Loop primers and loop-de-loop methods for detecting target nucleic acids

Abstract

The present disclosure provides a novel loop-de-loop method for detecting a target nucleic acid using a biosensor-labeled oligonucleotide. The present disclosure also provides a loop primer and a kit for use in the method.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 989,140, ​​filed March 13, 2020, which is incorporated by reference in its entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing with XXX sequences, which have been submitted via EFS-Web and are incorporated herein by reference in their entirety. The ASCII copy made to XXXX is named 48397WO_sequencelisting.txt and is XXX bytes in size. [Background technology]

[0003] 3.Background Methods for detecting target nucleic acids using the complementarity of nucleic acid sequences have been improved or modified in various ways from traditional Southern hybridization to the present day. In particular, the establishment of various in vitro nucleic acid amplification methods, such as polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), has made it possible to detect smaller amounts of target nucleic acids. These methods have been used for sequence-specific detection and quantification of target nucleic acids in samples for medical diagnosis of infectious diseases, determination of mutant genotypes, detection of single nucleotide polymorphisms (SNPs) and point mutations, etc. Nucleic acid amplification methods have been the gold standard of testing due to their high specificity and sensitivity.

[0004] However, current nucleic acid amplification methods are limited because amplification reactions and signal detection require precise measurements using controlled environments and expensive equipment. Thus, the methods are often cost-prohibitive for use in point-of-care situations. Furthermore, the methods are not optimized for detection of multiplexed targets in a single patient sample. Detection of multiplexed targets can be achieved by signal multiplexing in a single-pot reaction (fluorescence spectral multiplexing, arrays of electrochemical detectors), physical separation of multiple reactions into unique reaction vessels, or a combination thereof. However, for CLIA waiver testing, users should expect no more than three simple steps to simultaneously query a panel of nucleic acid targets using a single patient sample. Thus, unless complex equipment or disposables handle the process automatically, physical separation of samples into separate chambers quickly becomes infeasible for CLIA waiver testing. Although fluorescence spectral multiplexing can reduce the number of unique reactions required to target a panel of nucleic acid targets, spectral multiplexed LAMP reactions require dramatic sacrifices in assay speed or signal strength, which discourages successful application in POC testing.

[0005] Therefore, there is a need to develop new methods that allow easy amplification and detection of target nucleic acids, especially multiplexed targets, at low cost and with high sensitivity and specificity. Summary of the Invention

[0006] 4. Overview The present disclosure provides a new amplification method that allows easy detection of target nucleic acids in a closed system. The method allows detection of small amounts of target nucleic acids with high specificity and sensitivity by using a loop primer with a biosensor pair. The biosensor pair allows determination of loop-de-loop ("LDL") amplification of a target sequence by detecting conformational changes of the loop primer, for example, using fluorescence / quencher FRET technology. Furthermore, the use of multiple biosensors allows detection of multiplexed targets in a single tube. The loop primer can be used in combination with loop-mediated isothermal amplification (LAMP) as well as any other nucleic acid amplification method that utilizes a strand-displacing polymerase. This disclosure includes the following [1] to

[95] . [1] A loop primer for loop-de-loop amplification (LdL) of a target sequence, comprising, in a 5' to 3' direction: A first sensor molecule; a first clamping oligonucleotide; a spacing oligonucleotide; A second clamping oligonucleotide, The first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide are arranged to have a melting temperature (T m ) a second clamping oligonucleotide capable of forming a hairpin structure at a lower temperature; A second sensor molecule, a second sensor molecule, wherein the first sensor molecule and the second sensor molecule are a first biosensor pair; and a first primer sequence complementary to a first binding site on the target sequence; A loop primer comprising: [2] The loop primer described in [1] above, wherein the second clamping oligonucleotide is complementary to the first clamping oligonucleotide. [3] The loop primer according to [1] or [2] above, wherein the first biosensor pair is an energy donor and acceptor pair. [4] The loop primer described in [3] above, wherein the first biosensor pair is an energy donor and acceptor pair for fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET). [5] The loop primer according to [3] above, wherein the first sensor molecule is a FRET fluorophore and the second sensor molecule is a FRET quencher. [6] The loop primer described in [3] above, wherein the first sensor molecule is a FRET quencher and the second sensor molecule is a FRET fluorophore. [7] The loop primer described in [3] above, wherein the first sensor molecule is a BRET energy donor and the second sensor molecule is a BRET energy acceptor. [8] The loop primer described in [3] above, wherein the first sensor molecule is a BRET energy acceptor and the second sensor molecule is a BRET energy donor. [9] The loop primer described in [1] above, wherein the first sensor molecule and the second sensor molecule are capable of forming a complex that generates a detectable optical signal.

[10] The melting temperatures (T m ) is greater than 60°C.

[11] The melting temperatures (T m ) is greater than 65°C.

[12] The melting temperatures (T m ) is greater than 70°C.

[13] The melting temperatures (T m ) is greater than 80°C.

[14] The melting temperatures (T m ) is 70 to 80°C.

[15] The melting temperatures (T m ) is 72.5 to 77.5°C.

[16] The melting temperatures (T m ) is about 75°C.

[17] The melting temperatures (T m ) is less than 60°C.

[18] The melting temperatures (T m ) is 60 to 65°C.

[19] The loop primer described in any one of [1] to

[18] above, wherein the first clamping oligonucleotide and the second clamping oligonucleotide are 3 to 10 nucleotides in length.

[20] The loop primer described in

[19] above, wherein the first clamping oligonucleotide and the second clamping oligonucleotide are 3 to 7 nucleotides in length.

[21] The loop primer described in

[20] above, wherein the first clamping oligonucleotide and the second clamping oligonucleotide are 6 nucleotides in length.

[22] The loop primer described in any one of [1] to

[21] above, wherein the spacing oligonucleotide is 5 to 35 nucleotides in length.

[23] The loop primer according to

[22] above, wherein the spacing oligonucleotide is 10 to 20 nucleotides in length.

[24] The loop primer according to

[23] above, wherein the spacing oligonucleotide is 13 to 18 nucleotides in length.

[25] The loop primer according to

[24] above, wherein the spacing oligonucleotide is 13 nucleotides in length.

[26] The loop primer described in any one of [1] to

[25] above, wherein the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide are 15 to 35 nucleotides in length in total.

[27] The loop primer described in

[26] above, wherein the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide are 20 to 30 nucleotides in length in total.

[28] The loop primer described in

[27] above, wherein the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide are 23 to 28 nucleotides in length in total.

[29] The loop primer according to any one of [1] to

[28] above, wherein the first clamping oligonucleotide, the spacing oligonucleotide and the second clamping oligonucleotide comprise (i) a nucleobase selected from adenine, guanine, cytosine, thymine and uracil, (ii) a locked nucleic acid, (iii) a 2'O-methyl RNA base, (iv) a phosphorothioated DNA base, (v) a phosphorothioated RNA base, (vi) a phosphorothioated 2'-O-methyl RNA base, or (vii) a combination thereof.

[30] The loop primer described in any one of [1] to

[29] above, further comprising a first additional oligonucleotide at the 5' end of the loop primer.

[31] The loop primer described in any one of [1] to

[30] above, further comprising a second additional oligonucleotide between the first sensor molecule and the first clamping oligonucleotide.

[32] The loop primer according to

[30] or

[31] above, wherein the first or second further oligonucleotide is a barcode sequence.

[33] The loop primer described in any one of [1] to

[32] above, wherein the target sequence is specific to a pathogen genome.

[34] The loop primer described in

[33] above, wherein the target sequence is specific to Chlamydia trachomatis.

[35] The loop primer described in

[34] above, wherein the target sequence is derived from orf8 or cds2.

[36] The loop primer described in

[34] above, comprising the oligonucleotide of SEQ ID NO:15.

[37] The loop primer described in

[33] above, wherein the target sequence is specific to Neisseria gonorrhoeae.

[38] The loop primer according to

[37] above, wherein the target sequence is derived from porA or glnA.

[39] The loop primer described in

[37] above, comprising an oligonucleotide of SEQ ID NO:5 or SEQ ID NO:7.

[40] The loop primer described in

[33] above, wherein the target sequence is specific to a virus.

[41] The loop primer described in

[40] above, wherein the virus is SARS-CoV-2.

[42] The loop primer described in any one of [1] to

[30] above, wherein the target sequence is specific to Homo sapiens.

[43] The loop primer described in

[42] above, wherein the target sequence is derived from tbc1d3.

[44] The loop primer described in

[42] above, comprising the oligonucleotide of SEQ ID NO: 22.

[45] A primer mixture for loop-de-loop amplification of a target sequence, comprising the loop primer described in any one of [1] to

[44] above.

[46] The primer mixture described in

[45] above, further comprising: (i) a forward inner primer (FIP); (ii) a backward inner primer (BIP); and (iii) a forward primer (F3) and a backward primer (B3), wherein the FIP, the BIP, the F3 and the B3 bind to six different binding sites on the target sequence.

[47] The primer mixture described in

[46] above, further comprising (i) a loop-forward primer (LF) and (ii) a loop-backward primer (LB), wherein the LF and the LB bind to two different binding sites on the target sequence.

[48] ​​The primer mixture according to any one of

[45] to

[47] above, wherein the FIP, the BIP, the F3, the B3, the LF, or the LB binds to the first binding site on the target sequence.

[49] The primer mixture described in

[48] above, wherein the FIP binds to the first binding site, and the ratio of the amount of the FIP to the loop primer in the primer mixture is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1.

[50] The primer mixture described in

[48] above, wherein the BIP binds to the first binding site, and the ratio of the amount of the BIP to the loop primer in the primer mixture is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1.

[51] The primer mixture according to

[48] above, wherein the LF binds to the first binding site, and the ratio of the amount of the LF to the amount of the loop primer in the primer mixture is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1.

[52] The primer mixture described in

[48] above, wherein the LB binds to the first binding site, and the ratio of the amount of the LB to the loop primer in the primer mixture is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1.

[53] The primer mixture according to any one of

[46] to

[52] above, wherein F3 comprises an oligonucleotide of SEQ ID NO: 1, B3 comprises an oligonucleotide of SEQ ID NO: 2, FIP comprises an oligonucleotide of SEQ ID NO: 3, BIP comprises an oligonucleotide of SEQ ID NO: 4, LF comprises an oligonucleotide of SEQ ID NO: 6, or LB comprises an oligonucleotide of SEQ ID NO: 8.

[54] The primer mixture described in any one of

[46] to

[52] above, wherein F3 comprises an oligonucleotide of SEQ ID NO: 1, B3 comprises an oligonucleotide of SEQ ID NO: 2, FIP comprises an oligonucleotide of SEQ ID NO: 3, BIP comprises an oligonucleotide of SEQ ID NO: 4, LF comprises an oligonucleotide of SEQ ID NO: 6, and LB comprises an oligonucleotide of SEQ ID NO: 8.

[55] The primer mixture according to any one of

[46] to

[52] above, wherein F3 comprises an oligonucleotide of SEQ ID NO: 9, B3 comprises an oligonucleotide of SEQ ID NO: 10, FIP comprises an oligonucleotide of SEQ ID NO: 11, BIP comprises an oligonucleotide of SEQ ID NO: 12, LF comprises an oligonucleotide of SEQ ID NO: 13, or LB comprises an oligonucleotide of SEQ ID NO: 14.

[56] The primer mixture described in any one of

[46] to

[52] above, wherein F3 comprises an oligonucleotide of SEQ ID NO: 9, B3 comprises an oligonucleotide of SEQ ID NO: 10, FIP comprises an oligonucleotide of SEQ ID NO: 11, BIP comprises an oligonucleotide of SEQ ID NO: 12, LF comprises an oligonucleotide of SEQ ID NO: 13, and LB comprises an oligonucleotide of SEQ ID NO: 14.

[57] The primer mixture according to any one of

[46] to

[52] above, wherein F3 comprises an oligonucleotide of SEQ ID NO: 16, B3 comprises an oligonucleotide of SEQ ID NO: 17, FIP comprises an oligonucleotide of SEQ ID NO: 18, BIP comprises an oligonucleotide of SEQ ID NO: 19, LF comprises an oligonucleotide of SEQ ID NO: 20, or LB comprises an oligonucleotide of SEQ ID NO: 21.

[58] The primer mixture described in any one of

[46] to

[52] above, wherein F3 comprises an oligonucleotide of SEQ ID NO: 16, B3 comprises an oligonucleotide of SEQ ID NO: 17, FIP comprises an oligonucleotide of SEQ ID NO: 18, BIP comprises an oligonucleotide of SEQ ID NO: 19, LF comprises an oligonucleotide of SEQ ID NO: 20, and LB comprises an oligonucleotide of SEQ ID NO: 21.

[59] The method according to claim 1, further comprising the step of: a third sensor molecule; and a third clamping oligonucleotide; and a second spacing oligonucleotide; and a fourth clamping oligonucleotide, The third clamping oligonucleotide, the second spacing oligonucleotide, and the fourth clamping oligonucleotide are arranged to have a melting temperature (T m ) a fourth clamping oligonucleotide capable of forming a hairpin structure at a lower temperature; A fourth sensor molecule, a fourth sensor molecule, the third sensor molecule and the fourth sensor molecule being a second biosensor pair, the second biosensor pair being different from the first biosensor pair; and a second primer sequence that is complementary to the first binding site on the second target sequence; The primer mixture according to any one of the above

[45] to

[58] , comprising:

[60] The primer mixture described in

[59] above, wherein the third clamping oligonucleotide is complementary to the fourth clamping oligonucleotide.

[61] The primer mixture described in

[59] or

[60] above, wherein the target sequence and the second target sequence are identical.

[62] The primer mixture described in

[59] or

[60] above, wherein the target sequence and the second target sequence are different.

[63] The primer mixture described in any one of

[59] to

[62] above, further comprising: (i) a second forward inner primer (SFIP), (ii) a second backward inner primer (SBIP), (iii) a second forward primer (SF3) and (iv) a second backward primer (SB3), wherein the SFIP, the SBIP, the SF3 and the SB3 bind to six different binding sites on the second target sequence.

[64] The primer mixture described in any one of

[59] to

[60] above, further comprising (i) a second loop-forward primer (SLF) and (ii) a second loop-backward primer (SLB), wherein the SLF and the SLB bind to two different binding sites on the second target sequence.

