Loop-mediated isothermal nucleic acid amplification (LAMP) using LNA-modified primers and a metal-based colorimetric method for detecting amplified products

Abstract

The present invention relates to a loop-mediated isothermal amplification (LAMP) method, characterized in that the F3 nucleotide sequence and / or the B3 nucleotide sequence contain one or more locked nucleic acid (LNA) nucleotides located within the first third of the F3 nucleotide sequence or the B3 nucleotide sequence, respectively. The present invention further ... 2+ The present invention relates to an in vitro method for detecting nucleic acid sequence amplification products, characterized by the use of ions. The present invention further relates to a kit for carrying out the method of the present invention and the use of this kit.

Description

[Technical Field]

[0001] This application contains a Sequence Listing in computer readable format, which is incorporated herein by reference.

[0002] Technical field of the invention The present invention relates to a loop-mediated isothermal amplification (LAMP) method, characterized in that the F3 nucleotide sequence and / or the B3 nucleotide sequence contain one or more locked nucleic acid (LNA) nucleotides located within the first third of the F3 nucleotide sequence or the B3 nucleotide sequence, respectively. The present invention further ... LAMP method comprises a metal indicator, preferably 5-Br-PAPS, and a metal ion, preferably Zn 2+ The present invention relates to an in vitro method for detecting nucleic acid sequence amplification products, characterized by the use of ions. The present invention further relates to a kit for carrying out the method of the present invention and the use of this kit. [Background technology]

[0003] background Since the breakthrough development of polymerase chain reaction (PCR) in the 1980s, nucleic acid amplification tests (NAATs) have become an essential tool throughout the life sciences, becoming the gold standard for nucleic acid analysis, particularly in clinical applications, as well as for food quality control and environmental monitoring. A notable trend emerged between 1995 and 2005 in the development of isothermal NAATs, inspired by the limitations of PCR. The complex and expensive devices required for temperature cycling and real-time detection during PCR limit the use of this amplification method. Isothermal NAATs enable amplification reactions at a constant and moderate temperature. Simple and low-cost devices, as well as faster processing times compared to PCR, make isothermal NAATs increasingly attractive and open up new application opportunities in the field of point-of-care (POC) / point-of-need (PON) testing.

[0004] Loop-mediated isothermal amplification (LAMP) is one method for performing NAAT and was first described in Notomi et al. (2000), Nucleic Acids Res., 28(12):e63 (Non-Patent Document 1), see also EP 1020534 B2 (Patent Document 1), both of which are incorporated herein by reference.

[0005] However, there is a continuing need to improve LAMP, particularly by increasing the specificity of the primers. Furthermore, there is a continuing need for improved detection methods that can be used in NAAT. Thus, the technical challenge is to meet this need. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] EP 1020534 B2 [Non-patent literature]

[0007] [Non-Patent Document 1] Notomi et al. (2000), Nucleic Acids Res., 28(12):e63 Summary of the Invention

[0008] The technical problem is solved by the subject matter as defined in the claims.

[0009] Therefore, the present invention provides (i) a first inner primer (FIP) nucleotide sequence and a second inner primer (BIP) nucleotide sequence; (ii) a first outer primer (F3) nucleotide sequence and a second outer primer (B3) nucleotide sequence, wherein the F3 nucleotide sequence and / or the B3 nucleotide sequence comprises one or more locked nucleic acid (LNA) nucleotides located within the first third of the F3 nucleotide sequence or the B3 nucleotide sequence, respectively; (iii) a DNA polymerase that catalyzes a strand displacement reaction that synthesizes a complementary nucleic acid strand from a template nucleic acid; and (iv) a nucleotide that serves as a substrate for the DNA polymerase In a reaction mixture comprising at a temperature at which at least the FIP nucleotide sequence and the BIP nucleotide sequence can form stable base pairs with their complementary nucleotide sequences contained in the template nucleic acid, while maintaining DNA polymerase activity; The present invention relates to a loop-mediated isothermal amplification (LAMP) method, which comprises a step of synthesizing a nucleic acid sequence from a template nucleic acid sequence, thereby synthesizing a nucleic acid sequence.

[0010] The present invention further comprises: (a) contacting a reaction mixture comprising a template nucleic acid sequence, a primer nucleotide sequence, nucleotides, and a polymerase capable of amplifying a nucleic acid molecule with a metal indicator and a transition or post-transition metal ion, wherein the metal indicator and the transition or post-transition metal ion form a complex, but not with magnesium; (b) amplifying the nucleic acid sequence under suitable conditions to obtain a nucleic acid sequence amplification product; (c) detecting a change in the spectral or fluorescent properties of the complex resulting from amplification of the target DNA. The present invention relates to an in vitro method for detecting nucleic acid sequence amplification products, comprising:

[0011] The present invention further comprises: (i) a first outer primer (F3) nucleotide sequence and a second outer primer (B3) nucleotide sequence, each of which comprises a locked nucleic acid (LNA) nucleotide; and / or, optionally (ii) a first inner primer (FIP) nucleotide sequence and a second inner primer (BIP) nucleotide sequence, and / or optionally (iii) a DNA polymerase that catalyzes a strand displacement reaction to synthesize a complementary nucleic acid strand from a template nucleic acid, and / or optionally (iv) nucleotides that serve as substrates for said DNA polymerase, and / or optionally (v) a metal indicator, preferably 5-Br-PAPS; and / or, optionally (vi) a metal ion, preferably Zn 2+ ion The present invention relates to a kit for synthesizing a nucleic acid sequence by loop-mediated isothermal amplification (LAMP) on a template nucleic acid sequence, comprising:

[0012] The present invention further relates to the use of the kit of the present invention for carrying out the LAMP method of the present invention.

[0013] [The present invention 1001] (i) a first inner primer (FIP) nucleotide sequence and a second inner primer (BIP) nucleotide sequence; (ii) a first outer primer (F3) nucleotide sequence and a second outer primer (B3) nucleotide sequence, wherein the F3 nucleotide sequence and / or the B3 nucleotide sequence comprises one or more locked nucleic acid (LNA) nucleotides located within the first third of the F3 nucleotide sequence or the B3 nucleotide sequence, respectively; (iii) a DNA polymerase that catalyzes a strand displacement reaction that synthesizes a complementary nucleic acid strand from a template nucleic acid; and (iv) a nucleotide that serves as a substrate for the DNA polymerase In a reaction mixture comprising at a temperature at which at least the FIP nucleotide sequence and the BIP nucleotide sequence can form stable base pairs with their complementary nucleotide sequences contained in the template nucleic acid, while maintaining DNA polymerase activity; A loop-mediated isothermal amplification (LAMP) method includes a step of synthesizing a nucleic acid sequence from a template nucleic acid sequence, thereby synthesizing a nucleic acid sequence. [The present invention 1002] (i) the FIP nucleotide sequence comprises at least two regions, F2 and F1c; the F1c region is linked to the 5' side of the F2 region, the F2 region has a nucleotide sequence complementary to an optional region F2c in the template nucleic acid sequence; and the F1c region has substantially the same nucleotide sequence as a region F1c located 5' to the F2c region in the template nucleic acid sequence; (ii) the BIP nucleotide sequence comprises at least two regions, B2 and B1c; the B1c region is linked to the 5' side of the B2 region, the B2 region has a nucleotide sequence complementary to an arbitrary region B2c in the template nucleic acid sequence strand; and the B1c region has substantially the same nucleotide sequence as region B1c located 5' to region B2c in the template nucleic acid sequence; (iii) the F3 nucleotide sequence has a nucleotide sequence substantially complementary to a region F3c in the template nucleic acid sequence; a region F3c is located on the 3' side of the region F2c in the template nucleic acid sequence; a region F2c is located 3' to the region F1c in the template nucleic acid sequence; and / or (iv) the B3 nucleotide sequence has a nucleotide sequence substantially complementary to region B3c in the template nucleic acid sequence; a region B3c is located on the 3' side of the region B2c in the template nucleic acid sequence; the region B2c is located on the 3' side of the region B1c in the template nucleic acid sequence; The method of the present invention 1001. [The present invention 1003] (i) the reaction mixture further comprises loop primer nucleotide sequence F and / or loop primer nucleotide sequence B; (ii) the reaction mixture further comprises stem primer nucleotide sequence F and / or stem primer nucleotide sequence B; (iii) the reaction mixture further comprises swarm primer nucleotide sequence F1S and / or swarm primer nucleotide sequence B1S; (iv) the reaction mixture further comprises at least an additional first inner primer (FIP2) nucleotide sequence and an additional second inner primer (BIP2) nucleotide sequence, both of which are different from the FIP and BIP nucleotide sequences; (v) the reaction mixture further comprises primer nucleotide sequences for carrying out the method as a multiplex cross-displacement amplification (LAMP); and / or (vi) the reaction mixture further comprises primer nucleotide sequences for carrying out the method as a reverse transcription isothermal multiplex self-matching LAMP; Any method of the preceding invention. [The present invention 1004] (i) the template nucleic acid sequence is single-stranded or double-stranded; (ii) the template nucleic acid sequence is single-stranded or double-stranded DNA, RNA, or a DNA / RNA chimera; (iii) the DNA polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase; (iv) the DNA polymerase has reverse transcriptase activity; Any method of the preceding invention. [The present invention 1005] (i) the F3 nucleotide sequence has a length of 15 to 30, preferably 17 to 25, nucleotides; and / or (ii) the B3 nucleotide sequence has a length of 15 to 30, preferably 17 to 25, nucleotides; Any method of the preceding invention. [The present invention 1006] (i) the F3 nucleotide sequence comprises 1 to 5, preferably 3, LNA nucleotides; and / or (ii) the B3 nucleotide sequence contains 1 to 5, preferably 3, LNA nucleotides; Any method of the preceding invention. [The present invention 1007] Any of the preceding methods of the invention, wherein the reaction mixture further comprises a melting temperature regulator, for example, betaine, preferably at a concentration of 0.2M to 3.0M, Tween-20 / Triton-X, preferably at a concentration of 0.02% to 0.2%, guanidine thiocyanate or hydrochloride, preferably at a concentration of 20mM to 80mM, single-stranded binding protein (SSB), BSA, preferably at a concentration of 0.02mg / ml to 2mg / ml, TMAC, preferably at a concentration of 5mM to 80mM, and / or TCEP / DTT, preferably at a concentration of 0.5mM to 5mM. [The present invention 1008] Any of the preceding methods of the present invention, wherein the reaction mixture further comprises a detection agent for detecting the product of the nucleic acid sequence synthesis reaction, preferably wherein the detection agent is a metal indicator, more preferably wherein the detection agent further comprises a transition metal or post-transition metal. [The present invention 1009] The detection agent is a metal indicator, preferably 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS), PAR or Zincon, and a metal ion, preferably a transition metal or post-transition metal ion, more preferably Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, most preferably Zn 2+ ions, wherein the metal indicator and the metal ion each form a complex, but not with magnesium; and The method comprises: (b) detecting a change in the spectral or fluorescent properties of the complex resulting from amplification of said nucleic acid sequence. Any of the methods of the preceding invention further comprising: [The present invention 1010] (a) synthesizing a nucleic acid sequence; (b) detecting a nucleic acid sequence; or (c) diagnosing a disease, preferably a disease caused by a pathogen Any of the methods of the preceding invention, [The present invention 1011] (a) contacting a reaction mixture comprising a template nucleic acid sequence, a primer nucleotide sequence, nucleotides, and a polymerase capable of amplifying a nucleic acid molecule with a metal indicator and a transition or post-transition metal ion, wherein the metal indicator and the transition or post-transition metal ion form a complex, but not with magnesium; (b) amplifying the nucleic acid sequence under suitable conditions to obtain a nucleic acid sequence amplification product; (c) detecting a change in the spectral properties of a complex resulting from the amplification of the nucleic acid sequence, wherein the formation of the complex comprising the metal indicator and the transition or post-transition metal ion leads to a color change of the solution. 1. An in vitro method for detecting a nucleic acid sequence amplification product, comprising: [The present invention 1012] (i) the metal indicator is 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS); (ii) the metal ion is Zn 2+ is an ion; and / or (iii) the change in the spectral properties of the complex is a change in the spectrum of wavelengths of 380 to 740 nm; The method of the present invention 1011. [The present invention 1013] The method of any one of claims 1011 to 1012, wherein said amplification is carried out by the method defined in any one of claims 1001 to 1007. [The present invention 1014] (i) a first outer primer (F3) nucleotide sequence and a second outer primer (B3) nucleotide sequence, each of which comprises a locked nucleic acid (LNA) nucleotide; and / or, optionally (ii) a first inner primer (FIP) nucleotide sequence and a second inner primer (BIP) nucleotide sequence, and / or optionally (iii) a DNA polymerase that catalyzes a strand displacement reaction to synthesize a complementary nucleic acid strand from a template nucleic acid, and / or optionally (iv) nucleotides that serve as substrates for said DNA polymerase, and / or optionally (v) a metal indicator, preferably 5-Br-PAPS, PAR or Zincon; and / or, optionally (vi) a metal ion, preferably Zn 2+ ion A kit for synthesizing a nucleic acid sequence by loop-mediated isothermal amplification (LAMP) on a template nucleic acid sequence, comprising: [The present invention 1015] Use of the kit of the present invention 1014 for carrying out any of the methods of the present inventions 1001 to 1013. The present invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which: [Brief explanation of the drawings]

