Patent Application
20240026435
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
The present invention relates to a loop mediated isothermal amplification (LAMP) method characterized in that the F3 nucleotide sequence and/or said B3 nucleotide sequence comprises one or more locked nucleic acid (LNA) nucleotides which are located within the first third of said F3 nucleotide sequence or said B3 nucleotide sequence, respectively. The present invention further relates to an in vitro method for detecting a nucleic acid sequence amplification product characterized by the use of a metallochromic indicator, preferably 5-Br-PAPS, and metal ions, preferably Zn2+ ions. The present invention further relates to a kit for carrying out the methods of the invention and a use of the kit.
Since the revolutionary development of the polymerase chain reaction (PCR) in the 1980s, nucleic acid amplification tests (NAATs) have become an indispensable tool throughout the entire life sciences field, and have even grown to be the gold standard of nucleic acid analysis, especially in clinical applications, but also for food quality control and environmental monitoring. A remarkable trend, emerging between 1995 and 2005, can also be observed in the development of isothermal NAATs, which was provoked by the limitations of PCR. The complex and expensive devices required for thermal cycling and real-time detection during PCR restrict the use of this amplification method. Isothermal NAATs enable amplification reactions at constant and moderate temperatures. Simple and low-cost devices, as well as fast 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.
Loop-mediated isothermal amplification (LAMP), which was first described by Notomi et al. (2000), Nucleic Acids Res., 28(12):e63, see also EP 1020534 B2, both of which are incorporated by reference herein, is one of the methods for performing NAATs.
However, there is an ongoing need to improve LAMP, especially by increasing the specificity of the primers. Further, there is an ongoing need for improved detection methods that can be used in NAATs. The technical problem therefore is to comply with this need.
The technical problem is solved by the subject-matter as defined in the claims.
Accordingly, the present invention relates to a loop mediated isothermal amplification (LAMP) method, comprising synthesizing a nucleic acid sequence from a template nucleic acid sequence in a reaction mixture comprising
The present invention further relates to an in vitro method for detecting a nucleic acid sequence amplification product, comprising
The present invention further relates to a kit for the synthesis of a nucleic acid sequence by loop mediated isothermal amplification (LAMP) on a template nucleic acid sequence, comprising
The present invention further relates to the use of the kit of the invention for performing the method of the LAMP method of the invention.
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, respectively. The Figures show:
The present invention is described in detail in the following and is also illustrated by the appended examples and figures.
LAMP as such is e.g., already described in EP 1020534 B2, which is incorporated by reference herein. However, prior art has failed to show a successful application of locked nucleic acid (LNA) primers in LAMP. The inventors, however, surprisingly found that the use of locked nucleic acids (LNA) in the F3/B3 primer further improves LAMP. As shown in Example 5, the use of LNAs in the F3/B3 primers reduces reaction times and improves reproducibility and sensitivity. Interestingly, this effect is only present when the LNA are located within the first third of the F3 or B3 primer.
Accordingly, the present invention relates to a LAMP method, comprising synthesizing a nucleic acid sequence from a template nucleic acid sequence in a reaction mixture comprising
In one embodiment, said F3 nucleotide sequence comprises one or more LNA nucleotides. In a further embodiment, said B3 nucleotide sequence comprises one or more LNA nucleotides. In a further embodiment, said F3 nucleotide sequence and said B3 nucleotide sequence comprise one or more LNA nucleotides.
“Loop mediated isothermal amplification” (LAMP) as used herein relates to a (single-tube) technique for the amplification of nucleic acids. Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) combines LAMP with (concurrent) reverse transcription to allow the detection of RNA. LAMP is an isothermal nucleic acid amplification technique. In contrast to the polymerase chain reaction (PCR) technology, in which the reaction is carried out with a series of alternating temperature steps or cycles, isothermal amplification is carried out at a constant temperature, and does not require a thermal cycler. LAMP is known to a person skilled in the art. In this context, we exemplarily refer 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.
A locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), and often referred to as inaccessible RNA, as used herein may relate to a modified RNA or DNA, preferably DNA, nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2′ oxygen and 4′ carbon. The bridge “locks” the ribose in the 3′-endo (North) conformation. The bridge may be a methyl group covalently bound to the 2′ oxygen and 4′ oxygen, thereby bridging both oxygen atoms. LNAs are commercially available and a method of their synthesis is exemplarily 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, said F3 nucleotide sequence comprises 1-5, preferably 3 LNA nucleotides. Preferably, said B3 nucleotide sequence comprises 1-5, preferably 3 LNA nucleotides.
The nucleic acid synthesized by the LAMP method of the invention may result in a nucleic acid having complementary nucleotide sequences linked alternately in a single-stranded chain. Such a nucleic acid may have mutually complementary nucleotide sequences linked side by side in a single-stranded chain. Further, in the present invention, it may contain a nucleotide sequence for forming a loop between the complementary chains. In the present invention, this sequence is called the loop-forming sequence. The nucleic acid synthesized by the present invention is composed substantially of mutually complementary chains linked via the loop-forming sequence. In general, a strand not separated into 2 or more molecules upon dissociation of base pairing is called a single-stranded chain regardless of whether it partially involves base pairing or not. The complementary nucleotide sequence can form base pairing in the same chain. An intramolecular base-paired product, which can be obtained by permitting the nucleic acid having complementary nucleotide sequences linked alternately in a single-stranded chain according to the present invention to be base-paired in the same chain, gives a region constituting an apparently double-stranded chain and a loop not involving base pairing.
