Improved isothermal amplification

Abstract

The occurrence of false positives is reduced, the efficiency is increased, and / or the sensitivity is increased in loop-mediated isothermal amplification (LAMP) of a target nucleic acid. [Solution] 1. A method comprising: combining a plurality of LAMP primers for a target nucleic acid, the LAMP primers comprising an F3 primer and a B3 primer, wherein both the F3 and B3 primers or the F3 primer comprise a promoter for an RNA polymerase at its 5' end; dNTPs; a strand displacing DNA polymerase; a reverse transcriptase; an RNA polymerase; and rNTPs and optionally a nucleic acid binding dye or probe to form a LAMP reagent mix; incubating the LAMP reagent mix with a target nucleic acid under conditions suitable for amplification of the target nucleic acid, wherein the efficiency and / or sensitivity is increased compared to the same amplification performed in the absence of the promoter, RNA polymerase and rNTPs.

Description

[Technical field]

[0001] Technical Field

[0001] The present invention relates to a method and a kit for improving an isothermal amplification reaction. In particular, the present invention relates to a method and a kit for increasing the efficiency and / or sensitivity of a loop-mediated isothermal amplification (LAMP) reaction by using multiple LAMP primers, at least one of which includes a promoter for an RNA polymerase.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Australian Provisional Patent Application No. 2021903771, filed on November 23, 2021, which is incorporated by reference in its entirety. [Background technology]

[0003] background

[0003] Detection of a nucleic acid target sequence in a sample usually requires the target sequence to be amplified until the amount of DNA present is measurably high. Detection of an RNA target sequence, e.g., viral RNA, additionally requires transcription of the RNA to DNA prior to amplification. Polymerase chain reaction (PCR) plays a central role in nucleic acid amplification and detection methods. However, one drawback of PCR is the need for a thermal cycler to allow DNA denaturation and to repeat multiple cycles of denaturation and amplification, stepwise changing the temperature of the reaction mix every few seconds to minutes to amplify the DNA fragments.

[0004]

[0004] Isothermal amplification reactions allow detection of target nucleic acids at constant temperature, and therefore are not constrained by the need for thermal cycling. There are various isothermal amplification methods, all of which share some common features. For example, since DNA strands are not heat denatured, isothermal methods rely on polymerases with strand displacement activity to allow primer binding and initiation of the amplification reaction. Isothermal amplification methods have been applied to point-of-care diagnosis of diseases and commercial diagnostic platforms with great success.

[0005]

[0005] Loop-mediated isothermal amplification (LAMP) is an isothermal amplification method designed to detect target nucleic acids without requiring sophisticated equipment. LAMP reactions use a DNA polymerase with strand displacement activity and a set of four to six different primers that are specifically designed to recognize distinct regions of DNA or RNA target nucleic acids. The reaction can be performed with limited resources, for example, using a water bath for incubation (typically 60-70°C), and positive results can be identified visually by turbidity, colorimetric change, lateral flow readout, addition of fluorescent DNA-binding dyes or labeled probes. In the COVID-19 pandemic, LAMP reactions have been employed to rapidly detect SARS-CoV-2 RNA in clinical and residential settings.

[0006]

[0006] Although many isothermal methods and kits currently exist, there remains a need for improvements on existing methods and kits with improved efficiency and / or sensitivity.

[0007]

[0007] The present inventors have developed a method and a kit for improving the efficiency and / or sensitivity of existing isothermal amplification methods. In particular, the present inventors have unexpectedly found that the efficiency and / or sensitivity of LAMP can be improved by adding a LAMP primer containing an RNA polymerase promoter and by adding rNTPs together with RNA polymerase, compared to the same reaction (under the same conditions) in the absence of a LAMP primer containing an RNA polymerase promoter, rNTPs, and RNA polymerase.

[0008] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the specification is solely for the purpose of providing context for the present technology and is not to be construed as an admission that any or all of such matters formed part of the prior art body or were common general knowledge in the art relevant to the present technology by virtue of existing prior to the priority date of each claim herein. Summary of the Invention [Means for solving the problem]

[0009] overview In a first aspect, there is provided a method for reducing the occurrence of false positives, increasing the efficiency, and / or increasing the sensitivity of loop-mediated isothermal amplification (LAMP) of a target nucleic acid, comprising: a) i) a plurality of LAMP primers for a target nucleic acid, comprising an F3 primer and a B3 primer, wherein both the F3 and B3 primers or the F3 primer comprises a promoter for an RNA polymerase at the 5' end; ii) dNTPs; iii) strand-displacing DNA polymerases; iv) reverse transcriptase; v) RNA polymerase; and vi) rNTP and vii) Optionally, a nucleic acid binding dye or probe. combining to form a LAMP reagent mix; b) incubating the LAMP reagent mix with a target nucleic acid under conditions suitable for amplification of the target nucleic acid, which results in increased efficiency and / or sensitivity compared to the same amplification carried out in the absence of a promoter, RNA polymerase and rNTPs; A method is provided that includes:

[0010] In one embodiment, the probe is specific for the target, for example an Easybeacon™ probe (manufactured by Pentabase).

[0011] In one embodiment, a promoter for an RNA polymerase is present at the 5' end of the LAMP primer.

[0012]

[0012] Conditions suitable for amplifying a target nucleic acid may include incubating the LAMP reagent mix with the target nucleic acid at a temperature of about 60°C to about 70°C for about 10 minutes to about 40 minutes.

[0013]

[0013] Conditions suitable for amplifying the target nucleic acid may include incubating the LAMP reagent mix with the target nucleic acid at a temperature of about 65°C for about 10 minutes to about 40 minutes.

[0014]

[0014] In one embodiment, conditions suitable for amplifying a target nucleic acid may include incubating the LAMP reagent mix with the target nucleic acid at a temperature of about 42°C for about 1 minute to about 10 minutes, and then further incubating the LAMP reagent mix with the target nucleic acid at a temperature of about 60°C to about 70°C for about 10 minutes to about 40 minutes.

[0015]

[0015] In one embodiment, conditions suitable for amplifying a target nucleic acid may include incubating the LAMP reagent mix with the target nucleic acid at a temperature of about 42°C for about 1 minute to about 10 minutes, and then further incubating the LAMP reagent mix with the target nucleic acid at a temperature of about 65°C for about 10 minutes to about 40 minutes.

[0016]

[0016] In some embodiments, the promoter sequence is selected from the T7 promoter of SEQ ID NO: 1, the T3 promoter of SEQ ID NO: 2 to 4, and the SP6 promoter of SEQ ID NO: 5 to 6. In some embodiments, random nucleotides may be added to the 5' or 3' end of the promoter sequence to improve amplification.

[0017]

[0017] In some embodiments, the promoter is selected from a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:1, a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:2, a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:3, a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:4, a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:5, and a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:6.

[0018]

[0018] In some embodiments, the RNA polymerase is selected from T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerase.

[0019]

[0019] In some embodiments, the concentration of rNTP in the LAMP reagent mix is ​​about 0.25 mM, preferably less than 2.5 mM, and the concentration of RNA polymerase in the LAMP reagent mix is ​​about 7.5 units per reaction, preferably less than 50 units per reaction.

[0020]

[0020] In one embodiment, the LAMP reagent mix includes guanidinium hydrochloride (GuHCl). The concentration of GuHCl in the LAMP reagent mix is ​​at least about 30 mM to about 60 mM.

[0021]

[0021] In some embodiments, the target nucleic acid is bisulfite treated.

[0022] In some embodiments, the target nucleic acid is in its wild-type (WT) form.

[0023]

[0023] In one embodiment, the multiple LAMP primers include a FIP primer, a BIP primer, an LF primer and a LB primer.

[0024]

[0024] In some embodiments, the FIP primer, the BIP primer, the LF primer and the LB primer do not contain a promoter for an RNA polymerase.

[0025]

[0025] In some embodiments, both the F3 primer and the B3 primer contain a promoter sequence at the 5' end, and the promoter sequence is selected from a T7 promoter of SEQ ID NO: 1, a T3 promoter of SEQ ID NO: 2 to 4, and an SP6 promoter of SEQ ID NO: 5 to 6.

[0026]

[0026] In some embodiments, the F3 primer comprises a promoter sequence at the 5' end, and the promoter sequence is selected from the T7 promoter of SEQ ID NO: 1, the T3 promoters of SEQ ID NOs: 2-4, and the SP6 promoters of SEQ ID NOs: 5-6.