[65] The method according to claim 1, further comprising the step of: a fifth sensor molecule; and a fifth clamping oligonucleotide; and a third spacing oligonucleotide; and A sixth clamping oligonucleotide, The fifth clamping oligonucleotide, the third spacing oligonucleotide, and the sixth clamping oligonucleotide are arranged to have a melting temperature (T m ) a sixth clamping oligonucleotide capable of forming a hairpin structure at a lower temperature than A sixth sensor molecule, a sixth sensor molecule, the fifth sensor molecule and the sixth sensor molecule being a third biosensor pair, the third biosensor pair being different from the first biosensor pair and the second biosensor pair; a second primer sequence complementary to the first binding site on a third target sequence; The primer mixture according to any one of the above

[45] to

[64] , comprising:

[66] The primer mixture described in

[65] above, wherein the fifth clamping oligonucleotide is complementary to the sixth clamping oligonucleotide.

[67] The primer mixture described in

[65] or

[66] above, wherein the target sequence, the second target sequence and the third target sequence are identical.

[68] The primer mixture described in

[65] or

[66] above, wherein the target sequence, the second target sequence and the third target sequence are different.

[69] The primer mixture described in any one of

[65] to

[68] above, further comprising: (i) a third forward inner primer (TFIP), (ii) a third backward inner primer (TBIP), (iii) a third forward primer (TF3), and (iv) a third backward primer (TB3), wherein the TFIP, TBIP, TF3, and TB3 bind to six different binding sites on the third target sequence.

[70] The primer mixture described in any one of

[65] to

[69] above, further comprising (i) a third loop-forward primer (TLF) and (ii) a third loop-backward primer (TLB), wherein the TLF and the TLB bind to two different binding sites on the third target sequence.

[71] The primer mixture according to any one of

[65] to

[70] above, further comprising a fourth loop primer.

[72] The primer mixture described in

[71] above, further comprising a fifth loop primer.

[73] A dried primer mixture obtained by freeze-drying the loop primer described in any one of [1] to

[44] above or the primer mixture described in any one of

[45] to

[72] above.

[74] A kit for loop-de-loop amplification of a target sequence, comprising: A kit comprising the loop primer according to any one of [1] to

[44] above, the primer mixture according to any one of

[45] to

[72] above, or the dry primer mixture according to

[73] above.

[75] The kit of

[74] above, further comprising a polymerase, optionally comprising Bacillus stearothermophilus polymerase.

[76] dNTPs, MgSO 4 The kit according to any one of the above

[74] to

[75] , further comprising a buffer solution.

[77] The kit according to any one of

[74] to

[76] above, further comprising a reverse transcriptase.

[78] The kit according to any one of

[74] to

[77] above, further comprising an RNase inhibitor.

[79] The kit described in

[78] above, wherein the RNase inhibitor is a porcine or mouse RNase inhibitor.

[80] A method for detecting a target sequence in a sample, comprising: providing a sample; (i) adding a primer according to any one of the above [1] to

[44] , (ii) a primer mixture according to any one of the above

[45] to

[72] , or (iii) a reconstituted primer mixture obtained by rehydrating the dried primer mixture according to the above

[73] , and a polymerase to a sample, thereby generating a reaction mixture; Incubating the reaction mixture at 50 to 85° C. A method comprising:

[81] The method according to

[80] above, wherein the incubation is carried out at 50 to 70°C.

[82] The method according to

[81] above, wherein the incubation is carried out at 60 to 65°C.

[83] The method according to

[82] above, wherein the incubation is carried out at 62 to 65°C.

[84] The method according to any one of

[80] to

[83] above, wherein the polymerase is Bacillus stearothermophilus polymerase.

[85] The method according to any one of

[80] to

[84] above, further comprising the step of detecting a signal from the reaction mixture.

[86] The method according to

[85] above, wherein the signal is a fluorescent signal.

[87] The method according to any one of

[85] to

[86] above, wherein the detecting step is carried out during the incubation step.

[88] The method according to any one of

[80] to

[87] above, further comprising the step of examining the presence or absence of the target sequence in the sample.

[89] The method according to any one of

[80] to

[88] above, further comprising the preceding step of preparing the sample.

[90] The method according to

[89] above, wherein the step of preparing the sample comprises interacting reverse transcriptase with RNA molecules, thereby producing the sample containing DNA molecules.

[91] The method according to

[90] above, wherein the step of preparing the sample further comprises the step of pre-heating the RNA molecule before or during interaction with the reverse transcriptase.

[92] The method according to any one of

[80] to

[91] above, wherein the reaction mixture further contains an RNase inhibitor.

[93] The method according to

[92] above, wherein the RNase inhibitor is a porcine or mouse RNA inhibitor.

[94] The method according to any one of

[80] to

[93] above, wherein the sample comprises purified RNA, purified DNA, whole SARS-CoV-2 virus, whole human cells, saliva or a nasal swab, or a nasal or nasopharyngeal swab.

[95] The method according to any one of

[80] to

[93] above, wherein the sample comprises genomic DNA, synthetic DNA, total bacteria or total human cells derived from a vaginal swab.

[0007] Applicants have demonstrated that the loop-de-loop amplification method allows for sequence-specific amplification of target nucleic acid molecules with improved sensitivity and specificity with faster turnaround times compared to previously known methods including inhibitory fluorescent probes, e.g., DARQ (detection of amplification by emission of quenching) and OSD (single-step displacement) probes. Furthermore, the loop-de-loop amplification method allows for real-time detection of the amplified signal, unlike QUASR (quenching of unincorporated amplification signal reporter). The loop-de-loop method provides a strong signal even with crude samples, allowing the method to be performed with low-cost equipment.

[0008] Thus, the present invention provides a method for detecting one or more target nucleic acids present in a sample using loop primers (e.g., fluorophore-labeled primers). Fluorophore-labeled primers are fluorophore-labeled oligonucleotides having complementarity to a target nucleic acid and a biosensor pair, e.g., a continuous loop sequence internally labeled at or near the 5' end of the primer sequence that is not modified with a fluorophore or quencher molecule. The fluorophore-labeled primers are labeled with a quencher or fluorophore, respectively, at or near the 5' end of the added loop sequence to enable FRET with the internal label described in the previous point. In some embodiments, the fluorophore-labeled primer is labeled at an internal site with a fluorophore and a quencher at or near the 5' end of the added loop sequence. The fluorophore-labeled primer further comprises a first clamping sequence at the 3' end of the loop sequence (at the intersection with the unmodified primer sequence). This sequence may overlap with the unmodified primer sequence, may be directly adjacent to the unmodified primer sequence, or may be spaced apart from the unmodified primer sequence. The sequence may include dNTPs, locked nucleic acids, or any other form of nucleic acid modification or substitution.

[0009] The melting temperature of the clamping sequence is preferably about 10° C. higher than the extension temperature of the assay using the strand displacing polymerase, but can be lower than, equal to, or any amount higher than the extension temperature of the assay.

[0010] If the melting temperature of the clamping sequence is lower than the extension temperature of the reaction, real-time detection can be replaced in this method by end-point detection (cooling the reaction near or below the Tm of the clamping sequence), in which case there is no inhibition of the reaction even when loop primers are used at full strength (100% replacement of unmodified primers with loop primer analogs).

[0011] If the melting temperature of the clamping sequence is equal to the extension temperature of the reaction, real-time detection is still feasible, but there may be higher background fluorescence until the reaction is cooled for endpoint determination.

[0012] If the melting temperature of the clamping sequence is higher than the extension temperature of the reaction, real-time detection is the primary mode of operation and background fluorescence is minimized.

[0013] The fluorophore-labeled primer further comprises a spacing sequence that separates the internally conjugated fluorophore or quencher by an appropriate distance from the 5'-terminal quencher or fluorophore to inhibit FRET when the primer is in a linear (extended) conformation, resulting in increased fluorescence. The spacing sequence can be of any sequence and length and can include deoxyribonucleotides, locked nucleic acids, etc. Its length can be 25 nucleotides, but can be shorter or longer.

[0014] The fluorophore-labeled primer further comprises a second clamping sequence, i.e., the reverse complement of the first clamping sequence, at or near the 5' end of the loop sequence. The fluorophore-labeled primer may further comprise an additional DNA barcode, probe, or sequence at the 5' end of the loop-de-loop oligonucleotide. This sequence may include a dNTP, a locked nucleic acid, or any other form of nucleic acid modification or substitution.

[0015] The melting temperature of the second clamping sequence paired with the first clamping sequence is preferably 10° C. higher than the extension temperature of the assay using the strand displacement polymerase, but can be lower than, equal to, or any amount higher than the extension temperature of the assay.

[0016] The looped fluorophore-labeled primer can further comprise additional sequences, molecules, purification tags, beads or other moieties at the 5' end to allow for further applications, such as nucleic acid capture, molecular barcoding, magnetic separation, column purification, electrophoretic separation. Probe capture oligonucleotides patterned on a substrate can be used to capture the amplification products, thereby producing fluorescent, colorimetric, luminescent, or other bands or zones.

[0017] The loop primers can be titrated to various degrees in the assay to minimize cost or to increase sensitivity and specificity.

[0018] The loop primers described herein can be designed with sensor molecules other than fluorophores, such as reporter molecules that provide light emission, a color change, or other measurable signal when in close proximity or when moved sufficiently far apart. In some cases, biosensors can be used, such as NanoLuc, Nanobit, NonoBRET.

[0019] If a photoprotein is used, a decrease in signal can be an important indicator of a positive reaction. In some embodiments, a bioluminescent endpoint analysis can be performed.

[0020] Amplification of the target sequence can be accomplished using enzymes capable of strand displacement, and other reagents may be used as required by the nucleic acid amplification method chosen.

[0021] The method provided herein allows specific detection of fluorescent signals in high background conditions with high concentrations of non-target RNA or DNA, thereby reducing false positives and non-specific amplification detection. Fluorescence detection allows specific detection of only amplicons and products that incorporate labeled primers. This allows specific detection even when using crude or unprocessed samples, such as genital swabs, feces, saliva, urine, blood, plant material, soil, and environmental samples.

[0022] In some embodiments, the method can be applied to the detection of two or more unique nucleic acid targets. In such double, triple, or higher multiplexed LAMP assays, the targets can be differentially labeled. For example, one target is labeled with FAM and the other with Cy5. In some cases, the detection is spectrally multiplexed to detect a single nucleic acid target using multiple labeled primers to further reduce the ability to distinguish true positives from false positives. In some cases, the detection uses a single label, e.g., FAM, for multiple targets, and the identity of each target can be determined based on the analysis of real-time or end-point signals (e.g., relative signal intensity, time to result, etc.) depending on the particular circumstances of the assay. In some cases, multiplexing can be achieved by performing reactions in a physical reaction chamber. In some cases, multiplexing can be achieved in a single reaction chamber.

[0023] The method provided herein minimizes inhibition compared to other real-time LAMP displacement probe technologies, making the method highly sensitive. Titration into the assay demonstrates that full reaction rate is maintained at least 50% of loop primer displacements. Titration into the assay demonstrates that full reaction rate is maintained at least 25% of inner primer displacements.

[0024] The loop primers provided herein can be used in loop-mediated amplification (LAMP) that utilizes a strand-displacing polymerase, such as a polymerase isolated or adapted from Geobacilus stearothermophilus (formerly Bacillus stearothermophilus), in which case the loop primer can be used together with other primers for LAMP, such as the forward inner primer, backward inner primer, loop forward primer, loop backward primer, F3 primer, and B3 primer.

[0025] In some embodiments, the primers and optionally other reaction components are dried using a process such as, but not limited to, a process of lyophilization. The dried primers can be included in a diagnostic kit. In a preferred embodiment, the process of lyophilization does not affect the sensitivity of the LAMP primer set.

[0026] In one aspect, the disclosure provides a loop primer for loop-de-loop amplification (LdL) of a target sequence, the loop primer comprising, from 5' to 3', a first sensor molecule, a first clamping oligonucleotide, a spacing oligonucleotide, a second clamping oligonucleotide, where the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide are capable of forming a hairpin structure at a temperature below the melting temperatures (Tm) of the first and second clamping oligonucleotides, a second sensor molecule, where the first sensor molecule and the second sensor molecule are a first biosensor pair, and a first primer sequence complementary to a first binding site on the target sequence.

[0027] In some embodiments, the second clamping oligonucleotide is complementary to the first clamping oligonucleotide.

[0028] In some embodiments, the first biosensor pair is an energy donor and acceptor pair. In some embodiments, the first biosensor pair is an energy donor and acceptor pair for fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET). In some embodiments, the first sensor molecule is a FRET fluorophore and the second sensor molecule is a FRET quencher. In some embodiments, the first sensor molecule is a FRET quencher and the second sensor molecule is a FRET fluorophore. In some embodiments, the first sensor molecule is a BRET energy donor and the second sensor molecule is a BRET energy acceptor. In some embodiments, the first sensor molecule is a BRET energy acceptor and the second sensor molecule is a BRET energy donor. In some embodiments, the first sensor molecule and the second sensor molecule can form a complex that generates a detectable optical signal.

[0029] In some embodiments, the melting temperature (Tm) of the first and second clamping oligonucleotides is greater than 60° C. In some embodiments, the melting temperature (Tm) of the first and second clamping oligonucleotides is greater than 65° C. In some embodiments, the melting temperature (Tm) of the first and second clamping oligonucleotides is greater than 70° C. In some embodiments, the melting temperature (Tm) of the first and second clamping oligonucleotides is greater than 80° C. In some embodiments, the melting temperature (Tm) of the first and second clamping oligonucleotides is between 70 and 80° C. In some embodiments, the melting temperature (Tm) of the first and second clamping oligonucleotides is between 72.5 and 77.5° C. In some embodiments, the melting temperature (Tm) of the first and second clamping oligonucleotides is about 75° C. In some embodiments, the melting temperature (Tm) of the first and second clamping oligonucleotides is less than 60° C. In some embodiments, the melting temperature (Tm) of the first and second clamping oligonucleotides is between 60 and 65°C.

[0030] In some embodiments, the first and second clamping oligonucleotides are 3 to 10 nucleotides in length. In some embodiments, the first and second clamping oligonucleotides are 3 to 7 nucleotides in length. In some embodiments, the first and second clamping oligonucleotides are 6 nucleotides in length. In some embodiments, the spacing oligonucleotides are 5 to 35 nucleotides in length. In some embodiments, the spacing oligonucleotides are 10 to 20 nucleotides in length. In some embodiments, the spacing oligonucleotides are 13 to 18 nucleotides in length. In some embodiments, the spacing oligonucleotides are 13 nucleotides in length. In some embodiments, the first, spacing oligonucleotides and second clamping oligonucleotides are collectively 15 to 35 nucleotides in length. In some embodiments, the first, spacing oligonucleotides and second clamping oligonucleotides are collectively 20 to 30 nucleotides in length. In some embodiments, the spacing oligonucleotides and second clamping oligonucleotides are collectively 23 to 28 nucleotides in length.