That is, the nucleic acid having complementary nucleotide sequences linked alternately in a single-stranded chain according to the present invention contains complementary nucleotide sequences capable of annealing in the same chain, and its annealed product can be defined as single-stranded nucleic acid constituting a loop not involving base pairing at a bent hinged portion. A nucleotide having a nucleotide sequence complementary thereto can anneal to the loop not involving base pairing. The loop-forming sequence can be an arbitrary nucleotide sequence. The loop-forming sequence is capable of base pairing so as to initiate the synthesis of a complementary chain for displacement and is provided preferably with a sequence distinguishable from a nucleotide sequence located in the other region in order to achieve specific annealing. For example, in the present invention, the loop-forming sequence contains substantially the same nucleotide sequence as a region F2c (or B2c) located at the 3′-side of a region (i.e. F1c or B1c) derived from nucleic acid as a template and annealed in the same chain.
In the present invention, substantially the same nucleotide sequence is defined as follows. That is, when a complementary chain synthesized with a certain sequence as a template anneals to a target nucleotide sequence to give the origin of synthesizing a complementary chain, this certain sequence is substantially the same as the target nucleotide sequence. For example, substantially the same sequence as F2 includes not only absolutely the same nucleotide sequence as F2 but also a nucleotide sequence capable of functioning as a template giving a nucleotide sequence capable of annealing to F2 and acting as the origin of synthesizing complementary chain. The term “anneal” in the present invention means formation of a double-stranded structure of nucleic acid through base pairing based on the rules of Watson-Crick base pairing. Accordingly, even if a nucleic acid chain constituting base pairing is a single-stranded chain, annealing occurs if intramolecular complementary nucleotide sequences are base-paired. In the present invention, annealing and hybridization have the same meaning in that the nucleic acid constitutes a double-stranded structure through base pairing.
The number of pairs of complementary nucleotide sequences constituting the nucleic acid according to the present invention is at least 1. According to a desired mode of the present invention, it may be 2 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 the synthetic product of the present invention is constituted of plural sets of complementary nucleotide sequences, this nucleic acid is composed of repeated identical nucleotide sequences.
The nucleic acid (having complementary nucleotide sequences linked alternately in a single-stranded chain) synthesized by the present invention may not have the same structure as naturally occurring nucleic acid. It is known that if a nucleotide derivative is used as a substrate when nucleic acid is synthesized by the action of a DNA polymerase, a nucleic acid derivative can be synthesized. The nucleotide derivative used may include nucleotides labeled with a radioisotope or nucleotide derivatives labeled with a binding ligand such as biotin or digoxigenin. These nucleotide derivatives can be used to label nucleic acid derivatives as the product. Alternatively, if fluorescent nucleotides are used as a substrate, the nucleic acid as the product can be a fluorescent derivative. Further, this product may be either DNA or RNA. Which one is formed is determined by a combination of the structure of a primer, the type of substrate for polymerization, and the composition of polymerization reagents for carrying out polymerization of a nucleic acid.
Synthesis of the nucleic acid having the structure described above can be initiated by use of a DNA polymerase having strand displacement activity and nucleic acid which is provided at the 3′-terminal thereof with a region F1 capable of annealing to a part F1c in the same chain and which upon annealing of the region F1 to F1c, is capable of forming a loop containing a region F2c capable of base pairing. There are many reports on the reaction of synthesizing complementary chain wherein a hairpin loop is formed and a sample sequence itself is used as a template, while in the present invention the portion of the hairpin loop is provided with a region capable of base pairing, and there is a novel feature on utilization of this region in synthesizing complementary chain. By use of this region as the origin of synthesis, a complementary chain previously synthesized with a sample sequence itself as a template is displaced. Then, a region B1c (arbitrary region) located at the 3′-terminal of the displaced chain is in a state ready for base-pairing. A region having a complementary sequence to this B1c is annealed thereto, resulting in formation of the nucleic acid (2 molecules) having a nucleotide sequence extending from F1 to B1c and its complementary chain linked alternately via the loop-forming sequence. In the present invention, the arbitrary region such as B1c above can be selected arbitrarily provided that it can be annealed to a polynucleotide having a nucleotide sequence complementary to that region, and that a complementary chain synthesized with the polynucleotide as the origin of synthesis has necessary functions for the present invention.
In the present invention, the term “nucleic acid” is used. The nucleic acid in the present invention generally includes both DNA and RNA. However, nucleic acid whose nucleotide is replaced by an artificial derivative or modified nucleic acid from natural DNA or RNA is also included in the nucleic acid of the present invention insofar as it functions as a template for synthesis of complementary chain. The nucleic acid of the present invention may be contained in a biological sample. The biological sample includes animal, plant or microbial tissues, cells, cultures and excretions, or extracts therefrom. The biological sample of the present invention may include intracellular parasitic genomic DNA or RNA such as virus or mycoplasma. An exemplary virus may be SAR-CoV-2. The nucleic acid of the present invention may be derived from nucleic acid contained in said biological sample. For example, cDNA synthesized from mRNA, or nucleic acid amplified on the basis of nucleic acid derived from the biological sample, is a typical example of the nucleic acid of the present invention. The nucleic acid may be DNA.
The term “template” used in the present invention may relate to a nucleic acid serving as a template for synthesizing a complementary chain. A complementary chain having a nucleotide sequence complementary to the template relates to a chain corresponding to the template, but the relationship between the two is merely relative. That is, a chain synthesized as the complementary chain can function again as a template. That is, the complementary chain can become a template. Preferably, the template nucleic acid sequence is single stranded or double stranded. Preferably, the template nucleic acid sequence is single- or double-stranded DNA, RNA or a DNA/RNA chimera. In the context of RT-LAMP, the template preferably is RNA.