[0014] [Figure 1A] We present feasibility studies of preferred chelating dyes (5-Br-PAPS, PAR, and Zincon) and their complexation with preferred metal ions (Co2+, Cu2+, Fe2+, Ni2+, Rh2+, and Zn2+) in the presence of Mg2+. Significant color changes (Figure 1A-B), indicating complexation, were observed for almost all dye-metal ion combinations. Spectrophotometric analysis further demonstrated that complexes of the preferred dye, 5-Br-PAPS, exhibited the highest average absorbance values ​​at the same dye concentration (Figure 1C). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A] The stability of 5-Br-PAPS complexes with preferred metal ions is shown with respect to pyrophosphate (PPi) concentration. Both the visible (Figure 2A) and spectral (Figure 2B) changes indicated that increasing concentrations of PPi (0–20 mM, from left to right) could destabilize the complex and lead to an observable color change. The 5-Br-PAPS complex with Zn2+ was overwhelmingly sensitive to PPi, highlighting its potential for detecting PPi during ongoing nucleic acid amplification reactions. [Figure 2B] See legend to Figure 2A. [Figure 3A] The proposed mechanism behind the color change of the Zn2+ / 5-Br-PAPS detection reagent during the amplification reaction (Figure 3A) and supporting data are shown. Specifically, spectrophotometric data (Figure 3B) and visual data (Figure 3C) both show that either 25 μM or 50 μM of the reagent changes color from magenta to orange-yellow (left to right / darker to lighter) in a manner dependent on the concentration of pyrophosphate (PPi), which is normally produced by the action of DNA polymerase to incorporate nucleotides into nucleic acid chains. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4] The color transition of the Zn2+ / 5-Br-PAPS detection reagent before and after the LAMP amplification reaction is shown. Human RPP30 double-stranded (ds) DNA (Figure 4A) and SARS-CoV-2 N gene single-stranded (ss) RNA (Figure 4B) were amplified using LAMP and RT-LAMP, respectively, with either 25 μM or 50 μM Zn2+ / 5-Br-PAPS detection reagent in the reaction mix. After 30 minutes, a clear color change could be observed in samples with Bst 2.0 polymerase, regardless of the type of template used, demonstrating that the color change is specific to the amplification of the nucleic acid template and is not due to heating. [Figure 5] The time course of the color transition of the Zn2+ / 5-Br-PAPS detection reagent is shown. Here, human RPP30 dsDNA was amplified over a 90-minute period using LAMP. A gradual color change from magenta to red to orange-yellow was observed only in samples containing template dsDNA. Positively amplified samples were easily distinguishable from non-targeted (NTC) samples, even before the LAMP reaction was complete. Even though the color transition was complete at 25 minutes, the detection reagent retained its color throughout the entire 90 minutes for both NTC and template-containing samples. [Figure 6] We demonstrate the effect of Zn2+ / 5-Br-PAPS detection reagent on key LAMP amplification parameters. Here, we performed 45-minute RT-LAMP reactions targeting the SARS-CoV-2 RdRP gene with or without Zn2+ / 5-Br-PAPS detection reagent. A wide range of starting template amounts was used, and amplification was monitored fluorescently with the nucleic acid intercalating dye SYTO 59. The inclusion of 50 μM Zn2+ / 5-Br-PAPS detection reagent did not significantly alter the amplification curve (Figure 6A), while the time-to-reaction (TTR) was only slightly prolonged (Figure 6B). [Figure 7]Annotated images of the same experiment as described in Figures 6A-B. After LAMP amplification, in reactions with Zn2+ / 5-Br-PAPS detection reagent, the color of the NTC reaction remained magenta, while all samples changed color to orange-yellow regardless of the amount of starting template. [Figure 8] Figure 1 shows the performance of the Zn2+ / 5-Br-PAPS detection reagent using PCR amplification. After 45 cycles, reactions containing 25 μM Zn2+ / 5-Br-PAPS detection reagent changed color from magenta to orange-yellow only in samples containing dsDNA template followed by amplification of human RPP30. [Figure 9] Figure 9 shows the LAMP amplification performance of master mixes containing either Zn2+ / 5-Br-PAPS, phenol red (a pH-sensitive dye), or 5-Br-PAPS alone. Human RPP30 was amplified using a 30-minute LAMP reaction, with amplification monitored in real time with the nucleic acid intercalating dye SYTO 59. All variants showed amplification regardless of Tris-HCl concentration (Figure 9A), but higher Tris-HCl concentrations showed shorter reaction time (TTR, Figure 9B) for both Zn2+ / 5-Br-PAPS and phenol red. [Figure 10] Figure 9A-B shows an annotated image of the same experiment. Unlike pH-dependent detection exemplified by using phenol red as a colorimetric detector (metal indicator), the Zn2+ / 5-Br-PAPS detection reagent still changed color to orange-yellow upon successful LAMP amplification, even in the presence of up to 20 mM Tris-HCl (pH = 7.9). This color change was dependent on the presence of Zn2+, as 5-Br-PAPS itself did not change color. [Figure 11]We demonstrate that the sodium pyrophosphate-induced color transition of the Zn2+ / 5-Br-PAPS detection reagent is preserved upon addition of different pH buffers. Here, pseudo-LAMP samples were prepared such that samples without pyrophosphate (PPi) represented negative / pre-amplification samples, and samples with pyrophosphate mimicked samples where amplification had been performed. Both spectrophotometric data (Figure 11A) and visual data (Figure 11B) demonstrate that the behavior of the Zn2+ / 5-Br-PAPS detection reagent is unchanged upon addition of 25% (v / v) 10 mM buffer with various pH values. [Figure 12A] This figure shows the performance of the Zn2+ / 5-Br-PAPS detection reagent in LAMP amplification reactions using input samples buffered to different pH levels. A 30-minute LAMP amplification reaction was performed using a primer set targeting human RPP30 genomic DNA (gDNA), with amplification monitored in real time with the nucleic acid intercalating dye SYTO 59. Samples with templates prepared in different pH buffers did not negatively affect amplification (Figure 12A), reaction time to reaction (TTR, Figure 12B), or the resulting color change (Figure 12C). [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13]We demonstrate that LNA modification of the outer F3 / B3 primers in LAMP leads to a universal improvement in LAMP reaction performance. Four different F3 / B3 primer sets targeting three distinct SARS-CoV-2 genes demonstrate faster reaction kinetics (shorter reaction time, TTR), smaller amplification variability (standard deviation, SD; data point spread), and increased sensitivity (more replicates amplified). The numbers in parentheses indicate the number of replicates amplified out of the total number of replicates. The dotted line represents the average TTR without the LNA primer set; data points above and below the dotted line are slower and faster than the average TTR without the LNA primer set, respectively. E1 and E2, SARS-CoV-2 envelope gene primer sets 1 and 2; R1, SARS-CoV-2 RNA-dependent RNA polymerase gene primer set 1; N1, SARS-CoV-2 nucleoprotein gene primer set 1; NTC, no template control. [Figure 14] Multiple combinations of different modified F3 / B3 primers have been shown to lead to improved LAMP amplification, as indicated by faster turnaround times (TTR), reduced amplification variability (standard deviation, SD), and, in some cases, enhanced sensitivity. RT-LAMP was used to amplify a region in the SARS-CoV-2 E gene. The base sequences of the F3 and B3 primers tested were identical; only the position / number of LNA substitutions differed. E3, SARS-CoV-2 envelope gene primer set 3. [Figure 15] We demonstrate that the F3 / B3 primers are the only primers that respond favorably to the introduction of LNA modifications. We modified the FIP, BIP, LF, and LB primers in the same manner as the F3 / B3 primers, and used these modified sets to amplify pairs of SARS-CoV-2 E genes. All tested variant sets showed reduced assay performance, as evidenced by increased variability (standard deviation, SD), slower reaction time (TTR), and nonspecific amplification. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention The invention is described in detail below and illustrated by the accompanying examples and figures.

[0016] LAMP itself has already been described, for example, in EP 1020534 B2, which is incorporated herein by reference. However, the prior art has failed to demonstrate the successful application of locked nucleic acid (LNA) primers in LAMP. However, the present inventors have surprisingly found that the use of locked nucleic acid (LNA) in the F3 / B3 primer further improves LAMP. As shown in Example 5, the use of LNA in the F3 / B3 primer shortens the reaction time and improves reproducibility and sensitivity. Interestingly, this effect only exists when the LNA is located within the first third of the F3 or B3 primer.

[0017] Therefore, the present invention provides (i) a first inner primer (FIP) nucleotide sequence and a second inner primer (BIP) nucleotide sequence; (ii) a first outer primer (F3) nucleotide sequence and a second outer primer (B3) nucleotide sequence, wherein the F3 nucleotide sequence and / or the B3 nucleotide sequence comprises one or more locked nucleic acid (LNA) nucleotides located within the first third of the F3 nucleotide sequence or the B3 nucleotide sequence, respectively; (iii) a DNA polymerase that catalyzes a strand displacement reaction that synthesizes a complementary nucleic acid strand from a template nucleic acid; and (iv) a nucleotide that serves as a substrate for the DNA polymerase In a reaction mixture comprising at a temperature at which at least the FIP nucleotide sequence and the BIP nucleotide sequence can form stable base pairs with their complementary nucleotide sequences contained in the template nucleic acid, while maintaining DNA polymerase activity; The present invention relates to the LAMP method, which includes a step of synthesizing a nucleic acid sequence from a template nucleic acid sequence, thereby synthesizing a nucleic acid sequence.

[0018] In one embodiment, the F3 nucleotide sequence comprises one or more LNA nucleotides. In a further embodiment, the B3 nucleotide sequence comprises one or more LNA nucleotides. In a further embodiment, the F3 nucleotide sequence and the B3 nucleotide sequence comprise one or more LNA nucleotides.

[0019] "Loop-mediated isothermal amplification" (LAMP), as used herein, refers to a (single-tube) technique for amplifying nucleic acids. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP with (parallel) reverse transcription to enable RNA detection. LAMP is an isothermal nucleic acid amplification technique. In contrast to the polymerase chain reaction (PCR) technique, in which the reaction is carried out by alternating a series of temperature steps or cycles, isothermal amplification is carried out at a constant temperature and does not require a thermal cycler. LAMP is known to those skilled in the art. In this regard, we refer, by way of example, to EP 1 020 534 B2, which describes LAMP in detail. In particular, we refer to paragraphs

[0038] to

[0054] , which are incorporated by reference, including the figures cited in said paragraphs of EP 1 020 534 B2.

[0020] Locked nucleic acid (LNA), also known as bridged nucleic acid (BNA) and often referred to as inaccessible RNA, as used herein, refers to a modified RNA or DNA, preferably a DNA, nucleotide in which the ribose moiety is modified with an additional bridge connecting the 2' oxygen and the 4' carbon. This bridge "locks" the ribose in a 3'-endo (north) conformation. This bridge is a methyl group covalently attached to the 2' and 4' oxygens, thereby bridging both oxygen atoms. LNAs are commercially available, and their synthesis methods are illustratively described in Obika et al. (1997), Tetrahedron Letters, 38(50):8735-8738. LNA modified bases include adenine (+A), cytosine (+C), guanine (+G), thymine (+T), and uracil (+U), preferably +A and +T. Preferably, the F3 nucleotide sequence comprises 1 to 5, preferably 3 LNA nucleotides. Preferably, the B3 nucleotide sequence comprises 1 to 5, preferably 3 LNA nucleotides.

[0021] Nucleic acids synthesized by the LAMP method of the present invention can result in nucleic acids having complementary nucleotide sequences linked alternately in a single strand. Such nucleic acids can have mutually complementary nucleotide sequences linked side by side in a single strand. Furthermore, in the present invention, the nucleic acid can contain a nucleotide sequence for forming a loop between complementary strands. In the present invention, this sequence is referred to as a loop-forming sequence. Nucleic acids synthesized by the present invention essentially consist of mutually complementary strands linked via a loop-forming sequence. Generally, a strand that is not separated into two or more molecules upon dissociation of base pairing is called a single-stranded strand, regardless of whether it is partially base-paired or not. Complementary nucleotide sequences can form base pairs within the same strand. The intramolecular base-paired product obtainable by allowing nucleic acids having complementary nucleotide sequences linked alternately in a single strand according to the present invention to base-pair within the same strand results in a region that constitutes an apparent double strand and a loop without base pairing.

