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

Abstract

To provide a new amplification method that enables easy detection of a target nucleic acid in a closed system.SOLUTION: The present disclosure provides a novel loop-de-loop method of detecting a target nucleic acid using a biosensor-labeled oligonucleotide. Further provided herein is a looped primer and a kit for use in the method.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. Cross-reference of related applications This application is based on U.S. Provisional Patent Application No. 62 / 989,140, ​​filed on March 13, 2020. The priority of the title is claimed, and the entire title is incorporated by reference.

[0002] 2. Sequence Listing This application includes a sequence listing having the XXX sequence, which was submitted via EFS-Web. The entire AS created in XXXX is incorporated herein by reference. The name of the CII copy is 48397WO_sequencelisting.txt The size is XXX bytes. [Background technology]

[0003] 3.Background Methods for detecting target nucleic acids using nucleic acid sequence complementarity are traditional Southern hybridization From its initial development to the present day, it has been improved or modified in various ways. In particular, various in vitro Nucleic acid amplification methods, for example, polymerase chain reaction (PCR), strand substitution amplification (SDA), nucleic acid Array-based amplification (NASBA), rolling circle amplification (RCA), and loop-mediated isothermal amplification The establishment of amplification (LAMP) has made it possible to detect smaller amounts of target nucleic acids. These methods are used for the medical diagnosis of infectious diseases, determination of mutant genotypes, and single nucleotide polymorphisms (SNPs). It is also used for sequence-specific detection and quantification of target nucleic acids in samples, such as for the detection of point mutations. Nucleic acid amplification methods have been used in the Gold Standard tests due to their high specificity and sensitivity. It was dirt.

[0004] However, amplification reactions and signal detection require a controlled environment and expensive equipment. Because accurate measurement is required, current nucleic acid amplification methods have limitations. Therefore, this method In many cases, using it in a point-of-care setting is prohibitively expensive. Furthermore, this method is not optimized for detecting multiple targets in a single patient sample. The detection of multiplexed targets involves signal multiplexing in single-pot reactions (fluorescence spectral multiplexing). Chemical (array of electrochemical detectors), physical separation of multiple reactions into specific reaction vessels, or This can be achieved by a combination of these. However, in the case of the CLIA abandonment test, a single To simultaneously query a panel of nucleic acid targets using patient samples, the user can perform three steps or less. A simple process is desired. Therefore, complex equipment or disposable materials are not needed. Unless the sample is processed automatically, physically separating the sample into a separate chamber is not possible with CLI. In the A-type abandoned test, it quickly becomes impossible to perform. Spectral multiplexing by fluorescence is a method for targeting nucleic acid particles. This can reduce the number of specific reactions required to target Nell, but spectral multiplexing The LAMP reaction requires a dramatic sacrifice of assay rate or signal intensity, and this is This diminishes the prospects for successful application to proof-of-concept (POC) trials.

[0005] Therefore, target nucleic acids, especially multiplexed targets, can be produced at low cost with high sensitivity and specificity. There is a need to develop new methods that enable easy amplification and detection. [Overview of the project]

[0006] 4. Overview The present disclosure provides a new amplification method that enables easy detection of target nucleic acids in a closed system. This method uses loop primers having a biosensor pair to enable detection of a small amount of target nucleic acid with high specificity and sensitivity. The biosensor pair enables determination of loop-in-loop (「LDL」) amplification of a target sequence by detecting a conformational change of the loop primer, for example, using fluorescence / quencher FRET technology. Further, by using a plurality of biosensors, it becomes possible to detect multiplexed targets in a single tube. The loop primer can be used in combination with not only loop-mediated isothermal amplification (LAMP), but also any other nucleic acid amplification method that utilizes strand displacement polymerase. This disclosure includes the following [1] through

[95] . <0OO0091> [1] Loop primer for loop-de-loop amplification (LdL) of a target sequence, wherein the target sequence is 5' to 3', The first sensor molecule, The first clamping oligonucleotide, Spacing oligonucleotides and A second clamping oligonucleotide, The first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide have a melting temperature (T) of the first and second clamping oligonucleotides. A second clamping oligonucleotide, which can form a hairpin structure at a lower temperature, A second clamping oligonucleotide, which can form 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 constitute the first biosensor pair, A first primer sequence complementary to the first binding site on the target sequence, Loop primer containing [specific component]. [2] The loop primer according to [1], 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 a pair of an energy donor and a receptor. <00001II7> [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 according to [3] above, wherein the first sensor molecule is a FRET quencher and the second sensor molecule is a FRET fluorophore. [7] The loop primer according to [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 according to [3] above, wherein the first sensor molecule is a BRET energy receptor and the second sensor molecule is a BRET energy donor. [9] The loop primer according to [1], wherein the first sensor molecule and the second sensor molecule may form a complex that generates a detectable optical signal.

[10] Melting temperature of the first and second clamping oligonucleotides (T m A loop primer as described in any one of the above [1] to [9], wherein the temperature exceeds 60°C.

[11] Melting temperature of the first and second clamping oligonucleotides (T m A loop primer as described above

[10] , wherein the temperature exceeds 65°C.

