Application of arginine in preparation of product capable of improving LAMP (loop-mediated isothermal amplification) specificity

Abstract

The invention relates to application of arginine in preparation of a product capable of improving LAMP (loop-mediated isothermal amplification) specificity, and belongs to the technical field of biology. The invention provides an application of arginine (Arg) in preparation of a product capable of improving LAMP (loop-mediated isothermal amplification) specificity. Researches show that the addition of arginine in the LAMP reaction system can significantly improve the specificity of the reaction, reduce non-specific amplification, improve the sensitivity, stability and amplification efficiency of the reaction, shorten the detection time on the basis of ensuring the detection accuracy, and is suitable for the field of rapid diagnosis, so that the arginine can be used in the field of rapid diagnosis. As a product capable of improving the LAMP specificity, arginine has great application prospects in LAMP-based nucleic acid detection.

Description

Technical Field

[0001] The invention relates to application of arginine in preparing a product capable of improving the specificity of LAMP, and belongs to the field of biotechnology. Background Art

[0002] LAMP (Loop-mediated isothermal amplification) is a method for amplifying nucleic acids at a constant temperature. This method eliminates the thermal cycling steps of traditional PCR (polymerase chain reaction). Compared to other nucleic acid amplification techniques, it is rapid, efficient, highly specific, easily interpretable, requires no specialized equipment, and is independent of temperature fluctuations.

[0003] However, the isothermal amplification reaction process of LAMP often encounters the problem of non-specific amplification (NS), which leads to false positive results and affects the accuracy and reliability of LAMP detection. 2+ Methods such as increasing the concentration and adjusting the reaction conditions can be used to reduce nonspecific amplification, but these methods are often limited in effectiveness and may affect amplification efficiency while increasing experimental complexity. Therefore, it is urgent to find a method that can effectively improve the specificity of LAMP without affecting its amplification effect and experimental complexity. Summary of the Invention

[0004] To solve the above problems, the present invention provides the use of arginine (Arg) in preparing a product capable of improving the specificity of LAMP.

[0005] In one embodiment of the invention, the product comprises an additive.

[0006] The present invention also provides a product capable of improving the specificity of LAMP, wherein the ingredients of the product contain arginine.

[0007] In one embodiment of the invention, the product comprises an additive.

[0008] The present invention also provides a LAMP reagent, wherein the components of the LAMP reagent include arginine, an isothermal amplification buffer and an enzyme component.

[0009] In one embodiment of the present invention, the pH of the isothermal amplification buffer is 8.0-9.0.

[0010] In one embodiment of the present invention, the components of the isothermal amplification buffer include Tris-HCl, (NH4)2SO4, KCl, MgSO4, dNTPs and fluorescent dye.

[0011] In one embodiment of the present invention, the fluorescent dye includes at least one of SYTO-9 fluorescent dye, SYTO-11 fluorescent dye, SYTO-12 fluorescent dye, SYTO-14 fluorescent dye or SYTO-16 fluorescent dye.

[0012] In one embodiment of the present invention, the concentration of Tris-HCl in the LAMP reagent is 10 to 50 mM; the concentration of (NH4)2SO4 in the LAMP reagent is 10 to 50 mM; the concentration of KCl in the LAMP reagent is 50 to 150 mM; the concentration of dNTPs in the LAMP reagent is 0.8 to 1.6 mM; and the concentration of the fluorescent dye in the LAMP reagent is 0.02 to 2 μM.

[0013] In one embodiment of the present invention, the components of the enzyme component include DNA polymerase, or the components of the enzyme component include DNA polymerase and reverse transcriptase.

[0014] In one embodiment of the present invention, the concentration of the DNA polymerase in the LAMP reagent is 8 to 60 U; the concentration of the reverse transcriptase in the LAMP reagent is 40 to 80 U.

[0015] The present invention also provides a LAMP detection kit, which comprises the above-mentioned LAMP reagent and a LAMP primer set targeting a target nucleic acid.

[0016] In one embodiment of the present invention, the detection object of the LAMP detection kit is a virus; the virus includes parainfluenza virus, monkeypox virus, influenza A virus, influenza B virus and / or new coronavirus.