[0022] That is, a nucleic acid having complementary nucleotide sequences alternately linked in a single strand according to the present invention can be defined as a single-stranded nucleic acid containing complementary nucleotide sequences capable of annealing in the same strand, and the annealing product forming a loop without base pairing at the curved hinge junction. Nucleotides having a nucleotide sequence complementary to themselves can anneal to form a loop without base pairing. The loop-forming sequence can be any nucleotide sequence. The loop-forming sequence is capable of base pairing to initiate synthesis of a complementary strand for replacement, and preferably has a sequence distinguishable from nucleotide sequences located in other regions to achieve specific annealing. For example, in the present invention, the loop-forming sequence contains substantially the same nucleotide sequence as the region F2c (or B2c) derived from the template nucleic acid and located 3' to the region (i.e., F1c or B1c) that anneals in the same strand.

[0023] In the present invention, a substantially identical nucleotide sequence is defined as follows: if a complementary strand synthesized using a specific sequence as a template anneals to a target nucleotide sequence to provide a starting point for synthesis of a complementary strand, then the specific sequence is substantially identical to the target nucleotide sequence. For example, a sequence substantially identical to F2 includes not only a nucleotide sequence identical to F2, but also a nucleotide sequence that can function as a template to provide a nucleotide sequence that can anneal to F2 and act as a starting point for synthesis of a complementary strand. In the present invention, the term "annealing" refers to the formation of a double-stranded nucleic acid structure through base pairing according to the Watson-Crick base pairing rules. Therefore, even if the nucleic acid strand forming the base pairing is single-stranded, annealing occurs if complementary nucleotide sequences within the molecule are base-paired. In the present invention, annealing and hybridization have the same meaning in that a nucleic acid forms a double-stranded structure through base pairing.

[0024] The number of pairs of complementary nucleotide sequences constituting the nucleic acid of the present invention is at least one. According to a desired embodiment of the present invention, it can be two or more. In this case, there is theoretically no upper limit to the number of pairs of complementary nucleotide sequences constituting the nucleic acid. When the nucleic acid as a synthetic product of the present invention is composed of multiple pairs of complementary nucleotide sequences, this nucleic acid consists of repeated identical nucleotide sequences.

[0025] Nucleic acids synthesized by the present invention (having complementary nucleotide sequences alternately linked in a single strand) may not have the same structure as naturally occurring nucleic acids. It is known that nucleic acid derivatives can be synthesized by using nucleotide derivatives as substrates when nucleic acids are synthesized by the action of DNA polymerase. The nucleotide derivatives used may include nucleotides labeled with radioisotopes or nucleotide derivatives labeled with binding ligands such as biotin or digoxigenin. These nucleotide derivatives can be used to label the product nucleic acid derivative. Alternatively, if fluorescent nucleotides are used as substrates, the product nucleic acid can be a fluorescent derivative. Furthermore, the product can be either DNA or RNA. Which is formed depends on the combination of the structure of the primer, the type of polymerization substrate, and the composition of the polymerization reagent used to polymerize the nucleic acid.

[0026] Synthesis of a nucleic acid having the above-described structure can be initiated by using a DNA polymerase with strand displacement activity and a nucleic acid that has a region F1 at its 3' end that can anneal to the F1c site in the same strand and that can form a loop containing a base-pairing region F2c when region F1 anneals to F1c. Many reports have been published on reactions for synthesizing a complementary strand, in which a hairpin loop is formed and the sample sequence itself is used as a template. However, in the present invention, the hairpin loop portion contains a base-pairing region, and the use of this region in synthesizing a complementary strand is a novel feature. By using this region as the origin of synthesis, the complementary strand previously synthesized using the sample sequence itself as a template is displaced. Subsequently, region B1c (an arbitrary region) located at the 3' end of the displaced strand becomes ready for base pairing. The region having a sequence complementary to B1c anneals to B1c, resulting in the formation of a nucleic acid (two molecules) having a nucleotide sequence extending from F1 to B1c and its complementary strand alternately linked via a loop-forming sequence. In the present invention, any region such as B1c described above can be selected arbitrarily, provided that it can be annealed to a polynucleotide having a nucleotide sequence complementary to that region, and that the complementary strand synthesized using that polynucleotide as the origin of synthesis has the required function for the present invention.

[0027] The term "nucleic acid" is used in the present invention. Nucleic acids in the present invention generally include both DNA and RNA. However, nucleic acids in which nucleotides are replaced by artificial derivatives or modified nucleic acids derived from natural DNA or RNA are also included in the present invention, as long as they function as templates for the synthesis of complementary strands. Nucleic acids of the present invention can be contained in biological samples. Biological samples include tissues, cells, cultures, and excretions of animals, plants, or microorganisms, or extracts thereof. Biological samples of the present invention can contain genomic DNA or RNA of viruses or intracellular parasites such as mycoplasma. An exemplary virus is SAR-CoV-2. Nucleic acids of the present invention can be derived from nucleic acids contained in the biological sample. For example, cDNA synthesized from mRNA or nucleic acids amplified based on nucleic acids derived from a biological sample are typical examples of nucleic acids of the present invention. The nucleic acid can be DNA.

[0028] The term "template" as used in the present invention can refer to a nucleic acid that serves as a template for synthesizing a complementary strand. A complementary strand having a nucleotide sequence complementary to that of a template is related to the strand corresponding to the template, but the relationship between the two is merely relative. That is, a strand synthesized as a complementary strand can again function as a template. That is, a complementary strand can become a template. Preferably, the template nucleic acid sequence is single-stranded or double-stranded. Preferably, the template nucleic acid sequence is single-stranded or double-stranded DNA, RNA, or DNA / RNA chimera. In the context of RT-LAMP, the template is preferably RNA.

[0029] Preferably, the FIP nucleotide sequence comprises at least two regions, F2 and F1c, wherein the F1c region is linked to the 5' side of the F2 region, the F2 region has a nucleotide sequence complementary to an arbitrary region F2c in the template nucleic acid sequence, and the F1c region has substantially the same nucleotide sequence as the region F1c located 5' side of the F2c region in the template nucleic acid sequence.

[0030] Preferably, the BIP nucleotide sequence comprises at least two regions, B2 and B1c, wherein the B1c region is linked to the 5' side of the B2 region, the B2 region has a nucleotide sequence complementary to an arbitrary region B2c in the template nucleic acid sequence strand, and the B1c region has substantially the same nucleotide sequence as a region B1c located 5' to the region B2c in the template nucleic acid sequence.

[0031] Preferably, the F3 nucleotide sequence has a nucleotide sequence substantially complementary to region F3c in the template nucleic acid sequence, region F3c is located 3' to region F2c in the template nucleic acid sequence, and region F2c is located 3' to region F1c in the template nucleic acid sequence.

[0032] Preferably, the B3 nucleotide sequence has a nucleotide sequence substantially complementary to region B3c in the template nucleic acid sequence, region B3c being located 3' to region B2c in the template nucleic acid sequence, and region B2c being located 3' to region B1c in the template nucleic acid sequence.

[0033] Preferably, the reaction mixture further comprises loop primer nucleotide sequence F and / or loop primer nucleotide sequence B. Nagamine et al. (2002), Mol. Cell. Probes 16(3): 223-229, describe exemplary loop primer nucleotide sequences.

[0034] Preferably, the reaction mixture further comprises stem primer nucleotide sequence F and / or stem primer nucleotide sequence B. Gandelman et al. (2011), Int. J. Mol. Sci. 12: 9108-9124, describe exemplary stem primer nucleotide sequences.

[0035] Preferably, the reaction mixture further comprises a swarm primer nucleotide sequence F1S and / or a swarm primer nucleotide sequence B1S. Martineau et al. (2017), Anal. Chem. 89(1): 625-632 describes exemplary swarm primer nucleotide sequences.

[0036] Preferably, the reaction mixture further comprises at least an additional first inner primer (FIP2) nucleotide sequence and an additional second inner primer (BIP2) nucleotide sequence, both of which are different from the FIP and BIP nucleotide sequences. Wang et al. (2015), Molecules 20: 21515-21531, describes exemplary additional first or second inner primer nucleotide sequences.

[0037] Preferably, the reaction mixture further comprises primer nucleotide sequences for carrying out the method as a multiplex cross-displacement amplification (LAMP). Wang et al. (2015), Sci Rep. 5: 11902, describes an exemplary multiplex cross-displacement amplification (LAMP).

[0038] Preferably, the reaction mixture further comprises a primer nucleotide sequence for carrying out the method as a reverse transcription isothermal multiplex self-matching LAMP. Ding et al. (2014), J. Clin. Microbiol. 52(6): 1862-1870 discloses an exemplary reverse transcription isothermal multiplex self-matching LAMP.

[0039] The method for synthesizing nucleic acids according to the present invention is assisted by a DNA polymerase that catalyzes a strand-displacement reaction for the synthesis of a complementary strand. It is advantageous to use a single type of DNA polymerase. Exemplary and suitable polymerases are listed below. Furthermore, various mutants of these enzymes can be used in the present invention, as long as they have both sequence-dependent activity and strand-displacement activity in the synthesis of a complementary strand. The mutants referred to herein include those that only have a structure that confers the necessary catalytic activity on the enzyme, or those that have been modified in catalytic activity, stability, or thermostability, for example, by amino acid mutation. Exemplary polymerases include Bst DNA polymerase, Bst 2.0 WarmStart® DNA polymerase / Bst 3.0 DNA polymerase (New England Biolabs., Inc.), Saphir Bst2.0 polymerase (Jena Bioscience GmbH), GspSSD LF DNA polymerase / GspSSD2.0 LF DNA polymerase / GspM3.0 LF DNA polymerase (OptiGene Ltd.), Bca (exo-) DNA polymerase, DNA polymerase I Klenow fragment, Vent DNA polymerase, Vent (exo-) DNA polymerase (Vent DNA polymerase with exonuclease activity deleted), Deep Vent DNA polymerase, Deep Vent (exo-) DNA polymerase (Deep Vent DNA polymerase with exonuclease activity deleted), Φ29 phage DNA polymerase, MS-2 phage DNA polymerase, and Z-Taq These include, but are not limited to, DNA polymerase (Takara Shuzo Co., Ltd.), KOD DNA polymerase (Toyobo Co., Ltd.), OmniAmp® and LavaLAMP™ DNA / RNA enzymes (Lucigen Corp.), and SD polymerase (BIORON GmbH).

[0040] Among these enzymes, GspSSD2.0 LF DNA polymerase, LavaLAMP™ DNA / RNA enzyme, SD polymerase, and Bst DNA polymerase, more preferably Bst 2.0 WarmStart® DNA polymerase, are particularly desirable enzymes due to their certain thermostability and high catalytic activity. In a preferred embodiment, the reaction of the present invention can be carried out isothermally. Preferably, the enzyme is thermostable. While isothermal reactions are feasible, thermal denaturation may be performed to provide a nucleic acid as a first template. In this respect, the use of thermostable enzymes also broadens the options for assay protocols.

[0041] Vent (exo-) DNA polymerase is an enzyme that possesses both strand displacement activity and high thermostability. It is known that complementary strand synthesis, which involves strand displacement by DNA polymerase, can be promoted by the addition of a single-stranded binding protein (Paul M. Lizardi et al., Nature Genetics, 19, 225-232, July 1998). This effect can be applied to the present invention, and the addition of a single-stranded binding protein can be expected to promote the synthesis of complementary strands. For example, T4 gene 32 is effective as a single-stranded binding protein for Vent (exo-) DNA polymerase. Preferably, the DNA polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase. Preferably, the DNA polymerase has reverse transcriptase activity.

[0042] Preferably, the F3 nucleotide sequence has a length of 15 to 30, preferably 17 to 25 nucleotides.

[0043] Preferably, the B3 nucleotide sequence has a length of 15 to 30, preferably 17 to 25 nucleotides.

[0044] The LNA-LAMP method of the present invention is preferably carried out in the presence of a buffer that provides a suitable pH for the enzymatic reaction, salts necessary for annealing or maintaining the catalytic activity of the enzyme, enzyme protectors, and, if necessary, melting temperature (Tm) regulators. Exemplary buffers are known to those skilled in the art and include, for example, Tris-HCl or equivalents. The pH is adjusted depending on the DNA polymerase used. The buffer may be added at a concentration of 1 mM or more, for example, greater than 1 mM or 1.5 mM or more. Exemplary salts, such as KCl, NaCl, MgCl, (NH)SO, etc., are appropriately added to maintain enzymatic activity and adjust the melting temperature (Tm) of the nucleic acid. Protective agents for maintaining enzymatic activity include, for example, bovine serum albumin (BSA), tris(2-carboxyethyl)phosphine (TCEP), reducing agents such as (2S,3S)-1,4-bis(sulfanyl)butane-2,3-diol (DTT), and / or sugars. Additionally, dimethyl sulfoxide (DMSO) or formamide can be used as a melting temperature (Tm) adjuster. By using a melting temperature (Tm) adjuster, oligonucleotide annealing can be adjusted under limited temperature conditions. Furthermore, betaine (N,N,N-trimethylglycine) or tetraalkylammonium salts are also effective in improving the efficiency of strand displacement due to their isostabilizing properties. Betaine can be added to the reaction solution at a concentration of 0.2 to 3.0 M, preferably 0.5 to 1.5 M. Therefore, the reaction mixture preferably further contains a melting temperature adjuster, such as betaine, preferably at a concentration of 0.2 to 3.0 M, guanidine thiocyanate or hydrochloride, preferably at a concentration of 20 to 80 mM, and single-stranded binding protein (SSB) and / or tetramethylammonium chloride (TMAC), preferably at a concentration of 5 mM to 80 mM. In addition, the reaction mixture may contain stabilizers, such as BSA, preferably at a concentration of 0.02 mg / ml to 2 mg / ml, a non-ionic detergent such as Tween-20 / Triton X-100, preferably at a concentration of 0.02% to 0.2% v / v, and / or TCEP / DTT, preferably at a concentration of 0.5 mM to 5 mM.