[12] Melting temperature of the first and second clamping oligonucleotides (T m A loop primer as described in

[11] above, wherein the temperature exceeds 70°C.

[13] Melting temperature of the first and second clamping oligonucleotides (T m A loop primer as described in

[12] above, wherein the temperature exceeds 80°C.

[14] Melting temperature of the first and second clamping oligonucleotides (T m A loop primer as described in

[12] above, wherein the temperature is 70 to 80°C.

[15] Melting temperature of the first and second clamping oligonucleotides (T m A loop primer as described in

[13] above, wherein the temperature is 72.5 to 77.5°C.

[16] Melting temperature of the first and second clamping oligonucleotides (T m The loop primer described above

[14] , wherein the temperature is approximately 75°C.

[17] Melting temperature of the first and second clamping oligonucleotides (T m A loop primer as described in any one of the above [1] to [9], wherein the temperature is less than 60°C.

[18] Melting temperature of the first and second clamping oligonucleotides (T m A loop primer according to any one of the above [1] to [9], wherein the temperature is 60 to 65°C.

[19] The loop primer according to any one of the above [1] to

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

[20] The loop primer according to

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

[21] The loop primer according to

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

[22] The loop primer according to any one of the above [1] to

[21] , wherein the spacing oligonucleotide is 5 to 35 nucleotides long.

[23] The loop primer according to

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

[24] The loop primer according to

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

[25] The loop primer according to

[24] , wherein the spacing oligonucleotide is 13 nucleotides long.

[26] The loop primer according to any one of the above [1] to

[25] , wherein the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide have a total length of 15 to 35 nucleotides.

[27] The loop primer according to

[26] , wherein the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide have a total length of 20 to 30 nucleotides.

[28] The loop primer according to

[27] , wherein the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide have a total length of 23 to 28 nucleotides.

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

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

[30] The loop primer according to any one of the above [1] to

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

[31] The loop primer according to any one of the above [1] to

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

[32] The loop primer according to

[30] or

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

[33] A loop primer according to any one of the above [1] to

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

[34] The loop primer according to

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

[35] The loop primer according to

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

[36] The loop primer described in

[34] above, comprising the oligonucleotide of Sequence ID No. 15.

[37] The loop primer according to

[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 according to

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

[40] The loop primer according to

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

[41] The loop primer according to

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

[42] The loop primer according to any one of the above [1] to

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

[43] The loop primer according to

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

[44] The loop primer described in

[42] above, comprising the oligonucleotide of Sequence 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 the above [1] to

[44] .

[46] The primer mixture according to

[45] , 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.

[47] The primer mixture according to

[46] , 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 the above

[45] to

[47] , 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 according to

[48] , wherein the FIP is bound 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 according to

[48] , wherein the BIP is bound to the first binding site, and the ratio of the amount of 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] , wherein the LF is bound to the first binding site, and the ratio of the amount of the LF 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.

[52] The primer mixture according to

[48] , wherein the LB is bound 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] A primer mixture according to any one of the above

[46] to

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

[54] The primer mixture according to any one of the above

[46] to

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

[55] A primer mixture according to any one of the above

[46] to

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

[56] The primer mixture according to any one of the above

[46] to

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

[57] A primer mixture according to any one of the above

[46] to

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

[58] The primer mixture according to any one of the above

[46] to

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

[59] Further comprising a second loop primer, the second loop primer is The third sensor molecule, The third clamping oligonucleotide, The second spacing oligonucleotide, A fourth clamping oligonucleotide, The third clamping oligonucleotide, the second spacing oligonucleotide, and the fourth clamping oligonucleotide have a melting temperature (T) of the third and fourth clamping oligonucleotides. m A fourth clamping oligonucleotide that can form a hairpin structure at a lower temperature, It is a fourth sensor molecule, The third sensor molecule and the fourth sensor molecule constitute a second biosensor pair, and the second biosensor pair is different from the first biosensor pair, and the fourth sensor molecule A second primer sequence complementary to the first binding site on the second target sequence, A primer mixture according to any one of the above

[45] to

[58] , including the following:

[60] The primer mixture according to

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

[61] The primer mixture according to

[59] or

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

[62] The primer mixture according to

[59] or

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

[63] A primer mixture according to any one of the above

[59] to

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

[59] to

[60] , 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] Further comprising a third loop primer, the third loop primer is The fifth sensor molecule, The fifth clamping oligonucleotide, A third spacing oligonucleotide and A sixth clamping oligonucleotide, The fifth clamping oligonucleotide, the third spacing oligonucleotide, and the sixth clamping oligonucleotide have a melting temperature (T) of the fifth and sixth clamping oligonucleotides. m A sixth clamping oligonucleotide that can form a hairpin structure at a lower temperature, The sixth sensor molecule, The fifth sensor molecule and the sixth sensor molecule constitute a third biosensor pair, and the third biosensor pair is different from the first biosensor pair and the second biosensor pair, and the sixth sensor molecule is different from the first biosensor pair and the second biosensor pair. A second primer sequence complementary to the first binding site on the third target sequence, A primer mixture according to any one of the above

[45] to

[64] , including the following:

[66] The primer mixture according to

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

[67] The primer mixture according to

[65] or

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

[68] The primer mixture according to

[65] or

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

[69] A primer mixture according to any one of the above

[65] to

[68] , 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, the TBIP, the TF3, and the TB3 bind to six different binding sites on the third target sequence.