[0017] The present invention also provides a LAMP-based nucleic acid detection method, which comprises: using the above-mentioned LAMP detection kit to detect a sample to be detected.

[0018] In one embodiment of the present invention, the nucleic acid detection method includes: mixing a LAMP reagent, a LAMP primer set targeting a target nucleic acid, and a sample to be tested to obtain a LAMP reaction system; performing an amplification reaction on the LAMP reaction system; after the amplification reaction is completed, judging whether the target nucleic acid is present in the sample to be tested based on the amplification curve obtained by the reaction (if the fluorescence signal exceeds a set threshold and shows exponential growth, the target nucleic acid is present in the sample to be tested).

[0019] In one embodiment of the present invention, the concentration of arginine in the LAMP reaction system is 10-50 mM.

[0020] In one embodiment of the present invention, the incubation temperature is 60-65° C. and the incubation time is 30-60 min.

[0021] The present invention also provides the use of arginine or the above-mentioned product or the above-mentioned LAMP reagent or the above-mentioned LAMP detection kit in nucleic acid detection, wherein the use is for non-disease diagnosis and treatment purposes.

[0022] The technical solution of the present invention has the following advantages:

[0023] The present invention provides the use of arginine (Arg) in the preparation of products that can improve the specificity of LAMP. Studies have shown that when the working concentration of arginine in the LAMP reaction system is 25mM, for parainfluenza virus (PIV4), the amplification efficiency is significantly improved by 25.83%±6.83%, the detection rate is significantly improved by 20%±5%, and the detection limit is reduced to 15Copies / Reaction; for monkeypox virus (MPXV), while completely inhibiting nonspecific amplification, the amplification efficiency is significantly improved by 20.45%±3.45%, the detection rate is significantly improved by 25%±5%, and the detection limit is further reduced to 6.25Copies / Reaction. The above results were confirmed to have significant differences by statistical analysis (p<0.001), and the inter-batch coefficient of variation (CV) was less than 10%, indicating that arginine showed stable performance improvement in LAMP detection of RNA and DNA targets. It can be seen that the addition of arginine to LAMP can significantly improve the specificity of the reaction, reduce nonspecific amplification, and simultaneously enhance the sensitivity, stability, and amplification efficiency of the reaction. This shortens the detection time while ensuring detection accuracy, making it suitable for rapid diagnosis. Therefore, as a product that can enhance the specificity of LAMP, arginine has great application prospects in LAMP-based nucleic acid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Effect of arginine content on LAMP amplification kinetics. Figure 1 In the figure, A: amplification curve of NTC group; B: melting curve of NTC group.

[0025] Figure 2 : Effect of arginine content on LAMP amplification kinetics. Figure 2 a: amplification curve of the positive group (60 min); b: melting curve of the positive group (60 min); and c: melting curve of the positive group (25 min).

[0026] Figure 3 : Effect of arginine content on LAMP amplification kinetics (logarithmic phase linear fitting graph).

[0027] Figure 4: Effect of arginine content on LAMP amplification kinetics (capillary electrophoresis spectrum).

[0028] Figure 5 : Effect of arginine content on LAMP amplification kinetics (significance analysis).

[0029] Figure 6 : Effect of 25 mM arginine on the detection rate of parainfluenza virus. Figure 6 In the figure, A: amplification curve of 0 mM arginine; a: melting curve of 0 mM arginine; B: amplification curve of 25 mM arginine; D: melting curve of 25 mM arginine.

[0030] Figure 7 : Effect of 25 mM arginine on the detection rate of parainfluenza virus (significance analysis).

[0031] Figure 8 : Effect of arginine on NS (amplification curve).

[0032] Figure 9 : Effect of arginine on NS (capillary electrophoresis spectrum).

[0033] Figure 10 : Effect of arginine on monkeypox virus amplification kinetics (amplification curve).

[0034] Figure 11 : Effect of arginine on monkeypox virus amplification kinetics (significance analysis).