[0031] In some embodiments, the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide comprise (i) a nucleobase selected from adenine, guanine, cytosine, thymine, and uracil, (ii) a locked nucleic acid, (iii) a 2″O-methyl RNA base, (iv) a phosphorothioated DNA base, (v) a phosphorothioated RNA base, (vi) a phosphorothioated 2″-O-methyl RNA base, or (vii) a combination thereof.

[0032] In some embodiments, the loop primer further comprises a first additional oligonucleotide at the 5' end of the loop primer, hi some embodiments, the loop primer further comprises a second additional oligonucleotide between the first sensor molecule and the first clamping oligonucleotide.

[0033] In some embodiments, the first or second additional oligonucleotide is a barcode sequence.

[0034] In some embodiments, the target sequence is specific to a pathogen genome. In some embodiments, the target sequence is specific to Chlamydia trachomatis. In some embodiments, the target sequence is derived from orf8 or cds2. In some embodiments, the loop primer comprises an oligonucleotide of SEQ ID NO: 15.

[0035] In some embodiments, the target sequence is specific to Neisseria gonorrhoeae. In some embodiments, the target sequence is derived from porA or glnA. In some embodiments, the loop primer comprises an oligonucleotide of SEQ ID NO: 5 or 7.

[0036] In some embodiments, the target sequence is specific to a virus, hi some embodiments, the virus is SARS-CoV-2.

[0037] In some embodiments, the target sequence is specific to Homo sapiens. In some embodiments, the target sequence is derived from tbc1d3. In some embodiments, the loop primer comprises an oligonucleotide of SEQ ID NO:22.

[0038] In another aspect, the present disclosure provides a primer mixture for loop-de-loop amplification of a target sequence, comprising the loop primers provided herein.

[0039] In some embodiments, the primer mix further comprises (i) a forward inner primer (FIP), (ii) a backward inner primer (BIP), (iii) a forward primer (F3) and a backward primer (B3), where FIP, BIP, F3 and B3 bind to six different binding sites on the target sequence. In some embodiments, the primer mix comprises (i) a looped forward primer (LF) and (ii) a looped backward primer (LB), where LF and LB bind to two different binding sites on the target sequence. In some embodiments, FIP, BIP, F3, B3, LF or LB bind to a first binding site on the target sequence. In some embodiments, FIP binds to the first binding site and the ratio of the amount of FIP to the loop primer in the primer mix is ​​1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1. In some embodiments, BIP binds to the first binding site and the ratio of the amount of BIP to loop primer in the primer mixture is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1. In some embodiments, LF binds to the first binding site and the ratio of the amount of LF to loop primer in the primer mixture is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1.52. In some embodiments, LB binds to the first binding site and the ratio of the amount of LB to loop primer in the primer mixture is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1.

[0040] In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO:1, B3 comprises an oligonucleotide of SEQ ID NO:2, FIP comprises an oligonucleotide of SEQ ID NO:3, BIP comprises an oligonucleotide of SEQ ID NO:4, LF comprises an oligonucleotide of SEQ ID NO:6, or LB comprises an oligonucleotide of SEQ ID NO:8.

[0041] In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO:1, B3 comprises an oligonucleotide of SEQ ID NO:2, FIP comprises an oligonucleotide of SEQ ID NO:3, BIP comprises an oligonucleotide of SEQ ID NO:4, LF comprises an oligonucleotide of SEQ ID NO:6, and LB comprises an oligonucleotide of SEQ ID NO:8.

[0042] In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO:9, B3 comprises an oligonucleotide of SEQ ID NO:10, FIP comprises an oligonucleotide of SEQ ID NO:11, BIP comprises an oligonucleotide of SEQ ID NO:12, LF comprises an oligonucleotide of SEQ ID NO:13, or LB comprises an oligonucleotide of SEQ ID NO:14.

[0043] In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO:9, B3 comprises an oligonucleotide of SEQ ID NO:10, FIP comprises an oligonucleotide of SEQ ID NO:11, BIP comprises an oligonucleotide of SEQ ID NO:12, LF comprises an oligonucleotide of SEQ ID NO:13, and LB comprises an oligonucleotide of SEQ ID NO:14.

[0044] In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO: 16, B3 comprises an oligonucleotide of SEQ ID NO: 17, FIP comprises an oligonucleotide of SEQ ID NO: 18, BIP comprises an oligonucleotide of SEQ ID NO: 19, LF comprises an oligonucleotide of SEQ ID NO: 20, or LB comprises an oligonucleotide of SEQ ID NO: 21.

[0045] In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO: 16, B3 comprises an oligonucleotide of SEQ ID NO: 17, FIP comprises an oligonucleotide of SEQ ID NO: 18, BIP comprises an oligonucleotide of SEQ ID NO: 19, LF comprises an oligonucleotide of SEQ ID NO: 20, and LB comprises an oligonucleotide of SEQ ID NO: 21.

[0046] In some embodiments, the primer mix further comprises a second loop primer, the second loop primer comprising a third sensor molecule, a third clamping oligonucleotide, a second spacing oligonucleotide, a fourth clamping oligonucleotide, where the third clamping oligonucleotide, the second spacing oligonucleotide, and the fourth clamping oligonucleotide are capable of forming a hairpin structure at a temperature below the melting temperatures (Tm) of the third and fourth clamping oligonucleotides, a fourth sensor molecule, where the third sensor molecule and the fourth sensor molecule are a second biosensor pair, where the second biosensor pair is different from the first biosensor pair, and a second primer sequence complementary to a first binding site on the second target sequence.

[0047] In some embodiments, the third clamping oligonucleotide is complementary to the fourth clamping oligonucleotide. In some embodiments, the target sequence and the second target sequence are identical. In some embodiments, the target sequence and the second target sequence are different.

[0048] In some embodiments, the primer mix further comprises (i) a second forward inner primer (SFIP), (ii) a second backward inner primer (SBIP), (iii) a second forward primer (SF3) and (iv) a second backward primer (SB3), where SFIP, SBIP, SF3 and SB3 bind to six different binding sites on the second target sequence.

[0049] In some embodiments, the primer mix further comprises (i) a second loop-forward primer (SLF) and (ii) a second loop-backward primer (SLB), where the SLF and SLB bind to two different binding sites on the second target sequence. In some embodiments, the primer mix further comprises a third loop primer, the third loop primer comprising a fifth sensor molecule, a fifth clamping oligonucleotide, a third spacing oligonucleotide, a sixth clamping oligonucleotide, the fifth clamping oligonucleotide, the third spacing oligonucleotide, and the sixth clamping oligonucleotide being capable of forming a hairpin structure at a temperature below the melting temperature (Tm) of the fifth and sixth clamping oligonucleotides, the sixth sensor molecule, the fifth sensor molecule and the sixth sensor molecule being a third biosensor pair, the third biosensor pair being different from the first biosensor pair and the second biosensor pair, and a second primer sequence complementary to a first binding site on a third target sequence. In some embodiments, the fifth clamping oligonucleotide is complementary to the sixth clamping oligonucleotide. In some embodiments, the target sequence, the second target sequence, and the third target sequence are identical. In some embodiments, the target sequence, the second target sequence, and the third target sequence are different. In some embodiments, the primer mix further comprises (i) a third forward inner primer (TFIP), (ii) a third backward inner primer (TBIP), (iii) a third forward primer (TF3), and (iv) a third backward primer (TB3), where TFIP, TBIP, TF3, and TB3 bind to six different binding sites on the third target sequence. In some embodiments, the primer mix further comprises (i) a third loop forward primer (TLF), and (ii) a third loop backward primer (TLB), where TLF and TLB bind to two different binding sites on the third target sequence. In some embodiments, the primer mix further comprises a fourth loop primer.In some embodiments, the primer mix further comprises a fifth loop primer.

[0050] In yet another aspect, the present disclosure provides a dry primer mixture obtained by lyophilizing the loop primer or primer mixture provided herein.

[0051] In one aspect, the disclosure provides a kit for loop-de-loop amplification of a target sequence, the kit comprising a loop primer, a primer mixture, or a dry primer mixture provided herein. In some embodiments, the kit further comprises a polymerase, the polymerase optionally being a Bacillus stearothermophilus polymerase. In some embodiments, the kit further comprises dNTPs, MgSO4, and a buffer. In some embodiments, the kit further comprises a reverse transcriptase. In some embodiments, the kit further comprises an RNase inhibitor. In some embodiments, the RNase inhibitor is a porcine or mouse RNase inhibitor.

[0052] In another aspect, the disclosure provides a method of detecting a target sequence in a sample, the method comprising: providing a sample; adding (i) a primer, (ii) a primer mixture, or (iii) a reconstituted primer mixture obtained by rehydrating a dried primer mixture described herein, and a polymerase to the sample, thereby generating a reaction mixture; and incubating the reaction mixture at 50-85° C. In some embodiments, the incubation is at 50-70° C. In some embodiments, the incubation is at 60-65° C. In some embodiments, the incubation is at 62-65° C. In some embodiments, the polymerase is a Bacillus stearothermophilus polymerase. In some embodiments, the method further comprises detecting a signal from the reaction mixture. In some embodiments, the signal is a fluorescent signal. In some embodiments, the detecting step is performed during the incubation step. In some embodiments, the method further comprises determining the presence or absence of the target sequence in the sample.

[0053] In some embodiments, the method further comprises the preceding step of preparing a sample. In some embodiments, preparing the sample comprises interacting RNA molecules with a reverse transcriptase enzyme, thereby generating a sample comprising DNA molecules. In some embodiments, preparing the sample further comprises pre-heating the RNA molecules prior to or during interaction with the reverse transcriptase enzyme. In some embodiments, the reaction mixture further comprises an RNase inhibitor. In some embodiments, the RNase inhibitor is a porcine or mouse RNA inhibitor.

[0054] In some embodiments, the sample comprises purified RNA, purified DNA, whole SARS-CoV-2 virus, whole human cells, saliva or a nasal swab, or a nasal or nasopharyngeal swab. In some embodiments, the sample comprises genomic DNA, synthetic DNA, whole bacteria, or whole human cells from a vaginal swab. 5. Brief description of the drawings [Brief description of the drawings]

[0055] [Figure 1] Figure 1 shows the structure of the loop primer and the progress of DNA amplification in the loop-de-loop method.

[0056] [Figure 2A] Figure 2A provides the results of LAMP assays for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) visualized with an intercalating dye (SYTO). Amplification of [DNA] is rapid (<30 min) with at least 5 logs for CT and 6 logs for NG. The NG assay analytical sensitivity (LOD50) is 35 cp / 10 μL reaction by PROBIT analysis.

[0057] [Figure 2B] Figure 2B shows the readout from target amplification using the novel loop primer. The results demonstrate extremely bright real-time detection of the target with minimal inhibition and increased specificity over SYTO dyes.

[0058] [Figure 2C] Figure 2C shows detection of Neisseria gonorrhoeae using the novel loop primer. The results show that the method is reproducible and generates rapid, robust, high signal-to-noise amplification. The probes eliminate false positives.

[0059] [Figure 3-1] Figure 3 is a plot of real-time fluorescent signal over time showing amplification of Chlamydia trachomatis target nucleic acid using the loop-de-loop method with a 50% substitution FAM-labeled LF primer. Both positive and negative samples were tested, as shown in the table on the right. Each "cycle" on the y-axis represents 30 seconds of elapsed time at 65 degrees Celsius. [Figure 3-2] (As stated above.)

[0060] [Figure 4-1] Figure 4 is a plot of real-time fluorescent signal over time showing amplification of Neisseria gonorrhoeae target nucleic acid using the loop-de-loop method with a 50% substitution FAM-labeled LF primer. Both positive and negative samples were tested, as shown in the table on the right. Each "cycle" on the y-axis represents 30 seconds of elapsed time at 65 degrees Celsius. [Figure 4-2] (As stated above.)

[0061] [Figure 5-1] Figure 5 is a plot of real-time fluorescent signal over time showing amplification of Homo sapiens target nucleic acid using the loop-de-loop method with a 50% substitution FAM-labeled LF primer. Both positive and negative samples were tested, as shown in the table on the right. Each "cycle" on the y-axis represents 30 seconds of elapsed time at 65 degrees Celsius. [Figure 5-2] (As stated above.)

[0062] [Figure 6] Figure 6 shows images of tubes containing four positive reactions (left) and four negative reactions (right) using loop-de-loop primers. Fluorescence was excited with a blue LED and illuminated through a blue gel filter, and emission was visualized with an amber plastic filter held up to a camera phone.

[0063] [Figure 7] Figure 7 shows images of tubes containing dry (lyophilized) mixes for loop primer assays for Chlamydia trachomatis (top), Neisseria gonorrhoeae (middle) and Homo sapiens (bottom), prepared by lyophilization in PCR tubes.

[0064] [Figure 8]Figure 8 shows real-time fluorescent signals indicating amplification of Chlamydia trachomatis, Neisseria gonorrhoeae, and Homo sapiens target nucleic acids in loop-de-loop reactions using the dried mix from Figure 7 that was reconstituted prior to use. The results demonstrate that the assay activity and sensitivity of the reconstituted primers are maintained after drying.

[0065] [Figure 9] Figure 9A (first test) and Figure 9B (second test) plot the time required to obtain results from loop-de-loop LAMP reactions using primer sets for POP7b (Homo sapiens RNA transcript) or ORF1ab (SARS-CoV-2 genomic RNA) at various temperatures.

[0066] [Figure 10] Figure 10 shows the melting curves of loop-de-loop primers targeting DNA from Homo sapiens, C. Trachomatis, N. Gonorrhoeae, or SARS-CoV-2. The loop-de-loop primers are designed to unfold at approximately 10°C above the reaction temperature of 65°C. The curves show that the stem-loop sequence of the loop-de-loop primer is responsible for the fluorescent signal.