Preferably, said FIP nucleotide sequence comprises at least two regions F2 and F1c, wherein said F1c region is linked to the 5-side of the F2 region, wherein said F2 region has a nucleotide sequence complementary to an arbitrary region F2c in said template nucleic acid sequence, and wherein said F1c region has substantially the same nucleotide sequence as a region F1c located at the 5-side of said F2c region in said template nucleic acid sequence.
Preferably, said BIP nucleotide sequence comprises at least two regions B2 and B1c, wherein said B1c region is linked to the 5-side of the B2 region, wherein said B2 region has a nucleotide sequence complementary to an arbitrary region B2c in said template nucleic acid sequence strand, and wherein said B1c region has substantially the same nucleotide sequence as a region B1c located at the 5-side of said region B2c in said template nucleic acid sequence.
Preferably, said F3 nucleotide sequence has a nucleotide sequence substantially complementary to a region F3c in said template nucleic acid sequence, wherein region F3c is located at the 3′-side of region F2c in said template nucleic acid sequence, wherein region F2c is located at the 3′-side of region F1c in said template nucleic acid sequence.
Preferably, said B3 nucleotide sequence has a nucleotide sequence substantially complementary to a region B3c in the template nucleic acid sequence, wherein region B3c is located at the 3′-side of region B2c in said template nucleic acid sequence, wherein region B2c is located at the 3′-side of region B1c in said template nucleic acid sequence.
Preferably, said 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.
Preferably, said 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.
Preferably, said reaction mixture further comprises swarm primer nucleotide sequence F1S and/or swarm primer nucleotide sequence B1S. Martineau et al. (2017), Anal. Chem. 89(1): 625-632 describe exemplary swarm primer nucleotide sequences.
Preferably, said reaction mixture further comprises at least a further first inner primer (FIP2) nucleotide sequence and a further second inner primer (BIP2) nucleotide sequence, both of which are different from said FIP and BIP nucleotide sequence. Wang et al. (2015), Molecules 20: 21515-21531 describe exemplary further first or second inner primer nucleotide sequences.
Preferably, said reaction mixture further comprises primer nucleotide sequences for performing said method as multiple cross displacement amplification LAMP. Wang et al. (2015), Sci Rep. 5: 11902, describe an exemplary multiple cross displacement amplification LAMP.
Preferably, said reaction mixture further comprises primer nucleotide sequences for performing said method as reverse transcription isothermal multiple self matching LAMP. Ding et al. (2014), J. Clin. Microbiol. 52(6): 1862-1870, disclose an exemplary reverse transcription isothermal multiple self matching LAMP.
The method of synthesizing nucleic acid according to the present invention is supported by the DNA polymerase catalyzing the strand displacement-type reaction for synthesis of complementary chain. It is advantageous to use one kind of DNA polymerase. Exemplary suitable polymerases are listed in the following. Further, various mutants of these enzymes can be utilized in the present invention insofar as they have both the sequence-dependent activity for synthesis of complementary chain and the strand displacement activity. The mutants referred to herein include those having only a structure bringing about the catalytic activity required of the enzyme or those with modifications to catalytic activity, stability or thermostability by, e.g., mutations in amino acids. Exemplary polymerases include, but are not limited to, 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 deficient in exonuclease activity), Deep Vent DNA polymerase, Deep Vent(exo-)DNA polymerase (Deep Vent DNA polymerase deficient in exonuclease activity), ϕ29 phage DNA polymerase, MS-2 phage DNA polymerase, Z-Taq DNA polymerase (Takara Shuzo Co., Ltd.), KOD DNA polymerase (Toyobo Co., Ltd.), OmniAmp® and LavaLAMP™ DNA/RNA Enzyme (Lucigen Corp.), and SD Polymerase (BIORON GmbH).
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 desired enzymes because they have a certain degree of thermostability and high catalytic activity. The reaction of this invention can be carried isothermally in a preferred embodiment. Preferably, the enzyme is thermostable. Although the isothermal reaction is feasible, heat denaturation may be conducted to provide nucleic acid as a first template, and in this respect too, utilization of a thermostable enzyme broadens selection of assay protocol.
Vent (exo-)DNA polymerase is an enzyme having both strand displacement activity and a high degree of thermostability. It is known that the complementary chain synthetic reaction involving strand displacement by DNA polymerase is promoted by adding a single strand binding protein (Paul M. Lizardi et al., Nature Genetics, 19, 225-232, July, 1998). This action is applied to the present invention, and by adding the single strand binding protein, the effect of promoting the synthesis of complementary chain can be expected. For example, T4 gene 32 is effective as a single strand binding protein for Vent (exo-) DNA polymerase. Preferably, said DNA polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase. Preferably, said DNA polymerase has reverse transcriptase activity.
Preferably, said F3 nucleotide sequence has 15-30, preferably 17-25 nucleotides in length.
Preferably, said B3 nucleotide sequence has 15-30, preferably 17-25 nucleotides in length.