[0035] Figure 12 : Effect of arginine on the detection rate of monkeypox virus. Figure 12 In the figure, A: amplification curve of 0 mM arginine; a: melting curve of 0 mM arginine; B: amplification curve of 25 mM arginine; D: melting curve of 25 mM arginine.

[0036] Figure 13 : Effect of arginine on the detection rate of monkeypox virus (significance analysis).

[0037] Figure 14 : Effect of arginine on the detection rate of monkeypox virus (capillary electrophoresis spectrum). DETAILED DESCRIPTION

[0038] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0039] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0040] The Bst DNA polymerase, RNA reverse transcriptase, and dNTPs involved in the following examples were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., SYTO-16 fluorescent dye was purchased from Thermo Fisher Scientific, and MgSO4 was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0041] Example 1-1: A LAMP detection kit for detecting parainfluenza virus

[0042] This embodiment provides a LAMP detection kit for detecting parainfluenza virus (PIV4), the LAMP detection kit comprising 2.5× isothermal amplification buffer, 10× primers targeting PIV4, Bst DNA polymerase, RNA reverse transcriptase, and arginine;

[0043] The 2.5× isothermal amplification buffer is composed of 50 mM Tris-HCl, 25 mM (NH 4 ) 2 SO 4 , 125 mM KCl, 20 mM MgSO 4 , 1.2 mM dNTPs, and 1 μM SYTO-16 fluorescent dye (the solvent is nuclease-free pure water, with a pH of 8.8 at 25° C.);

[0044] The 10× primer targeting PIV4 consists of a FIP primer with a nucleotide sequence as shown in SEQ ID NO.1, a BIP primer with a nucleotide sequence as shown in SEQ ID NO.2, a Loop F primer with a nucleotide sequence as shown in SEQ ID NO.3, a Loop B primer with a nucleotide sequence as shown in SEQ ID NO.4, an F3 primer with a nucleotide sequence as shown in SEQ ID NO.5, and a B3 primer with a nucleotide sequence as shown in SEQ ID NO.6 (see Table 1 for the specific formula).

[0045] Table 110× Primer Formula

[0046]

[0047] Example 2-1: A nucleic acid detection method for detecting parainfluenza virus

[0048] This embodiment provides a nucleic acid detection method for detecting parainfluenza virus (PIV4), which comprises: preparing a LAMP reaction system (LAMP reaction system, arginine concentration is 25 mM) using the LAMP detection kit of Example 1-1 according to the formula in Table 2; mixing the LAMP reaction system and centrifuging it instantaneously, and then placing it in a Thermofisher ABI Quant Studio 1 real-time fluorescence quantitative PCR instrument to run an amplification reaction (LAMP reaction program is shown in Table 3); after the amplification reaction is completed, judging whether the parainfluenza virus is present in the sample to be tested based on the amplification curve obtained by the reaction.

[0049] Table 2 LAMP reaction system

[0050] Components Volume (μL) 2.5× Isothermal Amplification Buffer 10 Mixed enzyme (Bst:RT=1:1) 1 10× primer mix 2.5 template 5 500mM arginine stock solution 1.25 purified water to25 total 25

[0051] In Table 2, the solvent for the 500 mM arginine stock solution is nuclease-free pure water. 0.1742 g of arginine powder was weighed and dissolved in 2 mL of nuclease-free pure water to prepare a 500 mM arginine stock solution. The 0.22 μM solution was filtered and used for later use.

[0052] Table 3 LAMP reaction procedure

[0053]

[0054] Example 2-2 to Example 2-4: A Nucleic Acid Detection Method for Detecting Parainfluenza Virus

[0055] This embodiment provides a nucleic acid detection method for detecting parainfluenza virus (PIV4). The nucleic acid detection method is as follows: based on Example 2-1, the concentration of arginine in the LAMP reaction system is replaced from 25 mM to 12.5 mM, 37.5 mM, and 50 mM, respectively.

[0056] Comparative Example 1-1: A LAMP detection kit for detecting parainfluenza virus

[0057] This comparative example provides a LAMP detection kit for detecting parainfluenza virus (PIV4). The LAMP detection kit is: based on Example 1-1, except that arginine is removed.