[70] A primer mixture according to any one of the above

[65] to

[69] , 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 the above

[65] to

[70] , further comprising a fourth loop primer.

[72] The primer mixture according to

[71] , further comprising a fifth loop primer.

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

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

[45] to

[72] .

[74] A kit for loop-de-loop amplification of a target sequence, A kit comprising a loop primer as described in any one of items [1] to

[44] above, a primer mixture as described in any one of items

[45] to

[72] above, or a dry primer mixture as described in item

[73] above.

[75] The kit according to

[74] , further comprising a polymerase, wherein the polymerase is optionally Bacillus stearothermophilus polymerase.

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

[74] to

[75] , further comprising a buffer.

[77] The kit according to any one of the above

[74] to

[76] , further comprising reverse transcriptase.

[78] The kit according to any one of the above

[74] to

[77] , further comprising an RNase inhibitor.

[79] The kit according to

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

[80] A method for detecting a target sequence in a sample, The process of preparing the sample, (i) A step of adding a polymerase to a sample to generate a reaction mixture, which is obtained by rehydrating a primer according to any one of the above items [1] to

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

[45] to

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

[73] , The step of incubating the reaction mixture at 50-85°C Methods that include...

[81] The method according to

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

[82] The method according to

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

[83] The method according to

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

[84] The method according to any one of the above

[80] to

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

[85] The method according to any one of the above

[80] to

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

[86] The method according to

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

[87] The method according to any one of the above

[85] to

[86] , wherein the detection step is performed during the incubation step.

[88] The method according to any one of the above

[80] to

[87] , further comprising the step of examining whether or not the target sequence is present in the sample.

[89] The method according to any one of the above

[80] to

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

[90] The method according to

[89] , wherein the step of preparing the sample comprises interacting a reverse transcriptase with an RNA molecule to produce the sample containing a DNA molecule.

[91] The method according to

[90] , wherein the step of preparing the sample further includes a step of preheating the RNA molecule before or during the interaction with the reverse transcriptase.

[92] The method according to any one of the above

[80] to

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

[93] The method according to

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

[94] The method according to any one of the above

[80] to

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

[95] The method according to any one of the above

[80] to

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

[0007] The applicant has proposed a loop-de-loop amplification method that uses inhibitory fluorescent probes, such as DARQ (Detection of amplification by extinction emission) and previously known OSD (one-step displacement) probes Compared to the previous method, it offers faster turnaround time and improved sensitivity and specificity. Furthermore, it was demonstrated that this enables sequence-specific amplification of target nucleic acid molecules. The loop amplification method is different from QUASR (quenching of the unintegrated amplified signal reporter). This enables real-time detection of the amplified signal. According to the loop-de-loop method, the crude sample However, because a strong signal can be obtained, this method can be performed with low-cost equipment.

[0008] Therefore, the present invention relates to loop primers (e.g., fluorophore-labeled primers). This invention provides a method for detecting one or more target nucleic acids present in a sample using [a specific method]. Luorophore-labeled primers have complementarity to the target nucleic acid and a biosensor pair, for example, f The 5' end of a primer sequence that is not modified with a luolophore or quencher molecule or Fluorophor-labeled oligonucleotides having a continuous loop array internally labeled near them. It is othiocyanate. The fluorophore-labeled primer is attached to the 5' end of the attached loop sequence or Nearby, they are labeled with a quencher or fluorophore, respectively, as explained in the previous section. Partial labeling enables FRET. In some embodiments, fluorophore-labeled primer - is a fluorophore and quencher at or near the 5' end of the added loop sequence. The internal region having is labeled. The fluorophore-labeled primer is (unmodified primer (At the intersection with the sequence) the loop sequence further includes a first clamping sequence at its 3' end. This sequence may overlap with the unmodified primer sequence, and the unmodified sequence may also overlap. The primer sequence may be directly adjacent to it, or from an unmodified primer sequence. They may be spaced apart. This sequence is dNTP, locked nucleic acid, or any other form of nucleic acid. This may include modification or substitution.

[0009] The melting temperature of the above clamping sequence is preferably determined by using a chain substitution polymerase. The elongation temperature is about 10°C higher than that of Sei, but lower than or equal to that of Assay. Or, it can be increased by any amount greater than that.

[0010] If the melting temperature of the clamping sequence is lower than the reaction extension temperature, real-time detection is possible. This method involves (cooling the reaction to near or below the Tm of the clamping sequence) for endpoint detection. It can be replaced. In this case, use loop primer at maximum intensity (unmodified primer). Even when the lymer is 100% replaced with a loop primer analog, the reaction is not inhibited. stomach.