The method LNA-LAMP method of the invention is preferably carried out in the presence of a buffer giving suitable pH to the enzyme reaction, salts necessary for annealing or for maintaining the catalytic activity of the enzyme, a protective agent for the enzyme, and as necessary a regulator for melting temperature (Tm). Exemplary buffers are known to a person skilled in the art and include, e.g., 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, such as more than 1 mM or 1.5 mM or more. Exemplary salts such as KCl, NaCl, MgCl2, (NH4)2SO4 etc. are suitably added to maintain the activity of the enzyme and to regulate the melting temperature (Tm) of nucleic acid. Protective agents to maintain enzymatic activity include e.g., bovine serum albumin (BSA), reducing agents like tris(2-carboxyethyl)phosphine (TCEP), (2S,3S)-1,4-Bis(sulfanyl)butane-2,3-diol (DTT), and/or sugars. Further, dimethyl sulfoxide (DMSO) or formamide may be used as the regulator for melting temperature (Tm). By use of the regulator for melting temperature (Tm), annealing of the oligonucleotide can be regulated under limited temperature conditions. Further, betaine (N,N,N-trimethylglycine) or a tetraalkyl ammonium salt is also effective for improving the efficiency of strand displacement by virtue of its isostabilization. Betaine can be added at a concentration of 0.2 to 3.0 M, preferably 0.5 to 1.5 M to the reaction solution. Accordingly, the reaction mixture preferably further comprises a regulator of melting temperature, e.g., betaine, preferably in a concentration between 0.2 to 3.0 M, guanidine thiocyanate or hydrochloride in a concentration preferably between 20 and 80 mM, single-stranded binding protein (SSB), and/or tetramethyl ammonium chloride (TMAC) in a concentration preferably between 5 mM and 80 mM. In addition, the reaction mixture may comprise a stabilizer, e.g., BSA in a concentration preferably between 0.02 mg/ml and 2 mg/ml, a non-ionic surfactant such as Tween-20/Triton X-100 in a concentration preferably between 0.02% and 0.2% v/v and/or TCEP/DTT preferably in a concentration between 0.5 mM to 5 mM.
The methods of the present invention may be used for the detection of a given template such as a nucleic acid derived from a pathogen such as SARS-CoV-2. Accordingly, said reaction mixture may further comprise a detector for detecting the product of the nucleic acid sequence synthesis reaction.
The detector (for detecting the product of the nucleic acid sequence synthesis reaction) may be a metallochromic indicator. Further examples of a detector are a DNA-intercalating substance or a pH-sensitive dye, with a metallochromic indicator being preferred. As described herein, a metallochromic indicator is capable of building a complex with metal ions, e.g., magnesium. As also described herein, preferred metal ions are transition metal ions or post-transition metal ions.
A metallochromic indicator or, as also referred to herein, a complexometric indicator, is a molecule which can be bound by metal ions, i.e., it builds a complex with metal ions. Such a complex is a metallochromic indicator-metal ion complex, or, in short, as referred to herein “complex”. A complex has preferably a different colorimetric or fluorescence property than a non-complex/a metallochromic indicator not-complexed with metal ions, i.e., a metallochromic indicator in the absence of metal ions, i.e., not building a complex with metal ions, thus being in a non-complex or non-complexed. Accordingly, the detector preferably comprises a transition metal or post-transition metal. The detector preferably does not form or build a complex with magnesium.
Such a complex is reversible which means metal ions when bound to a metallochromic indictor can be/are released from the complex with the metallochromic indicator. A release of metal ions from the complex may occur, when a binding partner for the metal ions is at the same time present or will be added or generated, e.g., through a (chemical) reaction and which has a sufficient concentration and/or affinity for the metal ions bound by the metallochromic indicator.
A preferred metallochromic indicator provides in the context of the present invention, e.g., in the methods, uses or kits for the detection of a change of spectral or fluorescent properties of its complex with metal ions. Such a change occurs when metal ions become absent or are released from the complex. In the context of the present invention, during an amplification of a nucleic acid sequences, pyrophosphate occurs, which is a binding partner for a metal ion being in a complex with a metallochromic indicator. If pyrophosphate has a sufficient concentration and/or affinity for the metal ion bound by the metallochromic indicator, the metal ion is released from the complex and binds with/to pyrophosphate, thereby forming a metal ion-pyrophosphate salt.
A preferred change in the spectral properties of said complex is in the spectrum from 380 to 740 nm. Accordingly, the change in the spectral properties of said complex in its spectral properties is preferably a change of the color of the complex when compared to the metallochromic indicator not bound by metal ions, i.e., when metal ions no longer build a complex (with the metallochromic indicator) or are released from the complex. This may occur, as described above, since e.g., metal ions are released from the complex in the presence of, e.g., pyrophosphate which is generated during an amplification of a nucleic acid sequence as described herein.
In contrast to the present invention, the prior art applied for the detection of nucleic acid sequences metallochromic indicators, e.g., hydroxynaphtol blue (HNB) which build a complex the metal magnesium, i.e., a non-transition/non-post transition metal. Magnesium is an important ion for amplification methods (PCR, LAMP, etc.) as it serves as a cofactor and catalyst for enzyme activity and influences annealing of primers, stability of DNA, and incorporation of nucleotides. Because of this, using a colorimetric detection system that is dependent on Mg2+ ions is problematic as the levels of free Mg2+ for the enzyme and template vary during the course of the reaction. In addition, a colorimetric detection system that is dependent on Mg2+ prevents independent optimization of the detection system and the amplification reaction as both are tied to the concentration of the same ion. Accordingly, the detector or the metallochromic indicator preferably does not build a complex with magnesium. A detection system that uses a metal ion that is inert in the reaction is thus preferred. Suitable metal ions, in particular transition metal or post-transition metal ions, in the context of the invention may be Ba2+, Sr2+, Zn2+, Cd2+, Cu2+, Co2+, Ni2+, Hg2+, Pb2+, Pt2+, Ru2+, Rh2+, Fe2+, In3+, Al3+, Bi3+, La3+, Sc3+, Th3+, and/or Zr3+ ions, with Zn2+, Cu2+, C02+, Ni2+, Pt2+, Ru2+, Rh2+, Fe2+ ions being preferred, with Cu2+, Co2+, Ni2+, Zn2+ and/or Fe2+ ions being preferred, with Zn2+ ions being more preferred.