[0067] [Figure 11] Figure 11 shows the real-time fluorescent signal obtained from loop-de-loop reactions using primers of 25%, 50%, or 100% strength. In this context, "strength" is the degree to which the primer is replaced by the looped version of the loop-de-loop method. The data shows that stronger primers tend to show a larger signal at the expense of a 1-2 minute slower time to result. The 100% strength loop-de-loop primers slowed down the assay, but not to the same extent as other real-time LAMP displacement probe methods. Each "cycle" on the y-axis represents 30 seconds of elapsed time at 65 degrees Celsius.

[0068] [Figure 12] Figure 12 shows the relative fluorescence signal obtained from a loop-de-loop reaction containing both 0.4 μM loop-de-loop primer and 2 μM SYTO intercalating dye. The two-channel fluorescence data shows that the timing of onset of the intercalating dye (SYTO) and loop-de-loop signals is the same. There was no signal delay with loop-de-loop versus intercalating dye, and the loop-de-loop reaction showed a greater signal than SYTO.

[0069] [Figure 13] Figures 13A and 13B show the real-time fluorescent signal obtained from the amplification of a Chlamydia trachomatis target sequence using the loop-de-loop reaction. Figure 13A shows the results obtained from a freshly mixed reaction mixture, and Figure 13B shows the results obtained from a lyophilized reaction mixture. The lyophilized assay mixture was stable for over 3 months and gave a good readout. The assay was run in 14 replicates for each of the Ct E BOUR (Chlamydia trachomatis strains) at LoD95 (low positive) of the assay (20.7 copies / µL) plus two no template controls (NTC). There was no change in sensitivity (12 / 14 at LoD95, respectively) or mean time to result (16 min, T-test, P value = 0.66) between the fresh and lyophilized reaction mixtures.

[0070] [Figure 14]Figure 14A, Figure 14B and Figure 14C show the spectrally duplexed fluorescent signals obtained from loop-de-loop amplification of SARS-CoV-2 and human target sequences in a single-tube reaction (single pot). The dashed signal is from SARS-CoV-2 (FAM) and the solid signal is from human internal control (Cy5). Three types of samples were used: a control sample without target sequence (Figure 14A), a crude human nasal swab (Figure 14B), and a crude human nasal swab combined with heat-inactivated SARS-CoV-2 (intact virus with genomic RNA target sequence) (Figure 14C). The data show that the signal is specifically amplified only in the presence of the target sequence. The data further demonstrate the spectral multiplexing of the loop-de-loop reaction in a single reaction vessel.

[0071] [Figure 15A] Figure 15A shows the real-time fluorescent signal obtained from loop-de-loop amplification at various concentrations of POP7b primers. The signal intensity decreased as the concentration of POP7b primers was decreased (arrows). In multiplexed applications using two or more primer sets in a single reaction volume, the concentration of any given primer set is reduced compared to reactions where 100% of the primers belong to a single set. [Figure 15B] Figure 15B plots the time to result (min) for various concentrations of POP7b primers. Time to result is affected when primer concentration is reduced below 40%, which may be acceptable for many applications where the benefits of multiplexing more than two targets in a single tube outweigh the need for clinical or market-based speed. In this reaction, 10-4g of Block DNA was used in a 21 μL reaction volume.

[0072] [Figure 16]Figure 16 shows the real-time fluorescence signal obtained from loop-de-loop RT-LAMP amplification of either a SARS-CoV-2 specific RNA target sequence (ORF1ab), a Homo sapiens specific RNA target sequence (POP7b), both targets, or neither target in unprocessed nasal swabs obtained from coronavirus positive subjects. Nasal swabs were eluted directly into the loop-de-loop RT-LAMP reagent and diluted to four different concentrations to form reaction mixtures. 1x swab represents the standard concentration of samples used in this test configuration, in units of swabs eluted per unit volume. In this case, SARS-CoV-2 and human RNA primer sets were duplexed in a single tube. Each primer set contained one loop primer, each labeled with the same fluorophore and quencher pair (single fluorescent channel). As a result, reactions in which both SARS-CoV-2 and human RNA were detected were characterized by a dual amplification signal. Dilutions that resulted in detection of both targets were labeled as "double positives," those that resulted in detection of either target were labeled as "single positives," and those that resulted in detection of neither target were labeled as "double negatives." The data demonstrated that for this coronavirus-positive volunteer swab sample, the real-time loop-de-loop RT-LAMP assay was at least 370-fold more sensitive than required to detect both targets in the reaction.

[0073] [Figure 17] Figure 17 shows the real-time fluorescent signal obtained from loop-de-loop amplification of a target sequence specific for SARS-CoV-2 in a nasal swab obtained from a negative subject.

[0074] [Figure 18]Figure 18 shows the fluorescent signals obtained from multiplex loop-de-loop amplification of SARS-CoV-2 and human target sequences, demonstrating the specificity of the loop-de-loop reaction. Both SARS-CoV-2 and human primer sets were modified for loop-de-loop using FAM-labeled primers, so the double positive control shows two amplification events. RPPOS is Respiratory Pathogen Panel Positive (Exact Diagnostics LLC) containing genetic material from 22 non-target respiratory pathogens. PRNEG is a background matrix control of RPPOS products containing no nucleic acid. This data indicates that the loop-de-loop RT-LAMP reaction for detecting SARS-CoV-2 and human targets does not amplify off-target nucleic acids.

[0075] [Figure 19-1] FIG. 19 shows the fluorescent signal obtained from loop-de-loop amplification of samples containing high amounts of C. trachomatis (Ct) (10,000 copy equivalents per reaction) and high amounts of N. gonorrhoeae (Ng) (10,000 copy equivalents per reaction). [Figure 19-2] (As stated above.)

[0076] [Figure 20-1] Figure 20 shows the fluorescent signals obtained from loop-de-loop amplification of negative controls - swab only controls (left two panels) or buffer only controls (right two panels). [Figure 20-2] (As stated above.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] The drawings depict various embodiments of the present invention for purposes of illustration only. Those skilled in the art will readily appreciate from the following description that alternative embodiments of the structures and methods shown herein may be used without departing from the principles of the present invention described herein.

[0078] 6. Detailed Description 6.1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

[0079] The term "biosensor pair" as used herein refers to a pair of sensor molecules that can generate a detectable signal upon a specific physical interaction between two sensor molecules. For example, the biosensor pair can be a pair of donor and acceptor molecules used in Förster resonance energy transfer, e.g., fluorescence resonance energy transfer (FRET). In this case, a fluorescent signal can be generated by the distance-dependent transfer of energy from the donor molecule to the acceptor molecule. In other embodiments, the biosensor pair is a pair of sensor molecules used in bioluminescence resonance energy transfer (BRET). In this case, a bioluminescence signal can be generated by the distance-dependent transfer of energy from the donor molecule to the acceptor molecule. Other biosensor pairs known in the art can be used in various embodiments of the present disclosure.

[0080] As used herein, the term "loop-de-loop amplification" or "LdL amplification" refers to the amplification of a target nucleic acid using loop primers that are capable of generating a fluorescent signal by distance-dependent transfer of energy.

[0081] The term "LOD" as used herein refers to the limit of detection. For example, LOD95 is the 95th percentile limit of detection. This is the concentration of target at which an assay is statistically expected to detect a positive result 95% of the time.

[0082] 6.2. Other Rules of Interpretation Ranges described herein are understood to be shorthand for all values ​​within the range, including the endpoints described. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0083] Unless otherwise indicated, a reference to a compound having one or more stereocenters contemplates each and every stereoisomer thereof, and all combinations of stereoisomers.

[0084] 6.3. Loop primer In one embodiment, the present invention provides a loop primer for loop-de-loop amplification, the loop primer comprising, in a 5' to 3' direction: A first sensor molecule; a first clamping oligonucleotide; a spacing oligonucleotide; A second clamping oligonucleotide, The first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide are aligned such that the melting temperatures (T m ) a second clamping oligonucleotide capable of forming a hairpin structure at a lower temperature; A second sensor molecule, a second sensor molecule, wherein the first sensor molecule and the second sensor molecule are a first biosensor pair; and a first primer sequence complementary to a first binding site on the target sequence; Includes.

[0085] In some embodiments, the second clamping oligonucleotide is complementary to the first clamping oligonucleotide, hi some embodiments, the second clamping oligonucleotide is capable of binding to the first clamping oligonucleotide but is not completely complementary to the first clamping oligonucleotide.

[0086] The methods provided herein can use a variety of biosensors known in the art, such as a pair of molecules that change color or generate a measurable signal when in close proximity or when sufficiently far apart (e.g., NanoLuc, Nanobit, NonoBRET technology based on photoproteins).

[0087] In some embodiments, the first biosensor pair is an energy donor and acceptor pair. In some embodiments, the first biosensor pair is an energy donor and acceptor pair for Förster resonance energy transfer. In some embodiments, the first biosensor pair is an energy donor and acceptor pair for fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET). In some embodiments, the first sensor molecule is a FRET fluorophore and the second sensor molecule is a FRET quencher. In some embodiments, the first sensor molecule is a FRET quencher and the second sensor molecule is a FRET fluorophore. In some embodiments, the first sensor molecule is a BRET energy donor and the second sensor molecule is a BRET energy acceptor. In some embodiments, the first sensor molecule is a BRET energy acceptor and the second sensor molecule is a BRET energy donor.

[0088] In some embodiments, the FRET quencher is 5IABkFQ available from Integrated DNA technologies under the trade name 5'Iowa Black® FQ. 5'Iowa Black® FQ is a FRET quencher with a broad absorbance spectrum ranging from 420 to 620 nm with a peak absorbance at 531 nm. This quencher can be used with fluorescein and other fluorescent dyes that emit in the green to pink spectral range. In some embodiments, the quencher is either a Black Hole Quencher® (available from Biosearch Technologies), either an Iowa Black® quencher (available from Integrated DNA technologies), a Zen® quencher (available from Integrated DNA Technologies), an Onyx® quencher (available from Millipore-Sigma), or either an ATTO® quencher (available from ATTO-TECGmbH).

[0089] In some embodiments, the FRET fluorophore is i6-FAMK (FAM (fluorescein) azide), available from Integrated DNA technologies under the name Int 6-FAM (Azide). This form of FAM can be attached to oligonucleotides using click chemistry. An internal version of this modification is attached to the oligo via a dT base. A dT nucleotide can be added at the position of the modification. Alternatively, to avoid the addition of an extra nucleotide, an existing T nucleotide in the sequence can be replaced with the required modification. In some embodiments, the fluorophore is Cy3, Cy5, TAMRA, or Yakima Yellow® (available from Integrated DNA Technologies).

[0090] In one embodiment, the loop primer comprises an internal quencher (eg, Zen® or Onyx A®) and a 5' fluorophore (eg, Yakima Yellow® or HEX).

[0091] In some embodiments, the first sensor molecule and the second sensor molecule can form a complex that generates a detectable optical signal.

[0092] The first and second clamping oligonucleotides are complementary to each other and therefore can bind to each other. The first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide are aligned at a temperature that is equal to or greater than the melting temperature (T m ) hairpin structures can be formed at lower temperatures.

[0093] In some embodiments, the melting temperatures (T m ) is greater than 60° C. In some embodiments, the melting temperature (T m ) is greater than 65° C. In some embodiments, the melting temperatures (T m ) is greater than 70° C. In some embodiments, the melting temperatures (T m ) is greater than 80° C. In some embodiments, the melting temperature (T m ) is 70 to 80° C. In some embodiments, the melting temperature (T m ) is 72.5 to 77.5° C. In some embodiments, the melting temperature (T m ) is about 75° C. In some embodiments, the melting temperature (T m) is less than 60° C. In some embodiments, the melting temperature (T m ) is 60 to 65°C.

[0094] In some embodiments, the melting temperatures (T m ) is 10° C. higher than the extension temperature of the assay using the strand-displacing polymerase. In some embodiments, the melting temperature can be lower than, equal to, or any amount higher than the extension temperature of the assay.

[0095] T m If is lower than the extension temperature of the reaction, real-time detection is possible at the T m Nearby or T m Cooling the reaction until end-point detection (cooling the reaction until 100% substitution) can be substituted and there is no inhibition of the reaction, even when using loop-de-loop primers at full strength (100% substitution).

[0096] T m If t is equal to the extension temperature of the reaction, real-time detection may still be feasible, but higher background fluorescence may be present until the reaction is cooled for endpoint determination.

[0097] T m If is higher than the extension temperature of the reaction, real-time detection can be the basic mode of operation and background fluorescence will be minimized.

[0098] In some embodiments, the first and second clamping oligonucleotides are 3 to 10 nucleotides in length. In some embodiments, the first and second clamping oligonucleotides are 3 to 7 nucleotides in length. In some embodiments, the first and second clamping oligonucleotides are 6 nucleotides in length. In typical embodiments, the first and second clamping oligonucleotides have the same length.

[0099] In some embodiments, the spacing oligonucleotide is 5 to 35 nucleotides in length. In some embodiments, the spacing oligonucleotide is 10 to 20 nucleotides in length. In some embodiments, the spacing oligonucleotide is 13 to 18 nucleotides in length. In some embodiments, the spacing oligonucleotide is 13 nucleotides in length.

[0100] In some embodiments, the first clamping oligonucleotide, spacing oligonucleotide, and second clamping oligonucleotide, collectively, are 15 to 35 nucleotides in length. In some embodiments, the first clamping oligonucleotide, spacing oligonucleotide, and second clamping oligonucleotide, collectively, are 20 to 30 nucleotides in length. In some embodiments, the first clamping oligonucleotide, spacing oligonucleotide, and second clamping oligonucleotide, collectively, are 23 to 28 nucleotides in length.

[0101] The loop primer may comprise (i) a nucleobase selected from adenine, guanine, cytosine, thymine and uracil, (ii) a locked nucleic acid, (iii) a 2'O-methyl RNA base, (iv) a phosphorothioated DNA base, (v) a phosphorothioated RNA base, (vi) a phosphorothioated 2'-O-methyl RNA base, or (vii) a combination thereof. The first clamping oligonucleotide, the spacing oligonucleotide and the second clamping oligonucleotide comprise (i) a nucleobase selected from adenine, guanine, cytosine, thymine and uracil, (ii) a locked nucleic acid, (iii) a 2'O-methyl RNA base, (iv) a phosphorothioated DNA base, (v) a phosphorothioated RNA base, (vi) a phosphorothioated 2'-O-methyl RNA base, or (vii) a combination thereof.