[0027]

[0027] In some embodiments, the F3 primer or the B3 primer contains a promoter sequence at the 5' end, and the promoter sequence is selected from a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:1, a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:2, a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:3, a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:4, a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:5, and a sequence having at least 90% sequence identity to the sequence of SEQ ID NO:6.

[0028]

[0028] The target nucleic acid can be RNA or DNA. In some embodiments, the RNA or DNA is specific to a pathogen.

[0029]

[0029] The pathogen is selected from a bacterium, a virus, a fungus and a parasite.

[0030]

[0030] The bacteria may be Salmonella spp., Bordetella spp., Campylobacter spp., Clostridium spp., Chlamydia spp., Chlamydophila spp., Listeria spp.; Lymphogranuloma spp., Shigella spp., Neisseria spp., Staphylococcus spp., Streptococcus spp., Listeria spp., Leishmania spp., spp., Bacillus spp., Borrelia spp., Rickettsia spp., Corynebacterium spp., Gardenella spp., Haemophilus spp., Escherichia spp., Helicobacter spp., Klebsiella spp., Legionella spp., Mycobacerium spp., Mycoplasma spp.; Moraxella spp., Pasteurella spp. spp), Pneumocystis spp, Pseudomonas spp, Treponema spp, Ureaplasma spp, Vibrio spp, Aermonas spp, and Yersinia spp.

[0031]

[0031] Viruses include parechoviruses, rabies viruses, measles viruses, mumps viruses, rubella viruses, togaviridae, polyomaviruses, papillomaviruses, hepadnaviruses, poxviruses, adenoviruses, picornaviruses, hepeviruses, caliciviruses, reoviruses, retroviruses, orthomyxoviruses, paramyxoviruses, coronaviruses, Ebola viruses, The coronavirus may be selected from the families Filoviridae, Flaviviridae, Rhabdoviridae, Bunyavirales, Arenaviridae, and Hantaviridae, and Deltavirus. In one embodiment, the coronavirus is coronavirus HKU-1, coronavirus OC43, coronavirus NL63 / 229E, or SARS-CoV-2.

[0032]

[0032] The parasites include Blastocystis spp, Giardia spp, Cryptosporidium spp, Cyclospora spp, Blastocystis spp, Dientamoeba spp, Entamoeba spp, Cryptococcus spp, Enterocytozoon spp, Encephalitozoon spp, Babesia spp, Leishmania spp, Schistosoma spp, spp., Trypanosoma spp.; Trichimonas spp., Treponema spp., and Plasmdoium spp.

[0033]

[0033] The fungus may be selected from Aspergillus spp, Candida spp, Histoplasma spp, Fusarium spp, Pneumocystis spp, Paracoccoides spp, Coccidioides spp, and Scedosporium spp.

[0034] In some embodiments, the sensitivity of the LAMP reaction is increased by at least about 10% to about 30%. In one embodiment, the sensitivity of the LAMP reaction is improved to a single copy detection level in less than about 18 minutes.

[0035]

[0035] In some embodiments, the efficiency of the LAMP reaction is increased by at least about 5% to about 20%.

[0036]

[0036] In some embodiments, the LAMP is a multiplex reaction that includes, in step a), combining a second plurality of LAMP primers for a second target nucleic acid, the second plurality of LAMP primers including an F3 primer that includes a promoter for an RNA polymerase at the 5' end.

[0037]

[0037] In one embodiment, multiple LAMP primers for a target nucleic acid are provided, the multiple LAMP primers including an F3 primer and a B3 primer, each of which includes a promoter for an RNA polymerase at the 5' end, or an F3 primer including a promoter for an RNA polymerase and at least one sequence complementary to the target nucleic acid.

[0038] In a second aspect, the present invention provides a kit for detecting a loop-mediated isothermal amplification reaction (LAMP) using a target nucleic acid, comprising: i) a plurality of LAMP primers for a target nucleic acid, comprising an F3 primer and a B3 primer, wherein both the F3 and B3 primers or the F3 primer comprises a promoter for an RNA polymerase at the 5' end; ii) dNTPs; iii) strand-displacing DNA polymerases; iv) reverse transcriptase; v) RNA polymerase; vi) rNTP; and vii) Optionally, a nucleic acid binding dye or probe. The present invention relates to a kit comprising:

[0039]

[0039] LAMP reaction products can be detected using a variety of methods known to those of skill in the art, including visual examination or turbidity monitoring for precipitated magnesium pyrophosphate, fluorescent detection of double-stranded DNA (dsDNA) using intercalating fluorophores or fluorescent probes, bioluminescent reporting via pyrophosphate conversion, and by use of lateral flow devices.

[0040] In one embodiment, the kit of the second aspect detects an RNA or DNA specific pathogen.

[0041]

[0041] The multiple LAMP primers may further include a FIP primer, a BIP primer, an LF primer and a LB primer.

[0042]

[0042] In some embodiments, the FIP primer, BIP primer, LF primer and LB primer do not contain a promoter for an RNA polymerase.

[0043] In one embodiment, the kit of the second aspect is for detecting a multiplex LAMP reaction.

[0044]

[0044] In some embodiments, the second aspect of the kit for detecting a multiplex LAMP reaction further comprises a second plurality of LAMP primers for a second target nucleic acid, the second plurality of LAMP primers including an F3 primer comprising a promoter for an RNA polymerase at the 5' end. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] definition

[0045] Throughout this specification, unless the context otherwise requires, the word "comprise" or variations such as "comprises" or "comprising" are understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not to exclude any other element, integer or step, or group of elements, integers or steps.

[0046]

[0046] Unless the context specifically requires or unless specifically stated otherwise, integers, steps, or elements of the invention referred to in this specification as singular integers, steps or elements explicitly include both the singular and plural forms of the referred integer, step or element.

[0047] In the context of this specification, the terms "a," "an," and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, reference to "a sample" means one sample or more than one sample.

[0048]

[0048] "At least one", as used herein, relates to one or more, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0049]

[0049] In the context of this specification, the term "about" means that a reference to a number or value should not be interpreted as an absolute number or value, but includes an allowable variation above and below that number or value based on what a person skilled in the art would understand according to the art, including within typical tolerances or device limits. In other words, the use of the term "about" is understood to refer to a range or approximate value that a person skilled in the art would consider equivalent to the cited value in the context of achieving the same function or result.

[0050]

[0050] The phrase "and / or" as used herein should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present conjointly in some cases and unjoined in other cases. As a non-limiting example, a reference to "efficiency and / or sensitivity" may refer in one embodiment to efficiency only, in another embodiment to sensitivity only, and in yet another embodiment to both efficiency and sensitivity.

[0051]

[0051] As used herein, the term "isothermal amplification" refers to a process of repeatedly copying a target nucleic acid without using heat to separate any double strands formed during the process. In some embodiments, "isothermal amplification" refers to an amplification reaction carried out at a single temperature. In some embodiments, "isothermal amplification" refers to an amplification reaction carried out at two different temperatures. Isothermal amplification includes, but is not limited to, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), whole genome amplification (WGA), rolling circle amplification (RCA) and multiple displacement amplification (MDA). It is envisioned that the methods and kits described herein can be applied in various isothermal amplification techniques, such as LAMP, SDA, HDA, RPA, WGA, RCA and MDA.

[0052]

[0052] "Target nucleic acid" refers to a nucleic acid sequence or a subsequence of a larger nucleic acid (template) that is the subject of repeated copies. Target nucleic acids can be RNA or DNA and can be obtained from biological samples in vivo or in vitro. As used herein, the term "target nucleic acid" also encompasses mRNA and genomic DNA.

[0053]

[0053] As used herein, the term "sample" is used in its broadest sense. In one embodiment, it is meant to include representative portions or cultures obtained from any source, including biological and environmental sources. Biological samples can be obtained from animals (including humans) and include saliva, swab samples, bronchial lavage (BAL), body fluids (e.g., blood, plasma, serum, CSF, feces, or urine), organs, tissues, cells, sectioned portions of organs or tissues, or cells isolated from biological subjects (e.g., areas containing disease cells). Environmental samples include environmental materials, such as surface matter, soil, mud, sludge, sewage, biofilms, water, and industrial samples. However, such examples should not be construed as limiting the sample types applicable to the present invention.

[0054]

[0054] Target DNA or RNA can be of eukaryotic, prokaryotic, viral or bacteriophage origin.For example, target DNA or RNA can be obtained from insects, protozoa, birds, fish, reptiles, mammals (e.g., rats, mice, cows, dogs, guinea pigs or rabbits), or primates (e.g., chimpanzees or humans).Target DNA can be complementary DNA (cDNA) generated from RNA templates (e.g., mRNA, ribosomal RNA, siRNA and other variants) using reverse transcriptase enzyme.

