pathogens according to claim 1, wherein the 5 'end of the probe in the nucleic acid composition is labeled with fluorescein or a small molecule antigen, the middle position of the probe is labeled with tetrahydrofuran, and the 3' end is labeled with a spacer group; the upstream primer in the nucleic acid composition is unlabeled and the downstream primer is labeled with biotin or fluorescein.

3. The nucleic acid composition for detecting respiratory pathogens according to claim 2, wherein the luciferins labeled with the probes are FAM/FITC, AF488, TAMRA and Cy5.

4. The nucleic acid composition for detecting respiratory pathogens of claim 3, wherein the small molecule antigen is at least one of Dig, folic acid, glycocholic acid, cholesterol, estradiol, progesterone, penicillin, and tetracycline.

5. The nucleic acid composition for detecting respiratory pathogens according to claim 4, wherein the tetrahydrofuran is labeled at 30bp from the 5' end of the probe, and the Spacer group is C3 Spacer.

6. The nucleic acid composition for detecting respiratory pathogens of claim 5, wherein the fluorescein that labels the downstream primer is different from the fluorescein that labels the probe.

7. A kit for detecting a nucleic acid of a respiratory pathogen, comprising a nucleic acid detection test strip and an amplification reagent comprising the nucleic acid composition for detecting a respiratory pathogen of any of claims 1-6.

8. The kit for detecting a nucleic acid of a respiratory pathogen according to claim 7, wherein the nucleic acid detection test strip comprises a base plate, and a sample pad, a label-binding pad and a chromatographic reaction membrane disposed on the base plate;

the label binding pad is adsorbed with a specific complex, and the specific complex is obtained by coupling latex microspheres with one or more of SA, chicken IgY, folic acid and glycocholic acid.

9. The kit for detecting a nucleic acid of a respiratory pathogen of claim 8, wherein the latex microspheres comprise red, blue and purple.

10. The kit for detecting a nucleic acid of a respiratory pathogen according to claim 9, wherein a detection line is provided on the chromatographic reaction membrane, the detection line containing one or more of an anti-FAM antibody, an anti-DIG antibody, an anti-AF 488 antibody, an anti-CY 5 antibody, an anti-folate antibody or an anti-glycocholic acid antibody.

11. The kit for detecting nucleic acid of respiratory pathogens of claim 10, wherein the antibody concentration in the detection line is 0.25-0.5mg/mL.

12. The kit for detecting a nucleic acid of a respiratory pathogen according to claim 11, wherein a quality control line containing one or more of an anti-chicken IgY antibody, a Biotin, or an anti-SA antibody is further provided on the chromatographic reaction membrane.

13. The kit for detecting a nucleic acid of a respiratory pathogen of claim 12, wherein the concentration of the antibody in the quality control line is 0.25-0.5mg/mL.

14. The kit for detecting a nucleic acid of respiratory tract pathogen according to claim 7, wherein the amplification reagent contains a primer pair and a probe of the novel coronavirus N gene fragment, the novel coronavirus ORF gene fragment, the influenza a M1 gene fragment and the influenza b NS1 gene fragment.

15. The kit for detecting a nucleic acid of respiratory tract pathogen according to claim 14, wherein the concentration ratio of the primer pair and the probe of the N gene fragment of the novel coronavirus, the primer pair and the probe of the ORF gene of the novel coronavirus, the primer pair and the probe of the M1 gene fragment of the influenza a virus and the primer pair and the probe of the NS1 gene fragment of the influenza b virus in the amplification reagent is 1:1:1:1 to 1:2:2:2.

16. The kit for detecting a nucleic acid of respiratory tract pathogen according to claim 15, wherein the concentration ratio of the primer pair and probe of the N gene fragment of the novel coronavirus, the primer pair and probe of the ORF gene of the novel coronavirus, the primer pair and probe of the M1 gene fragment of influenza a virus and the primer pair and probe of the NS1 gene fragment of influenza b virus is 6:9:9:10.

17. the kit for detecting a nucleic acid of respiratory tract pathogen according to claim 16, wherein the amplification reagents have a molar ratio of the N gene fragment of the novel coronavirus, the ORF gene of the novel coronavirus, the M1 gene fragment of the influenza a virus and the NS1 gene fragment of the influenza b virus of 2:2:0.6-2:2:1.

18. the kit for detecting a nucleic acid of a respiratory pathogen of claim 17, wherein the amplification reagents further comprise a buffer and a magnesium acetate solution.

19. A method for detecting nucleic acid of respiratory pathogens for non-diagnostic therapeutic purposes, comprising: adding the mixed solution containing the sample into the amplification reagent according to any one of claims 14 to 18 for amplification reaction, diluting the amplification solution after the reaction is finished, taking out part of the diluted solution, adding the diluted solution onto a sample pad of the nucleic acid detection test strip according to any one of claims 7 to 13, detecting, and reading the result.

20. The method of claim 19, wherein the amplification reaction is carried out for a period of time ranging from 10 to 20 minutes at a temperature ranging from 37 to 42 ℃.

21. The method of claim 20, wherein the amplification solution is diluted 5-10 fold.

22. The method of claim 21, wherein the volume of the partially diluted solution is 40-80 μl.

23. The method of claim 22, wherein the time of detection is 10-20 minutes.

24. The method of claim 23, wherein the 50 μl reaction system of the amplification reaction is: 2.5. Mu.L of magnesium acetate solution, 2. Mu.L of 10. Mu.M upstream and downstream primer, 0.6. Mu.L of probe, 2. Mu.L of template, and buffer to 50. Mu.L.