[0102] In some embodiments, the loop primer further comprises a first additional oligonucleotide at the 5' end of the loop primer. In some embodiments, the loop primer further comprises a second additional oligonucleotide between the first sensor molecule and the first clamping oligonucleotide. In some embodiments, the first or second additional oligonucleotide is a barcode sequence.

[0103] In some embodiments, the loop primer further comprises a barcode sequence, a probe sequence, or other sequence at the 5' end of the loop primer. The further sequence may comprise a nucleobase or a modification thereof.

[0104] In some embodiments, the target sequence is specific to a pathogen genome. In some embodiments, the target sequence is specific to Chlamydia trachomatis. In some embodiments, the target sequence is from orf8 or cds2. Specifically, the target binding site can have the sequence of SEQ ID NO:15.

[0105] In some embodiments, the target sequence is specific for Neisseria gonorrhoeae. In some embodiments, the target sequence is derived from porA or glnA. Specifically, the target binding site can have the sequence of SEQ ID NO:5 or SEQ ID NO:7.

[0106] In some embodiments, the target sequence is specific to Homo sapiens. In some embodiments, the target sequence is derived from tbc1d3. Specifically, the target binding site can have the sequence of SEQ ID NO:22.

[0107] 6.4. Primer Mixture for Loop-de-Loop Amplification In another aspect, the present invention provides a primer mixture for loop-de-loop amplification, the primer mixture comprising a loop primer as provided herein.

[0108] In some embodiments, the primer mixture includes one loop primer. In some embodiments, the primer mixture includes two or more loop primers. When it includes two or more loop primers, the primers in the mixture can bind to a single target sequence or multiple target sequences. In some embodiments, the multiple loop primers are designed to detect target sequences from multiple sources. For example, the mixture can include multiple loop primers designed to detect target sequences from multiple pathogens.

[0109] The primer mixture can further comprise additional primers for the amplification reaction. For example, the primer mixture can further comprise (i) forward inner primer (FIP), (ii) backward inner primer (BIP), (iii) forward primer (F3) and backward primer (B3), where FIP, BIP, F3 and B3 bind to six different binding sites on the target sequence. In some embodiments, the primer mixture further comprises (i) loop forward primer (LF) and (ii) loop backward primer (LB), where LF and LB bind to two different binding sites on the target sequence. In some embodiments, one of the additional primers, for example FIP, BIP, F3, B3, LF or LB, binds to the first binding site, i.e., the same binding site as the loop primer on the target sequence.

[0110] In some embodiments, the primer mix comprises one primer set. In some embodiments, the primer set comprises loop primers for loop-de-loop amplification provided herein: (i) forward inner primer (FIP), (ii) backward inner primer (BIP), (iii) forward primer (F3) and (iv) backward primer (B3). In some embodiments, the primer set further comprises (i) loop forward primer (LF) and (ii) loop backward primer (LB).

[0111] In some embodiments, the primer set comprises a loop primer for loop-de-loop amplification provided herein and three primers selected from (i) a forward inner primer (FIP), (ii) a backward inner primer (BIP), (iii) a forward primer (F3) and (iv) a backward primer (B3). In some embodiments, the primer set comprises loop primers, BIP, F3 and B3. In some embodiments, the primer set comprises loop primers, FIP, F3 and B3. In some embodiments, the primer set comprises loop primers, FIP, BIP and B3. In some embodiments, the primer set comprises loop primers, FIP, BIP and F3.

[0112] In some embodiments, the primer set comprises a loop primer for loop-de-loop amplification provided herein and five primers selected from (i) forward inner primer (FIP), (ii) backward inner primer (BIP), (iii) forward primer (F3), (iv) backward primer (B3), (v) loop forward primer (LF) and (vi) loop backward primer (LB). In some embodiments, the primer set comprises loop primers, BIP, F3, B3, LF and LB. In some embodiments, the primer set comprises loop primers, FIP, F3, B3, LF and LB. In some embodiments, the primer set comprises loop primers, FIP, BIP, B3, LF and LB. In some embodiments, the primer set comprises loop primers, FIP, BIP, F3, LF and LB. In some embodiments, the primer set comprises loop primers, FIP, BIP, F3, B3 and LF. In some embodiments, the primer set comprises loop primers, FIP, BIP, F3, B3 and LF. In some embodiments, the primer set comprises loop primers, FIP, BIP, F3, B3 and LB.

[0113] In some embodiments, the primer mix comprises two primer sets, in some embodiments, the primer mix comprises three primer sets, in some embodiments, the primer mix comprises four or five primer sets.

[0114] In some embodiments, each primer set is for amplifying a unique target sequence. In some embodiments, the primer mix includes two or more primer sets for amplifying the same target sequence. In some embodiments, the primer mix includes two or more loop primers that bind to the same binding site on the same target sequence.

[0115] The loop primer can be mixed with the additional primer in any ratio optimized for the amplification reaction. In some embodiments, FIP binds to the first binding site, and the ratio of the amount of FIP to loop primer in the primer mixture is 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1. In some embodiments, BIP binds to the first binding site, and the ratio of the amount of BIP to loop primer in the primer mixture is 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1. In some embodiments, LF binds to the first binding site, and the ratio of the amount of LF to loop primer in the primer mixture is 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, or 9:1. In some embodiments, the LB binds to the first binding site and the ratio of the amount of LB to loop primer in the primer mix is ​​0:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1.

[0116] In some embodiments, the primer mix is ​​designed to detect a target sequence specific to Neisseria gonorrhoeae. In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO: 1, B3 comprises an oligonucleotide of SEQ ID NO: 2, FIP comprises an oligonucleotide of SEQ ID NO: 3, BIP comprises an oligonucleotide of SEQ ID NO: 4, LF comprises an oligonucleotide of SEQ ID NO: 6, or LB comprises an oligonucleotide of SEQ ID NO: 8. In one embodiment, F3 comprises an oligonucleotide of SEQ ID NO: 1, B3 comprises an oligonucleotide of SEQ ID NO: 2, FIP comprises an oligonucleotide of SEQ ID NO: 3, BIP comprises an oligonucleotide of SEQ ID NO: 4, LF comprises an oligonucleotide of SEQ ID NO: 6, and LB comprises an oligonucleotide of SEQ ID NO: 8. In some embodiments, the loop primer is an oligonucleotide of SEQ ID NO: 5 or SEQ ID NO: 7.

[0117] In some embodiments, the primer mix is ​​designed to detect a target sequence specific to Chlamydia trachomatis. In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO:9, B3 comprises an oligonucleotide of SEQ ID NO:10, FIP comprises an oligonucleotide of SEQ ID NO:11, BIP comprises an oligonucleotide of SEQ ID NO:12, LF comprises an oligonucleotide of SEQ ID NO:13, or LB comprises an oligonucleotide of SEQ ID NO:14. In one embodiment, F3 comprises an oligonucleotide of SEQ ID NO:9, B3 comprises an oligonucleotide of SEQ ID NO:10, FIP comprises an oligonucleotide of SEQ ID NO:11, BIP comprises an oligonucleotide of SEQ ID NO:12, LF comprises an oligonucleotide of SEQ ID NO:13, and LB comprises an oligonucleotide of SEQ ID NO:14. In some embodiments, the loop primer is an oligonucleotide of SEQ ID NO:15.

[0118] In some embodiments, the primer mix is ​​designed to detect a target sequence specific to Homo sapiens. In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO: 16, B3 comprises an oligonucleotide of SEQ ID NO: 17, FIP comprises an oligonucleotide of SEQ ID NO: 18, BIP comprises an oligonucleotide of SEQ ID NO: 19, LF comprises an oligonucleotide of SEQ ID NO: 20, or LB comprises an oligonucleotide of SEQ ID NO: 21. In one embodiment, F3 comprises an oligonucleotide of SEQ ID NO: 16, B3 comprises an oligonucleotide of SEQ ID NO: 17, FIP comprises an oligonucleotide of SEQ ID NO: 18, BIP comprises an oligonucleotide of SEQ ID NO: 19, LF comprises an oligonucleotide of SEQ ID NO: 20, and LB comprises an oligonucleotide of SEQ ID NO: 21. In some embodiments, the loop primer is an oligonucleotide of SEQ ID NO: 22.

[0119] In some embodiments, the primer mix is ​​designed to detect a target sequence specific to a virus, in some embodiments, the virus is SARS-CoV-2.

[0120] In some embodiments, the primer mixtures provided herein are further combined for the detection of multiple target sequences. In some embodiments, the multiple target sequences are specific to different organisms. For example, the multiple target sequences are specific to different pathogens.

[0121] Thus, in some embodiments, the primer mix further comprises a second loop primer, the second loop primer comprising: a third sensor molecule; and a third clamping oligonucleotide; and a second spacing oligonucleotide; and a fourth clamping oligonucleotide, The third clamping oligonucleotide, the second spacing oligonucleotide, and the fourth clamping oligonucleotide are arranged to have a melting temperature (Tm ) a fourth clamping oligonucleotide capable of forming a hairpin structure at a lower temperature; A fourth sensor molecule, a fourth sensor molecule, the third sensor molecule and the fourth sensor molecule being a second biosensor pair, the second biosensor pair being different from the first biosensor pair; a second primer sequence that is complementary to the first binding site on the second target sequence; Includes.

[0122] In some embodiments, the third clamping oligonucleotide is complementary to the fourth clamping oligonucleotide, in some embodiments, the third clamping oligonucleotide is capable of binding to the fourth clamping oligonucleotide but is not fully complementary to the fourth clamping oligonucleotide.

[0123] In some embodiments, the primer mix further comprises (i) a second forward inner primer (SFIP), (ii) a second backward inner primer (SBIP), (iii) a second forward primer (SF3) and a second backward primer (SB3), where SFIP, SBIP, SF3 and SB3 bind to six different binding sites on the second target sequence. In some embodiments, the primer mix further comprises (i) a second looped forward primer (SLF) and (ii) a second looped backward primer (SLB), where SLF and SLB bind to two different binding sites on the second target sequence.

[0124] In some embodiments, the primer mix further comprises a third loop primer, the third loop primer comprising: a fifth sensor molecule; and a fifth clamping oligonucleotide; and a third spacing oligonucleotide; and A sixth clamping oligonucleotide, The fifth clamping oligonucleotide, the third spacing oligonucleotide, and the sixth clamping oligonucleotide are arranged to have a melting temperature (T m ) a sixth clamping oligonucleotide capable of forming a hairpin structure at a lower temperature than A sixth sensor molecule, a sixth sensor molecule, the fifth sensor molecule and the sixth sensor molecule being a third biosensor pair, the third biosensor pair being different from the first biosensor pair and the second biosensor pair; a second primer sequence complementary to the first binding site on a third target sequence; Includes.

[0125] In some embodiments, the fifth clamping oligonucleotide is complementary to the sixth clamping oligonucleotide, in some embodiments, the fifth clamping oligonucleotide binds to the sixth clamping oligonucleotide, but the fifth clamping oligonucleotide is not completely complementary to the sixth clamping oligonucleotide.

[0126] In some embodiments, the primer mix further comprises (i) a third forward inner primer (TFIP), (ii) a third backward inner primer (TBIP), (iii) a third forward primer (TF3) and a third backward primer (TB3), where TFIP, TBIP, TF3 and TB3 bind to six different binding sites on a third target sequence.

[0127] In some embodiments, the primer mix comprises (i) a third loop-forward primer (TLF) and (ii) a third loop-backward primer (TLB), where the TLF and TLB bind to two different binding sites on a third target sequence.

[0128] In some embodiments, the primer mix includes two, three, four, five, or six loop primers. When the primer mix includes two or more loop primers, each loop primer may include a unique biosensor pair, each of which provides a unique signal for detection. In some embodiments, each biosensor pair provides a unique visual signal (e.g., color) for detection. In some embodiments, each biosensor pair includes a unique dye molecule.

[0129] In some embodiments, two or more loop primers in a primer mix comprise the same biosensor pair. In some embodiments, two or more loop primers in a primer mix are labeled with FAM. In some embodiments, two different loop primers in a primer mix are labeled with FAM.

[0130] In some embodiments, the primer mixture provided herein is lyophilized. The dried primer mixture can include any of the looper primers or primer mixtures described herein. In some embodiments, a primer mixture including two or more loop primers is lyophilized. In some embodiments, the primer mixture is in the form of lyophilized beads.

[0131] 6.5. Loop-de-loop amplification kit In another aspect, a kit for loop-de-loop amplification is provided, which may include any of the loop primers or primer mixtures provided herein.

[0132] In some embodiments, the kit comprises one primer set. In some embodiments, the primer set comprises loop primers for loop-de-loop amplification provided herein: (i) forward inner primer (FIP), (ii) backward inner primer (BIP), (iii) forward primer (F3) and backward primer (B3). In some embodiments, the primer set further comprises (i) loop forward primer (LF) and (ii) loop backward primer (LB).

[0133] In some embodiments, the kit comprises two primer sets, in some embodiments, the kit comprises three primer sets, in some embodiments, the kit comprises four or five primer sets.

[0134] In some embodiments, the kit comprises multiple primer sets in a single container. In some embodiments, the kit comprises multiple primer sets, each primer set individually contained in a separate container.

[0135] In some embodiments, the kit further comprises a polymerase. In some embodiments, the polymerase is a strand displacing DNA polymerase. In some embodiments, the polymerase is Bacillus stearothermophilus polymerase. In some embodiments, the polymerase is Bst 2.0 WarnStart® DNA Polymerase (available from NEB). In some embodiments, the kit further comprises other reaction enzymes, for example, a reverse transcriptase. In some embodiments, the reverse transcriptase is WarmStart® RTx reverse transcriptase (available from NEB). In some embodiments, the kit further comprises an RNase inhibitor. In some embodiments, the RNase inhibitor is a porcine or mouse RNase inhibitor.

[0136] In some embodiments, the kit further comprises reagents for the amplification reaction. In some embodiments, the reagents comprise dNTPs, MgSO4, and a buffer. In some embodiments, the buffer comprises a detergent. In some embodiments, the buffer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% Tween-20. In some embodiments, the reagents comprise trehalose. In some embodiments, the reagents comprise sucrose. In some embodiments, the reagents comprise a polymer for stabilization. Amplification reagents can be selected and optimized depending on the polymerase.

[0137] In some embodiments, the kit comprises a mixture comprising dNTPs, MgSO4, a buffer, one or more primer sets for loop-de-loop amplification, and a polymerase. In some embodiments, the kit comprises a mixture comprising dNTPs, one or more primer sets for loop-de-loop amplification, a polymerase, a reverse transcriptase, and an RNase inhibitor.