[0055]

[0055] The term "primer" or "primer sequence" refers to an oligonucleotide that hybridizes to a target nucleic acid template to generate a target nucleic acid:primer hybrid and initiate nucleic acid synthesis. A person skilled in the art can design and identify suitable primers for any isothermal amplification reaction. Tools for designing primers are known in the art. Primers can be RNA oligonucleotides, DNA oligonucleotides, or chimeric sequences. In one embodiment, a plurality of LAMP primers are provided for detecting LAMP of a target nucleic acid sequence. The term "LAMP primers" as used herein refers to a plurality of primers including a first outer primer F3, a second outer primer B3, a first inner primer FIP (or primers F1c and F2), a second inner primer BIP (or primers B1c and B2), a first loop primer LF, and a second loop-primer LB. In addition to the standard set of LAMP primers, in one embodiment, at least one of the LAMP primers includes a promoter sequence for RNA polymerase. For example, the F3 or B3 primer is designed to have an RNA polymerase binding site along with a target recognition sequence. In one embodiment, the RNA polymerase binding site is located at the 5' end of the LAMP primer sequence.

[0056]

[0056] "RNA polymerase" as used herein may include, but is not limited to, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerase. For example, the term "T7 promoter" refers to a sequence recognized by T7 RNA polymerase, the term "T3 promoter" refers to a sequence recognized by T3 RNA polymerase, and the term "SP6 promoter" refers to a sequence recognized by SP6 RNA polymerase. When the promoter sequence is recognized by the respective RNA polymerase, the RNA polymerase initiates transcription of RNA from a DNA template. Any suitable RNA polymerase may be included in the methods and kits described herein. In one embodiment, the nucleic acid sequence of the T7 promoter is 20 nucleotides in length and is set forth in SEQ ID NO: 1. In one embodiment, the nucleic acid sequence of the T3 promoter is either 17, 20 or 24 nucleotides in length and is set forth in SEQ ID NOs: 2-4, respectively. In one embodiment, the nucleic acid sequence of the SP6 promoter is 24 or 18 nucleotides in length and is set forth in SEQ ID NOs: 5 and 6, respectively.

[0057]

[0057] As used herein, the term "strand-displacing DNA polymerase" refers to a DNA polymerase that has strand-displacing activity apart from its DNA synthesis activity. Strand-displacing DNA polymerase can continue DNA synthesis by reading the template strand based on the sequence of the nucleic acid template strand, while displacing the complementary strand that is annealed to the template strand. The strand-displacing DNA polymerase used in the isothermal amplification method can be a proofreading or non-proofreading DNA polymerase, which can be thermophilic or mesophilic. Any suitable strand-displacing DNA polymerase can be included in the methods and kits described herein. For example, the strand displacing DNA polymerase may be selected from Bst large fragment polymerase, Bst2.0, Bst3.0, Bca(exo-), Vent, Vent(exo-), Deep Vent, Deep Vent(exo-), Φ29 phage, MS-2 phage, Z-Taq, KOD, Klenow fragment, GspSSD, GspF, OmniAmp polymerase, SD polymerase, and any combination thereof.

[0058]

[0058] The terms "dNTP" and "rNTP" refer to nucleotides, i.e., deoxyribonucleotide triphosphates and ribonucleotide triphosphates, respectively, and are known to those skilled in the art. The terms also encompass modified forms of dNTPs and rNTPs, provided that the modified forms are recognized by enzymes having DNA polymerase activity and / or RNA polymerase activity.

[0059]

[0059] As used herein, the term "reverse transcriptase" refers to any DNA polymerase that can copy a first strand complementary DNA (cDNA) from an RNA template. Such enzymes are commonly referred to as RNA-directed or RNA-dependent DNA polymerases. In some embodiments, the reverse transcriptase can copy a cDNA strand using either single-stranded RNA or DNA as a template.

[0060]

[0060] Amplification reagents suitable for various isothermal amplification methods are known in the art. For example, a reagent mix for a conventional LAMP reaction includes a strand-displacing DNA polymerase, a LAMP primer, and dNTPs that are added to the target DNA. A reagent mix for a conventional LAMP reaction may further include a reverse transcriptase when the target nucleic acid is an RNA target. In the context of this specification, the term "LAMP reagent mix" refers to a reagent mix that includes: i) a plurality of LAMP primers for a target nucleic acid, at least one of the LAMP primers includes a promoter for an RNA polymerase; ii) dNTPs; iii) a strand-displacing DNA polymerase; iv) a reverse transcriptase; v) an RNA polymerase; and vi) rNTPs.

[0061]

[0061] As used herein, "efficiency" is measured based on the time required to reach a detectable level of amplification. For example, the higher the amplification efficiency, the shorter the time required to reach an amplification result.

[0062]

[0062] As used herein, "sensitivity" refers to the detection limit, i.e., the minimum amount of target nucleic acid that must be present to reliably detect and quantify under given amplification conditions. In one embodiment, sensitivity is expressed as a threshold cycle (Ct) value. The Ct value serves as a tool for calculation of the starting amount of nucleic acid template in a sample and represents the cycle number at which the fluorescent signal begins to increase significantly from the baseline (base signal). In one embodiment, the method and kit of the present invention improve the sensitivity of isothermal amplification reaction down to single copy detection level in less than 18 minutes.

[0063] In order that this invention may be more clearly understood, preferred embodiments will now be described with reference to the following examples.

[0064] Description of the embodiments method

[0064] The present inventors have found that the speed and sensitivity of various isothermal amplification methods can be improved by adding a promoter for RNA polymerase to the LAMP primer and adding rNTPs together with the RNA polymerase.

[0065]

[0065] Conventional LAMP is usually performed at a constant temperature of 60°C to 70°C and uses a DNA polymerase with strand displacement activity and a set of four oligonucleotides called inner and outer primers that are specifically designed to recognize six different recognition sites on the target nucleic acid. The two outer primers play a role in strand displacement only during the non-cycling step, while the inner primer contains both sense and antisense sequences and contributes to the formation of a typical LAMP amplification product with a stem-loop structure. In addition to the four oligonucleotide primers, the LAMP assay may contain two additional primers, so-called loop primers, to improve the amplification efficiency, thereby resulting in a total of six primers per target sequence. Such a combination of different LAMP primers, spanning eight distinct sequences on the target nucleic acid, provides better specificity.

[0066]

[0066] The present invention provides a method for increasing the efficiency and / or sensitivity of LAMP by using a plurality of LAMP primers, including i) a plurality of LAMP primers for a target nucleic acid, comprising an F3 primer and a B3 primer, where both the F3 and B3 primers or the F3 primer comprises a promoter for an RNA polymerase at the 5' end. The method also includes ii) dNTPs, iii) a strand-displacing DNA polymerase, iv) a reverse transcriptase, v) an RNA polymerase, vi) rNTPs, and optionally a nucleic acid-binding dye or probe. The components are combined to form a LAMP reagent mix. The reagent mix is ​​usually prepared immediately before use. However, it is envisioned that the reagent mix or some components of the reagent mix may be prepared in advance, for example, the primers, dNTPs and rNTPs may be premixed and the enzyme may be added immediately before use.

[0067]

[0067] The multiple LAMP primers further include a FIP primer, a BIP primer, an LF primer and a LB primer.

[0068]

[0068] Preferably, the F3 and B3 primers or only the F3 primer contain a promoter for RNA polymerase at the 5' end. That is, the other primers do not contain a promoter for RNA polymerase. For example, the FIP primer, the BIP primer, the LF primer and the LB primer do not contain a promoter for RNA polymerase.

[0069]

[0069] The reagent mix also includes a buffer solution. Suitable buffer solutions for LAMP reactions are known in the art. One suitable buffer is a pH 8.8 Tris-HCl buffer containing 20 mM Tris-HCL, 10 mM (NH4)2SO4, 50 mM KCL, 2 mM MgSO4, 0.1% Tween® 20. Those skilled in the art will recognize alternative buffers and understand that the choice of buffer is partially dependent on the polymerase used. For example, the above buffer is optimized for use with Bst2.0 DNA polymerase.

[0070]

[0070] Once the LAMP reagent mix is ​​formed, the target nucleic acid is added and the reagent mix and nucleic acid are incubated under conditions suitable for amplification of the target nucleic acid.