[0058] Comparative Example 2-1: A nucleic acid detection method for detecting parainfluenza virus

[0059] This comparative example provides a nucleic acid detection method for detecting parainfluenza virus (PIV4), which is as follows: on the basis of Example 2-1, the LAMP detection kit of Comparative Example 1-1 is used, and at this time, the concentration of arginine in the LAMP reaction system is 0 mM.

[0060] Experimental Example 1: Effect of Arginine on LAMP Isothermal Amplification

[0061] Target: Parainfluenza virus (PIV4)

[0062] Experiment 1: Effect of Arginine on Amplification Performance (0 Copies / Reaction + 50 Copies / Reaction)

[0063] The experimental process is as follows:

[0064] The parainfluenza virus template used in this experiment is a standard product purchased from Tovisit Biotechnology, with an original concentration of 1×10^6 copies / μL. It was gradiently diluted with nuclease-free pure water to 10 copies / μL for later use, thus obtaining the sample to be tested.

[0065] In this experiment, nucleic acid detection was performed using the nucleic acid detection methods of Examples 2-1 to 2-4 and Comparative Example 2-1. After the detection, the fluorescence signal results obtained by the real-time fluorescence quantitative PCR instrument were analyzed.

[0066] In this experiment, the template concentrations were 0 and 50 Copies / Reaction, respectively. The 0 Copies / Reaction group was the no-template negative control (NTC), in which nuclease-free pure water was used to replace the template solution in the LAMP reaction system. The 50 Copies / Reaction group was the test sample group, in which the test sample was used as the template solution in the LAMP reaction system.

[0067] The experimental results are shown in Tables 4 to 5 and Figures 1 to 5 This experimental result adds a melting curve and introduces the product Tm value to assist in the interpretation of specific and nonspecific amplification. When the Tm value cannot be accurately interpreted, capillary electrophoresis is combined to assist in interpretation.

[0068] Table 4 is the data of the fluorescence quantitative PCR instrument. The CT value is the amplification time / cycle number when the fluorescence intensity reaches the set threshold. Under the conditions of the same template concentration, the smaller the CT value, the higher the amplification efficiency. The binding specificity of the primer and the template is good, and the reaction conditions are suitable (such as temperature, buffer composition, etc.), so that the Bst enzyme and the RT enzyme can more efficiently copy the target DNA sequence, thereby accumulating enough fluorescence signals under less amplification time, reducing the CT value. Undetermined means undetected, that is, the fluorescence signal does not reach the threshold until the end of the reaction. In this experiment, Comparative Example 2-1 is used as the control group of the experiment, and Examples 2-1 to 2-4 are used as experimental groups. The T / C ratio represents the ratio of the CT value of the experimental group to the CT value of the control group. T / C <1 indicates that the amplification efficiency of the experimental group is better than that of the control group, and T / C >1 indicates that the amplification efficiency of the experimental group is weaker than that of the control group. In this experiment, the introduction of T / C as the basis for judging the amplification efficiency in the isothermal amplification reaction can eliminate the slight error caused by the dilution of the template solution in the batch experiment and perform homogenization on the experimental results.

[0069] Figures 1 and 2 Figure 3 is the amplification curve obtained by fluorescent quantitative PCR. Figure 3 The figure is a logarithmic linear fitting graph obtained by fitting the fluorescence signal data of the amplification curve exported by Thermofisher ABI Quant Studio 1. Figure 4 This is the capillary electrophoresis spectrum of the LAMP amplification product. Figure 5 The results of significance analysis were obtained based on the results of 5 independent repeated experiments.

[0070] The experimental results are analyzed as follows:

[0071] In the fluorescence amplification curve of the negative control group (NTC, 0 Copies / Reaction), no significant fluorescence signal fluctuation was observed in Examples 2-1 to 2-4 and Comparative Example 2-1 (arginine concentration gradient: 0-50 mM) ( Figure 1 ), that is, in the absence of target template, no DNA amplification occurred in the reaction system regardless of whether arginine was added (the product Tm value had no characteristic peak, Figure 4 ), and the baseline fluorescence signal was consistent across experimental groups. These data confirm that the introduction of arginine does not induce nonspecific amplification in the NTC group, indicating that this additive is highly specific in suppressing false-positive results.