[0138] In some embodiments, the mixture is in a liquid form. In some embodiments, the mixture is in a dry form. In some embodiments, the mixture is formulated into lyophilized beads or pellets.

[0139] In some embodiments, the kit further comprises an apparatus for an amplification reaction. In some embodiments, the kit comprises an apparatus for loop-mediated isothermal amplification.

[0140] In some embodiments, the kit further comprises a reaction tube for carrying out the amplification reaction, hi some embodiments, the kit further comprises components for filtering or purifying the sample prior to the amplification reaction.

[0141] In some embodiments, the kit is for diagnosis of infectious diseases. In some embodiments, the kit is for diagnosis of pathogenic infectious diseases, such as Chlamydia trachomatis and Neisseria gonorrhoeae. In some embodiments, the kit is used for determination of single nucleotide polymorphisms (SNPs) and point mutations. In some embodiments, the kit is used for determination of mutant genotypes. In some embodiments, the kit is used for determination of mutant genotypes associated with drug resistance phenotypes. For example, drug resistance markers, such as ceftriaxone / cefixime resistance markers, quinolone (ciprofloxacin) resistance markers, macrolide resistance markers (azithromycin) can be detected.

[0142] 6.6. Loop-de-loop amplification method In another aspect, a loop de-loop amplification method is provided, the method comprising: providing a sample; (i) adding a primer, a primer mixture, or a reconstituted primer mixture obtained by rehydrating the dried primer mixture provided herein, and (ii) a polymerase to the sample, thereby generating a reaction mixture; incubating the reaction mixture at 50-85°C; may include.

[0143] The reaction temperature can be adjusted depending on the polymerase and the target sequence. In some embodiments, the incubation is performed at 50-70°C. In some embodiments, the incubation is performed at 55-70°C. In some embodiments, the incubation is performed at 60-65°C. In some embodiments, the incubation is performed at 62-65°C. In some embodiments, the incubation is performed at 60, 61, 62, 63, 64 or 65°C.

[0144] In some embodiments, the method further comprises detecting a signal from the reaction mixture. In some embodiments, the method comprises detecting a fluorescent signal. In some embodiments, the method comprises detecting a change in color or turbidity. In some embodiments, the method comprises detecting a non-visual signal. In some embodiments, the detecting step occurs during the incubation step. In some embodiments, the detecting step occurs after completion of the incubation step. In some embodiments, the signal is detected in real time. In some embodiments, the signal is recorded in real time and analyzed after completion of the incubation step.

[0145] In some embodiments, the method further comprises preparing a sample for loop-de-loop amplification. In some embodiments, preparing the sample comprises interacting an RNA molecule with a reverse transcriptase, thereby generating a sample comprising DNA molecules. In some embodiments, preparing the sample further comprises pre-heating the sample or reaction mixture containing the RNA molecule prior to interacting with the reverse transcriptase.

[0146] In some embodiments, the sample for loop de loop amplification comprises purified polynucleotide molecules. In some embodiments, the sample comprises purified RNA, purified DNA, whole SARS-CoV-2 virus, whole human cells, saliva or a nasal swab, or a nasal or nasopharyngeal swab. In some embodiments, the sample comprises genomic DNA, synthetic DNA, whole bacteria, or whole human cells from a vaginal swab. In some embodiments, the sample for loop de loop amplification is a crude sample. In some embodiments, the sample for loop de loop amplification is a purified sample.

[0147] In some embodiments, more than one signal is detected. In some embodiments, multiple fluorescent or other visual signals are detected. In some embodiments, multiple signals are detected to determine the presence or absence of multiple target sequences. In some embodiments, multiple signals are detected to confirm the presence or absence of a single target sequence. In some embodiments, multiple signals are detected to provide additional sensitivity and specificity to the method.

[0148] A variety of amplification methods known in the art can be used to amplify the target sequence.

[0149] In a typical embodiment, loop-mediated isothermal amplification ("LAMP") is used for loop-de-loop amplification of target nucleic acids. LAMP is an isothermal DNA amplification method that relies on the strand-displacing activity of enzymes known as polymerases to add nucleotide bases to a growing DNA or RNA strand in a base-specific manner to form a double-stranded nucleic acid with a complementary sequence. In isothermal amplification, strand-displacing polymerases, such as the polymerase from Geobacillus stearothermophilus bacteria (Bst polymerase and its variants), displace one strand of double-stranded DNA as they polymerize the complementary strand, thus eliminating the need for thermal cycling.

[0150] The LAMP method can use four different primers (F3, B3, inner forward primer or FIP, and inner backward primer or BIP) that are specifically designed to recognize six different regions of the target DNA sequence. Two additional "loop" primers may be added to improve the speed of the reaction. The concentration of primers in the reaction mixture can vary, but is typically set at 1.6 μM for FIP and BIP primers, 0.8 μM for forward and reverse loop primers (LF, LB), and 0.2 μM for F3 and B3 primers. In some embodiments of the LAMP method, five primers can be utilized (using only one of the two possible LAMP primers). The LAMP reaction proceeds at a constant temperature (around 65° C.) utilizing a strand displacement reaction. The amplification and detection of the target can be completed in one step by incubating the sample, primers, DNA polymerase with strand displacement activity, buffer, and substrate at a constant temperature. A typical mixture composition of LAMP contains the following reagents: 20 mM Tris-HCL, 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween® 20, 1.4 mM dNTPs, 0.32 U / μL Bst polymerase, primers at the aforementioned concentrations, and water, with the pH adjusted to 8.8 at 20° C. The reaction volume is typically 5 μL to 50 μL. The reaction temperature is optimized for the particular enzyme and primers used, and the reaction proceeds for 5 to 60 minutes. LAMP has high sensitivity, specificity, and efficiency.

[0151] LAMP relies on at least four primers that recognize six target sites (e.g., F3, B3, FIP, and BIP) to amplify a specific DNA or RNA target (RNA targets require first reverse transcription to DNA). When loop primers (e.g., LF and LB) are included, a total of eight unique sites in the target nucleic acid are recognized by the six primers. In various embodiments provided herein, one of the total eight unique sites can be recognized by the loop primers described herein. If the target is present in the sample, an amplification reaction can occur, resulting in a large amount of DNA.

[0152] The novel loop-de-loop method described herein may be applied to other isothermal amplification methods besides LAMP. A number of isothermal amplification methods have been developed to address the temperature cycle dependency of the polymerase chain reaction (PCR). Although these methods can vary considerably, they all share some features in common. For example, DNA strands are not heat denatured, so all isothermal methods rely on alternative approaches to allow primer binding and initiation of the amplification reaction. Once the reaction is initiated, the polymerase must also displace the strand that is still annealed to the sequence of interest. Isothermal methods typically use the strand displacement activity of DNA polymerases to separate double-stranded DNA. Polymerases with this ability include Klenow fragment (3'-5'exo-), Bsu large fragment and phi29 for moderate temperature reactions (25-40°C), and the large fragment of Bst DNA polymerase for higher temperature (50-65°C) reactions. To detect RNA species, a reverse transcriptase compatible with the reaction temperature is added to maintain the isothermal nature of the amplification. In addition to a strand displacement mechanism to separate dsDNA, isothermal methods may require the design of enzymes or primers to avoid the requirement of initial denaturation for initiation.

[0153] As mentioned above, loop-mediated isothermal amplification (LAMP) uses 4-6 primers that recognize 6-8 different regions of the target DNA. A strand-displacing DNA polymerase initiates synthesis, and two primers form a loop structure to facilitate subsequent amplification. LAMP is rapid, sensitive, and the amplification is very large, making LAMP well suited for on-site diagnostics. Loop-de-loop primers can be used single or in any combination or inner and / or loop primers.

[0154] Strand displacement amplification (SDA) relies on a strand-displacing DNA polymerase, typically Bst DNA polymerase, large fragment or Klenow fragment (3'-5'exo-), to initiate at a nick made by a strand restriction endonuclease or nicking enzyme at a site contained in the primer. SDA requires one forward and one reverse primer, as well as one bumping forward and one bumping reverse primer. The nicking site is regenerated with each polymerase displacement step, resulting in exponential amplification. SDA is typically used in clinical diagnostics. Existing fluorescence monitoring techniques exist for SDA (Nadeau et al., Real-Time, Sequence-specific detection of nucleic acids during strand displacement amplification, 276m 2 177-187 (1999)), but rely on the action of a restriction endonuclease enzyme to generate fluorescence. Either forward or reverse SDA primers can be adapted for use in the loop-de-loop method, which does not require the location of a cleavage site between the fluorophore and quencher pairs. The cleavage site resides next to the clamping sequence of the loop-de-loop primer, toward the 3' end of the primer, such that fluorescence is generated upon complete extension of the 5' end of the primer by polymerase on the complementary strand prior to primer cleavage by the restriction endonuclease.

[0155] Helicase-dependent amplification (HDA) utilizes the double-stranded DNA unwinding activity of helicase to separate the strands, allowing primer annealing and extension by a strand-displacing DNA polymerase. Similar to PCR, the system requires only two primers, one forward and one reverse. HDA is used in several diagnostic devices and FDA-approved tests. Either primer in HDA can be adapted for use in the loop-de-loop method to allow real-time closed-tube monitoring of the reaction in real time. In HDA, the helicase enzyme can open the loop structure of the loop-de-loop primer, which is stabilized by a single-stranded binding protein and can then be converted into a double-stranded fluorescent amplicon by a DNA polymerase.

[0156] Nicking enzyme amplification reaction (NEAR) uses a strand-displacing DNA polymerase that initiates at a nick made by a nicking enzyme to rapidly generate many short nucleic acids from a target sequence. The process is very rapid and sensitive, and minute amounts of target can be detected in minutes. NEAR is commonly used for pathogen detection in clinical and biosafety applications. Either the forward or reverse primer for NEAR can use the loop-de-loop method to generate real-time fluorescence via extension of the loop by a strand-displacing DNA polymerase.

[0157] 6.7.How to use The loop-de-loop amplification method provided herein can be used to detect target sequences from a variety of sources. For example, it can be used to detect target sequences specific to viral genomes, bacterial genomes, archaeal genomes, plant genomes, animal genomes, protist genomes, prokaryotic genomes, or eukaryotic genomes. In some embodiments, the method is used to detect RNA (e.g., positive-sense RNA, negative-sense RNA) or DNA. In some embodiments, the method is used to detect synthetically generated target sequences.

[0158] In some embodiments, the loop-de-loop method is used to detect DNA specific for a pathogen. In some embodiments, the pathogen is a virus, a bacterium, a fungus, a protozoan, or a parasite. In some embodiments, the loop-de-loop method is used to detect a pathogen associated with an STD. In some embodiments, the pathogen is Chlamydia trachomatis. In some embodiments, the pathogen is Neisseria gonorrhoeae. In some embodiments, the pathogen is SARS-CoV-2.

[0159] In some embodiments, the loop-de-loop method is used to diagnose infection. In some embodiments, the loop-de-loop method is used to determine mutant genotypes. In some embodiments, the loop-de-loop method is used to determine mutant genotypes associated with drug resistance phenotypes. For example, drug resistance markers, such as ceftriaxone / cefixime resistance markers, quinolone (ciprofloxacin) resistance markers, macrolide resistance markers (azithromycin) can be detected.

[0160] In some embodiments, the loop-de-loop method is used for determining single nucleotide polymorphisms (SNPs). In some embodiments, the loop-de-loop method is used for determining mutations.

[0161] In some embodiments, the loop-de-loop method is used for the detection of a single target. In some embodiments, the loop-de-loop method is used for the detection of two or more targets. In some embodiments, the loop-de-loop method is used for the detection of two, three, four or five targets.

[0162] In some embodiments, the loop-de-loop method is used for the analysis or characterization of a sample. In some embodiments, the loop-de-loop method is used to identify the source of a sample. For example, the loop-de-loop method is used to identify a human sample.

[0163] The loop-de-loop method described herein can be used to analyze a variety of samples. In some embodiments, blood, urine, semen, tissue, or saliva samples are analyzed. In some embodiments, the sample is taken from an animal or human patient. In some embodiments, a purified sample is analyzed. In some embodiments, a crude sample is analyzed. In some embodiments, the sample comprises purified RNA, purified DNA, whole SARS-CoV-2 virus, whole human cells, saliva or a nasal swab, or a middle turbinate or nasopharyngeal swab. In some embodiments, the sample comprises genomic DNA, synthetic DNA, whole bacteria, or whole human cells from a vaginal swab. EXAMPLES

[0164] 6.8.Example The following examples are offered by way of illustration and not by way of limitation. 6.8.1. Example 1: LAMP assay for Chlamydia trachomatis and Neisseria gonorrhoeae using an intercalating dye (SYTO)

[0165] A LAMP reaction mixture was prepared to detect Chlamydia trachomatis genomic DNA and Niesseria gonorrhea genomic DNA separately. The reaction was prepared in a volume of 10 μL and contained the following reagents: 20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween® 20, 1.4 mM dNTPs, 0.32 U / μL Bst2.0 WarmStart® polymerase, 1.6 μM FIP and BIP, 0.8 μM LF and LB, 0.2 μM primers with F3 and B3 (SEQ ID NOs: 1-4, 6, 8-14), 2.5 μM SYTO85 intercalating dye, and water, and was adjusted to pH 8.8 at 20 °C. Target genomic DNA was diluted 10-fold in pH 8.0 Tris-HCL buffer from the stock solution purchased from ATCC. 1 μL of target DNA or buffer without DNA (for no template control) was added to the 9 μL solution mixture in each PCR tube. The temperature of the reaction was 65 °C and the reaction was monitored by SYTO85 fluorescence. A real-time PCR machine was used to heat the reaction and measure the fluorescence in real time. The reaction was run for 60 min. The data shown in Figure 2A shows representative curves of real-time fluorescence (arbitrary units) on the ordinate versus time on the abscissa obtained from LAMP reactions for Chlamydia trachomatis (green) and Neisseria gonorrhoeae (blue), monitored by an intercalating dye, for three levels of genomic DNA target—high (stock concentration), low (10% of stock DNA for Ct), and high (10% of stock DNA for Ct). -5 Dilutions, 10 Ng of stock DNA -6 dilutions), and no template control (no DNA (NTC)).