[0071]

[0071] In some embodiments, the target nucleic acid or a sample suspected of containing the target nucleic acid is bisulfite treated using any method known in the art, which may facilitate detection of specific methylated forms of the target nucleic acid.

[0072]

[0072] In embodiments in which bisulfite treatment is used, the reagent mix and sample may optionally be incubated at about 42°C for a period of 1 to 15 minutes and then incubated at an elevated temperature of 50 to 55°C (e.g., 53°C) for 5 to 40 minutes, e.g., 15, 20, 30 minutes.

[0073]

[0073] In other embodiments, the reagent mix and sample are optionally incubated at about 42°C for a period of 1 to 15 minutes and then incubated at an elevated temperature of 60 to 70°C (e.g., 65°C) for 5 to 40 minutes, e.g., 15, 20, or 30 minutes.

[0074]

[0074] Preferably, the method is carried out using guanidinium hydrochloride (GuHCl) if required for the particular primer set added to the LAMP reagent mix. GuHCl can be present at a concentration of about 30 nM to about 90 nM, for example, about 30 mM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, or about 90 nM. In some embodiments, GuHCl is present in the LAMP reagent mix at a concentration of about 60 nM.

[0075]

[0075] The concentration of rNTP and RNA polymerase in the LAMP reagent mix can be optimized. In some embodiments, the concentration of rNTP in the LAMP reagent mix is ​​about 0.25 mM, preferably less than about 2.5 mM, and the concentration of RNA polymerase in the LAMP reagent mix is ​​about 7.5 units per reaction, preferably less than about 50 units per reaction.

[0076] As exemplified herein, the use of an RNA polymerase promoter sequence as part of a primer sequence improves the efficiency and / or sensitivity of LAMP reaction.It is envisioned that the same approach can be used to improve the efficiency and / or sensitivity of other isothermal amplification techniques, such as strand displacement amplification (SDA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), whole genome amplification (WGA), rolling circle amplification (RCA) and multiple displacement amplification (MDA).

[0077] Detection of isothermal amplification products

[0077] The progress of the reaction can be measured using any method known in the art for detecting nucleic acids. For example, LAMP amplification products can be detected using direct or indirect approaches, such as nucleic acid binding dyes or probes.

[0078]

[0078] Direct detection of LAMP amplification products can use fluorescent reporting. This approach is based on the use of intercalating dyes, such as ethidium bromide, SYBR Green, EvaGreen and YO-PRO-I. In general, intercalating dyes are non-sequence-specific fluorescent dyes that exhibit a large increase in fluorescence emission when bound to double-stranded DNA. This property can be used to monitor nucleic acid amplification in real time by continuously measuring fluorescence during the LAMP reaction.

[0079]

[0079] More specific detection approaches have been developed and are known in the art (i.e., florescence only in the presence of the target nucleic acid sequence, see e.g., Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes. Gadkar VJ, Goldfarb DM, Gantt S, Tilley PAG. Sci Rep. 2018 Apr 3;8(1):5548).

[0080]

[0080] Indirect detection of LAMP amplification is well known and essentially relies on the formation of pyrophosphate as a reaction by-product. As the LAMP reaction proceeds, pyrophosphate ions are released by incorporation of dNTPs into the DNA strand during nucleic acid polymerization. These ions react with divalent metal ions, such as magnesium ions (present in the LAMP reaction mix), to produce a white, insoluble magnesium pyrophosphate precipitate. This product results in a cumulative increase in the turbidity of the reaction solution, and pyrophosphate precipitate can be measured in units of turbidity. Alternatively, detection of LAMP amplification can be achieved via incorporation of manganese ions and calcein (also known as fluorescein) in the reaction. Calcein fluorescence is naturally quenched by binding of manganese ions. Pyrophosphate produced as a by-product of the LAMP reaction removes manganese ions from the buffer via precipitation, and the increase in turbidity coupled with restored calcein fluorescence allows for easy visual readout upon excitation with visible or UV light. Another detection format is the enzymatic conversion of pyrophosphate to ATP, which is produced during DNA synthesis, and monitored via bioluminescence generated by thermostable firefly luciferase.

[0081] In some embodiments, a probe (nucleic acid probe) is used to measure the progress of the reaction. Preferably, the probe is specific for the target sequence to be amplified (e.g., a target sequence from SARS-CoV-2). Nucleic acid probes are often generated by conjugation of one or more fluorophores and quenchers to a nucleic acid that can bind to the target sequence. Thus, this type of probe is used for real-time sequence-specific quantification of nucleic acids and may contain a central target-specific single-stranded loop flanked by a stretch of 5-7 complementary nucleotides that can base-pair to form a stem terminating in a paired fluorophore and quencher. In the absence of a specific sequence target, the fluorescence remains quenched due to the proximity of the fluorophore and quencher at the 5' and 3' ends of the probe. Binding of a complementary nucleic acid to the loop results in a conformational change that forces the fluorophore and stem to unpair, and separation of the fluorophore from the quencher results in sequence-specific fluorescence. The probe produces a signal only when bound to the target, thus providing a direct measure of the amplification product.

[0082]

[0082] The methods described herein may be used with any probe known in the art, and one of skill in the art will be able to identify or design target-specific probes suitable for use with the methods.

[0083] In one embodiment, the method utilises one or more Easybeacon™ probes.

[0084]

[0084] Multiplexing for simultaneously amplifying several different target nucleic acid sequences is known and can be accomplished by one of skill in the art.

[0085] In one embodiment, the LAMP reaction described herein allows for multiplex detection of two different target nucleic acid sequences in a single tube / reaction by combining a first and a second plurality of LAMP primers with probes targeting the first and the second target nucleic acid. Preferably, at least one of the first plurality of LAMP primers includes a promoter for an RNA polymerase at its 5' end, and at least one of the second plurality of LAMP primers includes a promoter for an RNA polymerase at its 5' end.

[0086] In one embodiment, the LAMP reaction described herein allows for multiplex detection of three different target nucleic acid sequences in a single tube / reaction by combining a first, second, and third plurality of LAMP primers with probes targeting the first, second, and third target nucleic acids. Preferably, at least one of the first plurality of LAMP primers includes a promoter for an RNA polymerase at its 5' end, at least one of the second plurality of LAMP primers includes a promoter for an RNA polymerase at its 5' end, and at least one of the third plurality of LAMP primers includes a promoter for an RNA polymerase at its 5' end.

[0087]

[0087] In one embodiment, the LAMP reaction is suitable for multiplexing with multiple Easybeacon™ probes.

[0088]

[0088] In some embodiments, the LAMP reaction described herein is performed without extraction of DNA or RNA from the sample and is referred to as "extraction-free LAMP."

[0089] target nucleic acid

[0089] The methods described herein can be used with any target nucleic acid, for example, an RNA or DNA sequence specific to a virus or bacteria, or an RNA or DNA sequence specifically associated with a disease or pathology.

[0090]

[0090] In some embodiments, the target nucleic acid is from a pathogen or infectious agent, for example, when the biological sample contains or is suspected to contain a pathogen. Thus, the methods and kits provided herein are useful for detecting any known pathogen or infectious agent.

[0091] The methods and kits include those for detecting viral, bacterial, fungal or parasitic pathogens and infectious agents, such as viruses, e.g., single-stranded RNA viruses, single-stranded DNA viruses, Zika virus, HIV, Hepatitis A, B, and C viruses, HSV, CMV, It can be used to detect EBV, HPV, SARS-CoV-2, influenza A, influenza B, RSV, dengue virus types 1-4, chikungunya, parainfluenza types 1-4, adenovirus, human rhinovirus, enterovirus, chickenpox, e.g., VZV, Herpesvirsus, e.g., HSV-1, HSV-2, Epstein-Barr virus, human metapneumovirus, rotavirus, norovirus groups 1 and 2, sapovirus, astrovirus, bocavirus, Ebola virus, Filoviridae, Flaviviridae, Rhabdoviridae, Bunyavirales, Arenaviridae, and Hantaviridae.

[0092] In one embodiment, the viral pathogen is a parechovirus, rabies virus, measles virus, mumps virus, rubella virus, togaviridae, polyomavirus, papillomavirus, hepadnavirus, poxvirus, adenovirus, picornavirus, hepevirus, calicivirus, reovirus, retrovirus orthomyxovirus, paramyxovirus, coronavirus, Ebola virus, The virus is selected from the family Filoviridae, Flaviviridae, Rhabdoviridae, Bunyavirales, Arenaviridae, Hantaviridae and Deltavirus.