[0072] In the positive experimental group with a target concentration of 50 copies / reaction, the fluorescence amplification curve (horizontal axis: amplification time; vertical axis: △Rn, the difference between the real-time fluorescence intensity and the baseline value) directly reflects the dynamic accumulation process of the target DNA in the LAMP reaction. Experimental data show that with the increase of amplification time, the △Rn value shows a significant upward trend, indicating that the synthesis rate of the target DNA is positively correlated with the fluorescence signal intensity. In other words, the greater the change in the fluorescence signal, the more target DNA is produced ( Figure 2 ).

[0073] With the increase of arginine working concentration in the reaction system, the melting temperature (Tm value) of LAMP amplification products showed a gradual downward trend ( Figure 2 , Table 4). The reason for this phenomenon is that the amplified product is a double-stranded DNA, whose phosphate backbone is negatively charged, while arginine has a strongly positively charged guanidine group at physiological pH. The bulky guanidine group of arginine may insert into the minor groove of the DNA double helix or interfere with the hydrogen bond network of base pairs, destroying the regularity of the local double helix, resulting in a weakening of hydrogen bonds or base stacking forces, thereby reducing thermal stability. Figure 4 The capillary electrophoresis results show that although the Tm of the experimental group with added arginine is slightly reduced, the electrophoresis spectrum of its product is exactly the same as that of the control group, indicating that the addition of arginine does not change the sequence specificity of the amplified product, but only regulates the stability of the DNA secondary structure through physical and chemical effects.

[0074] When the working concentration of arginine was 25 mM, the reaction system showed the best amplification performance: (1) the CT value was significantly reduced to 5.862 ± 0.408 (n = 3), which was 23.53% shorter than that of the control group (7.663 ± 0.661); (2) the T / C ratio reached the lowest value of 0.76, indicating that the amplification efficiency was improved by about 24.0% (calculated as (1-T / C) × 100%); (3) the fluorescence signal accumulation rate increased by 27.23% (calculated by the slope of the ΔRn curve); (4) the intra-assay reproducibility was good (CV < 10%) (Tables 4, 5 and Figure 2 、 Figure 3 ).

[0075] The repeatability and stability of the effect of arginine concentration on LAMP amplification kinetics were verified by five independent repeated experiments. The experimental results showed that the T / C values ​​of the 12.5mM and 25mM arginine experimental groups were significantly less than 1 (0.884±0.055 and 0.752±0.044, respectively), indicating that their CT values ​​were significantly shorter than those of the control group (1.00±0.00) (p<0.05), and the amplification efficiency was increased by 11.60%±0.05 and 24.76%±0.05, respectively. The results of the five experiments were consistent, and the inter-assay CV values ​​were all less than 10%, indicating that this experiment is repeatable and the results are reliable ( Figure 5The 12.5mM arginine experimental group showed significant differences compared to the control group (marked with *), and the 25mM arginine experimental group showed significant differences compared to the control group (marked with ****). Considering the improvement in amplification efficiency (25.83% ± 6.83%) and the statistical significance, 25mM arginine was determined to be the optimal working concentration, with good stability.

[0076] Experiment 2: Effect of Arginine on Amplification Performance (15 Copies / Reaction)

[0077] The experimental process is as follows:

[0078] Based on Experiment 1, the experiment was conducted at a template concentration of 15 copies / reaction. During the experiment, the template stock solution was gradient diluted to 3 copies / μL with nuclease-free pure water to obtain the sample to be tested. The experimental results are shown in Table 6 and Figures 6 and 7 shown.