[0045] The methods of the present invention can be used to detect a given template, such as a nucleic acid derived from a pathogen such as SARS-CoV-2. Accordingly, the reaction mixture can further comprise a detection agent for detecting the product of the nucleic acid sequence synthesis reaction.

[0046] The detection agent (for detecting the product of the nucleic acid sequence synthesis reaction) can be a metal indicator. Further examples of the detection agent are DNA intercalators or pH-sensitive dyes, with metal indicators being preferred. As described herein, the metal indicator can form a complex with a metal ion, for example, magnesium. Also as described herein, preferred metal ions are transition metal ions or post-transition metal ions.

[0047] A metal indicator (also referred to herein as a complexometric indicator) is a molecule capable of binding a metal ion, i.e., the indicator forms a complex with the metal ion. Such a complex is referred to herein as a metal indicator-metal ion complex, or simply as a "complex." The complex preferably has colorimetric or fluorescent properties different from those of a non-complexed / uncomplexed metal indicator, i.e., a metal indicator in which no metal ion is present, i.e., not complexed with the metal ion, and therefore in an uncomplexed or uncomplexed state. Thus, the detecting agent preferably includes a transition metal or a post-transition metal. The detecting agent preferably does not complex or form a complex with magnesium.

[0048] Such complexes are reversible, meaning that the metal ion, when bound to the metal indicator, can be / is released from the complex with the metal indicator. Release of the metal ion from the complex can occur if a binding partner for the metal ion is simultaneously present or is added or generated (e.g., through a (chemical) reaction), and if the metal indicator has sufficient concentration and / or affinity for the bound metal ion.

[0049] In the context of the present invention, a preferred metal indicator is provided in a method, use, or kit for detecting, for example, changes in the spectral or fluorescent properties of its complex with a metal ion. Such changes occur when the metal ion disappears or is released from the complex. In the context of the present invention, during the amplification of a nucleic acid sequence, pyrophosphate, which is a binding partner for the metal ion in a complex with the metal indicator, is generated. When the pyrophosphate has sufficient concentration and / or affinity for the metal ion bound to the metal indicator, the metal ion is released from the complex and binds to the pyrophosphate, thereby forming a metal ion-pyrophosphate salt.

[0050] The preferred change in the spectral properties of the complex is in the 380-740 nm spectrum. Thus, the change in the spectral properties of the complex is preferably a change in color of the complex compared to when the metal ion is no longer bound to the metal indicator, i.e., when the metal ion is no longer complexed with the metal indicator or released from the complex. This can occur, for example, after the metal ion is released from the complex in the presence of pyrophosphate, as described above, for example, during amplification of nucleic acid sequences as described herein.

[0051] In contrast to the present invention, the prior art has applied metal indicators, such as hydroxynaphthol blue (HNB), which forms complexes with metallic magnesium, i.e., non-transition / non-post-transition metals, to detect nucleic acid sequences. Magnesium is an important ion for amplification methods (e.g., PCR, LAMP), as it acts as a cofactor and catalyst for enzyme activity and affects primer annealing, DNA stability, and nucleotide incorporation. Therefore, Mg 2+ The use of an ion-dependent colorimetric detection system allows for the detection of free Mg to the enzyme and template. 2+ This is problematic because the level of Mg fluctuates during the reaction. 2+Colorimetric detection systems that rely on magnesium ions prevent independent optimization of the detection system and the amplification reaction (since both are related to the concentration of the same ion). Therefore, it is preferred that the detection agent or metal indicator does not complex with magnesium. Therefore, detection systems that use metal ions that are inert in the reaction are preferred. Suitable metal ions in the context of the present invention, particularly transition metal or post-transition metal ions, are Ba. 2+ , Sr 2+ , Zn 2+ , Cd 2+ , Cu 2+ , Co 2+ , Ni 2+ , Hg 2+ , Pb 2+ , Pt 2+ , Ru 2+ , Rh 2+ , Fe 2+ , In 3+ , Al 3+ , Bi 3+ , La 3+ ,Sc. 3+ , Th 3+ , and / or Zr 3+ ions, but Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ , Fe 2+ ions are preferred, Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ and / or Fe 2+ ions are preferred, Zn 2+ Ions are more preferred.

[0052] Also, in contrast to the present invention, the prior art has applied metal indicators, such as calcein, which forms complexes with the non-transition metal manganese, to detect nucleic acid sequences. Such complexes change their fluorescent properties upon release of the metal ion. Manganese is not typically included in PCR / LAMP reaction buffers, but it is known to have a dramatic effect on amplification and should therefore be avoided. 2+ is Mg in PCR2+ While Mn has been shown to be a good substitute for , it is well known to reduce the fidelity of DNA polymerases, thereby inducing random mutations during PCR. At high concentrations (e.g., above 500 μM), Mn 2+ may also be inhibitory to PCR (calcein / Mn 2+ The detection system was 1 mM Mn 2+ (Use

[0053] In the context of the present invention, a metal indicator is used to bind to a metal ion added to a reaction mixture containing a template nucleic acid sequence, a primer nucleotide sequence, nucleotides, and a polymerase capable of amplifying a nucleic acid molecule. Naturally, the metal indicator and the metal ion are both included in the reaction mixture. Thus, the metal indicator binds to the metal ion to form a complex. Without being bound by theory, during the amplification of the nucleotide sequence, more and more pyrophosphate is released. Subsequently, increasing amounts of pyrophosphate bind to the metal ion to which the metal indicator is bound. As a result, the metal indicator changes its spectral and / or fluorescent properties. This change serves as an indicator of whether the amplification reaction is (successfully) occurring or has occurred.

[0054] Preferably, the pyrophosphate metal ion complex or salt has a higher affinity for the metal ion than the metal indicator. This means that the metal ion equilibrium is preferably shifted toward the pyrophosphate rather than the metal indicator. Alternatively, the pyrophosphate metal ion complex or salt is preferably at a concentration sufficient to shift the metal ion equilibrium toward complexing with the pyrophosphate rather than the metal indicator. This can be easily determined by one skilled in the art.

[0055] Additionally, one skilled in the art can determine the amount of metal indicator and / or metal ion to use in a reaction mixture as described herein so as to detect a desired change in the spectral or fluorescent properties of the metal indicator-metal ion complex, which occurs upon release of the metal ion from the metal indicator-metal ion complex, because more and more pyrophosphate is produced during amplification of a nucleic acid sequence as described herein, and because it is generally known in the art that pyrophosphate is released when a polymerase incorporates a nucleotide into DNA or RNA.

[0056] In the context of the present invention, for example, in the context of the methods, uses, or kits described herein, preferred metal ions are transition metal ions or post-transition metal ions. As used herein, "transition metal" refers to an element selected from the group consisting of Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, La, Hf, Ta, W, Re, Os, Ir, Pt, Au, Th, Rf, Db, Sg, Bh, and Hs. Sr and Ba can be considered as additional elements of the transition metal group. As used herein, "post-transition metal" refers to an element selected from the group consisting of Al, Zn, Ga, Cd, In, Sn, Hg, Tl, Pb, Bi, Po, and At. A preferred example of a metal ion in general, and a preferred example of a transition or post-transition metal ion in particular, is Ba. 2+ , Sr 2+ , Zn 2+ , Cd 2+ , Cu 2+ , Co 2+ , Ni 2+ , Hg 2+ , Pb 2+ , Pt 2+ , Ru 2+ , Rh 2+ , Fe 2+ , In 3+ , Al 3+ , Bi 3+ , La 3+ ,Sc. 3+ , Th 3+ , Zr3+ ions, Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ , Fe 2+ ions are preferred, Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ and Fe 2+ ions are preferred, Zn 2+ Ions are more preferred.

[0057] Preferred examples of metal indicators are those capable of binding to metal ions, preferably transition metal ions or post-transition metal ions. Examples of preferred metal indicators include pyrocatechol violet, dithizone, zincon, eriochrome black T, murexide, PAN, phthalein purple, xylenol orange, and 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS). More preferred examples of metal indicators include pyrocatechol violet, zincon, dithizone, PAN, and 5-Br-PAPS. Even more preferred examples of metal indicators include zincon and 5-Br-PAPS. 5-Br-PAPS is the most preferred.

[0058] The structures of the more preferred metal indicators Zincon and Zincon derivatives are depicted below. In the case of Zincon derivatives, R1, R2, and R3 can represent one or more functional groups attached to the corresponding phenyl ring: TIFF0007801330000001.tif32134

[0059] The structures of the more preferred metal indicators PAN and PAR and PAN / PAR derivatives are depicted below. In the case of PAN / PAR derivatives, R can represent one or more functional groups attached to the phenol ring: TIFF0007801330000002.tif24145

[0060] The term "5-Br-PAPS" also encompasses, and is also preferred, derivatives of 5-Br-PAPS, which may have one or more modifications but are still capable of forming complexes with metal ions and having the same or at least similar spectral properties as 5-Br-PAPS when bound to metal ions. Exemplary 5-Br-PAPS (X=Br) derivatives are 2-(5-fluoro-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (X=F), 2-(5-chloro-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (X=Cl), 2-(5-iodo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (X=I), 2-(5-nitro-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (X=NO), or 2-(5-cyano-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (X=CN). The following structure shows the PAPS backbone in its disodium salt anhydrous form, where X may be substituted as described above: TIFF0007801330000003.tif24128

[0061] As exemplified in the following structures, X may be substituted as described above or may occupy other positions on the pyridine ring (e.g., positions 3-6) as shown in the PAPS derivatives below: TIFF0007801330000004.tif38128

[0062] Table 1 below shows some additional exemplary metal indicators and their changes in spectral properties with and without metal ions.

[0063] Table 1. Exemplary metal indicators and their corresponding changes in spectral properties with and without metal ions. TIFF0007801330000005.tif69165

[0064] Table 2 below shows the visible and spectral properties of more preferred metal indicators, with and without preferred metal ions in the presence of magnesium ions.

[0065] Table 2. Visible and spectral properties of preferred metal indicators, with and without preferred metal ions in the presence of magnesium ions. TIFF0007801330000006.tif63163

[0066] For example, in one embodiment of the LAMP method of the present invention, the detection reagent comprises a metal indicator, preferably 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS), and a metal ion, preferably a transition metal or post-transition metal, more preferably Zn. 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, most preferably Zn 2+ ion, wherein the metal indicator and the metal ion each form a complex, but not with magnesium, and the method comprises: (b) detecting a change in the spectral or fluorescent properties of the complex resulting from amplification of said nucleic acid sequence. Further includes:

[0067] For example, in one embodiment of the LAMP method of the present invention, the detection agent is a combination of 5-Br-PAPS and a metal ion. The metal ion in this context is Zn, which is chelated by both 5-Br-PAPS and pyrophosphate. 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, preferably Zn 2+The reaction mixture may contain any transition or post-transition metal, including, but not limited to, ions. Thus, for example, the reaction mixture used in the method of the present invention may contain 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS) and Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, preferably Zn 2+ ions, and the 5-Br-PAPS and the ions each form a complex. In this regard, the method of the present invention may further comprise: (b) detecting a change in the spectral or fluorescent properties of the complex resulting from amplification of said nucleic acid sequence. Further includes:

[0068] 5-Br-PAPS is a Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ These compounds have the ability to bind with high affinity to transition or post-transition metals, including but not limited to ions, thereby forming complexes that lead to a color change or, in other words, a change in spectral or fluorescent properties. 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, preferably Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ and Fe 2+ ions, more preferably Zn 2+ If the metal ion is Zn, the reaction mixture will develop a color.2+ If the reaction mixture is magenta, the color of the reaction mixture will be magenta (see also FIG. 1). Without being bound by theory, the synthesis reaction of the LNA-LAMP method of the present invention or any other nucleic acid-producing polymerase (chain) reaction generates pyrophosphate (PP) from triphosphate nucleotides (e.g., ATP, GTP, CTP, TTP, UTP, etc.) during the extension reaction. i ) leads to the release of PP i Also, Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, preferably Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ and Fe 2+ ions, more preferably Zn 2+ 5-Br-PAPS and Zn are chelating agents for metal ions, including but not limited to transition and / or post-transition metal ions. 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, preferably Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ and Fe 2+ ions, more preferably Zn 2+ The complex with the ion can be disrupted, leading to a "color transition" of the reaction mixture from magenta to orange-yellow (see Examples 1-4) and changing the spectral or fluorescent properties of the complex. Thus, if the template to be verified is present, an extension reaction will occur and PP i This leads to the release of PP i Then, the Zn complexed with 5-Br-PAPS was 2+ , Cu 2+ , Co 2+ , Ni2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, preferably Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ and Fe 2+ ions, more preferably Zn 2+ ions. A corresponding color change in spectral or fluorescent properties can be used as an indicator of the presence of the template. Specifically, the metal ion is Zn. 2+ In this case, increasing the PPi concentration weakens the absorption peak at 560 nm (±10 nm) of the reaction mixture ("magenta") while simultaneously enhancing a new peak at 448 nm (±10 nm) ("orange-yellow"). This color transition can be monitored in real time.