[0093]

[0093] In one embodiment, the virus is a coronavirus, such as coronavirus HKU-1, coronavirus OC43, coronavirus NL63 / 229E, SARS-CoV-2.

[0094] In one embodiment, the bacteria is Salmonella spp.; Bordetella spp., e.g., B pertussis, B. holmesii, B. parapertussis, Campylobacter spp.; Clostridium spp., e.g., C. difficile, C. difficile ribotype 027, C difficile ribotype 078; Chlamydia spp., e.g., C. pneumoniae, C. trachomatis; Chlamydophila spp. spp., for example C. psittaci; Listeria spp.; Lymphogranuloma spp., for example Shigella spp.; Neisseria spp., for example N. gonorrhoaea; Staphylococcus spp.; Streptococcus spp., for example S agalactiae; Listeria spp.; Leishmania spp.; Bacillus spp.; Borrelia spp.; Rickettsia spp.; Corynebacterium spp. spp; Gardenella spp; Haemophilus spp., for example H. influenzae; Escherichia spp., for example E. coli; Helicobacter spp., for example H. pylori.pylori; Klebsiella spp.; Legionella spp., for example, L pnemophila; Mycobacerium spp., for example, M. tuberculosis; Mycoplasma spp., for example, M, pnemoniae, M. genitalium, M. homini; Moraxella spp.; Pasteurella spp.; Pneumocystis spp., for example, P jirovecii; Pseudomonas spp.; Treponema spp. spp; Ureaplasma spp., e.g., U. urealyticum; Vibrio spp., Aermonas spp., and Yersinia spp., e.g., Y pestis.

[0095]

[0095] The parasitic pathogen may be a protozoan or metazoan pathogen, such as Plasmodia spp., Leishmania spp., Schistosoma spp., and Trypanosoma spp., bacteria (e.g., Mycobacteria, in particular M. tuberculosis, Salmonella spp., Streptococci, E. coli, and Staphylococci), and fungi (e.g., Candida spp. and Aspergillus spp.).

[0096] In one embodiment, the parasitic pathogen is Blastocystis spp., e.g., B. hominis; Giardia spp., e.g., G. intestinalis, G. lamblia; Cryptosporidium spp., Cyclospora spp., e.g., C. cayetanensis; Blastocystis spp., Dientamoeba spp., e.g., D. fragilis; Entamoeba spp. spp., for example E. histolytica; Cryptococcus spp., Enterocytozoon spp., for example E. bieneusi; Encephalitozoon spp., for example E. intestinalis; Babesia spp.; Leishmania spp., Schistosoma spp., Trypanosoma spp.; Trichimonas spp., for example T. vaginalis; Treponema spp. spp., for example, T. pallidum; and Plasmdoium spp.

[0097]

[0097] In one embodiment, the fungal pathogen is an Aspergillus spp, Candida spp, Histoplasma spp, Fusarium spp, Pneumocystis spp, Paracoccoides spp, Coccidioides spp, or Scedosporium spp.

[0098]

[0098] The methods and kits described herein can be applied to the detection and identification of essentially any nucleic acid-containing organism or free nucleic acid in the environment. Thus, the pathogen or infectious agent can be substantially any pathogen or infectious agent for which genetic information (e.g., gene sequence) is available. In some embodiments, the target nucleic acid is from human origin. In such cases, the methods and kits can be used to detect target nucleic acid in biological samples, such as biological samples obtained for forensic analysis, genotyping, etc.

[0099] In some embodiments, the disease may include, for example, cancer, diabetes, heart disease, hypertension, neurogenerative and infectious diseases. In some embodiments, the target nucleic acid is associated with a particular genetic condition. For example, the target nucleic acid includes a single nucleotide polymorphism (SNP) that is favorable for PAM discrimination, including, but not limited to, BRCA1 / BRCA2 mutations, cystic fibrosis, Duchenne muscular dystrophy and hemochromatosis.

[0100]

[0100] Conventional LAMP utilizes multiple primers for target nucleic acid, but the relatively high occurrence of false positive results is a known limitation. Conventional LAMP assays may use indirect methods of amplification detection, whether they are real-time or end-point, and rely on careful primer design, optimal reaction conditions, and robustness testing to negate false positives. In contrast, the method described herein uses LAMP primers that contain a promoter for RNA polymerase, and is demonstrated to reduce the occurrence of false positive results compared to FDA-approved LAMP reactions (see Example 9). Thus, the T7 modified LAMP protocol described herein may improve the usefulness of LAMP method and reduce the occurrence of false positives.

[0101] kit

[0101] The present invention also provides a kit for carrying out the method disclosed herein. Typically, the kit for carrying out the method of the present invention contains all the necessary reagents for carrying out the method. For example, in one embodiment, the kit includes: i) a plurality of LAMP primers for target nucleic acid, comprising F3 primer and B3 primer, and both F3 and B3 primers or F3 primer comprises a promoter for RNA polymerase at the 5' end; ii) dNTPs; iii) strand-displacing DNA polymerase; iv) reverse transcriptase; v) RNA polymerase; vi) rNTPs; and optionally vii) nucleic acid binding dye and / or vii) buffer. The kit may further include instructions for carrying out the detection and / or identification method provided herein.

[0102]

[0102] In one embodiment, the kit may include one or more containers containing components for a reaction mix, where the addition of target nucleic acid to the reaction mix promotes nucleic acid amplification when the reaction mix and target nucleic acid are incubated at an appropriate temperature as described herein.

[0103] Typically, kits of the invention will also include one or more other containers containing, for example, washing reagents and / or other reagents required in the performance of the methods of the invention.

[0104] In the context of the present invention, a kit includes any kit in which reagents are contained in separate containers, which may include small glass containers, plastic containers, or pieces of plastic or paper. Such containers may allow efficient transfer of reagents from one compartment to another, while avoiding cross-contamination of samples and reagents, and quantitative addition of drugs or solutions from each container from one compartment to another.

[0105]

[0105] Such kits may also include containers for receiving test samples, containers containing reagents used in the methods, and containers containing reagents required for detection of amplified nucleic acids.

[0106]

[0106] Typically, the kits of the invention also include instructions for carrying out a suitable method using the kit components. The kits and methods of the invention can be used in conjunction with automated analytical equipment and systems.

[0107]

[0107] For application to detection, identification or quantification of different target nucleic acids, a single kit of the present invention can be applied, or, for example, different kits containing specific reagents for each target may be required.The method and kit of the present invention are applied in any situation where it is desired to detect, identify or quantify any target nucleic acid.

[0108]

[0108] The present invention will now be described in more detail with reference to the following specific examples, which should not be construed in any way as limiting the scope of the invention. EXAMPLES

[0109] Working Example Example 1: Materials and Methods

[0109] Primer

[0110] A 100 mM stock solution of each primer (IDT) was prepared in molecular grade water (Sigma RNBJ2199). The primers were then mixed in the ratios shown in Table 1 to obtain a 10x working solution.

[0110] [Table 1]

[0111]

[0111] The promoters tested include T7, T3 and SP6 promoters. As provided in Table 2, the nucleic acid sequence of the T7 promoter is 20 nucleotides in length and is set forth in SEQ ID NO:1, the nucleic acid sequence of the T3 promoter is either 17, 20 or 24 nucleotides in length and is set forth in SEQ ID NOs:2-4, and the nucleic acid sequence of the SP6 promoter is 24 or 18 nucleotides in length and is set forth in SEQ ID NOs:5 and 6. The promoter sequences are shown in bold and underlined in Table 2. Random nucleotides can be added at the 5' or 3' end of the promoter sequence to improve amplification.

[0112] [Table 2]

[0113]

[0112] Primers B3 and F3 were synthesized containing 5' tails containing binding sites for T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, and were used in place of the conventional LAMP F3 and B3 primers in the following reactions.

[0114] LAMP primer sets were designed for the M, N, E and RdRP genes of SARS CoV-2, and for the hemagglutinin gene of influenza A to detect influenza A H3 targets, as listed in Table 3. Other primers used herein are also listed in Table 3.

[0115] [Table 3]

[0116] [Table 4]

[0117] [Table 5]

[0118] Sample preparation Both bisulfite (B) and wild type (WT) reactions were successful. Bisulfite treatment of samples was performed as follows: step 1) 150 μL of quantitative culture, QAP, RNA or positive clinical sample was added to 250 μL of combined reagent 1+2 from the EasyScreen SP006 Sample Processing Kit (Genetic Signatures); step 2) samples were heated at 95°C for 20 minutes, vortexed briefly, then spun for 2 seconds and allowed to cool to room temperature; step 3) material was then purified on a GS-mini according to the manufacturer's instructions (Genetic Signatures) with a final elution volume of 60-200 μL; step 4) samples were frozen at -70°C after purification.