[0011] If the melting temperature of the clamping sequence is equal to the reaction extension temperature, real-time detection is still possible. It is feasible as such, but a higher background is needed until the reaction is cooled for endpoint determination. Do-fluorescence may be present.

[0012] If the melting temperature of the clamping sequence is higher than the reaction extension temperature, real-time detection is based on In this operating mode, background fluorescence is minimized.

[0013] Fluorophore-labeled primers are Fluorophore-labeled when the primer is in a linear (extended) conformation. To inhibit RET, only at an appropriate distance from the 5' terminal quencher or fluorophore. Internally conjugated fluorophores or quenchers are separated, and fluorescence is detected as a result. Further includes spacing arrays that increase the number of elements. The spacing arrays have arbitrary arrangement and length. It can include deoxyribonucleotides, locked nucleic acids, etc. Its length is It can be 25 nucleotides, but it can be shorter or longer.

[0014] The fluorophore-labeled primer has a second clan at or near the 5' end of the loop sequence. Further includes a pinning sequence, i.e., the reverse complement of the first clamping sequence. Fluorophores The labeled primer has an additional D at the 5' end of the loop-de-loop oligonucleotide. It may further include NA barcodes, probes, or sequences. This sequence is dNTP. This may include locked nucleic acids, or any other form of nucleic acid modification or substitution.

[0015] The melting temperature of the second clamping array paired with the first clamping array is preferably The extension temperature is 10°C higher than that of assays using chain substitution polymerases, but the assay It can be lower than, equal to, or any amount higher than the extension temperature. .

[0016] Loop-shaped fluorophore-labeled primers have further applications, such as nucleic acid capture and molecular capture. To enable barcoding, magnetic separation, column purification, and electrophoretic separation, the 5' end is The following may further be included on the substrate: a sequence, molecules, purified tags, beads, or other parts. Patterned probe-capture oligonucleotides are used to capture the amplification product, and this This can produce fluorescence, colorimetric effects, emission, or other bands or zones.

[0017] Loop primers are used to minimize costs or to enhance sensitivity and specificity. Therefore, it can be titrated to various degrees in the assay.

[0018] The loop primers described herein are suitable for sensor molecules other than phosphors, such as luminescence and color. Changes, or other measurable signals, when very close or when moving at a sufficiently large distance. It can be designed using the provided reporter molecule. In some cases, a biosensor can be used. For example, NanoLuc, Nanobit, and NonoBRET can be used. .

[0019] When luminescent proteins are used, a decrease in signal can be an important indicator of a positive reaction. In some embodiments, endpoint analysis can be performed using bioluminescence.

[0020] For amplification of the target sequence, enzymes capable of strand substitution can be used. Selected nucleic acid amplification Depending on the method, other reagents may be used as needed.

[0021] The method provided herein involves high backgrounds with high concentrations of non-target RNA or DNA. This enables specific detection of fluorescence signals under specific conditions, thus eliminating false positives and nonspecific amplification detection. It can be reduced. Fluorescence detection involves an amplicon incorporating a labeled primer and This enables the specific detection of only the substance. This allows for the detection of crude or untreated samples, such as genitalia. Even when using swabs, feces, saliva, urine, blood, plant materials, soil, or environmental samples, This enables specific detection.

[0022] In some embodiments, this method is used to detect two or more unique nucleic acid targets. It can be applied. In such double, triple, or higher-order multiplexed LAMP assays, Targets can be labeled differentially. For example, one target is labeled with FAM, and the other is labeled with FAM. Labeled with Cy5. In some cases, detection has the ability to further distinguish between true positives and false positives. To reduce the detection rate, multiple labeled primers are used to detect a single nucleic acid target. The vectors are multiplexed. In some cases, detection is performed using a single label for multiple targets, for example. If FAM is used, the identity of each target can be determined in real time or depending on the specific circumstances of the assay. The determination is based on an analysis of the endpoint signal (e.g., relative signal strength, time to the result, etc.). This is possible. In some cases, multiplexing involves carrying out the reaction within a physical reaction chamber. Therefore, this can be achieved. In some cases, multiplexing can be achieved within a single reaction chamber.

[0023] The method provided herein is compared to other real-time LAMP displacement probe techniques. Minimize inhibition and increase the sensitivity of this method. Titration to the assay is performed when the complete reaction rate is reached. The assay demonstrates that the effect is maintained in at least 50% of loop primer substitutions. Titration to ensure that the complete reaction rate is maintained at least 25% of the inner primer substitution. This demonstrates that it will be done.

[0024] The loop primers provided herein are chain-substituted polymerases, such as geobatyl Geobacilus stearothermophilus (formerly known as Bacillus stearothermophilus) Poly(Bacillus stearothermophilus) isolated or adapted from thearothermophilus It can be used in loop-mediated amplification (LAMP) using merase. In this case, The primers are forward inner primer, backward inner primer, Loop forward primer, loop backward primer, F3 primer and B3 It can be used with other primers for LAMP, such as primers.