[0079] The experimental results are analyzed as follows:

[0080] The experimental results show that the addition of 25mM arginine can significantly improve the performance of the LAMP detection system. Under the low template concentration condition of 15Copies / Reaction, the detection rate of the experimental group (25mM arginine) reached 85.00% (17 / 20), which was 20.00% higher than that of the control group (65.00%, 13 / 20) (Table 6). At the same time, the detection sensitivity of the system was significantly improved, the detection limit was reduced to 15Copies / Reaction, and the signal-to-noise ratio (S / N) was increased by 4.25 times. Amplification efficiency analysis showed that the T / C ratio of the experimental group was 0.77 (n=20), corresponding to an increase in amplification efficiency of approximately 23.00% (Table 6). This result is highly consistent with the previous experimental data (efficiency increase of 25.83%±6.83%). Melting curve analysis confirmed that the Tm values ​​of all positive products were concentrated at 84.2±0.3℃, and no non-specific peaks appeared ( Figure 6 ), indicating that the system maintained good specificity while improving sensitivity. Overall, the addition of 25 mM arginine enabled the LAMP detection system to achieve simultaneous optimization of sensitivity and specificity at low template concentrations.

[0081] After 5 independent repeated experiments, the experimental data showed excellent repeatability and statistical significance. The inter-batch CV was less than 10%, and the consistency was good, which confirmed that the experimental system has high repeatability ( Figure 7 There was a significant difference between the 25 mM arginine experimental group and the control group (marked with ****). Quantitative analysis showed that arginine treatment increased the parainfluenza virus detection rate by 20% ± 5% and reduced the detection limit to 15 copies / reaction.

[0082] Table 4 Effect of arginine concentration gradient on LAMP amplification kinetics

[0083]

[0084]

[0085] Table 5 Effect of arginine concentration gradient on LAMP amplification kinetics (logarithmic phase linear fitting)

[0086] Slope Y-intercept X-intercept equation R-squared Comparative Example 2-1 225871 -1813668 8.03 Y=225871*X-1813668 0.9918 Example 2-2 250794 -1725824 6.881 Y=250794*X-1725824 0.9919 Example 2-1 287394 -1958339 6.814 Y=287394*X-1958339 0.9914 Example 2-3 225948 -2133141 9.441 Y=225948*X-2133141 0.9931 Examples 2-4 225364 -2496186 11.08 Y=225364*X-2496186 0.9935

[0087] Table 6 Effect of 25mM arginine on parainfluenza virus detection rate

[0088]

[0089]

[0090] Example 3-1: A LAMP detection kit for detecting monkeypox virus

[0091] This embodiment provides a LAMP detection kit for detecting monkeypox virus (MPXV), and the LAMP detection kit is: based on Example 1-1, RNA reverse transcriptase is removed (reverse transcription is not required for the detection of DNA viruses), and the 10× primer targeting PIV4 is replaced with a 10× primer targeting MPXV; the 10× primer targeting MPXV is composed of a FIP primer with a nucleotide sequence as shown in SEQ ID NO.7, a BIP primer with a nucleotide sequence as shown in SEQ ID NO.8, a Loop F primer with a nucleotide sequence as shown in SEQ ID NO.9, an F3 primer with a nucleotide sequence as shown in SEQ ID NO.10, and a B3 primer with a nucleotide sequence as shown in SEQ ID NO.11.

[0092] Example 4-1: A nucleic acid detection method for detecting monkeypox virus

[0093] This embodiment provides a nucleic acid detection method for detecting monkeypox virus (MPXV). The nucleic acid detection method is: based on Example 2-1, the LAMP detection kit of Example 1-1 is replaced with the LAMP detection kit of Example 3-1.

[0094] Comparative Example 3-1: A LAMP detection kit for detecting monkeypox virus

[0095] This comparative example provides a LAMP detection kit for detecting monkeypox virus (MPXV). The LAMP detection kit is: based on Example 3-1, except that arginine is removed.

[0096] Comparative Example 4-1: A nucleic acid detection method for detecting monkeypox virus

[0097] This comparative example provides a nucleic acid detection method for detecting monkeypox virus (MPXV), wherein the nucleic acid detection method is as follows: on the basis of Example 4-1, the LAMP detection kit of Comparative Example 3-1 is used, and at this time, the concentration of arginine in the LAMP reaction system is 0 mM.