[0119]

[0115] Wild-type 4-base purification was performed on a GS-mini according to the manufacturer's instructions (Genetic Signatures) with a final elution volume of 100-400 μL; samples were frozen at -70°C after purification.

[0120] Reactions were prepared in WarmStart Colorimetric LAMP 2x LAMP Master Mix (New England Biolabs:NEB), which contained 16 mM MgSO4, hence 8 mM MgSO4 in the 1x final reaction. Additional components included SYTO-9 Green Fluorescent Nucleic Acid Stain Mix (Invitrogen), Firescript Reverse Transcriptase (Solis BioDyne), 10x Primer Mix (IDT), rNTPs (NEB), RNA Polymerase (NEB), water and sample. Reagents are listed in Table 4 and the volumes of each component are listed in Table 5.

[0121]

[0117] The test LAMP reaction contained RNA polymerase, rNTPs and a primer with an RNA polymerase tail (promoter). The control LAMP reaction contained no RNA polymerase or rNTPs and included a primer without an RNA polymerase tail. The negative sample "No Template Control (NTC)" contained no template (nucleic acid).

[0122] Bisulfite treatment (bisulfite LAMP) reactions were optionally run first at 42°C for 1-10 min in a PCR thermal cycler (BioRad CFX96) and then at 53°C for 30-60 min in the same PCR thermal cycler. Alternatively, samples can be run on a heat block, water bath or incubator. Fluorescence was measured once per minute to assess reaction progress.

[0123] Wild-type 4-base LAMP reactions were optionally run in a PCR thermal cycler (BioRad CFX96) first at 42°C for 1-10 minutes and then at 65°C for 30 minutes in the same PCR thermal cycler. Alternatively, samples can be run on a heat block, water bath or incubator. Fluorescence was measured once per minute to assess reaction progress.

[0124]

[0120] The target nucleic acids comprising the LAMP primer sets are listed in Table 3.

[0125] [Table 6]

[0126] [Table 7]

[0127] Example 2: Influenza A LAMP

[0121] LAMP reactions were performed on a number of influenza A positive clinical samples from St Vincent's Hospital Sydney, Australia. Table 6 shows the results of the wild-type 4-base LAMP influenza H3 typing assay performed on these samples.

[0128] [Table 8]

[0129] LAMP primers F3 and B3 were synthesized with either a T7 or SP6 tail (T7 or SP6 promoter) and compared to control LAMP reactions. As shown in Table 6, the data using tailed primers resulted in both an increase in the number of positive samples detected and a decrease in the time to result.

[0130] Example 3: SARS-CoV-2 LAMP LAMP reactions were performed on the 2021 Quality Control for Molecular Diagnostics (QCMD) SARS-CoV-2 panel. Table 7 shows the results of the wild-type 4-base LAMP assay performed on these samples.

[0131] [Table 9]

[0132]

[0124] The quantitative QCMD panel was amplified using wild-type 4-base LAMP primers. F3 primers containing T7, SP6 or T3 5' tails were synthesized and compared with conventional LAMP assays. As shown in Table 7, reactions performed using LAMP with RNA polymerase tails resulted in faster time to results. More importantly, an increase in sensitivity was generated for the SP6 assay, which detected 4 of 5 samples, while conventional LAMP only detected 2 of 5 samples.

[0133] Example 4: Cultured SARS-CoV-2 LAMP

[0125] LAMP reactions were performed using bisulfite-converted cultured SARS-CoV-2 samples from St Vincent's Hospital Sydney, Australia.

[0134] [Table 10]

[0135] LAMP primers containing T7, SP6 or T3 5' tails were synthesized and compared to control (i.e., conventional) LAMP reactions. As shown in Table 8, the data using tailed primers resulted in both an increase in the number of positive samples detected and a decrease in the time to result. Table 8 shows that at least a 10-fold increase in sensitivity can be achieved when using tailed primers.

[0136] Example 5: Primer titration Optimization of T7-LAMP (detection of N gene) primer concentration reduced the time of first positive result using 75,000 copies from about 17 minutes to 10.76 minutes, and improved the sensitivity to single copy detection level in less than 18 minutes (Table 9). The data show that when using 1.75x optimal primer concentration, a 10-fold increase in sensitivity can be achieved compared to control LAMP. Bisulfite and wild-type T7-LAMP reactions have similar sensitivity and can detect single copy targets when optimized.

[0137] [Table 11]

[0138] Example 6: Guanidinium hydrochloride

[0128] The addition of guanidinium hydrochloride (GuHCl) had a favorable effect in increasing the rate and sensitivity of the T7-LAMP reaction.

[0139] [Table 12]

[0140] Example 7: Optimization of rNTP and RNA polymerase concentrations Optimization of T7-LAMP (detection of N gene) using T7 RNA polymerase (50,000U / mL) titration showed that there was an increase in positivity when 10, 7.5 and 5 units of polymerase were used in the reaction compared to 12.5 units and no T7 added when compared to the control reaction. However, there was a time lag of more than 8 minutes for sample #8 when 10 units were added to the reaction.

[0141] [Table 13]

[0142] Optimization of T7-LAMP (detection of N gene) using rNTP (25 mM) titration showed that adding about 0.175 μl of rNTP to the reaction was the optimal range, resulting in detection of 5 samples compared to 0.2 μl, which detected 4 samples. When no rNTP was added to the reaction, the time to result was delayed by a maximum of 15 minutes, and only 3 samples were detected as positive. Sample #2 was detected at all concentrations of rNTP except for the highest concentration of rNTP (0.2 μl) and the sample that had no added rNTP. A maximum delay of 12 minutes was shown when 0.1 μl was added.

[0143] [Table 14]

[0144] Example 8: Probe-based LAMP-multiplexing

[0131] Multiplexing of LAMP reaction was performed, which involves attaching a fluorescent dye to the 5' end of the FIP primer, and synthesizing a second primer complementary to FIP containing a 3' quencher. When amplification occurs, the complementary oligo is displaced and fluorescence is released. See the example for the N gene below. [ka]

[0145]

[0132] Another approach is to design conventional Easybeacon™ probes against regions in the target nucleic acid that do not have a priming site, or using non-essential primers, such as the LB and LF primers, as probe binding sites. These probes then bind and are subsequently displaced upon synthesis of a new strand of DNA.

[0146] To determine the compatibility of the LAMP assay with multiplexing, Easybeacon™ probes were synthesized for the following SARS-CoV-2 regions: RdRP, Orf1a, As1e, N gene, E gene, and M gene, as well as an endogenous human control (12S rRNA). Easybeacon™ probes contain an Intercalating Pseudo Nucleotide (IPN) that improves the efficiency of binding to the target sequence. The composition of the probe-based singleplex reaction is shown in Table 13.

[0147] [Table 15]

[0148] Probes were designed for both the LB (loop-back) and LF (loop-forward) regions of the LAMP reaction. All probes were first tested in singleplex to determine the level of fluorescence obtained using probe-based chemistry with SARS-CoV-2 clinical samples. The performance (measured in minutes) of five different fluorescent probes in singleplex with SARS-CoV-2 clinical samples is shown in Table 14. The data below show that all regions and fluorophores produce strong signals in the LAMP reaction, some of which are stronger than typical RT-PCR signals.

[0149] [Table 16]

[0150] To determine whether the LAMP assay can be multiplexed, various combinations of primers and probes were tested in singleplex, duplex, and triplex. The probes used in the assay were Easybeacon™ probes (Pentabase). The composition of the LAMP multiplex mix is ​​listed in Table 15. The data below shows that the multiplex reaction worked well in triplex, but the time to result was slightly delayed.

[0151] [Table 17]

[0152] [Table 18]

[0153] [Table 19]

[0154]

[0136] The LAMP assay was also tested by incorporation of an endogenous human control 12S human rRNA target (Table 18).

[0155] [Table 20]

[0156]

[0137] Table 18 shows that it is possible to multiplex three individual targets (E gene, RdRP and N gene of SARS-CoV-2) and an internal process control (human 12S rRNA) to produce a complete assay.