[0025] In some embodiments, the primer and optionally other reaction components are limited. However, they are dried using processes such as freeze-drying. The MA can be included in the diagnostic kit. In a preferred embodiment, the freeze-drying process This does not affect the sensitivity of the LAMP primer set.

[0026] In one embodiment, the present disclosure relates to a loop-de-loop amplification (LdL) of a target sequence. A clamp primer, with a first sensor molecule and a first clamp from 5' to 3'. Spacing oligonucleotides and spacing oligonucleotides e) and a second clamping oligonucleotide , first clamping oligonucleotide, spacing oligonucleotide and second The clamping oligonucleotides are the first and second clamping oligonucleotides A second clamping mechanism that can form a hairpin structure at a temperature lower than its melting temperature (Tm). A first-sensor oligonucleotide and a second sensor molecule, the first sensor molecule and the second sensor molecule The first biosensor molecule is the second sensor molecule, and the first binding occurs on the target sequence. The present invention provides a loop primer containing a first primer sequence complementary to the site.

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

[0028] In some embodiments, the first biosensor pair is a pair of an energy donor and a receptor. In some embodiments, the first biosensor pair uses fluorescence resonance energy transfer (FR Energy donors and receptors for ET or bioluminescent resonance energy transfer (BRET). It is a pair of. In some embodiments, the first sensor molecule is a FRET fluorophore. The second sensor molecule is a FRET quencher. In some embodiments, the first 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. The second sensor molecule is a BRET energy receptor. In some embodiments, the first The first sensor molecule is a BRET energy receptor, and the second sensor molecule is a BRET energy - It is a donor. In some embodiments, the first sensor molecule and the second sensor molecule are A complex can be formed that generates a detectable optical signal.

[0029] In some embodiments, the melting temperatures of the first and second clamping oligonucleotides (Tm) exceeds 60°C. In some embodiments, first and second clamping The melting temperature (Tm) of ligonucleotides is above 65°C. In some embodiments, The melting temperatures (Tm) of clamping oligonucleotides 1 and 2 are above 70°C. In some embodiments, the melting temperatures of the first and second clamping oligonucleotides ( Tm) exceeds 80°C. In some embodiments, the first and second clamping olives The melting temperature (Tm) of the gonucleotide is 70 to 80°C. In some embodiments, The melting temperatures (Tm) of the first and second clamping oligonucleotides are 72.5°C. The temperature is 77.5°C. In some embodiments, the first and second clamping oligonucleotides The melting temperature (Tm) of ocide is approximately 75°C. In some embodiments, the first and second The melting temperature (Tm) of the clamping oligonucleotide is less than 60°C. In this embodiment, the melting temperatures (Tm) of the first and second clamping oligonucleotides are The temperature is between 60 and 65 degrees Celsius.

[0030] In some embodiments, a first clamping oligonucleotide and a second clamping oligonucleotide The oligonucleotides are 3 to 10 nucleotides long. In some embodiments, The first clamping oligonucleotide and the second clamping oligonucleotide are , 3 to 7 nucleotides in length. In some embodiments, the first clamping oligo The nucleotide and the second clamping oligonucleotide are 6 nucleotides long. In some embodiments, the spacing oligonucleotides are 5 to 35 nucleotides. It is long. In some embodiments, the spacing oligonucleotides are 10 to 20 This is the nucleotide length. In some embodiments, the spacing oligonucleotide is They are 13 to 18 nucleotides long. In some embodiments, spacing oligonucleotides The rheotide is 13 nucleotides long. In some embodiments, the first clamping Oligonucleotides, spacing oligonucleotides and second clamping oligonucleotides The creotide is 15 to 35 nucleotides in length in total. In some embodiments, First clamping oligonucleotide, spacing oligonucleotide and second clamping oligonucleotide Ramping oligonucleotides are 20 to 30 nucleotides long in total. In one embodiment, a spacing oligonucleotide and a second clamping oligonucleotide Rheotide is 23 to 28 nucleotides long in total.

[0031] In some embodiments, a first clamping oligonucleotide, a spacing oligonucleotide The gonucleotide and the second clamping oligonucleotide are (i) adenine, guani (ii) Nucleic acid base selected from cytosine, thymine and uracil, (ii) Locked nucleic acid, ( iii) 2''O-methyl RNA base, (iv) phosphorothioate-modified DNA base, (v ) Phosphothioated RNA base, (vi) Phosphothioated 2''-O-methyl (vii) Includes RNA bases, or combinations thereof.

[0032] In some embodiments, the loop primer has a first at the 5' end of the loop primer. Further comprising oligonucleotides. In some embodiments, loop primers. This involves a second further connection between the first sensor molecule and the first clamping oligonucleotide. It further contains oligonucleotides.

[0033] In some embodiments, a first or second additional alkyl group is used in the barcode It is an array.

[0034] In some embodiments, the target sequence is specific to the pathogen genome. Morphologically, the target sequence is specific to Chlamydia trachomatis. In some embodiments, the target sequence is derived from orf8 or cds2. In one embodiment, the loop primer contains the 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 this embodiment, the loop primer comprises the oligonucleotide of SEQ ID NO: 5 or 7.