Also in contrast to the present invention, the prior art applied for the detection of nucleic acid sequences metallochromic indicators, e.g., calcein which build a complex with the non-transition metal manganese. Such a complex, when releasing metal ions provides for a change in its fluorescent properties. Manganese, while not normally included in PCR/LAMP reaction buffer, is known to have dramatic effects on amplification and thus should be avoided. While Mn2+ has been found to substitute for Mg2+ in PCR, it is well known to reduce the fidelity of DNA polymerases and consequently induce random mutagenesis during PCR. At high concentrations (e.g., 500 μM and above), Mn2+ can also be inhitory to PCR—the calcein/Mn2+ detection system uses 1 mM Mn2+.
In the context of the present invention, a metallochromic indicator is used to bind to metal ions added to a reaction mixture comprising a template nucleic acid sequence, primer nucleotide sequences, nucleotides and a polymerase capable of amplifying the nucleic acid molecule. Of course, the metallochromic indicator and metal ions are also comprised by the reaction mixture. Accordingly, the metallochromic indicator binds metal ions to build a complex. Without being bound by theory, during the amplification of nucleotide sequences, more and more pyrophosphate is released. The increasing amount of pyrophosphate will then bind the metal ions bound by the metallochromic indicator. As a result, the metallochromic indicator will change its spectral and/or fluorescent properties. This change serves as the indicator that the amplification reaction (successfully) takes place or has taken place.
Preferably, a pyrophosphate metal ion complex or salt has a higher affinity for the metal ion than the metallochromic indicator. This means the equilibrium of the metal ions is preferably shifted to the pyrophosphate than to the metallochromic indicator. Or, preferably, a pyrophosphate metal ion complex or salt is at a sufficient concentration to shift the equilibrium of the metal ions to complexing with pyrophosphate rather than the metallochromic indicator. The skilled person can readily determine this.
Also, the skilled person can determine the amount of metallochromic indicator and/or metal ions when used in a reaction mixture as described herein such that the desired change in spectral or fluorescent properties of the complex between a metallochromic indicator and metal ions can be detected. A change occurs when metal ions are released from the complex between a metallochromic indicator and metal ions, because more and more pyrophosphate is generated during the amplification of nucleic acid sequences as described herein and is commonly known in the art that pyrophosphate is released if a polymerase incorporates a nucleotide into DNA or RNA.
Preferred metal ions used in the context of the present invention, e.g., in the context of the methods, uses or kits described herein are transition metal ions or post-transition metal ions. A “transition metal” as used herein is 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 may be seen as being further elements of the transition metal group. A “post-transition metal” as used herein is an element selected from the group consisting of Al, Zn, Ga, Cd, In, Sn, Hg, TI, Pb, Bi, Po and At. Preferred examples of metal ions in general and of transition or post-transition metal ions in particular are Ba2+, Sr2+, Zn2+, Cd2+, Cu2+, C02+, Ni2+, Hg2+, Pb2+, Pt2+, Ru2+, Rh2+, Fe2+, In3+, Al3+, Bi3+, La3+, Sc3+, Th3+, Zr3+ ions, with Zn2+, Cu2+, Co2+, Ni2+, Pt2+, Ru2+, Rh2+, Fe2+ ions being preferred, with Cu2+, Co2+, Ni2+, Zn2+ and Fe2+ ions being preferred, with Zn2+ ions being more preferred.
Preferred examples of metallochromic indicators are those which are able to bind metal ions, preferably transition metal ions or post-transition metal ions. Examples of preferred metallochromic indicators are pyrocatechol violet, dithizone, zincon, eriochrome black T, murexide, PAN, phthalein purple, xylenol orange, 2-(5-Bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS). Examples of more preferred metallochromic indicators are pyrocatechol violet, zincon, dithizone, PAN, 5-Br-PAPS. Examples of even more preferred metallochromic indicators are zincon and 5-Br-PAPS. 5-Br-PAPS is most preferred.
The structures of the more preferred metallochromic indicators zincon as well as 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 associated phenyl rings:
The structures of the more preferred metallochromic 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 connected to the phenol ring:
The term “5-Br-PAPS” also encompasses derivatives of 5-Br-PAPS which are also preferred. Such derivatives may have one or more modifications, but are still capable of forming a complex with metal ions, and having identical, but 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=NO2), or 2-(5-Cyano-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (X=CN). The following structure shows a PAPS backbone in form of its disodium salt dehydrate, in which X can be replaced as described above:
As illustrated in the structure below, X can be replaced as described above and can also occupy alternative positions in the pyridine ring (e.g., positions 3-6) as a PAPS derivative:
The following Table 1 shows some further exemplary metallochromic indicators and its change in spectral properties with and without metal ions.
The following Table 2 shows more preferred metallochromic indicators, visible and spectral properties, with and without preferred metal ions in the presence of magnesium ions. PGP-26J12
In one embodiment, e.g., of the LAMP method of the invention, the detector is a metallochromic indicator, preferably 2-(5-Bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS) in combination with a metal ion, preferably a transition metal or post-transition metal, more preferably Zn2+, Cu2+, Co2+, Ni2+, Pt2+, Ru2+, Rh2+, and/or Fe2+ ions, most preferably Zn2+ ions, said metallochromic indicator and each of said metal ions building a complex, but not with magnesium, and wherein the method further comprises (b) detecting a change in spectral or fluorescent properties of the complex resulting from the amplification of said nucleic acid sequence.