[0157] Comparison of multiplexed GSL LAMP and real-time PCR Prospective study In a prospective study, 121 SARS-CoV-2 positive samples were freshly collected from a local hospital, diluted in UTM, and split into two. One set was purified using the TGA approved GSL sample preparation method (SP012) and then amplified by RT-PCR using the TGA approved RP012 kit. The second set was purified using GS-mini according to the manufacturer's instructions and amplified with a QuadPlex GSL real-time LAMP assay containing the As1e, RdRP, N, and M genes as targets (see Tables 20 and 21). Presumptive positives were samples that were positive for only one of two genes in RT-PCR or one of three genes in the LAMP assay.

[0158] [Table 21]

[0159] [Table 22]

[0160] [Table 23]

[0161] The prospective study consisted of 121 positive SARS-CoV-2 samples retrieved from a local pathology laboratory. Presumptive positives are samples that are positive for only one of the SARS-CoV-2 targets in the assay. Using the GSL LAMP assay, three samples that were presumptively positive by PCR were called strictly positive in the LAMP assay. Presumptive positive samples were not generally called positive, and thus GSL LAMP gave a sensitivity of 100% (121 / 121), while PCR gave a sensitivity of 97.5% (118 / 121).

[0162] Retrospective study In the retrospective study, 82 SARS-CoV-2 samples stored at -80°C for over 18 months were diluted in UTM and split into two. Samples were then amplified with GSL real-time PCR and Quadplex GSL modified RT-LAMP assays as described above. Presumptive positive samples were not included in the total count if only one gene was positive. There were still samples detected with Ct>40 with GSL LAMP. There was 100% concordance when used with qRT-PCR using samples collected in the prospective study. Detection of samples with Ct values ​​>40 generally indicates the presence of only one or two copies of the target sequence in the sample, demonstrating the improved sensitivity of the GSL LAMP procedure. The data showed a slightly reduced performance of the retrospective samples, which could be caused by sample degradation and the large size (250bp) of the LAMP amplicon compared to qRT-PCR (<100bp).

[0163] [Table 24]

[0164] Example 9: Comparison with Color Genomics' N gene primers The specificity of Color's (https: / / www.color.com) LAMP N primers was tested against a series of clinical extracts and non-template controls. The N gene primers used were the "Color SARS-CoV-2 LAMP Diagnostic Assay" primers, which have been provided with Emergency use Authorisation (EUA) by the FDA. For this experiment, primers were synthesised based on the sequences listed in Dudley DM, Newman CM, Weiler AM, Ramuta MD, Shortreed CG, Heffron AS, et al. (2020) Optimising direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples. PLoS ONE 15(12):e0244882, and a T7-tailed F3 primer was added. As can be seen from the data in Table 23 (below) after approximately 18 minutes, a false positive amplification curve was generated using the non-template control (NTC) assay using Color's N gene primers. In contrast, using the T7LAMP variant assay described herein, no false positive signals were generated even after 25 minutes of amplification.

[0165]

[0141] False positive amplification signals are a well-documented drawback of the LAMP method. As shown in Table 23, the T7 modified LAMP protocol reduces the occurrence of false positives.

[0166] [Table 25]

[0167] The specificity of the LAMP As1e assay was tested against a series of SARS-CoV-2 clinical extracts, non-template controls and positive influenza A samples. As can be seen from the data after about 15 minutes, false positive amplification curves were generated using the control assay. Additionally, false positive signals were seen using the influenza A clinical samples already tested. However, using the T7 LAMP variant assay, no false positive signals were generated even after 20 minutes of amplification for both NTC and influenza A samples. False positive amplification signals are a well-documented drawback of the LAMP method. Thus, the T7 modified LAMP protocol improves the utility of the LAMP method, resulting in increased detection of low positive signals. Additionally, samples 8, 10 and 29 were positive after 6-11 minutes in the T7lamp As1e assay but negative in the control assay.

[0168] [Table 26]

[0169] Example 10: Comparison with known LAMP reaction

[0143] The ability of LAMP primers containing a promoter for RNA polymerase to improve the LAMP reaction was tested by adding the T7 promoter to various published promoter sequences (see Table 25 for promoter sequences), specifically, the T7 promoter was added to the F3 primer.

[0170] [Table 27]

[0171] [Table 28]

[0172]

[0144] The Color-ORF1a primers in Table 26 are described in Color Genomics. SARS-CoV-2 LAMP Diagnostic Assay. 2020 https: / / www.color.com / wp-content / uploads / 2020 / 05 / LAMP-Diagnostic-Assay.pdf.

[0173]

[0145] The gene-NA primers in Table 27 are described in Broughton JP, Deng X, Yu G, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020 Jul;38:870-874.

[0174]

[0146] The Yu-ORF1a primer in Table 28 is described in Yu L, Wu S, Hao X, et al. Rapid Detection of COVID-19 Coronavirus Using a Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) Diagnostic Platform. [letter]. Clin Chem 2020 Jul 1; 66(7): 975-977.

[0175]

[0147] El-Tholoth-ORF1a in Table 29 is described in El-Tholoth M, Bau HH, Song JA single and two-stage, closed tube, molecular test for the 2019 novel coronavirus (COVID-19) at home, clinic, and points of entry. ChemRxiv. 2020.

[0176]

[0148] The Lamb-ORF1a primers in Table 30 are described in Lamb LE, Bartolone SN, Ward E, Chancellor MB. Rapid detection of novel coronavirus / Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification. PLoS One. 2020; 15.

[0177]

[0149] As can be seen from Tables 26-30, the T7LAMP assay provides improved performance in terms of sensitivity and reduced false positives.

[0178] [Table 29]

[0179] [Table 30]

[0180] [Table 31]

[0181] [Table 32]

[0182] [Table 33]

[0183] Example 11. Comparison of T7LAMP and real-time PCR Table 31 shows the results of 28 randomly selected pre-tested SARS CoV-2 samples. Due to limited availability, samples were diluted at least 10-fold before testing. As can be seen, the T7-LAMP procedure is currently approaching the sensitivity of the "gold standard" PCR method.

[0184] [Table 34]

[0185] Example 12. Extraction-free LAMP Eighty clinical samples from a prospective study were diluted in extraction-free buffer containing 400 mM GuHCl and proteinase K (7.5 μL buffer and 12.5 μL sample). The samples were then heated at 80°C for 10 minutes and then added directly to the GSL LAMP reaction containing modified primers for the lateral flow device. A positive signal was seen 2-3 minutes after closing the detection chamber. The advantages of the extraction-free LAMP method are that it only requires the use of a heat block, results can be obtained within 30 minutes, and it does not require extraction of RNA or DNA, so it is particularly useful in resource-limited situations. The data also show that samples were still positive at Ct values ​​>35, but negative at Ct >37.

[0186] [Table 35]

[0187] Example 13. Sample Further details of the samples identified in the above examples are provided in Table 33. Samples with the same description but different sample numbers (eg, clinical samples, influenza A positive) are from different patients or sources.

[0188] [Table 36]

[0189] Example 14. T7 placement position As shown above, LAMP reactions using LAMP primers F3 and B3 synthesized with either T7 or SP6 tails (T7 or SP6 promoter) showed improved amplification sensitivity and efficiency compared to the control LAMP reaction. As shown in Table 6, the data using tailed primers resulted in both an increase in the number of positive samples detected and a decrease in the time to result, thus demonstrating that both F3 and B3 outer primers can be used for RNA polymerase promoter placement.

[0190]

[0154] Upon further testing, the inventors found that a mixture of F3-T7 and B3-T7 did not further improve the sensitivity of the LAMP assay, and when a larger number of samples were tested, F3-T7 was found to perform slightly better overall than B3-T7.

[0191] Table 34 shows the effect of T7 promoter placement on various LAMP primers. The data shows that the optimal placement for the T7 tail (at the 5' end of the primer sequence) is on the outer F3 primer set. When the T7 tail is placed on the inner FIP primer, there is essentially no difference in time to result using the RdRP (SARS-CoV-2 gene) region 1 primer set, and with the RdRP primer set 2, the reaction is substantially inhibited. When the T7 tail is placed on the inner loop LF primer, there is little difference compared to the control reaction without the T7 tail. In contrast, when the T7 tail is placed on the F3 (outer primer), there is a decrease in time to result and even an increase in positivity for the RdRP region 1 (F3-T7 results in bold).

[0192] [Table 37]

[0193] Example 15. Neisseria gonorrohoea LAMP The LAMP reaction described herein can also be used for bacterial detection. Table 35 shows the results of GSL LAMP when used to detect the presence of N. gonorrohoea. LAMP primers against the unique region of 16S rRNA were designed and amplified as usual (Table 36). As can be seen from the data, as few as 5-10 copies can be detected in the LAMP amplification reaction (Table 35).