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

[0037] In some embodiments, the target sequence is specific to Homo sapiens. Morphologically, the target sequence is derived from tbc1d3. In some embodiments, loopplant The IMER contains the oligonucleotide of sequence number 22.

[0038] In another embodiment, the Disclosure relates to targeting, including loop primers provided herein. Provides a primer mixture for loop-de-loop amplification of columns.

[0039] In some embodiments, the primer mixture is (i) forward inner primer (FIP), (ii) Backward Inner Primer (BIP), (iii) Forward Further comprising a doprimer (F3) and a backward primer (B3), FIP, BI P, F3, and B3 bind to six different binding sites on the target sequence. Several implementations In this state, the primer mixture consists of (i) loop forward primer (LF) and (ii) The primer contains a loopbackward primer (LB), and LF and LB are two different primers on the target sequence. It binds to the binding site. In some embodiments, FIP, BIP, F3, B3, LF or LB binds to a first binding site on the target sequence. In some embodiments, FIP is , binds to the first binding site, and the amount of FIP and loop primer in the primer mixture The ratios are 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 a first binding site in the primer mixture. The ratio of BIP to loop primer amounts is 1:1, 2:1, 3:1, 4:1, 5:1, 6 The ratios are 1, 7:1, 8:1, or 9:1. In some embodiments, LF is the first coupling. The binding occurs at the 1:1 position, and the ratio 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 one embodiment, LB is bound to the first binding site, and LB in the primer mixture and lu The ratios of the amounts of primer are 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1. It is 8:1 or 9:1.

[0040] In some embodiments, F3 contains the alkyl group of sequence number 1, and B3 is sequence FIP contains oligonucleotide number 2, FIP contains oligonucleotide number 3, BIP contains oligonucleotides of SEQ ID NO: 4, LF contains oligonucleotides of SEQ ID NO: 6 D contains, or LB contains the oligonucleotide of SEQ ID NO: 8.

[0041] In some embodiments, F3 contains the oligonucleotide of sequence number 1, and B3 is sequence FIP contains oligonucleotide number 2, and FIP contains oligonucleotide number 3. BIP contains oligonucleotides of SEQ ID NO: 4, and LF contains oligonucleotides of SEQ ID NO: 6. It contains D, and LB contains the oligonucleotide of SEQ ID NO: 8.

[0042] In some embodiments, F3 contains the oligonucleotide of sequence number 9, and B3 is the sequence. FIP contains oligonucleotide number 10, and FIP contains oligonucleotide number 11. Hmm, BIP contains the oligonucleotide of SEQ ID NO: 12, LF contains the oligonucleotide of SEQ ID NO: 13 Contains a cleotide, or LB contains the oligonucleotide of SEQ ID NO: 14.

[0043] In some embodiments, F3 contains the oligonucleotide of sequence number 9, and B3 is sequence FIP contains oligonucleotide number 10, and FIP contains oligonucleotide number 11. BIP contains the oligonucleotide of SEQ ID NO: 12, and LF contains the oligonucleotide of SEQ ID NO: 13. It contains a cleotide, and LB contains the oligonucleotide of SEQ ID NO: 14.

[0044] In some embodiments, F3 contains the oligonucleotide of SEQ ID NO: 16, and B3 contains FIP contains the oligonucleotide of sequence number 18, which is included in column number 17. BIP contains the oligonucleotide of SEQ ID NO: 19, LF contains the oligonucleotide of SEQ ID NO: 20 Contains a nucleotide, or LB contains the oligonucleotide of SEQ ID NO: 21.

[0045] In some embodiments, F3 comprises the oligonucleotide of sequence number 16, and B3 comprises Including the oligonucleotide in column number 17, FIP contains the oligonucleotide of sequence number 18. BIP contains the oligonucleotide of SEQ ID NO: 19, and LF contains the oligonucleotide of SEQ ID NO: 20. It contains nucleotides, and LB contains the oligonucleotide of SEQ ID NO: 21.

[0046] In some embodiments, the primer mixture further comprises a second loop primer. The second loop primer connects to the third sensor molecule and the third clamping oligonucleotide. Tide, a second spacing oligonucleotide, and a fourth clamping oligonucleotide It is a thiodioxide, and the third clamping oligonucleotide, the second spacing oligonucleotide The leotide and the fourth clamping oligonucleotide are the third and fourth clamping oligonucleotides Hairpin structures can be formed at temperatures lower than the melting temperature (Tm) of ligonucleotides. a fourth clamping oligonucleotide and a fourth sensor molecule, the third sensor The S molecule and the fourth sensor molecule constitute the second biosensor pair, and the second biosensor pair is the A fourth sensor molecule, distinct from the first biosensor pair, and a first binding site on the second target sequence. It includes a second primer sequence complementary to the first position.

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

[0048] In some embodiments, the primer mixture is (i) a second forward inner primer Immer (SFIP), (ii) Second backward inner primer (SBIP), ( iii) Second forward primer (SF3) and (iv) Second backward primer Further including MAR (SB3), SFIP, SBIP, SF3, and SB3 are second target distributions. It binds to six different binding sites on the column.