[0098] Experimental Example 2: Effect of Arginine on LAMP Isothermal Amplification

[0099] Target: Monkeypox virus (MPXV)

[0100] Experiment 1: Effect of Arginine on Amplification Performance (0 Copies / Reaction + 12.5 Copies / Reaction)

[0101] The experimental process is as follows:

[0102] The monkeypox virus template used in this experiment was purchased from the National Center for Standard Materials Research. The original concentration was (2.74±0.46)×10^3 copies / μL. It was gradiently diluted with nuclease-free pure water to 2.5 copies / μL for later use, thus obtaining the sample to be tested.

[0103] In this experiment, nucleic acid detection was performed using the nucleic acid detection methods of Example 4-1 and Comparative Example 4-1. After the detection, the fluorescence signal results obtained by the real-time fluorescence quantitative PCR instrument were analyzed.

[0104] In this experiment, the template concentrations were 0 and 12.5 copies / reaction, respectively. 0 copies / reaction was the no-template negative control (NTC), where nuclease-free pure water was used to replace the template solution in the LAMP reaction system. 12.5 copies / reaction was the test sample group, where the test sample was used as the template solution in the LAMP reaction system. The experimental results are shown in Tables 7 and 8. Figures 8 to 11 shown.

[0105] The experimental results are analyzed as follows:

[0106] Experimental data showed that in the monkeypox virus (MPXV) detection system, Comparative Example 4-1-NTC was used as a negative control group (no arginine was added), and the monkeypox virus target mixed primer set produced nonspecific amplification under the condition of no template addition, and its Tm value was about 74 ° C, which was significantly different from the specific amplification Tm value of about 84 ° C in Comparative Example 4-1 (Table 7, Figure 8 ). Capillary electrophoresis analysis further confirmed this result. The products of comparative example 4-1-NTC were all mixed peaks, showing random amplification, which was inconsistent with the spectra of specific amplification products. It is worth noting that the experimental group (Example 4-1) with the addition of 25mM arginine completely inhibited nonspecific amplification, and the capillary electrophoresis spectrum results showed no amplification products ( Figure 9 ).

[0107] After five independent replicates, all experimental batches demonstrated consistent inhibitory effects. Analysis showed that the experimental group supplemented with 25 mM arginine completely suppressed nonspecific amplification in the monkeypox virus detection system.

[0108] Under standardized reaction conditions, the introduction of 25 mM arginine significantly improved the performance parameters of the LAMP amplification system and significantly increased amplification efficiency. Real-time fluorescence quantitative analysis showed that the CT value of the arginine-treated group was significantly reduced to 10.252 ± 0.526, approximately 20% higher than the 13.097 ± 2.503 value of the control group (p < 0.001). The coefficient of variation (CV) between parallel reactions within the experimental group remained at a low level of 5.13%, indicating that the optimized protocol has excellent reproducibility (Table 8, Figure 10 Notably, this result is highly consistent with previous research data targeting RNA targets (parainfluenza virus) (efficiency increased by 25.83% ± 6.83%, CV = 5.79%).

[0109] Statistical analysis of five independent replicates revealed that the addition of 25 mM arginine significantly improved the amplification performance of the LAMP system and was highly reproducible. The arginine-treated group showed a highly significant difference compared to the control group (marked with ****, p<0.0001), with amplification efficiency increased by 20.45% ± 3.45%, and the inter-assay coefficient of variation (CV) remained below 5%, confirming the good reproducibility of the experimental results ( Figure 11Notably, this optimization effect was consistent across different nucleic acid target types (DNA / RNA), with the average amplification efficiency increase remaining stable within a range of 20-23%. These systematic research results statistically confirm the broad-spectrum applicability of arginine as a nucleic acid amplification enhancer. Its ability to significantly improve amplification efficiency (p<0.0001) and excellent experimental repeatability (CV<10%) provide important theoretical basis and technical support for the development of efficient isothermal amplification detection systems.

[0110] Experiment 2: Effect of Arginine on Amplification Performance (6.25 Copies / Reaction)

[0111] The experimental process is as follows:

[0112] Based on Experiment 1, the experiment was conducted at a template concentration of 6.25 copies / reaction. During the experiment, the template mother solution was gradient diluted to 1.25 copies / μL with nuclease-free pure water to obtain the sample to be tested. The experimental results are shown in Table 9 and Figures 12 to 14 shown.