[0069] In this regard, the present invention further comprises: (a) contacting a reaction mixture comprising a template nucleic acid sequence, a primer nucleotide sequence, nucleotides, and a polymerase capable of amplifying a nucleic acid molecule with a metal indicator and a metal ion, wherein the metal indicator and the ion form a complex; (b) amplifying the nucleic acid sequence under suitable conditions to obtain a nucleic acid sequence amplification product; (c) detecting a change in the spectral or fluorescent properties of the complex resulting from amplification of said nucleic acid sequence. The present invention relates to an in vitro method for detecting nucleic acid sequence amplification products, comprising:

[0070] Preferably, the metal indicator is 5-Br-PAPS. Preferably, the metal ion is a transition metal ion or a post-transition metal ion, preferably Zn. 2+ions. Preferably, the change in the spectral properties of the complex is a change in the spectrum between 380 and 740 nm wavelengths. More preferably, the spectral change can be monitored at 560 nm (±10 nm) ("magenta") and / or 448 nm (±10 nm) ("orange-yellow"). In other words, if a nucleic acid amplification product is generated in an in vitro method for detecting nucleic acid sequence amplification products or other methods, uses, and kits described herein, a change in the spectral properties of the reaction mixture from magenta to orange-yellow can be visually observed.

[0071] Importantly, the changes in the spectral or fluorescent properties of the (5-Br-PAPS) complex are independent of changes in the pH of the reaction mixture (see Example 4, Figures 7 and 8). 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, preferably Zn 2+ The reaction mixture is then heated to 1000°C, which is then heated to 100°C. i The reaction mixture depends on the amount of Tris (tris(hydroxymethyl)aminomethane) buffer or an equivalent buffer, but preferably does not depend on pH. This is in contrast to the methods described in the prior art, such as those shown in EP2888374B1. As a result, the reaction mixture may contain 1 mM or more Tris (tris(hydroxymethyl)aminomethane) buffer or an equivalent buffer, for example, more than 1 mM, more than 1.5 mM, more than 5 mM, more than 10 mM, more than 50 mM, more than 100 mM, or more than 250 mM Tris buffer or an equivalent buffer. In this regard, pH-sensitive polymerases may also be used.

[0072] The nucleic acid sequence amplification product of step (b) in the in vitro method for detecting a nucleic acid sequence amplification product can be obtained by any nucleic acid amplification method, including but not limited to polymerase chain reaction (PCR), LAMP, LNA-LAMP, nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), and helicase-dependent amplification (HAD), preferably PCR or LNA-LAMP. Step (b) of the in vitro method for detecting a (template) nucleic acid sequence amplification product can be performed by implementing the LNA-LAMP method of the present invention.

[0073] Detection of changes in the spectral properties of the (5-Br-PAPS) complexes can be achieved by their photochemical properties, for example, using an operator's eye, a fluorometer, or a spectrophotometer. The term "detecting" can be used interchangeably with the term "monitoring."

[0074] A metal indicator, preferably 5-Br-PAPS, and a metal ion, preferably Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, preferably Zn 2+ The final molar ratio of ions to ions in aqueous solution can be 2:1. 2+ / 5-Br-PAPS complex stoichiometry. 2+ The / 5-Br-PAPS complex may be added to a final concentration of about 1 to about 250 μM, about 10 to about 100 μM, about 15 to about 75 μM, about 25 to about 75 μM, about 25 μM, or about 50 μM.

[0075] The LAMP method of the present invention can be used for different purposes. In one embodiment, the LAMP method of the present invention is for synthesizing a nucleic acid sequence. In one embodiment, the LAMP method of the present invention is for detecting a nucleic acid sequence. In one embodiment, the LAMP method of the present invention is for diagnosing a disease, preferably a disease caused by a pathogen.

[0076] The present invention further comprises: (i) a first outer primer (F3) nucleotide sequence and a second outer primer (B3) nucleotide sequence, each of which comprises a locked nucleic acid (LNA) nucleotide; and / or, optionally (ii) a first inner primer (FIP) nucleotide sequence and a second inner primer (BIP) nucleotide sequence, and / or optionally (iii) a DNA polymerase that catalyzes a strand displacement reaction to synthesize a complementary nucleic acid strand from a template nucleic acid, and / or optionally (iv) a nucleotide that serves as a substrate for the DNA polymerase The present invention relates to a kit for synthesizing a nucleic acid sequence by loop-mediated isothermal amplification (LAMP) on a template nucleic acid sequence, comprising:

[0077] The F3 nucleotide sequence and / or the B3 nucleotide sequence may comprise one or more locked nucleic acid (LNA) nucleotides located within the first third of the F3 nucleotide sequence or the B3 nucleotide sequence. The F3 nucleotide sequence may comprise one or more locked nucleic acid (LNA) nucleotides located within the first third of the F3 nucleotide sequence. The B3 nucleotide sequence may comprise one or more locked nucleic acid (LNA) nucleotides located within the first third of the B3 nucleotide sequence. As used herein, "first third" may refer to the first third of the nucleotide sequence in the 5' to 3' direction, i.e., the one-third of the nucleotides of the nucleotide sequence that are at the 5' end of the nucleotide sequence.

[0078] 5-Br-PAPS and Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, preferably Zn 2+ The ions are further useful components of the kit, since they can be used to detect the presence of a template nucleic acid sequence. Thus, the kit of the present invention includes (v) 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS); and optionally (vi) Zn 2+ , Cu 2+ , Co 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, preferably Zn 2+ The kits of the present invention may further comprise a metal indicator, preferably 5-Br-PAPS; and / or a metal ion, preferably Zn. 2+ It may further comprise ions.

[0079] The present disclosure further relates to an aqueous preparation comprising 5-Br-PAPS, (DNA) polymerase, and dNTPs in a formulation containing a buffer of 1 mM or more Tris or an equivalent buffer, for example, more than 1 mM, more than 1.5 mM, more than 5 mM, more than 10 mM, more than 50 mM, more than 100 mM, or more than 250 mM Tris buffer or an equivalent buffer. The aqueous preparation of the present invention may further comprise a primer, preferably the primer is the F3 nucleotide sequence and / or the B3 nucleotide sequence as defined herein. The aqueous preparation may further comprise a nucleotide that serves as a substrate for the DNA polymerase. The aqueous preparation of the present invention may further comprise a Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh2+ and / or Fe 2+ ions, preferably Zn 2+ It may further comprise ions.

[0080] The present disclosure further relates to an aqueous preparation comprising a first outer primer (F3) nucleotide sequence and a second outer primer (B3) nucleotide sequence as defined herein. The aqueous preparation may further comprise a first inner primer (FIP) nucleotide sequence and a second inner primer (BIP) nucleotide sequence as defined herein. The aqueous preparation may further comprise a DNA polymerase that catalyzes a strand displacement reaction to synthesize a complementary nucleic acid strand from the template nucleic acid. The aqueous preparation may further comprise a nucleotide that serves as a substrate for the DNA polymerase.

[0081] The present invention further relates to the following items:

[0082] 1. (i) a first inner primer (FIP) nucleotide sequence and a second inner primer (BIP) nucleotide sequence; (ii) a first outer primer (F3) nucleotide sequence and a second outer primer (B3) nucleotide sequence, wherein the F3 nucleotide sequence and / or the B3 nucleotide sequence comprises one or more locked nucleic acid (LNA) nucleotides located within the first third of the F3 nucleotide sequence or the B3 nucleotide sequence, respectively; (iii) a DNA polymerase that catalyzes a strand displacement reaction that synthesizes a complementary nucleic acid strand from a template nucleic acid; and (iv) a nucleotide that serves as a substrate for the DNA polymerase In a reaction mixture comprising at a temperature at which at least the FIP nucleotide sequence and the BIP nucleotide sequence can form stable base pairs with their complementary nucleotide sequences contained in the template nucleic acid, while maintaining DNA polymerase activity; A loop-mediated isothermal amplification (LAMP) method includes a step of synthesizing a nucleic acid sequence from a template nucleic acid sequence, thereby synthesizing a nucleic acid sequence.

[0083] 2. The FIP nucleotide sequence comprises at least two regions, F2 and F1c; the F1c region is linked to the 5' side of the F2 region, the F2 region has a nucleotide sequence complementary to an optional region F2c in the template nucleic acid sequence; and the F1c region has substantially the same nucleotide sequence as the F1c region located 5' to the F2c region in the template nucleic acid sequence; 10. The method of any one of the preceding items.

[0084] 3. The BIP nucleotide sequence comprises at least two regions, B2 and B1c; the B1c region is linked to the 5' side of the B2 region, the B2 region has a nucleotide sequence complementary to an arbitrary region B2c in the template nucleic acid sequence strand; and the B1c region has substantially the same nucleotide sequence as the region B1c located 5' to the region B2c in the template nucleic acid sequence; 10. The method of any one of the preceding items.

[0085] 4. the F3 nucleotide sequence has a nucleotide sequence substantially complementary to a region F3c in the template nucleic acid sequence; a region F3c is located on the 3' side of the region F2c in the template nucleic acid sequence; the region F2c is located on the 3' side of the region F1c in the template nucleic acid sequence; 10. The method of any one of the preceding items.

[0086] 5. the B3 nucleotide sequence has a nucleotide sequence substantially complementary to region B3c in the template nucleic acid sequence; a region B3c is located on the 3' side of the region B2c in the template nucleic acid sequence; the region B2c is located on the 3' side of the region B1c in the template nucleic acid sequence; 10. The method of any one of the preceding items.

[0087] 6. The method of any one of the preceding items, wherein the reaction mixture further comprises loop primer nucleotide sequence F and / or loop primer nucleotide sequence B.

[0088] 7. The method of any one of the preceding items, wherein the reaction mixture further comprises stem primer nucleotide sequence F and / or stem primer nucleotide sequence B.

[0089] 8. The method of any one of the preceding items, wherein the reaction mixture further comprises swarm primer nucleotide sequence F1S and / or swarm primer nucleotide sequence B1S.

[0090] 9. The method of any one of the preceding items, wherein the reaction mixture further comprises at least an additional first inner primer (FIP2) nucleotide sequence and an additional second inner primer (BIP2) nucleotide sequence, both of which are different from the FIP and BIP nucleotide sequences.

[0091] 10. The method of any one of the preceding items, wherein the reaction mixture further comprises primer nucleotide sequences for carrying out the method as a multiplex cross-displacement amplification (LAMP).

[0092] 11. The method of any one of the preceding items, wherein the reaction mixture further comprises primer nucleotide sequences for carrying out the method as reverse transcription isothermal multiplex self-matching LAMP.

[0093] 12. The method of any one of the preceding items, wherein the template nucleic acid sequence is single-stranded or double-stranded.

[0094] 13. The method of any one of the preceding items, wherein the template nucleic acid sequence is single-stranded or double-stranded DNA, RNA, or a DNA / RNA chimera.

[0095] 14. The method of any one of the preceding items, wherein the DNA polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.

[0096] 15. The method of any one of the preceding paragraphs, wherein the DNA polymerase has reverse transcriptase activity.

[0097] 16. The method of any one of the preceding items, wherein the F3 nucleotide sequence has a length of 15 to 30, preferably 17 to 25, nucleotides.

[0098] 17. The method of any one of the preceding items, wherein the B3 nucleotide sequence has a length of 15 to 30, preferably 17 to 25, nucleotides.

[0099] 18. The method of any one of the preceding items, wherein the F3 nucleotide sequence comprises 1 to 5, preferably 3, LNA nucleotides.

[0100] 19. The method of any one of the preceding items, wherein the B3 nucleotide sequence comprises 1 to 5, preferably 3, LNA nucleotides.