[0166] 6.8.2. Example 2: Detection of Chlamydia trachomatis by Loop-de-Loop Amplification A loop-de-loop LAMP reaction mixture was prepared to detect Chlamydia trachomatis genomic DNA. The reaction was prepared in a 10 μL volume and contained the following reagents: 20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween® 20, 1.4 mM dNTPs, 0.32 U / μL Bst2.0 WarmStart® polymerase, 1.6 μM FIP and BIP, 0.4 μM LF and LF-LdL, 0.8 μM LB, 0.2 μM primers with F3 and B3 (SEQ ID NOs: 9-15), and water, and was adjusted to pH 8.8 at 20 °C. Quantified target genomic DNA was diluted 10-fold or 2-fold (for finer resolution) in Tris-HCL buffer at pH 8.0 from the stock solution purchased from ATCC. 1 μL of target DNA dilutions or no DNA-containing buffer (for no template control) was added to the 9 μL solution mixture in each PCR tube, and up to 20 replicates per concentration were used over a several log concentration range to examine the assay sensitivity. The temperature of the reaction was 65 °C and the reaction was monitored by FAM fluorescence emitted by the loop-de-loop primer. Reactions were heated and fluorescence was measured in real time using a real-time PCR machine. Reactions were run for 60 min. Data shown in Figure 3 show representative curves of real-time fluorescence (arbitrary units) versus time on the horizontal axis (each "cycle" represents 30 s) obtained from a loop-de-loop LAMP reaction of Chlamydia trachomatis against a 10-fold dilution of genomic DNA target. The assay sensitivity (limits of detection, 50% and 95% probability) was then estimated by PROBIT analysis based on the assay endpoint determinations. [Table 1]

[0167] 6.8.3. Example 3: Detection of Neisseria gonorrhoeae by loop-de-loop amplification A loop-de-loop LAMP reaction mixture was prepared to detect Neisseria gonorrhoeae genomic DNA. The reaction was prepared in a 10 μL volume and contained the following reagents: 20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween® 20, 1.4 mM dNTPs, 0.32 U / μL Bst2.0 WarmStart® polymerase, 1.6 μM FIP and BIP, 0.4 μM LF and LF-LdL, 0.8 μM LB, 0.2 μM primers with F3 and B3 (SEQ ID NOs: 1-4, 6-8), and water, and was adjusted to pH 8.8 at 20 °C. Quantified target genomic DNA was diluted 10-fold or 2-fold (for finer resolution) in Tris-HCL buffer at pH 8.0 from the stock solution purchased from ATCC. 1 μL of target DNA dilutions or DNA-free buffer (for no template control) was added to the 9 μL solution mixture in each PCR tube, and up to 20 replicates per concentration were used over a several-log concentration range to examine the assay sensitivity. The temperature of the reaction was 65 °C, and the reaction was monitored by FAM fluorescence emitted by the loop-de-loop primer. A real-time PCR machine was used to heat the reaction and measure the fluorescence in real time. The reaction was run for 60 min. Data shown in Figure 2B-C show representative curves of real-time fluorescence (arbitrary units) versus time on the horizontal axis obtained from a loop-de-loop LAMP reaction of Neisseria gonorrhoeae against a 10-fold dilution of genomic DNA target. Figure 2B compares the signal obtained from the loop-de-loop assay with that of a LAMP assay performed with SYTO85 dye without the loop-de-loop primer as shown in Figure 2A. The loop-de-loop assay provides a much larger signal in case of positive amplification. In Figure 2C, the reproducibility of the loop-de-loop assay is shown, as well as the negligible background fluorescence and reduced late spurious amplification products in the no template control.The data shown in Figure 4 shows a representative curve of real-time fluorescence (arbitrary units) versus time on the horizontal axis (each "cycle" represents 30 seconds) obtained from a loop-de-loop LAMP reaction of Neisseria gonorrhoeae for 10-fold dilutions of genomic DNA target. The assay sensitivity (limits of detection, 50% and 95% probability) was then estimated from the serial dilution test results by PROBIT analysis based on the assay endpoint determination. [Table 2]

[0168] 6.8.4. Example 4: Detection of Homo sapiens by loop-de-loop amplification A loop-de-loop LAMP reaction mixture was prepared to detect Homo sapiens genomic DNA. The reaction was prepared in a 10 μL volume and contained the following reagents: 20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween® 20, 1.4 mM dNTPs, 0.32 U / μL Bst2.0 WarmStart® polymerase, 1.6 μM FIP and BIP, 0.4 μM LF and LF-LdL, 0.8 μM LB, 0.2 μM primers with F3 and B3 (SEQ ID NOs: 16-22), and water, adjusted to pH 8.8 at 20 °C. Quantified target genomic DNA was diluted 10-fold or 2-fold (for finer resolution) in Tris-HCL buffer at pH 8.0 from the stock solution purchased from ATCC. Target DNA dilutions or DNA-free buffer (for no template controls) were added at 1 μL into a 9 μL solution mixture in each PCR tube, with up to 20 replicates per concentration over a several-log concentration range to examine assay sensitivity. The temperature of the reaction was 65 °C, and the reaction was monitored by FAM fluorescence emitted by the loop-de-loop primer. A real-time PCR machine was used to heat the reaction and measure fluorescence in real time. The reaction was run for 60 min. The data shown in Figure 3 shows a representative curve of real-time fluorescence (arbitrary units) versus time on the horizontal axis (each "cycle" represents 30 s) obtained from a Homo sapiens loop-de-loop LAMP reaction for a 10-fold dilution of genomic DNA target. The assay sensitivity (limits of detection, 50% and 95% probability) was then estimated by PROBIT analysis based on the assay end point determination. [Table 3]

[0169] 6.8.5. Example 5: Dry primer mix for loop-de-loop amplification Formulation into lyophilized reagents was performed in an in-house lyophilization study using a five-step lyophilization protocol. A loop-de-loop LAMP reaction mixture designed to detect Neisseria gonorrhoeae was prepared in 25 μL volume / tube and dispensed into each tube. The lyophilization mixture contained the following reagents: 1.4 mM dNTPs, 0.32 U / μL Bst 2.0 WarmStart® polymerase as a glycerol-free formulation, 1.6 μM FIP and BIP, 0.4 μM LF and LF-LdL, 0.8 μM LB, primers (SEQ ID NOs: 1-4, 6-8) with F3 and B3 at 0.2 μM, 5% trehalose, and water (maximum 25 μL per reaction). Tube caps were removed for lyophilization. Tube strips were placed on metal shelves in a heated shelf lyophilizer unit, which is standard equipment in the pharmaceutical and biotechnology industries. The freeze dryer was programmed to run in 5 stages. Stage 1: Condenser ON, vacuum OFF, cool shelves and reagents to 41°F, 30 min. Stage 2: Condenser ON, vacuum OFF, cool shelves and reagents to 23F, 30 min. Stage 3: Condenser ON, vacuum OFF, cool shelves and reagents to -23F, 2 hr. Stage 4: Condenser ON, vacuum ON, hold shelves and reagents at -23F, 10 hr. Stage 5: Condenser ON, vacuum ON, heat shelves and reagents to 77F, 5 hr. Once this process was complete, the tubes were removed and capped to obtain the product shown in Figure 7. The activity of the freeze-dried assay was tested over time after incubation in various environmental conditions. Figure 8 shows a representative real-time loop-de-loop LAMP assay activity of the rehydration reaction. The rehydration protocol consisted of adding 24 μL of rehydration buffer to the dried reagents along with 1 μL of Neisseria gonorrhoeae target genomic DNA. The rehydration buffer consisted of 20 mM Tris-HCL, 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween® 20, and water, with the pH adjusted to 8.8 at 20° C. The buffer was added to the tube, which was then resealed and placed directly into the real-time qPCR machine without vortexing or mixing.The temperature of the reaction was 65 °C, and the reaction was monitored by FAM fluorescence emitted by the loop-de-loop primer. A real-time PCR machine was used to heat the reaction and measure the fluorescence in real time. The reaction was run for 60 min. The data shown in Figure 8 shows a representative curve of real-time fluorescence (arbitrary units) versus time on the horizontal axis (each "cycle" represents 30 seconds) obtained from a loop-de-loop LAMP reaction of Neisseria gonorrhoeae against a 10-fold dilution of the genomic DNA target. The speed, sensitivity, and specificity of the lyophilized loop-de-loop assay were found to be unchanged from that of the freshly formulated assay. Furthermore, the magnitude of fluorescence was not affected by drying and rehydration, indicating that the loop-de-loop method may provide a storage-stable in vitro diagnostic kit for pathogen detection.

[0170] 6.8.6. Example 6: Temperature for Loop-de-Loop Amplification Loop-de-loop LAMP reaction mixtures were prepared as described above, one containing the ORF1ab primer set and the other containing the POP7b primer set. The ORF1ab primer set is specific for the SARS-CoV-2 virus, which has a positive-sense single-stranded RNA genome. The POP7b primer set is specific for a human RNA target that does not naturally occur as a DNA template, and therefore this primer set is useful as a specific indicator of human RNA in the sample. In both cases, loop-de-loop was used to modify one of the six constituent primers used for LAMP to create a seventh loop primer. The loop primers utilize a fluorophore and quencher pair to generate an observable signal. In this experiment, moderately concentrated synthetic double-stranded DNA templates containing sequences on their positive-sense strands corresponding to the sequences of the RNA targets of each primer set were utilized as targets for LAMP reaction temperature optimization to minimize variation due to stochastic noise encountered in the reverse transcription step or dilute targets. Target DNA dilutions were added to the mixtures in a 384-well plate. They were incubated at various temperatures ranging from 55 to 70 °C, and the reactions were monitored by the FAM fluorescence emitted by the loop-de-loop primers. A real-time PCR machine was used to heat the reactions and measure the fluorescence in real time. The reactions were run for 60 min. Two experiments (first and second tests) were performed for overlapping temperature ranges, and the data are shown in Figure 9A and Figure 9B. The figure shows the time required to obtain sufficient signal for detection. The results show that the primer sets are active over a wide range of temperatures. For example, about 57-70 °C was the acceptable range for both the POP7b and ORF1ab primer sets. Optimal performance was achieved between 60 °C and 68 °C.

[0171] 6.8.7. Example 7: Multiplex Loop-de-Loop Reaction for Detection of Both Purified and Crude Targets Figure 14A, Figure 14B and Figure 14C show two fluorescent signals obtained from loop-de-loop amplification of SARS-CoV-2 and human target sequences using ORF1ab and POP7b LdL primer sets, respectively. The signals obtained from SARS-CoV-2 ORF1ab (FAM) and POP7b human internal control (Cy5) are shown. Three types of samples were used: a control sample without target sequence (no template control) (Figure 14A), a human nasal swab (Figure 14B), and a human nasal swab combined with the SARS-CoV-2 target sequence in the form of heat-inactivated virus (Figure 14C). Human nasal swabs were self-collected from volunteers and added directly to the reaction without sample processing or nucleic acid extraction. The swabs were eluted into the reaction mixture by twisting for a few seconds. The SARS-CoV-2 target sequence was introduced into the reaction as intact heat-inactivated virus (ATCC VR-1986HK) added to the SARS-CoV-2 positive reaction. The ORF1ab LdL-FAM and POP7b LdL-Cy5 primer sets were duplexed in a 1:1 ratio in the replicate reaction volume. Both primer sets utilized the LdL primer in a 1:3 ratio to the unlabeled primer analog (25% intensity). The reaction contained reverse transcriptase, strand-displacing polymerase and RNase inhibitor. The reaction was incubated at 55.6 °C for 2.5 min and then at 63.5 °C for 60 min while recording fluorescence measurements of FAM and Cy5 using a real-time PCR instrument (Bio-Rad CFX-384®). As expected, the no template control replicates showed no loop-de-loop fluorescent signal over the 60 min period. Reactions containing COVID-19 negative nasal swab samples showed amplification of the POP7b signal as evidenced by an increase in Cy5 fluorescence, while the ORF1ab signal remained flat (negative). Samples spiked with heat-inactivated virus showed spectral dual detection of both RNA targets in a single reaction vessel. This result indicates that loop-de-loop RT-LAMP allows single-tube spectral multiplexing of SARS-CoV-2 and human targets.

[0172] The multiplex loop-de-loop test for SARS-CoV-2 using the ORF1ab LdL primer set was as sensitive as the PCR test and did not require extraction. This is because the reaction with crude samples gave good results as shown in the table below. The POP7b LdL primer set was used as an internal control. Serial dilutions of intact heat-inactivated SARS-CoV-2 virus (ATCC VR-1986HK) were added to the dual loop-de-loop reaction and monitored for real-time signal development. The detection limit of the assay is LoD95=400cp / swab=2.7×10 for the specific format of the test kit. 3 was estimated to be cp / mL. [Table 4]

[0173] A triplexed loop-de-loop reaction was also tested in a single tube. It showed specific amplification of three targets and maintained a fast time to result. Two loop-de-loop primer sets labeled with FAM fluorophores were used to detect two separate targets in SARS-CoV-2 viral RNA. A human internal control loop-de-loop primer set labeled with Cy5 detected a third RNA target. The internal Cy5 fluorophore was paired with a 5' Iowa Black® RQ quencher. The reaction contained crude nasal swab eluate and was spiked with heat-inactivated SARS-CoV-2.

[0174] Additional loop primers were also tested for use in the loop-de-loop primer set for the POP7b human internal control. In one case, the second sensor molecule, an internal TAMRA fluorophore, was paired with the first sensor molecule, a 5' Iowa Black® FQ quencher. In another case, the first sensor molecule, a 5' Yakima Yellow® (Epoch Biosciences), was paired with the second sensor molecule, an internal Zen™ (Integrated DNA Technologies) quencher. Three variations of loop primers were made and tested for the Yakima Yellow and Zen arrangements. In the first variation, the first and second clamping oligonucleotides were fully complementary and each was 6 bases long. The spacing oligonucleotide was 13 bases long. In the second variation, the first clamping oligonucleotide featured an additional base at its 5' end, such that the first clamping oligonucleotide was 7 bases long and the second clamping oligonucleotide was 6 bases long. There were 6 complementary bases between the first and second clamping oligonucleotides. The spacing oligonucleotide was 13 bases long. In a third variation, the first and second clamping oligonucleotides were both 7 bases long and were perfectly complementary in sequence. The spacing oligonucleotide was 10 bases long.

[0175] These additional loop primers are used in the LAMP reaction, which provides a specific amplification signal of the target sequence.