[0194] [Table 38]

[0195] [Table 39]

[0196] [Table 40]

[0197]

[0157] Table 37 shows the results of a cross-reactivity study using the N. gonorrohoea LAMP reaction. No cross-reactivity was observed with any of the bacterial or viral non-target organisms tested.

[0198]

[0158] It will be appreciated by those skilled in the art that numerous variations and / or modifications can be made to the present invention as shown in the specific embodiments and examples above without departing from the spirit and scope of the present invention as broadly described. The present embodiments should therefore be considered in all respects as illustrative and not restrictive. In particular, it should be understood that those skilled in the art, having regard to the benefit of this disclosure, will be able to identify, select, optimize or modify conditions and / or parameters suitable for use of the methods and kits according to the principles of the present invention and suitable for these and other types of applications. The exact use, choice of reagents, variables such as concentrations, volumes, incubation times, incubation temperatures, etc., may depend greatly on the particular application for which it is intended.

Claims

1. 1. A method for reducing the incidence of false positives, increasing the efficiency, and / or increasing the sensitivity of loop-mediated isothermal amplification (LAMP) of a target nucleic acid, comprising: a) i) a plurality of LAMP primers for a target nucleic acid, comprising an F3 primer and a B3 primer, wherein both the F3 and B3 primers or the F3 primer comprise a promoter for an RNA polymerase at the 5' end; ii) dNTPs; iii) a strand-displacing DNA polymerase; iv) reverse transcriptase; v) RNA polymerase; and iv) rNTP; and vii) optionally a nucleic acid binding dye or probe; combining to form a LAMP reagent mix; b) incubating said LAMP reagent mix with said target nucleic acid under conditions suitable for amplification of said target nucleic acid, resulting in an increase in efficiency and / or sensitivity compared to the same amplification carried out in the absence of said promoter, RNA polymerase and rNTPs; A method comprising:

2. The method of claim 1 , wherein the probe is a fluorescent probe specific for the target.

3. 2. The method of claim 1, wherein the promoter sequence is selected from the group consisting of a T7 promoter of SEQ ID NO: 1, a T3 promoter of SEQ ID NO: 2-4, and a SP6 promoter of SEQ ID NO: 5-6.

4. 2. The method of claim 1, wherein the RNA polymerase is selected from T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.

5. 2. The method of claim 1, wherein the concentration of the rNTP is from about 0.25 mM to about 2.5 mM and the concentration of RNA polymerase is from about 7.5 units per reaction to about 50 units per reaction.

6. The method of claim 1 , wherein the LAMP reagent mix comprises guanidinium hydrochloride (GuHCl).

7. 7. The method of claim 6, wherein the GuHCl is at a concentration of at least about 30 mM.

8. 10. The method of claim 1, wherein the target nucleic acid is bisulfite treated.

9. The method of claim 1 , wherein the plurality of LAMP primers further comprises a FIP primer, a BIP primer, a LF primer, and a LB primer.

10. The method of claim 9 , wherein the FIP primer, the BIP primer, the LF primer, and the LB primer do not contain a promoter for an RNA polymerase.

11. 10. The method of claim 9, wherein both the F3 primer and the B3 primer comprise a promoter sequence at the 5' end, and the promoter sequence is selected from a T7 promoter of SEQ ID NO: 1, a T3 promoter of SEQ ID NOs: 2-4, and an SP6 promoter of SEQ ID NOs: 5-6.

12. 10. The method of claim 9, wherein the F3 primer comprises a promoter sequence at the 5' end, and the promoter sequence is selected from the group consisting of a T7 promoter of SEQ ID NO: 1, a T3 promoter of SEQ ID NOs: 2 to 4, and a SP6 promoter of SEQ ID NOs: 5 to 6.

13. 10. The method of claim 1, wherein the target nucleic acid is pathogen-specific RNA or DNA.

14. 14. The method of claim 13, wherein the pathogen is selected from bacteria, viruses, fungi and parasites.

15. Said bacteria may be Salmonella spp., Bordetella spp., Campylobacter spp., Clostridium spp., Chlamydia spp., Chlamydophila spp., Listeria spp.; Lymphogranuloma spp., Shigella spp., Neisseria spp., Staphylococcus spp., Streptococcus spp., Listeria spp., Leishmania spp., Bacillus spp., Borrelia spp. spp), Rickettsia spp, Corynebacterium spp, Gardenella spp, Haemophilus spp, Escherichia spp, Helicobacter spp, Klebsiella spp, Legionella spp, Mycobacerium spp, Mycoplasma spp, Moraxella spp, Pasteurella spp, Pneumocystis spp, Pseudomonas spp, Treponema spp spp), Ureaplasma spp, Vibrio spp, Aermonas spp, and Yersinia spp.

16. The viruses include parechoviruses, rabies viruses, measles viruses, mumps viruses, rubella viruses, togaviridae, polyomaviruses, papillomaviruses, hepadnaviruses, poxviruses, adenoviruses, picornaviruses, hepeviruses, caliciviruses, reoviruses, retroviruses, orthomyxoviruses, paramyxoviruses, coronaviruses, Ebola viruses, 15. The method of claim 14, wherein the virus is selected from the family Filoviridae, Flaviviridae, Rhabdoviridae, order Bunyavirales, family Arenaviridae, family Hantaviridae, and Deltavirus.

17. 17. The method of claim 16, wherein the coronavirus is coronavirus HKU-1, coronavirus OC43, coronavirus NL63 / 229E, or SARS-CoV-2.

18. The parasites include Blastocystis spp., Giardia spp., Cryptosporidium spp., Cyclospora spp., Blastocystis spp., Dientamoeba spp., Entamoeba spp., Cryptococcus spp., Enterocytozoon spp., Encephalitozoon spp., Babesia spp.; Leishmania spp., Schistosoma spp., Trypanosoma spp. spp), Trichimonas spp, Treponema spp, and Plasmdoium spp.

19. 15. The method of claim 14, wherein the fungus is selected from Aspergillus spp, Candida spp, Histoplasma spp, Fusarium spp, Pneumocystis spp, Paracoccoides spp, Coccidioides spp, and Scedosporium spp.

20. 10. The method of claim 1, wherein the sensitivity of the LAMP reaction is increased by at least about 10% to about 30%.

21. 10. The method of claim 1, wherein the efficiency of the LAMP reaction is increased by at least about 5% to about 20%.

22. 2. The method of claim 1, wherein the loop-mediated isothermal amplification (LAMP) is a multiplex reaction, and in step a) a second plurality of LAMP primers for a second target nucleic acid is combined, the second plurality of LAMP primers including an F3 primer comprising a promoter for an RNA polymerase at its 5' end.

23. A plurality of LAMP primers for a target nucleic acid, the LAMP primers comprising an F3 primer and a B3 primer, each of which comprises a promoter for an RNA polymerase at its 5' end, or an F3 primer comprising a promoter for an RNA polymerase at its 5' end and at least one sequence complementary to the target nucleic acid.

24. 1. A kit for detecting a target nucleic acid using a loop-mediated isothermal amplification (LAMP) reaction, comprising: i) a plurality of LAMP primers for the target nucleic acid, comprising an F3 primer and a B3 primer, wherein both the F3 and B3 primers or the F3 primer comprise a promoter for an RNA polymerase at the 5' end; ii) dNTPs; iii) a strand-displacing DNA polymerase; iv) reverse transcriptase; v) RNA polymerase; iv) rNTP; and vii) optionally a nucleic acid binding dye or probe; Kit including:

25. 25. The kit of claim 24, wherein the probe is a fluorescent probe specific for the target.

26. 25. The kit of claim 24, wherein the target nucleic acid is an RNA or DNA specific pathogen.

27. 25. The kit of claim 24, wherein the plurality of LAMP primers further comprises a FIP primer, a BIP primer, a LF primer, and a LB primer.

28. 28. The kit of claim 27, wherein the FIP primer, the BIP primer, the LF primer, and the LB primer do not contain a promoter for an RNA polymerase.

29. 25. The kit of claim 24, wherein the loop-mediated isothermal amplification (LAMP) is a multiplex reaction.

30. 30. The kit of claim 29, further comprising a second plurality of LAMP primers for a second target nucleic acid, the second plurality of LAMP primers including F3 primers comprising a promoter for an RNA polymerase at the 5' end.