[0101] 20. The method of any one of the preceding items, wherein the reaction mixture further comprises a melting temperature regulator, for example, betaine, preferably at a concentration of 0.2M to 3.0M, Tween-20 / Triton-X, preferably at a concentration of 0.02% to 0.2%, guanidine thiocyanate or hydrochloride, preferably at a concentration of 20mM to 80mM, single-stranded binding protein (SSB), BSA, preferably at a concentration of 0.02mg / ml to 2mg / ml, TMAC, preferably at a concentration of 5mM to 80mM, or TCEP / DTT, preferably at a concentration of 0.5mM to 5mM.

[0102] 21. The method of any one of the preceding items, wherein the reaction mixture further comprises a detection agent for detecting the product of the nucleic acid sequence synthesis reaction.

[0103] 22. The method according to item 21, wherein the detection reagent is a metal indicator.

[0104] 23. The method according to item 21 or 22, wherein the detection agent further comprises a metal (ion), preferably a transition metal (ion) or a post-transition metal (ion).

[0105] 24. The detection reagent comprises a metal indicator, preferably 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS), and a metal ion, preferably a transition metal or post-transition metal ion, more preferably Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, most preferably Zn 2+ ions, wherein the metal indicator and the metal ion each form a complex, but not with magnesium; and The method comprises: (b) detecting a change in the spectral or fluorescent properties of the complex resulting from amplification of said nucleic acid sequence. 8. The method of any one of the preceding items, further comprising:

[0106] 25. The method of item 24, wherein the reaction mixture further comprises 1 mM or more Tris (tris(hydroxymethyl)aminomethane) buffer or an equivalent buffer, for example, more than 1 mM, more than 1.5 mM, more than 5 mM, more than 10 mM, more than 50 mM, more than 100 mM, or more than 250 mM Tris buffer or an equivalent buffer.

[0107] 26. (a) synthesizing a nucleic acid sequence; (b) detecting a nucleic acid sequence; or (c) Diagnosing diseases caused by pathogens 2. The method of any one of the preceding items, for

[0108] 27. (a) contacting a reaction mixture containing a template nucleic acid sequence, a primer nucleotide sequence, nucleotides, and a polymerase capable of amplifying a nucleic acid molecule with a metal indicator and a metal ion, wherein the metal indicator and the metal ion, the detection agent and the ion form a complex; (b) amplifying the nucleic acid sequence under suitable conditions to obtain a nucleic acid sequence amplification product; (c) detecting a change in the spectral or fluorescent properties of the complex resulting from amplification of said nucleic acid sequence. 1. An in vitro method for detecting a nucleic acid sequence amplification product, comprising:

[0109] 28. The method of item 27, wherein the metal ion is a transition metal ion or a post-transition metal ion, and / or the transition or post-transition metal ion and the detection agent form a complex, but do not form a complex with magnesium.

[0110] 29. (a) contacting a reaction mixture comprising a template nucleic acid sequence, a primer nucleotide sequence, nucleotides, and a polymerase capable of amplifying a nucleic acid molecule with a metal indicator and a transition or post-transition metal ion, wherein the metal indicator and the transition or post-transition metal ion form a complex, but not with magnesium; (b) amplifying the nucleic acid sequence under suitable conditions to obtain a nucleic acid sequence amplification product; (c) detecting a change in the spectral or fluorescent properties of a complex resulting from the amplification of said nucleic acid sequence, preferably detecting a change in the spectral properties of a complex resulting from the amplification of said nucleic acid sequence, wherein the formation of said complex comprising said metal indicator and said transition or post-transition metal ion leads to a color change of the solution. 1. An in vitro method for detecting a nucleic acid sequence amplification product, comprising:

[0111] 30. The method according to any one of items 27 to 29, wherein the metal indicator is 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS).

[0112] 31. The metal ion is Zn 2+ 31. The method according to any one of items 27 to 30, wherein the ion is a cation.

[0113] 32. The method according to any one of items 27 to 31, wherein the change in the spectral properties of the complex is a change in the spectrum of wavelengths from 380 to 740 nm.

[0114] 33. The method of any one of items 27 to 32, wherein the reaction mixture further comprises 1 mM or more Tris (tris(hydroxymethyl)aminomethane) buffer or an equivalent buffer, for example, more than 1.5 mM, more than 5 mM, more than 10 mM, more than 50 mM, more than 100 mM, or more than 250 mM Tris buffer or an equivalent buffer.

[0115] 34. The method of any one of items 27 to 33, wherein the amplification is carried out by the method defined in any one of items 1 to 23.

[0116] 35. (i) a first outer primer (F3) nucleotide sequence and a second outer primer (B3) nucleotide sequence, each of which comprises a locked nucleic acid (LNA) nucleotide; and / or, optionally (ii) a first inner primer (FIP) nucleotide sequence and a second inner primer (BIP) nucleotide sequence, and / or optionally (iii) a DNA polymerase that catalyzes a strand displacement reaction to synthesize a complementary nucleic acid strand from a template nucleic acid, and / or optionally (iv) a nucleotide that serves as a substrate for the DNA polymerase A kit for synthesizing a nucleic acid sequence by loop-mediated isothermal amplification (LAMP) on a template nucleic acid sequence, comprising:

[0117] 36. The kit of item 35, wherein the F3 nucleotide sequence and / or the B3 nucleotide sequence comprises one or more locked nucleic acid (LNA) nucleotides located within the first third of the F3 nucleotide sequence or the B3 nucleotide sequence.

[0118] 37. (v) a metal indicator, preferably 5-Br-PAPS; and optionally (vi) a metal ion, preferably Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, more preferably Zn 2+ ion 37. The kit according to item 35 or 36, further comprising:

[0119] 38. Use of the kit according to any one of items 35 to 37 for carrying out the method according to any one of items 1 to 34.

[0120] 39. An aqueous preparation comprising 5-Br-PAPS, (DNA) polymerase, dNTPs in a formulation containing a buffer in an amount of 1 mM or more of Tris or an equivalent buffer, for example, greater than 1 mM, greater than 1.5 mM, greater than 5 mM, greater than 10 mM, greater than 50 mM, greater than 100 mM, or greater than 250 mM Tris buffer or an equivalent buffer.

[0121] 40. The aqueous preparation according to item 37, further comprising a primer, preferably the primer being the F3 nucleotide sequence and / or the B3 nucleotide sequence as defined in item 36.

[0122] 41. The aqueous preparation according to item 39 or 40, further comprising nucleotides that serve as substrates for the DNA polymerase.

[0123] 42. An aqueous preparation comprising a first outer primer (F3) nucleotide sequence and a second outer primer (B3) nucleotide sequence as defined in item 1 or 36.

[0124] 43. The aqueous preparation according to item 42, further comprising a first inner primer (FIP) nucleotide sequence and a second inner primer (BIP) nucleotide sequence.

[0125] 44. The aqueous preparation according to item 42 or 43, further comprising a DNA polymerase that catalyzes a strand displacement reaction to synthesize a complementary nucleic acid strand from the template nucleic acid.

[0126] 45. The aqueous preparation of any one of items 42 to 44, further comprising nucleotides that serve as substrates for the DNA polymerase.

[0127] 46. ​​The method of any one of the preceding items, comprising a step of detecting a change in the spectral properties of a complex resulting from amplification of the nucleic acid sequence, wherein the formation of the complex comprising the metal indicator and the transition or post-transition metal ion leads to a color change in the solution.

[0128] 47. The method of any one of the preceding items, carried out in an aqueous solution.

[0129] 48. The method of any one of the preceding items, carried out at a pH range of about 7 to about 9, preferably at a pH range of 7.5 to 8.5.

[0130] 49. The method of any one of the preceding items, which does not involve luminescence, bioluminescence, or fluorescence detection.

[0131] 50. The method of any one of the preceding items, comprising only colorimetric detection.

[0132] 51. The method of any one of the preceding items, wherein the metal indicator is water-soluble.

[0133] 52. The method of any one of the preceding items, wherein the metal indicator is 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS), PAR, or Zincon.

[0134] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such various reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that can modify or substitute for the methods described herein.

[0135] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0136] The term "and / or" whenever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said item".

[0137] The terms "less than" or conversely "more than" do not include actual numbers.

[0138] For example, less than 20 means less than the stated number. Similarly, more or greater than means more or greater than the stated number, for example, more than 80% means more or greater than 80% of the stated number.

[0139] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of the specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including," or, as sometimes used herein, with the term "having." As used herein, "consisting of" excludes any element, step, or ingredient not specified.

[0140] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0141] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0142] All publications (including all patents, patent applications, scientific publications, manuals, etc.), cited throughout the text of this specification, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will supersede any such material.

[0143] The contents of all papers and patent documents cited herein are incorporated by reference in their entirety. [Example]

[0144] A still further understanding of the present invention and its advantages will become apparent from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0145] Example 1: Comparison of preferred detection reagents Our preferred detection reagents were selected based on initial testing identifying chelating dyes with desirable spectral properties, i.e., the ability to form colored complexes with preferred metal ions in the presence of magnesium, the vividness of the observed color change, and the high absorbance of the dye-metal complexes formed. 2+ The insensitive detection reagent combination consisted of one of the preferred complexometric dyes (5-Br-PAPS, PAR, or Zincon; all supplied by Sigma-Aldrich, Missouri, USA) and a preferred metal ion (Co) under buffer conditions (20 mM Tris-HCl pH = 7.9, 50 mM KCl) in the presence of a high magnesium concentration (8 mM MgSO). 2+ , Cu 2+ , Fe 2+ , Ni 2+ , Rh 2+ , Zn 2+) were mixed to give a final complexometric dye concentration of 100 μM, reflecting the expected stoichiometry of the dye-metal complex formed, while the metal ion concentration was either 50 μM (for 5-Br-PAPS, PAR) or 100 μM (for Zincon).

[0146] Spectroscopic analysis on a Tecan Infinite M1000 Pro Multi-mode Reader (Tecan, Switzerland) was used to measure the λ of the free dye and its dye-metal complexes. max As can be seen from Figure 1A-B, the Zinc / Rh 2+ All combinations except for 5-Br-PAPS showed dye-metal complex formation in the presence of high concentrations of magnesium, demonstrating their potential use for colorimetric detection in the presence of magnesium. However, as shown in Figure 1C, the 5-Br-PAPS complex had a much higher average absorbance value than the PAR and zincon complexes, resulting in a sharper color shift between the free and complexed forms of the dye. For this reason, 5-Br-PAPS was chosen as the most preferred chelating dye, as higher absorbance values ​​require lower concentrations of both the dye and the metal, both of which can interfere with enzyme activity in the assay.

[0147] The 5-Br-PAPS / metal complexes were further compared for their ability to release chelated metal ions in the presence of pyrophosphate (PPi). 2+ , Cu 2+ , Fe 2+ , Ni 2+ , Rh 2+ , Zn 2+ , 50 μM each) to gradually higher concentrations of PPi (from 0 to 20 mM), and the absorbance λ of the dye-metal complex was measured. maxwas measured spectrophotometrically using a Tecan Infinite M1000 Pro Multi-mode Reader (Tecan, Switzerland). The results (Figure 2A-B) show that all 5-Br-PAPS / metal complexes reacted with PPi to release the complexed metal from 5-Br-PAPS, but only Zn 2+ It was demonstrated that the Zn / 5-Br-PAPS complex was by far the most readily disrupted by PPi (almost complete complex disruption was observed at the lowest tested concentration). This should provide the highest sensitivity in the PPi generation assay, so we investigated the effect of Zn on the PPi formation. 2+ The / 5-Br-PAPS complex was chosen as our most preferred detection reagent and was used for further testing.

[0148] Example 2: Zn 2+ Characterization of 5-Br-PAPS detection reagent Most preferred Zn 2+ To prepare the 5-Br-PAPS detection reagent, a solution of 5-Br-PAPS and a solution of ZnSO4 (both in PCR-grade water) were mixed together such that the final molar ratio of 5-Br-PAPS to ZnSO4 was 1:1. 2+ Mix them in a ratio of 2:1, which corresponds to the stoichiometry of Zn / 5-Br-PAPS complex. 2+ A 1.5 mM solution of 5-Br-PAPS detection reagent (1.5 mM 5-Br-PAPS, 750 μM ZnSO 4 ) is stable at room temperature in the dark and can be used to prepare the reaction mix.

[0149] Zn 2+ The 5-Br-PAPS detection reagent is characterized by the fact that PPi (generated as a by-product of dNTP hydrolysis by polymerase) competes with 5-Br-PAPS to detect Zn. 2+ PPi changes color during DNA amplification due to binding to Zn (Figure 3A). As amplification progresses, PPi accumulates and increasing concentrations of PPi react with Zn. 2+This mechanism is demonstrated by the gradual shift of the 5-Br-PAPS complex (magenta) to free 5-Br-PAPS (orange-yellow) and zinc(II) pyrophosphate. This mechanism is demonstrated by the addition of the basic LAMP reaction mix components (20 mM Tris-HCl pH = 7.9, 20 mM KCl, 60 mM GuCl, 9 mM MgSO, 0.1% Tween-20, 0.05% Triton X-100), 25 or 50 μM Zn 2+ This was easily demonstrated under the target assay conditions (maximum PPi generation, approximately 5 mM) by preparing pseudo-samples containing 5-Br-PAPS detection reagent and sodium pyrophosphate (concentration range 0-5 mM).