[0176] 6.8.8. Example 8: Detection of SARS-CoV-2 in human samples by loop-de-loop amplification A loop-de-loop LAMP reaction mixture was prepared to detect SARS-CoV-2 from unprocessed human saliva. Reactions were prepared in PCR tubes by rehydrating lyophilized enzyme, dNTP, and oligonucleotide primer mixtures with a 10% volume / volume mixture of human saliva in a pH-buffered salt solution. The lyophilized primer mixture contained primer sets for SARS-CoV-2 and human internal control RNA sequences. After rehydration with the saliva sample, the reactions were incubated at a preheated temperature for a predetermined period of time to promote viral lysis, RNase inhibition, and reverse transcription, and then incubated at a higher reaction temperature for LAMP DNA amplification. Custom instrumentation was used to collect temperature-controlled and real-time fluorescence data.

[0177] Heat-inactivated SARS-CoV-2 was spiked into a pool of fresh saliva collected from an anonymous donor. Three-fold serial dilutions of the saliva were prepared. Twenty samples were tested using the loop-de-loop amplification method. Readouts by mobile application, visual estimation, or real-time curve testing are summarized below. These results indicate an LoD of approximately 2,500 cp / mL. [Table 5]

[0178] A self-collected nasal swab was obtained from a volunteer subject and added directly to the reaction mixture by twisting it 10 times. Figure 16 shows the amplification results obtained from a nasal swab obtained from a symptomatic volunteer who was later confirmed to be COVID positive by PCR testing. The results obtained from the positive / negative control samples are also shown. These results indicate that the sample (1x swab) was 365 times more concentrated than the concentration required to detect a positive sample in the loop-de-loop assay. With an estimated LoD of approximately 2,500 cp / mL, a given sample had a nasal concentration of approximately 9.1x10 5 It was estimated to contain 100 cp / mL of SARS-CoV-2 viral RNA.

[0179] Figure 17 shows the amplification results obtained from a nasal swab obtained from a negative volunteer who was detected as negative by both the loop-de-loop reaction and PCR testing.

[0180] The reaction mixture for detecting SARS-CoV-2 was multiplexed with primers for detecting human genome sequences at a 1:1 ratio. The multiplexed amplification results are shown in Figure 18. These results demonstrate specific and sensitive detection of the two target sequences without cross-reactivity.

[0181] 6.8.9. Example 9: Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in human samples by loop-de-loop amplification Three vaginal swabs (BD BBL Culture Swabs, polyurethane foam-tipped swabs purchased from Lee Biosolutions, MO, sourced from unique individual donors) were eluted in 1,294 μL of rehydration buffer (431 μL per swab) using a 30 s, 1 Hz spinning method (Panpradist et al., 2016). After some fluid was lost in the swabs, 1,095 μL of pooled vaginal swab eluate was obtained. Fluid recovery was 85%. This swab eluate was pipetted into injection molded prototype disposables containing the lyophilized reaction mixture for loop-de-loop LAMP (one for Ct, one for Ng, and one for human process control).

[0182] Each reaction was rehydrated with: · 18 μL swab eluate (swab swirled in rehydration buffer); 1 μL of total Ct pathogen suspended in rehydration buffer 1 μL of total Ng pathogen suspended in rehydration buffer Ct and Ng pathogen samples were in separate disposable reaction chambers, so for example, the Ct assay was tasked with detecting Ct in the simultaneous presence of Ng and human targets as well as any bacterial milieu present in the swab samples.

[0183] Amplification results are shown in Figures 19-22. Figure 19 shows the fluorescent signal obtained from a sample containing high Ct (10,000 copy equivalents per reaction) and high Ng (10,000 copy equivalents per reaction). Figure 20 shows the signal obtained from the negative controls - swab only controls (left two panels) or buffer only controls (right two panels). There was no amplification of Ct or Ng in the swab only controls, but the human genome sequence was amplified as expected. There was no amplification of Ct or Ng in the buffer only controls. Homo sapiens amplification was detected with a delay in one of the buffer only reactions (Test 20), but given the delayed signal this is likely a spurious amplification.

[0184] These results indicate that the assay was sensitive to detect Ct at approximately 100 copies per reaction and Ng at more than approximately 1,000 copies per reaction. 7. Arrays [Table 6]

[0185] 8. INCORPORATION BY REFERENCE All publications, patents, patent applications, and other documents cited in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0186] 9. Equivalents The present disclosure provides, inter alia, compositions of cannabinoids and associated compositions. The present disclosure also provides methods for treating neurodegenerative diseases by administering cannabinoid compositions and associated compositions. Although various specific embodiments have been shown and described, the above specification is not intended to be limiting. It will be understood that various modifications can be made without departing from the spirit and scope of the present invention. Many variations will be apparent to those skilled in the art upon review of this specification.

Claims

1. 1. A primer mixture for loop-de-loop amplification of a target sequence, comprising: (i) from 5' to 3', A first sensor molecule; a first clamping oligonucleotide; a spacing oligonucleotide that is not complementary to the target sequence; A second clamping oligonucleotide, a second clamping oligonucleotide, wherein the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide are capable of forming a hairpin structure at a temperature lower than the melting temperature (Tm) of the first and second clamping oligonucleotides; A second sensor molecule, a second sensor molecule, the first sensor molecule and the second sensor molecule being a first biosensor pair; and a first primer sequence complementary to a first binding site on the target sequence; wherein the loop primer is configured to be amplified by a strand displacing polymerase when bound to the target sequence; (ii) a forward inner primer (FIP), (iii) a backward inner primer (BIP), (iv) a forward primer (F3) and (v) a backward primer (B3), (vi) a looped forward primer (LF) and (vii) a looped backward primer (LB), wherein the FIP, the BIP, the F3, the B3, the LF and the LB bind to six different binding sites on the target sequence, and at least one of the FIP, the BIP, the F3, the B3, the LF and the LB binds to the first binding site.

2. and a second loop primer, the second loop primer comprising: a third sensor molecule; and a third clamping oligonucleotide; and a second spacing oligonucleotide; and a fourth clamping oligonucleotide, a fourth clamping oligonucleotide, wherein the third clamping oligonucleotide, the second spacing oligonucleotide, and the fourth clamping oligonucleotide are capable of forming a hairpin structure at a temperature lower than the melting temperature (Tm) of the third and fourth clamping oligonucleotides; A fourth sensor molecule, a fourth sensor molecule, the third sensor molecule and the fourth sensor molecule being a second biosensor pair, the second biosensor pair being different from the first biosensor pair; and a second primer sequence complementary to the first binding site on the second target sequence; The primer mixture of claim 1 , comprising:

3. The primer mix of claim 2 , wherein the target sequence and the second target sequence are different.

4. 3. The primer mix of claim 2, further comprising: (i) a second forward inner primer (SFIP), (ii) a second backward inner primer (SBIP), (iii) a second forward primer (SF3) and (iv) a second backward primer (SB3), wherein the SFIP, the SBIP, the SF3 and the SB3 bind to six different binding sites on the second target sequence.

5. 3. The primer mix of claim 2, further comprising: (i) a second loop-forward primer (SLF) and (ii) a second loop-backward primer (SLB), wherein the SLF and the SLB bind to two different binding sites on the second target sequence.

6. and a third loop primer, the third loop primer comprising: A fifth sensor molecule; and a fifth clamping oligonucleotide; and a third spacing oligonucleotide; and a sixth clamping oligonucleotide, a sixth clamping oligonucleotide, wherein the fifth clamping oligonucleotide, the third spacing oligonucleotide, and the sixth clamping oligonucleotide are capable of forming a hairpin structure at a temperature lower than the melting temperature (Tm) of the fifth and sixth clamping oligonucleotides; A sixth sensor molecule, a sixth sensor molecule, the fifth sensor molecule and the sixth sensor molecule being a third biosensor pair, the third biosensor pair being different from the first biosensor pair and the second biosensor pair; a third primer sequence complementary to the first binding site on a third target sequence; The primer mixture of claim 2 , comprising:

7. 1. A kit for loop-de-loop amplification of a target sequence, comprising:

10. A kit comprising the primer mixture of claim 1 and a polymerase, wherein the polymerase is a strand-displacing polymerase.

8. The kit of claim 7 further comprising a reverse transcriptase.

9. 1. A method for detecting a target sequence in a sample, comprising: providing the sample; (i) adding a primer mixture according to claim 1 and a polymerase to the sample, thereby forming a reaction mixture; incubating the reaction mixture at 50-85°C; detecting a fluorescent signal from the reaction mixture; A method comprising:

10. A loop primer for loop-de-loop amplification (LdL) of a target sequence, comprising, from 5' to 3': A first sensor molecule; a first clamping oligonucleotide; a spacing oligonucleotide; A second clamping oligonucleotide, a second clamping oligonucleotide, wherein the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide are capable of forming a hairpin structure at a temperature lower than the melting temperature (Tm) of the first and second clamping oligonucleotides; A second sensor molecule, a second sensor molecule, the first sensor molecule and the second sensor molecule being a first biosensor pair; and a first primer sequence complementary to a first binding site on the target sequence; Including, a first additional oligonucleotide, said first additional oligonucleotide being at a 5' end of said loop primer, said first additional oligonucleotide being a barcode sequence; Loop primer.

11. The loop primer of claim 10, wherein the second clamping oligonucleotide is complementary to the first clamping oligonucleotide.

12. The loop primer of claim 10, wherein the first biosensor pair is an energy donor and acceptor pair.

13. The loop primer of claim 12, wherein the first biosensor pair is an energy donor and acceptor pair for fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET).

14. The loop primer of claim 10 , wherein the first sensor molecule and the second sensor molecule are capable of forming a complex that generates a detectable optical signal.

15. 11. The loop primer of claim 10, wherein the melting temperature (Tm) of the first and second clamping oligonucleotides is greater than 60°C.

16. 16. The loop primer of claim 15, wherein the melting temperature (Tm) of the first and second clamping oligonucleotides is greater than 70°C.

17. 17. The loop primer of claim 16, wherein the melting temperature (Tm) of the first and second clamping oligonucleotides is greater than 80°C.

18. 16. The loop primer of claim 15, wherein the melting temperature (Tm) of the first and second clamping oligonucleotides is between 70 and 80°C.

19. 11. The loop primer of claim 10, wherein the melting temperature (Tm) of the first and second clamping oligonucleotides is less than 60°C.

20. 11. The loop primer of claim 10, wherein each of the first clamping oligonucleotide and the second clamping oligonucleotide is 3 to 10 nucleotides in length.

21. The loop primer of claim 10, wherein the spacing oligonucleotide is 5 to 35 nucleotides in length.

22. 11. The loop primer of claim 10, wherein the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide are collectively 15 to 35 nucleotides in length.

23. 11. The loop primer of claim 10, wherein the first clamping oligonucleotide, the spacing oligonucleotide and the second clamping oligonucleotide comprise (i) a nucleobase selected from adenine, guanine, cytosine, thymine and uracil, (ii) a locked nucleic acid, (iii) a 2'O-methyl RNA base, (iv) a phosphorothioated DNA base, (v) a phosphorothioated RNA base, (vi) a phosphorothioated 2'-O-methyl RNA base, or (vii) a combination thereof.

24. 11. The loop primer of claim 10, further comprising a second additional oligonucleotide, wherein the second additional oligonucleotide is between the first sensor molecule and the first clamping oligonucleotide, the first additional oligonucleotide is a barcode sequence, and the second additional oligonucleotide is a barcode sequence.

25. The loop primer of claim 10, wherein the target sequence is specific to a pathogen genome.

26. A primer mixture for loop-de-loop amplification of a target sequence, comprising the loop primers according to claim 10.

27. 27. The primer mix of claim 26, further comprising: (i) a forward inner primer (FIP); (ii) a backward inner primer (BIP); (iii) a forward primer (F3) and a backward primer (B3), wherein the FIP, the BIP, the F3 and the B3 bind to six different binding sites on the target sequence.

28. 28. The primer mixture of claim 27, further comprising: (i) a loop forward primer (LF) and (ii) a loop backward primer (LB), wherein the LF and the LB bind to two different binding sites on the target sequence.

29. and a second loop primer, the second loop primer comprising: a third sensor molecule; and a third clamping oligonucleotide; and a second spacing oligonucleotide; and a fourth clamping oligonucleotide, a fourth clamping oligonucleotide, wherein the third clamping oligonucleotide, the second spacing oligonucleotide, and the fourth clamping oligonucleotide are capable of forming a hairpin structure at a temperature lower than the melting temperature (Tm) of the third and fourth clamping oligonucleotides; A fourth sensor molecule, a fourth sensor molecule, the third sensor molecule and the fourth sensor molecule being a second biosensor pair, the second biosensor pair being different from the first biosensor pair; and a second primer sequence complementary to the first binding site on the second target sequence; 27. The primer mixture of claim 26, comprising:

30. 30. The primer mix of claim 29, wherein the target sequence and the second target sequence are different.

31. 30. The primer mix of claim 29, further comprising: (i) a second forward inner primer (SFIP), (ii) a second backward inner primer (SBIP), (iii) a second forward primer (SF3), and (iv) a second backward primer (SB3), wherein the SFIP, SBIP, SF3, and SB3 bind to six different binding sites on the second target sequence.

32. 30. The primer mix of claim 29, further comprising: (i) a second loop-forward primer (SLF) and (ii) a second loop-backward primer (SLB), wherein the SLF and the SLB bind to two different binding sites on the second target sequence.

33. and a third loop primer, the third loop primer comprising: A fifth sensor molecule; and a fifth clamping oligonucleotide; and a third spacing oligonucleotide; and a sixth clamping oligonucleotide, a sixth clamping oligonucleotide, wherein the fifth clamping oligonucleotide, the third spacing oligonucleotide, and the sixth clamping oligonucleotide are capable of forming a hairpin structure at a temperature lower than the melting temperature (Tm) of the fifth and sixth clamping oligonucleotides; A sixth sensor molecule, a sixth sensor molecule, the fifth sensor molecule and the sixth sensor molecule being a third biosensor pair, the third biosensor pair being different from the first biosensor pair and the second biosensor pair; a second primer sequence complementary to the first binding site on a third target sequence; 30. The primer mixture of claim 29, comprising:

34. 1. A kit for loop-de-loop amplification of a target sequence, comprising: A kit comprising the loop primer of claim 10 and a polymerase.

35. 35. The kit of claim 34, further comprising a reverse transcriptase.

36. 35. The kit of claim 34, wherein the polymerase is Geobacillus stearothermophilus polymerase.

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