[0049] In some embodiments, the primer mixture is (i) a second loop forward primer (ii) a primer (SLF) and (ii) a second loopback primer (SLB) Furthermore, SLF and SLB bind to two different binding sites on the second target sequence. In that embodiment, the primer mixture further comprises a third loop primer, and the third The loop primer consists of a fifth sensor molecule and a fifth clamping oligonucleotide. The third spacing oligonucleotide and the sixth clamping oligonucleotide are That is, the fifth clamping oligonucleotide, the third spacing oligonucleotide. The 6th clamping oligonucleotide is the 5th and 6th clamping oligonucleotide The sixth type can form a hairpin structure at a temperature lower than the melting temperature (Tm) of rheotide. The clamping oligonucleotide and the sixth sensor molecule, which is the fifth sensor molecule and The sixth sensor molecule is the third biosensor pair, and the third biosensor pair is the first bio A sixth sensor molecule distinct from the Osensor pair and the second biosensor pair, and a third target sequence. It includes a second primer sequence complementary to the first binding site above. In some embodiments, The fifth clamping oligonucleotide is the sixth clamping oligonucleotide. It is complementary to the target sequence, the second target sequence and the third target sequence. The columns are identical. In some embodiments, the target sequence, the second target sequence and the third target sequence The columns are different. In some embodiments, the primer mixture is (i) a third forward (ii) Inner primer (TFIP), (ii) Third backward inner primer (TB IP), (iii) third forward primer (TF3) and (iv) third back primer Further comprising a lead primer (TB3), TFIP, TBIP, TF3, and TB3 are the first It binds to six different binding sites on the target sequence 3. In some embodiments, the primer - The mixture contains (i) a third loop-forward primer (TLF) and (ii) a third loop Further comprising a backward primer (TLB), TLF and TLB are third target components. It binds to two different binding sites on the row. In some embodiments, the primer mixture is , further comprising a fourth loop primer. In some embodiments, the primer mixture is , further including a fifth loop primer.

[0050] In yet another embodiment, the Disclosure relates to loop primers or plastics provided herein. This provides a dried primer mixture obtained by freeze-drying an immer mixture.

[0051] In one embodiment, the present disclosure is a kit for loop-de-loop amplification of a target sequence, Loop primers, primer mixtures, or dry primer mixtures provided herein A kit containing is provided. In some embodiments, the kit further contains polymerase, Polymerase is optionally selected from Bacillus stearothermophilus. It is an rmophilus polymerase. In some embodiments, the kit contains dNTPs, M The kit further comprises gSO4 and buffer. In some embodiments, the kit includes reverse transcriptase. It also includes. In some embodiments, the kit further includes an RNase inhibitor. In that embodiment, the RNase inhibitor is a porcine or mouse RNase inhibitor.

[0052] In another embodiment, the present disclosure relates to a method for detecting a target sequence in a sample, wherein the sample is prepared The process involves (i) a primer, (ii) a primer mixture, or (iii) as specified herein. Reconstituted primer mixture obtained by rehydrating the described dried primer mixture The process involves adding polymerase to the sample to generate a reaction mixture, and then mixing the reaction mixture. The present invention provides a method comprising the step of incubating a material at 50-85°C. Several implementations In some embodiments, incubation is carried out at 50-70°C. Incubation is carried out at 60-65°C. In some embodiments, incubation The procedure is carried out at 62-65°C. In some embodiments, polymerase is used for Bacillus... This is stearothermophilus polymerase. In some embodiments, the method further includes the step of detecting a signal from the reaction mixture. In some embodiments, the signal is a fluorescent signal. In some embodiments, the detection process is This is done during the incubation process. In some embodiments, this method is used for the sample The process further includes a step of checking for the presence or absence of a target sequence within.

[0053] In some embodiments, the method further includes a preceding step of preparing a sample. In one embodiment, the step of preparing the sample involves interacting RNA molecules with reverse transcriptase, This includes the step of generating a sample containing DNA molecules. In some embodiments, the sample The preparation step involves preheating the RNA molecule before or during interaction with reverse transcriptase. The process further includes the step of adding an RNase inhibitor to the reaction mixture. In some embodiments, the reaction mixture contains an RNase inhibitor. It also includes. In some embodiments, the RNase inhibitor is a porcine or mouse RNA inhibitor. That is the case.

[0054] In some embodiments, the sample is purified RNA, purified DNA, and total SARS-CoV-2 This includes viruses, whole human cells, saliva or nasal swabs, or nasal or nasopharyngeal swabs. In several embodiments, the sample is genomic DNA, synthetic DNA, whole bacteria, or derived from a vaginal swab. Includes all human cells. 5. Brief description of the drawing [Brief explanation of the drawing]

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

[0056] [Figure 2A] Figure 2A shows the results of LAMP assays for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) visualized with intercalating dye (SYTO). Amplification of at least 5 log [DNA] for CT and 6 log for NG is rapid (less than 30 minutes). The analytical sensitivity (LOD50) of the NG assay is 35 cp / 10 μL reaction according to PROBIT analysis.