[0150] Zn 2+ The PPi concentration-dependent effect on the color of the / 5-Br-PAPS complex was analyzed spectrophotometrically by performing spectral analysis in the wavelength range of 330–600 nm on a Tecan Infinite M1000 Pro Multi-mode Reader (Tecan, Switzerland), and simultaneously, the visible color change was recorded by a mobile camera.

[0151] Figure 3B shows that increasing PPi concentrations affect the activity of the reaction mix (25 μM Zn 2+ The blank-adjusted absorption spectrum (with 5-Br-PAPS detection reagent) gradually changes. Specifically, with increasing PPi concentration, the absorption peak at 560 nm weakened, while a new peak at 448 nm increased. Figure 3C shows this at 25 μM and 50 μM Zn. 2+ This corresponds to a readily discernible visible color change (magenta to orange-yellow) with both the 5-Br-PAPS detection reagent and the 5-Br-PAPS detection reagent.

[0152] Example 3: Zn 2+ / LAMP amplification detection using 5-Br-PAPS detection reagent LAMP amplification reactions (all in a final volume of 20 μl) were carried out in a reaction buffer containing 20 mM Tris-HCl pH 7.9, 20 mM KCl, 60 mM GuCl (guanidine hydrochloride), 1.4 mM dNTPs, 0.1% Tween-20, 0.05% Triton X-100, and 320 U / ml Bst 2.0 WarmStart® DNA polymerase (M0538, New England Biolabs, Massachusetts, USA). RT-LAMP reactions in which the template strand was RNA also contained 300 U / ml WarmStart® RTx reverse transcriptase (M0380, New England Biolabs, Massachusetts, USA) and 0.8 M betaine. The LAMP primer concentrations were the same for all tested primer sets, specifically 1600 nM for FIP / BIP, 200 nM for F3 / B3, and 600 nM for LF / LB.

[0153] In Figures 4A-B, LAMP amplification was performed using primers against human RPP30 and a template input of 0.375 ng / μl human gDNA (G3041, Promega, Wisconsin, USA). RT-LAMP reactions were also performed using primers against the SARS-CoV-2 N gene and a template input of 25 copies / μl synthetic SARS-CoV-2 RNA (102019, Twist Bioscience, California, USA). In both cases, reactions were performed at 65°C for 30 min. The color transition (magenta to orange-yellow), easily observed with the naked eye, was specific to nucleic acid amplification, as only samples with a polymerase (e.g., Bst 2.0 WarmStart® DNA Polymerase and / or WarmStart® RTx Reverse Transcriptase) changed color (Figure 4A), whereas those lacking the polymerase did not (Figure 4B). This indicates that the amount of pyrophosphate generated during LAMP is surprisingly similar to that of Zn from 5-Br-PAPS. 2+This suggests that the pH is sufficient to displace the nucleotides. Additionally, the type of template used (double-stranded DNA / single-stranded RNA) did not affect the success of the amplification / color change. Note that the strong buffering of the reaction mix (20 mM Tris-HCl, pH = 7.9) suggests that pH changes do not contribute to the observed color change.

[0154] In Figure 5, a LAMP reaction targeting human RPP30 was performed with a template input of 0.375 ng / μl human gDNA and 50 μM Zn 2+ The experiments were carried out at 65°C using the 5-Br-PAPS detection reagent. Unlike the experiment depicted in Figures 4A-B, all reactions contained Bst 2.0 WarmStart® DNA polymerase, and a true non-template control (NTC) reaction was run in parallel instead of the no-polymerase sample. Color change was monitored at intervals throughout the 90-minute reaction period. The color began to change at the 15-minute mark and gradually changed from red to orange-yellow over the next 10 minutes, demonstrating that the reagent continuously changes color during nucleic acid amplification. At the end of the 90-minute period, the template-positive reactions were still orange-yellow, whereas the NTC reactions remained magenta, indicating the presence of the chelating dye 5-Br-PAPS and its Zn. 2+ The complexes are demonstrated to be stable for extended periods under typical LAMP reaction conditions.

[0155] Figures 6A-B and 7 show the addition of Zn to the reaction mix. 2+ We demonstrate that the inclusion of 5-Br-PAPS detection does not impede amplification speed or sensitivity. RT-LAMP targeting the SARS-CoV-2 RdRP gene was performed in a standard reaction mix as described above supplemented with 0.5 μM nucleic acid intercalating fluorescent dye SYTO 59 (Thermo Fisher Scientific, Massachusetts, USA) and 50 μM Zn. 2+The experiments were performed with or without Zn / 5-Br-PAPS detection reagent. Different amounts of input template (100, 50, 25, 12.5, and 0 copies / μl of SARS-CoV-2 RNA) were tested, and target amplification was monitored in real time (Cy5 channel) on an AriaMx Real-Time PCR system (Agilent, California, USA). Reactions were run at 65°C and monitored in real time for 45 minutes. Figure 6A-B shows the effect of 50 μM Zn at all template concentrations tested. 2+ The results show that reactions containing Zn / 5-Br-PAPS reagent (Figure 6A) exhibit similar amplification profiles to those without (Figure 6B, shaded areas ± SEM). Although the mean ± standard deviation (SD) of the time to reaction (TTR) was slightly higher, all samples with template changed color to orange-yellow, regardless of the amount of input template. This suggests that Zn 2+ We demonstrate that colorimetric detection using the 5-Br-PAPS detection reagent is as sensitive as real-time fluorescent detection.

[0156] The following primers were used, the sequence between the hyphens being a linker that is not complementary to the template nucleic acid: TIFF0007801330000007.tif171163

[0157] Example 4: Zn 2+ / PCR amplification detection using 5-Br-PAPS detection reagent 25 μM Zn 2+Nucleic acid amplification during PCR was detected using a SensoQuest Gradient Labcycler (SensoQuest, Germany) with a reaction containing 5-Br-PAPS detection reagent. PCR was performed in a reaction buffer containing 70 mM Tris-HCl pH 7.9, 17.5 mM (NH4)2SO4, 1 mM dNTPs, 3 mM MgCl2, 0.02% Tween-20, and 50 U / ml HOT FIREpol DNA polymerase (01-02-1000, Solis Biodyne, Estonia). Amplification was performed using 0.375 ng / μl human gDNA template input and primers targeting human RPP30 (both forward and reverse primers, 500 nM). PCR reactions were performed as follows: a 10-minute heating period at 95°C, followed by 45 cycles of melting (95°C for 15 seconds) and annealing / extension (60°C for 30 seconds). As can be seen in Figure 8, unlike the NTC reaction which remained magenta, the reaction with the gDNA template changed color from magenta to orange-yellow, indicating that Zn 2+ We demonstrate that the 5-Br-PAPS detection reagent can be used to detect nucleic acid amplification under non-isothermal conditions, such as during PCR. Furthermore, the preservation of color in both the NTC and template reactions demonstrates the thermostability of the reagent to repeated cycles of heating up to 95°C.

[0158] The following primers were used: TIFF0007801330000008.tif15128

[0159] Example 5: Zn for reaction buffering 2+ Tolerance of 5-Br-PAPS detection reagent and its use in LAMP amplification of buffered samples It is known from the prior art that colorimetric detection systems for nucleic acid amplification detection, especially those that rely on pH-based detection, are prone to reaction failure in buffered samples or those containing variable initial pH (e.g., biological samples such as saliva), which prevents their use with unpurified samples. This limitation also precludes proper buffering of the reaction mix throughout the amplification, which can result in suboptimal amplification kinetics and / or sensitivity. 2+ Because the 5-Br-PAPS detection reagent functions without relying on the accumulation of free hydronium ions and the resulting pH drop, reactions utilizing this reagent can tolerate not only strong buffering in the reaction mix, but also extension of the input sample. This combination of properties makes it particularly suitable for techniques such as direct pathogen detection without prior nucleic acid isolation in biological matrices with varying levels of pH / buffering capacity. The following reaction was set up to demonstrate that the colorimetric assay of the present invention is robust to pH changes, as it tolerates buffers preferably higher than 1.5 mM Tris or equivalent, in both the reaction mix and the input sample.

[0160] LAMP reactions were performed similarly to those described in Example 3, with some minor modifications in certain experiments (noted in the description). Pseudo-LAMP reactions have the same base composition as regular LAMP, with the addition of a defined amount of Zn 2+ To prepare mock samples, 50% of the 2x master mix (40 mM Tris-HCl pH = 7.9, 40 mM KCl, 120 mM GuCl, 18 mM MgSO, 0.2% Tween-20, 0.1% Triton X-100) was mixed with 25% water or 10 mM buffers of different pHs (10 mM sodium citrate at pH = 5 or 6; 10 mM Tris-HCl at pH = 7, 8, or 9; final concentration 2.5 mM) and 25% water (for mock-negative samples) or 16 mM sodium pyrophosphate (for mock-positive samples, final concentration 4 mM).

[0161] 9A-B and 10 show the effect of Zn on pH change. 2+This figure shows the proposed advantages of the 5-Br-PAPS detection reagent. Human RPP30 was amplified using LAMP with 0.375 ng / μl human gDNA as input and 0.5 μM SYTO 59 at 65°C for 30 minutes. The reaction was monitored in real time (Cy5 channel) using the AriaMx Real-Time PCR system. 50 μM Zn 2+ The 5-Br-PAPS detection reagent was prepared by adding 50 μM 5-Br-PAPS only (Zn 2+ We compared the amplification of Zn with 100 μM phenol red (pH-sensitive indicator) and 100 μM phenol red at different Tris-HCl buffer concentrations in the reaction mix. As Figure 9A-B demonstrates, under all conditions tested, amplification occurred in the presence of template (as assessed by real-time fluorescence detection). In addition, higher concentrations of Tris-HCl buffer increased the amplification of Zn. 2+ Both 5-Br-PAPS-based and phenol red-based detection resulted in faster amplification times (shorter TTR) (Figure 9B), and therefore maintaining the reaction in a strongly buffered state proved advantageous. However, at strong buffering (20 mM Tris-HCl, pH = 7.9), the pH-dependent detection did not result in a color change (Figure 10), despite successful amplification by real-time detection (Figure 9A). In contrast, Zn 2+ The Zn / 5-Br-PAPS detection reagent exhibited a consistent color transition regardless of Tris-HCl concentration. 5-Br-PAPS itself is orange-yellow and does not change color with amplification. 2+ The observed color change of the 5-Br-PAPS detection reagent indicates the presence of Zn in the starting reaction mix. 2+ Note that the total dependence of the Zn 2+ The 5-Br-PAPS detection reagent does not rely on pH changes to detect nucleic acid amplification and works in both very low and high strength buffers.

[0162] In Figure 11A, Zn 2+Spectral analysis of the Zn / 5-Br-PAPS detection reagent (25 μM) (performed as described in Example 1) can be confirmed with either a false-positive sample (solid line, with 4 mM PPi) or a false-negative sample (dotted line, without PPi). Replacing 25% of the final LAMP reaction mix volume with a 10 mM buffer solution at a commonly used pH level did not significantly affect the absorption spectrum, with or without PPi. As can be seen in Figure 9B, this lack of color change is due to the presence of Zn at both lower and higher concentrations. 2+ The same was visually observable with the 5-Br-PAPS detection reagent.

[0163] In Figures 12A-C, LAMP targeting human RPP30 was performed with a template input of 0.375 ng / µl human gDNA, 0.5 µM SYTO 59, and 50 µM Zn. 2+ The reaction was carried out at 65°C for 30 minutes using Zn / 5-Br-PAPS detection reagent. Input gDNA (25% of the final reaction volume) was diluted with either water or 10 mM buffer as used for mock sample preparation, and target amplification was monitored in real time (Cy5 channel) on the AriaMx Real-Time PCR system. All replicates with gDNA template were successfully amplified regardless of diluent (Figure 12A). Although there were some small differences in reaction arrival time (TTR, Figure 12B) between individual dissolution media, the inclusion of buffer did not prevent amplification and color change from occurring (Figure 12C), suggesting a relationship between input sample buffering capacity and Zn / 5-Br-PAPS detection reagent relative to pH. 2+ This confirms the robustness of the 5-Br-PAPS detection reagent. Indeed, all buffers tested matched or exceeded the performance of the water-only control in terms of TTR and reproducibility (i.e., variability between replicates).

[0164] In summary, the present inventors have surprisingly been able to show that a combination of 5-Br-PAPS and zinc ions can be used to detect nucleic acid amplification in a reaction mixture.