In one embodiment, e.g., of the LAMP method of the invention, the detector is 5-Br-PAPS in combination with metal ions. Metal ions in this context may include any transition or post-transition metal that is chelated by both 5-Br-PAPS and pyrophosphate, including, but not limited to, Zn2+, Cu2+, Co2+, Ni2+, Pt2+, Ru2+, Rh2+, and/or Fe2+ ions, preferably Zn2+ ions.
Accordingly, the reaction mixture, e.g., used in the methods of the invention, may further comprise 2-(5-Bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS) and Zn2+, Cu2+, Co2+, Ni2+, Pt2+, Ru2+, Rh2+, and/or Fe2+ ions, preferably Zn2+ ions, said 5-Br-PAPS and each of said ions building a complex. In this context, the method of the invention further comprises
5-Br-PAPS is a compound that has the ability to bind transition or post-transition metals including, but not limited to, Zn2+, Cu2+, Co2+, Ni2+, Pt2+, Ru2+, Rh2+, and/or Fe2+ ions with high affinity, thereby building a complex, leading to a color change or in other words, a change in spectral or fluorescent properties. If the reaction mixture contains Zn2+, Cu2+, Co2+, Ni2+, Pt2+, Ru2+, Rh2+, and/or Fe2+ ions, preferably Cu2+, Co2+, Ni2+, Zn2+ and Fe2+ ions, more preferably Zn2+ ions, the reaction mixture is colored In case the metal ion is Zn2+, the reaction mixture is colored magenta (see also
In this context, the present invention further relates to an in vitro method for detecting a nucleic acid sequence amplification product, comprising
Preferably, the metallochromic indicator is 5-Br-PAPS. Preferably, the metal ion is a transition metal ion or post-transition metal ion, preferably a Zn2+ ion. Preferably, the change in spectral properties of said complex is in the spectrum from 380 to 740 nm wavelength. 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, a change in spectral properties of the reaction mixture from magenta to orange-yellow is visible, if a nucleic acid amplification product is produced in the in vitro method for detecting a nucleic acid sequence amplification product or other methods, uses and kits described herein.
Importantly, the change in spectral or fluorescent properties of the (5-Br-PAPS) complex is not dependent on a pH change of the reaction mixture (see Example 4,
The nucleic acid sequence amplification product of step (b) in the in vitro method for detecting a nucleic acid sequence amplification product may be obtained by any nucleic acid amplification method, including, but not limited to, polymerase chain reaction (PCR), LAMP, LNA-LAMP of the invention, nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), 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 may be done by carrying out the LNA-LAMP method of the present invention.
The detection of changes in the spectral properties of the (5-Br-PAPS) complex can be achieved by their photochemical properties using for example, the eyes of the operator, a fluorimeter, or a spectrophotometer. The term “detecting” may be used interchangeably with the term “monitoring”.
The final molar ratio of metallochromic indicator, preferably 5-Br-PAPS, to the metal ion, preferably Zn2+, Cu2+, Co2+, Ni2+, Pt2+, Ru2+, Rh2+, and/or Fe2+ ions, preferably Zn2+ ions, may be 2:1. This corresponds to the stoichiometry of the Ion2+/5-Br-PAPS complex in aqueous solutions. The Ion2+/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.
The LAMP method of the present invention may be used for different purposes. In one embodiment, the LAMP method of the invention is for synthesizing a nucleic acid sequence. In one embodiment, the LAMP method of the invention is for detecting a nucleic acid sequence. In one embodiment, the LAMP method of the invention is for diagnosing a disease, preferably caused by a pathogen.
The present invention further relates to a kit for the synthesis of a nucleic acid sequence by loop mediated isothermal amplification (LAMP) on a template nucleic acid sequence, comprising
The F3 nucleotide sequence and/or said B3 nucleotide sequence may comprise one or more locked nucleic acid (LNA) nucleotides which are located within the first third of said F3 nucleotide sequence or said B3 nucleotide sequence. The F3 nucleotide sequence may comprise one or more locked nucleic acid (LNA) nucleotides which are located within the first third of said F3 nucleotide sequence. The B3 nucleotide sequence may comprise one or more locked nucleic acid (LNA) nucleotides which are located within the first third of said B3 nucleotide sequence. “First third” as used herein may relate to the first third of a nucleotide sequence in 5′- to 3′-orientation, i.e., the nucleotides of the third of a nucleotide sequence, which is at the 5′-end of the nucleotide sequence.
5-Br-PAPS and Zn2+, Cu2+, Co2+, Ni2+, Pt2+, Ru2+, Rh2+, and/or Fe2+ ions, preferably Zn2+ ions, are further useful components of a kit since they will enable to carry out the detection of the presence of the template nucleic acid sequence. Accordingly, the kit of the invention may further comprise (v) 2-(5-Bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]phenol (5-Br-PAPS); and optionally a transition or post-transition metal that includes, but is not limited to, (vi) Zn2+, Cu2+, Co2+, Pt2+, Ru2+, Rh2+, and/or Fe2+ ions, preferably Zn2+ ions. The kit of the invention may further comprise a metallochromic indicator, preferably 5-Br-PAPS; and/or metal ions, preferably Zn2+ ions.