[0113] The experimental results are analyzed as follows:

[0114] Under the same template concentration conditions, the addition of 25mM arginine significantly improved the performance of the detection system. In the control group (Comparative Example 4-1), 6 of the 20 parallel reactions showed nonspecific amplification (30%), which was manifested as abnormal Tm values ​​and capillary electrophoresis miscellaneous peaks ( Figure 12 、 Figure 14 ), while all 20 reactions in the arginine-treated group (Example 4-1) exhibited specific amplification, with consistent Tm values ​​and a single electrophoretic pattern. Quantitative analysis showed that arginine treatment significantly increased the detection rate from 70.0% to 100.0% (p < 0.001), lowering the limit of detection to 6.25 copies / reaction. A concomitant decrease in the T / C ratio indicated an approximately 40% increase in amplification efficiency (Table 9). The low coefficient of variation of Ct values ​​within the experimental groups demonstrated good intra-assay consistency.

[0115] Statistical analysis of five independent repeated experiments confirmed that the addition of 25mM arginine significantly improved detection sensitivity and repeatability. Experimental data showed that the arginine-treated group had a significant difference compared with the control group (marked with ***, p<0.001), with the monkeypox virus detection rate increased by 25%±5%, and the inter-assay coefficient of variation (CV) remained below 10%, indicating that the experimental results had good repeatability and reliability ( Figure 13This optimization effect is highly consistent with previous research results on RNA targets (parainfluenza virus), both of which showed a 25-30% increase in detection rate and complete inhibition of nonspecific amplification (p < 0.001). These systematic research data confirm the broad applicability of 25mM arginine as a nucleic acid amplification enhancer from multiple dimensions, including amplification kinetics, detection rate, and detection limit, providing important experimental basis and methodological reference for establishing a highly sensitive and specific isothermal amplification detection system.

[0116] Table 7 Effect of 25mM arginine on NS

[0117]

[0118]

[0119] Table 8 Effect of arginine on monkeypox virus amplification kinetics

[0120]

[0121] Table 9 Effect of arginine on monkeypox virus detection rate

[0122]

[0123]

[0124] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Application of arginine in the preparation of products capable of improving LAMP specificity.

2. A product capable of improving the specificity of LAMP, characterized in that: The ingredients of the product include arginine.

3. A LAMP reagent, characterized in that The components of the LAMP reagent include arginine, isothermal amplification buffer and enzyme components.

4. The LAMP reagent according to claim 3, wherein The pH of the isothermal amplification buffer is 8.0-9.

0.

5. The LAMP reagent according to claim 3 or 4, wherein The components of the enzyme component include DNA polymerase, or the components of the enzyme component include DNA polymerase and reverse transcriptase.

6. The LAMP reagent according to claim 5, wherein The concentration of the DNA polymerase in the LAMP reagent is 8 to 60 U; the concentration of the reverse transcriptase in the LAMP reagent is 40 to 80 U.

7. A LAMP detection kit, characterized in that The components of the LAMP detection kit include the LAMP reagent according to any one of claims 3 to 6 and a LAMP primer set targeting a target nucleic acid.

8. A method for nucleic acid detection based on LAMP, characterized in that: The nucleic acid detection method comprises: using the LAMP detection kit according to claim 7 to detect the sample to be tested.

9. The nucleic acid detection method according to claim 8, wherein The nucleic acid detection method includes: mixing a LAMP reagent, a LAMP primer set targeting a target nucleic acid, and a sample to be tested to obtain a LAMP reaction system; performing an amplification reaction on the LAMP reaction system; after the amplification reaction is completed, judging whether the target nucleic acid is present in the sample to be tested based on the amplification curve obtained by the reaction.

10. Use of arginine or the product according to claim 2 or the LAMP reagent according to any one of claims 3 to 7 or the LAMP detection kit according to claim 8 in nucleic acid detection, characterized in that: The application is not for the purpose of diagnosis and treatment of diseases.

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