[0165] Example 6: Use of LNA-containing primers in LAMP improves reaction time and specificity Replacing specific bases in the F3 / B3 LAMP primer described herein with their LNA variants can significantly alter LAMP reaction kinetics and target recognition. Specifically, the introduction of a predetermined number of LNA bases near the 5' end of the F3 / B3 primer significantly shortens the time to reach reaction (TTR) while simultaneously increasing specificity, both properties desirable in potential isothermal diagnostic tests. This is useful for shortening the time to positive reaction and thus widening the window between true-positive and false-positive results, which can be problematic for isothermal methods such as LAMP. Incorporation of LNA-modified bases into LAMP primers is also beneficial for normalizing primer melting temperatures (Tm) and providing flexibility in LAMP primer design when targeting difficult A,T-rich or G,C-rich templates or organisms with high mutation rates (e.g., SARS-CoV-2 and influenza). Those skilled in the art may be aware of the fact that incorporation of LNA into primers can lead to improved target hybridization rates, primarily by increasing the melting temperature (Tm) of the primers; however, due to the complexity of primer interactions during LAMP target amplification, inadvertent changes to primer base composition are not expected to lead to beneficial effects during LAMP amplification and may promote non-specific interactions and amplification, and may interfere with the strand displacement activity of the polymerase.

[0166] Unless otherwise specified, LAMP amplification was performed in all experiments described below as follows: A reaction mix was prepared by mixing WarmStart Colorimetric RT-LAMP 2X MasterMix (M1800, New England Biolabs, Massachusetts, USA) with a 10x primer mix consisting of six specific LAMP primers. Basic LAMP primer sets targeting SARS-CoV-2 genes E (set E1, E2, E3), RdRP (set R1), and N (set N1) were designed according to recommendations as known from the prior art. The final reaction concentrations of LAMP primers were FIP / BIP - 1600 nM, F3 / B3 - 200 nM, and LF / LB - 600 nM. All assays utilized a reaction volume of 20 μl. All reactions were also supplemented with 0.05% Triton X-100, 1 mM extra MgCl2 (final concentration 9 mM), and 1 μM nucleic acid intercalating dye SYTO 9 (Thermo Fischer Scientific, Massachusetts, USA). Reactions performed at 65°C also contained 40 mM guanidine isothiocyanate and 0.8 M betaine. Positive-testing reactions contained either SARS-CoV-2 RNA mixed with human genomic DNA (COV019, Exact Diagnostics, California, USA; if RNA copies / rxn was <200) or pure SARS-CoV-2 RNA (102019, Twist Bioscience, California, USA; if RNA copies / rxn was ≥200). Once reactions were set up, they were run at temperatures specific to the primer set / conditions and amplified on an Agilent AriaMx Real-Time PCR System (Agilent, California, USA). The course of amplification was continuously monitored by measuring the change in SYTO 9 fluorescence (FAM channel) every 30 seconds, and the reaction was terminated after 90 minutes.The reaction arrival time (TTR, threshold cycle number / 2) was automatically determined using default settings using Agilent Aria 1.7.1 software (Agilent, California, USA). Melting curve analysis of the resulting amplicons was also used to distinguish specific from nonspecific products. Specifically, products were considered specific if their Tm was within ±0.5°C of the mean of the products observed in internally established high template concentration reactions for each primer set tested. Replicates without amplification are not depicted in any of the figures and were excluded from the calculation of descriptive statistics, e.g., mean ± SD.

[0167] In Figure 13, four different LAMP primer sets (E1, E2, R1, and N1) were used in RT-LAMP to amplify target sequences from the SARS-CoV-2 genome. LNA base substitution at the 5' end of the outer primers F3 / B3 was compared with the standard LAMP primer set. Template-bearing reactions contained 400 (E1, E2), 100 (R1), or 25 (N1) template copies per reaction. The reaction temperature was either 60°C (E1, E2) or 65°C (R1, N1), and amplification proceeded for 90 minutes. Solid symbols represent samples with template, while open symbols represent NTC reactions. The number in parentheses indicates the number of replicates with successfully amplified template. The figure clearly shows that for all primer sets tested, the introduction of LNA near the 5' end improved all investigated metrics of LAMP set performance. The sets with LNAs showed reduced TTR (portrayed as fold change relative to the unmodified set) and variability (SD represented by error bars), as well as unchanged or increased amplification success, suggesting improved sensitivity. Importantly, the propensity for nonspecific amplification was not significantly increased in sets carrying LNA substitutions.

[0168] The beneficial effect of incorporating 5' LNA into the outer LAMP primers is robust and independent of the specific base modified, as demonstrated in Figure 14. RT-LAMP was used with set E3 to amplify the SARS-CoV-2 E gene (400 template copies per reaction for the modified set and 800 copies for the basic E3 set) at 60°C for 90 minutes. Four different combinations of LNA-modified F3 / B3 primers (two F3 primers and two B3 primers, labeled [1] or [2]), differing only in the number / position of LNA bases in the sequence, were compared with the basic set without LNA modifications. As can be seen, each variant exhibited the improved performance characteristics described above, even when the unmodified set was run with twice as many templates.

[0169] In Figure 15, RT-LAMP was performed using set E1, comparing the basic set with sets in which FIP, BIP, LF, and LB were modified with LNAs near their 5' ends. For the FIP / BIP primers, both the F2 / B2 and F1c / B1c segments were modified. Reactions contained 400 copies of template and were run at 60°C for 90 minutes. As can be seen, LNA modification in these primer sets led to either unchanged or significantly reduced LAMP amplification performance. TTR was higher (slower), variability was greater, and sensitivity (number of amplified replicates) was lower or unchanged. This demonstrates that the LNA substitution effect is LAMP primer-specific and not the result of a general effect of LNA on primer hybridization behavior. Similarly, LNAs in primers other than F3 / B3 impair LAMP amplification and / or target recognition; therefore, unlike the F3 / B3 primer, modification of other LAMP primers is not expected to consistently lead to the desired effect.

[0170] The following primers were used, where (+N) depicts the corresponding base in the form of LNA, and the sequence between the hyphens is a linker that is not complementary to the template nucleic acid: TIFF0007801330000009.tif181164TIFF0007801330000010.tif250161TIFF0007801330000011.tif44162

Claims

1. (i) a first inner primer (FIP) nucleotide and a second inner primer (BIP) nucleotide; (ii) a first outer primer (F3) nucleotide and a second outer primer (B3) nucleotide, wherein the F3 nucleotide and / or the B3 nucleotide comprises one or more locked nucleic acid (LNA) nucleotides located within the first third of the F3 nucleotide or the B3 nucleotide, respectively, and the first third is the first third of nucleotides in the 5' to 3' direction; (iii) a DNA polymerase that catalyzes a strand displacement reaction that synthesizes a complementary nucleic acid strand from a template nucleic acid; and (iv) a nucleotide that serves as a substrate for the DNA polymerase In a reaction mixture comprising at a temperature at which at least FIP ​​nucleotides and BIP nucleotides can form stable base pairs with their complementary nucleotides contained in the template nucleic acid, while maintaining DNA polymerase activity; A loop-mediated isothermal amplification (LAMP) method includes a step of synthesizing a nucleic acid from a template nucleic acid, thereby synthesizing a nucleic acid.

2. (i) the FIP nucleotide comprises at least two regions, F2 and F1c; the F1c region is linked to the 5' side of the F2 region, the F2 region has nucleotides complementary to an arbitrary region F2c in the template nucleic acid; and the F1c region has substantially the same nucleotides as the F1c region located 5' to the F2c region in the template nucleic acid; (ii) the BIP nucleotide comprises at least two regions, B2 and B1c; the B1c region is linked to the 5' side of the B2 region, the B2 region has nucleotides complementary to an arbitrary region B2c in the template nucleic acid; and the B1c region has substantially the same nucleotides as the region B1c located 5' to the region B2c in the template nucleic acid; (iii) the F3 nucleotide has a nucleotide that is substantially complementary to a region F3c in the template nucleic acid; a region F3c is located on the 3' side of the region F2c in the template nucleic acid; the region F2c is located 3' to the region F1c in the template nucleic acid; and (iv) the B3 nucleotide has a nucleotide that is substantially complementary to a region B3c in the template nucleic acid; a region B3c is located on the 3' side of the region B2c in the template nucleic acid; the region B2c is located on the 3' side of the region B1c in the template nucleic acid; 10. The method of claim 1.

3. (i) the reaction mixture further comprises loop primer nucleotide F and / or loop primer nucleotide B; (ii) the reaction mixture further comprises stem primer nucleotide F and / or stem primer nucleotide B; (iii) the reaction mixture further comprises swarm primer nucleotide F1S and / or swarm primer nucleotide B1S; (iv) the reaction mixture further comprises at least an additional first inner primer (FIP2) nucleotide and an additional second inner primer (BIP2) nucleotide, both of which are different from the FIP and BIP nucleotides; (v) the reaction mixture further comprises primer nucleotides for carrying out the method as a multiplex cross-displacement amplification (LAMP); and / or (vi) the reaction mixture further comprises primer nucleotides for carrying out the method as reverse transcription isothermal multiplex self-matching LAMP; 3. The method of claim 1 or 2.

4. (i) the template nucleic acid is single-stranded or double-stranded; (ii) the template nucleic acid is single-stranded or double-stranded DNA, RNA, or DNA / RNA chimera; (iii) the DNA polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase; or (iv) the DNA polymerase has reverse transcriptase activity; 3. The method of claim 1 or 2.

5. (i) the F3 nucleotide has a length of 15 to 30, preferably 17 to 25, nucleotides; and / or (ii) the B3 nucleotide has a length of 15 to 30, preferably 17 to 25, nucleotides; 3. The method of claim 1 or 2.

6. (i) the F3 nucleotides comprise 1 to 5, preferably 3, LNA nucleotides; and / or (ii) the B3 nucleotides comprise 1 to 5, preferably 3, LNA nucleotides; 3. The method of claim 1 or 2.

7. 3. The method of claim 1 or 2, wherein the reaction mixture further comprises a melting temperature regulator.

8. 3. The method of claim 1, wherein the reaction mixture further comprises a detection agent for detecting the product of the nucleic acid synthesis reaction, preferably the detection agent is a metal indicator, more preferably the detection agent further comprises a transition metal or a post-transition metal.

9. The reaction mixture further comprises a detection agent for detecting a product of the nucleic acid synthesis reaction, the detection agent comprising a metal indicator, preferably 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS), PAR, or Zincon, and a metal ion, preferably a transition metal or post-transition metal ion, more preferably Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Pt 2+ , Ru 2+ , Rh 2+ and / or Fe 2+ ions, most preferably Zn 2+ ions, wherein the metal indicator and the metal ions each form a complex, but do not form a complex with magnesium; and The method comprises: (b) detecting a change in the spectral or fluorescent properties of the complex resulting from amplification of said nucleic acid.

3. The method of claim 1 or 2, further comprising:

10. (a) synthesizing nucleic acids, or (b) detecting nucleic acids; The method according to claim 1 or 2, wherein the method is for the purpose of

11. (a) contacting a reaction mixture comprising a template nucleic acid, primer nucleotides, nucleotides, and a polymerase capable of amplifying said nucleic acid with a metal indicator and a transition or post-transition metal ion, wherein said metal indicator and said transition or post-transition metal ion form a complex, but not with magnesium; (b) amplifying the nucleic acid under appropriate conditions to obtain a nucleic acid amplification product; (c) detecting a change in the spectral properties of a complex resulting from the amplification of the nucleic acid, wherein the formation of the complex comprising the metal indicator and the transition or post-transition metal ion leads to a color change of the solution. Including, The amplification is carried out by a method as defined in claim 1 or 2, In vitro methods for detecting nucleic acid amplification products.

12. (i) The metal indicator is 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS); (ii) The metal ion is Zn 2+ is an ion; and / or (iii) the change in the spectral properties of the complex is a change in the spectrum of 380 to 740 nm wavelength; 12. The method of claim 11.

13. (i) a first outer primer (F3) nucleotide and a second outer primer (B3) nucleotide, each of which comprises a locked nucleic acid (LNA) nucleotide located within a first third of the F3 nucleotide or the B3 nucleotide, respectively, wherein the first third is the first third of nucleotides in a 5' to 3' direction; (ii) a first inner primer (FIP) nucleotide and a second inner primer (BIP) nucleotide; (iii) a DNA polymerase that catalyzes a strand displacement reaction that synthesizes a complementary nucleic acid strand from a template nucleic acid; (iv) nucleotides that serve as substrates for the DNA polymerase, and optionally (v) a metal indicator, preferably 5-Br-PAPS, PAR or Zincon; and / or, optionally (vi) a metal ion, preferably Zn 2+ ion A kit for synthesizing nucleic acids by loop-mediated isothermal amplification (LAMP) on a template nucleic acid, comprising:

14. Use of the kit according to claim 13 for carrying out the method according to any one of claims 1 to 12.

Images