The present disclosure further relates to an aqueous preparation comprising: 5-Br-PAPS, a (DNA) polymerase, dNTPs, in a formulation that contains a buffering agent in an amount of 1 mM or more Tris or equivalent buffering agent such as 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 equivalent buffer. The aqueous preparation of the invention may further comprise primers, preferably the primers are F3 nucleotide sequence and/or said B3 nucleotide sequence as defined herein. The aqueous preparation may further comprise nucleotides serving as substrates for said DNA polymerase. The aqueous preparation of the invention may further comprise Zn2+, Cu2+, Co2+, Ni2+, Pt2+, Ru2+, Rh2+, and/or Fe2+ ions, preferably Zn2+ ions.
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 catalyzing a strand-displacement-type reaction of synthesizing a complementary nucleic acid strand from said template nucleic acid. The aqueous preparation may further comprise nucleotides serving as substrates for said DNA polymerase.
The present invention further relates to the following items:
It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
Unless otherwise indicated, the term “at least” preceding a series of elements is to 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 the present invention.
The term “and/or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.
The term “less than” or in turn “more than” does not include the concrete number.
For example, less than 20 means less than the number indicated. Similarly, more than or greater than means more than or greater than the indicated number, e.g., more than 80% means more than or greater than the indicated number of 80%.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated 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. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. When used herein “consisting of” excludes any element, step, or ingredient not specified.
The term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can 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.
All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.
The content of all documents and patent documents cited herein is incorporated by reference in their entirety.
An even better understanding of the present invention and of its advantages will be evident from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.
Our preferred detection reagent was selected in an initial screen to identify chelatometric dyes with desired spectral characteristics, namely ability to form colored complexes with preferred metal ions in the presence of magnesium, lucidness of observed color change and high absorbance of the formed dye-metal complex. Combinations of potential Mg2+-insensitive detection reagents were prepared under buffered conditions (20 mM Tris-HCl pH=7.9, 50 mM KCl) in the presence of high magnesium concentrations (8 mM MgSO4) by mixing one of the preferred complexometric dyes (5-Br-PAPS, PAR or zincon; all sourced from Sigma-Aldrich, Missouri, USA) with one of the preferred metal ions (Co2+, Cu2+, Fe2+, Ni2+, Rh2+, Zn2+) in such a way that the final concentration of complexometric dyes was 100 μM while the concentration of metal ions was either 50 μM (with 5-Br-PAPS, PAR) or 100 μM (with zincon) reflecting the predicted stoichiometry of the formed dye-metal complexes.
Spectral analysis on a Tecan Infinite M1000 Pro Multi-mode Reader (Tecan, Switzerland) was used to determine the Amax of free dyes and their dye-metal complexes while pictures were taken with a cell phone camera. As can be seen in
5-Br-PAPS/metal complexes were further compared with respect to their ability to release chelated metal ions in the presence of pyrophosphate (PPi). 100 μM complexes of 5-Br-PAPS and preferred metal ions (Co2+, Cu2+, Fe2+, Ni2+, Rh2+, Zn2+, 50 μM each) were exposed to progressively higher concentrations of PPi (from 0 to 20 mM) and absorbances at dye-metal complex Amax were measured spectrophotometrically on a Tecan Infinite M1000 Pro Multi-mode Reader (Tecan, Switzerland). Results (
To prepare the most preferred Zn2+/5-Br-PAPS detection reagent, a solution of 5-Br-PAPS is mixed with a solution of ZnSO4 (both in PCR-grade water) in such a way, that the final molar ratio of 5-Br-PAPS to ZnSO4 is 2:1 corresponding to the stoichiometry of the Zn2+/5-Br-PAPS complex in aqueous solutions. A 1.5 mM solution of the Zn2+/5-Br-PAPS detection reagent (1.5 mM 5-Br-PAPS, 750 μM ZnSO4) is stable at room temperature in the dark and can be used to prepare reaction mixes.
The Zn2+/5-Br-PAPS detection reagent changes color during DNA amplification due to PPi (produced as a side-product of dNTP hydrolysis by a polymerase) competing with 5-Br-PAPS to bind Zn2+ (
The PPi concentration-dependent effect on the color of the Zn2+/5-Br-PAPS complex was analyzed spectrophotometrically on a Tecan Infinite M1000 Pro Multi-mode Reader (Tecan, Switzerland) by performing a spectral analysis in the 330-600 nm wavelength range while visual color changes were recorded by a cell phone camera.
LAMP amplification reactions (all 20 μl final volume) were performed 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, where the template strand is RNA, also included 300 U/ml WarmStart® RTx reverse transcriptase (M0380, New England Biolabs, Massachusetts, USA) and 0.8 M betaine. 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.
In
In
The following primers were used and sequences between hypens are linkers not complementary to the template nucleic acid:
Reactions containing 25 μM of Zn2+/5-Br-PAPS detection reagent were used to detect nucleic acid amplification during PCR on a SensoQuest Gradient Labcycler (SensoQuest, Germany). 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). A template input of 0.375 ng/μl of human gDNA and primers targeting human RPP30 (both forward and reverse primers at 500 nM) were used to perform the amplification. The PCR reaction was performed as follows: a 10-minute hot-start at 95° C. was followed by 45 cycles of melting (95° C. for 15 seconds) and annealing/extension (60° C. for 30 seconds). As can be seen in
The following primers were used:
It is known from the prior art that colorimetric detection systems for nucleic acid amplification detection, especially those relying on pH-based detection, are susceptible to reaction failure with buffered samples or those containing variable initial pH (e.g., biological samples like saliva), prohibiting their use with unpurified samples. This limitation also precludes proper reaction mix buffering throughout amplification which can result in suboptimal amplification kinetics and/or sensitivity. As the Zn2+/5-Br-PAPS detection reagent does not rely on free hydronium ion accumulation with its associated pH decrease to function, reactions utilizing this reagent can not only tolerate strong buffering in the reaction mix but by extension also of the input sample. Such combination of properties lends itself especially well to techniques such as direct pathogen detection with no prior nucleic acid isolation in biological matrices with varying levels of pH/buffering capacity. The following reactions were set up to show that the colorimetric assay of the present invention is robust with respect to pH change, since it accepts buffers preferably higher than 1.5 mM Tris or an equivalent thereto both in the reaction mix and in input samples.
LAMP reactions were performed in the same manner as it is described in Example 3 with some minor modifications in certain experiments which are pointed out in their descriptions. Mock LAMP reactions had the same basic composition as in regular LAMP to which specified amounts of the Zn2+/5-Br-PAPS detection reagent was added. To prepare the mock samples, 50% volume of 2× master mix (40 mM Tris-HCl pH=7.9, 40 mM KCl, 120 mM, GuCl, 18 mM MgSO4, 0.2% Tween-20, 0.1% Triton X-100) was mixed with 25% volume of water or different pH 10 mM buffers (pH=5, 6-10 mM sodium citrate; pH=7, 8, 9-10 mM Tris-HCl; 2.5 mM final concentration) and 25% volume of either water (mock negative sample) or 16 mM sodium pyrophosphate (mock positive sample, 4 mM final concentration).
In
In
In sum, the inventors could surprisingly show that 5-Br-PAPS in combination with Zinc ions can be used to detect nucleic acid amplification in a reaction mixture.
The replacement of specific bases with their LNA variants in F3/B3 LAMP primers described herein may significantly alter LAMP reaction kinetics and target recognition. Specifically, introduction of a predefined number of LNA bases near the 5′ end of F3/B3 primers significantly reduces time-to-reaction (TTR) while simultaneously increasing specificity, both traits desirable in potential isothermal diagnostic tests. This may be useful to shorten positive reaction times thus widening the window between true and false positive results, which can be problematic for isothermal methods like LAMP. Incorporating LNA-modified bases in LAMP primers is also beneficial for normalizing primer melting temperatures (Tm) and providing flexibility in LAMP primer design when targeting challenging templates that are either A,T or G,C rich or organisms that have high rates of mutations (e.g., SARS-CoV-2 and Influenza). While those skilled in the art may appreciate the fact that LNA incorporation into primers can lead to improved target hybridization rates chiefly by increasing the primer's melting temperature (Tm), due to the complex nature of primer interactions during LAMP target amplification, unthoughtful changes to primer base composition would not be expected to lead to beneficial effects during LAMP amplification and may facilitate non-specific interactions and amplification and impede strand displacement activity of the polymerase.
Unless specified otherwise, in all experiments described below, LAMP amplification was performed as follows. Reaction mixes were prepared by mixing WarmStart Colorimetric RT-LAMP 2× MasterMix (M1800, New England Biolabs, Massachusetts, USA) and 10× primer mix consisting of 6 specific LAMP primers. Base LAMP primer sets targeting SARS-CoV-2 genes E (sets E1, E2, E3), RdRP (set R1) and N (set N1) were designed to follow the recommendations as known from the prior art. Final reaction concentrations of LAMP primers were: FIP/BIP—1600 nM, F3/B3—200 nM, LF/LB—600 nM. All assays utilized 20 μl reaction volumes. All reactions were also supplemented with 0.05% Triton X-100, 1 mM extra of MgCl2 (final concentration 9 mM), and 1 μM of the nucleic acid-intercalating dye, SYTO 9 (Thermo Fischer Scientific, Massachusetts, USA). Reactions performed at 65° C. also included 40 mM guanidine isothiocyanate and 0.8 M betaine. Positive test reactions either contained SARS-CoV-2 RNA mixed with human genomic DNA (COV019, Exact Diagnostics, California, USA; when RNA copy/rxn was <200) or pure SARS-CoV-2 RNA (102019, Twist Bioscience, California, USA; when RNA copy/rxn was ≥200). Once the reactions were set up, they were run at a primer set-/condition-specific temperature and allowed to amplify on an Agilent AriaMx Real-Time PCR System (Agilent, California, USA). The process of amplification was continuously monitored every 30 seconds by measuring changes in SYTO 9 fluorescence (FAM channel) and the reaction was terminated after 90 minutes. Time-to-reaction (TTR, cycle threshold/2) was determined automatically by the Agilent Aria 1.7.1 software (Agilent, California, USA) using default settings. Melt curve analysis of the resulting amplicons was also used to distinguish specific from non-specific products. Specifically, products were considered specific if their Tm was in the range mean±0.5° C. of products observed in high template concentration reactions as internally established for each tested primer set. Replicates with no amplification are not depicted in any of the figures and were omitted from calculating descriptive statistics, e.g., mean±SD.
In
Beneficial effects of 5′ LNA incorporation into outer LAMP primers are robust and not dependent on specific bases modified as
In
The following primers were used, wherein (+N) depicts the corresponding base in form of an LNA and sequences between hypens are linkers not complementary to the template nucleic acid:
| Number | Date | Country | Kind |
|---|---|---|---|
| 20206643.7 | Nov 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/081230 | 11/10/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63120652 | Dec 2020 | US |