[0057] [Figure 2B]Figure 2B shows readout from target amplification using a novel loop primer. This result demonstrates extremely bright real-time detection of the target with minimal inhibition and increased specificity compared to SYTO dyes.

[0058] [Figure 2C] Figure 2C shows the detection of Neisseria gonorrhoeae using a novel loop primer. These results demonstrate that this method is reproducible and generates rapid, robust, high signal-to-noise ratio amplification. The probe eliminates false positives.

[0059] [Figure 3-1] Figure 3 is a plot of real-time fluorescence signals over time showing the amplification of the target nucleic acid of Chlamydia trachomatis using the loop-de-loop method with 50% substitution FAM-labeled LF primers. 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 time-course plot of real-time fluorescence signals showing amplification of target nucleic acids in Neisseria gonorrhoeae using the loop-de-loop method with 50% substituted FAM-labeled LF primers. 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 time elapsed at 65 degrees Celsius. [Figure 4-2] (As stated above.)

[0061] [Figure 5-1]Figure 5 is a time-course plot of real-time fluorescence signals showing amplification of Homo sapiens target nucleic acids using the loop-de-loop method with 50% substituted FAM-labeled LF primers. 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 the emission was visualized with an amber plastic filter held over a cameraphone.

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

[0064] [Figure 8] Figure 8 shows real-time fluorescence signals indicating amplification of target nucleic acids from Chlamydia trachomatis, Neisseria gonorrhoeae, and Homo sapiens in a loop-de-loop reaction using the reconstituted dried mixture from Figure 7. These results demonstrate that the assay activity and sensitivity of the primers reconstituted after drying are maintained.

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

[0066] [Figure 10] Figure 10 shows the melting curves of loop-de-loop primers targeting DNA from Homo sapiens, Chlamydia trachomatis (C. Trachomatis), Neisseria gonorrhoeae (N. Gonorrhoeae), or SARS-CoV-2. The loop-de-loop primers are designed to unhold at a temperature approximately 10°C higher than the reaction temperature of 65°C. This curve demonstrates that the stem-loop sequence of the loop-de-loop primer is responsible for the fluorescence signal.

[0067] [Figure 11] Figure 11 shows real-time fluorescence signals obtained from loop-de-loop reactions using primers of 25%, 50%, or 100% intensity. In this context, "intensity" refers to the degree to which the primer is replaced by a loop version of the loop-de-loop method. This data indicates that stronger primers tend to produce larger signals, at the cost of a 1–2 minute delay in the time to results. 100% intensity loop-de-loop primers slowed the assay, but not to the same extent as other real-time LAMP displacement probe methods. Each "cycle" on the y-axis represents a 30-second elapsed time at 65 degrees Celsius.

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

[0069] [Figure 13] Figures 13A and 13B show real-time fluorescence signals obtained from amplification of the target sequence of Chlamydia trachomatis using a loop-de-loop reaction. Figure 13A shows results obtained from a freshly mixed reaction mixture, and Figure 13B shows results obtained from a lyophilized reaction mixture. The lyophilized assay mixture was stable for more than 3 months and yielded good readouts. This assay was performed on 14 copies of each of the LoD95 (low-positive) Ct E BOUR (Chlamydia trachomatis) strains of the assay (20.7 copies / μL), each with two template-free controls (NTCs). There was no change in sensitivity (12 / 14, respectively, for LoD95) or in the mean time to results (16 minutes, t-test, P-value = 0.66) between the fresh and lyophilized reaction mixtures.

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

[0071] [Figure 15A] Figure 15A shows real-time fluorescence signals obtained from loop-de-loop amplification at various concentrations of POP7b primer. The signal intensity decreased as the concentration of POP7b primer decreased (arrows). In multiplexing applications using two or more primer sets in a single reaction volume, the concentration of any given primer set is lower compared to a reaction where 100% of the primers belong to a single set. [Figure 15B] Figure 15B plots the time to result (in minutes) at various concentrations of POP7b primer. The time to result is affected when the primer concentration drops below 40%, which may be acceptable in many applications where the benefit of multiplexing more than two targets in a single tube outweighs the clinical or market-based rate requirements. In this reaction, 10⁴ g of block DNA was used in a reaction volume of 21 μL.

[0072] [Figure 16]Figure 16 shows real-time fluorescence signals 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 untreated nasal swabs obtained from coronavirus-positive subjects. Nasal swabs were directly eluted into the loop-de-loop RT-LAMP reagent and diluted to four different concentrations to prepare reaction mixtures. 1× swab represents the unit of swab eluted per unit volume and is a standard concentration of the sample used in this test configuration. In this case, primer sets for SARS-CoV-2 and human RNA were duplicated in a single tube. Each primer set contained one loop primer labeled with the same fluorophore-quencher pair (single fluorescence 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 the detection of both targets were labeled "double positive," those that resulted in the detection of either target were labeled "single positive," and those that resulted in the detection of neither target were labeled "double negative." This data showed that for swab samples from these coronavirus-positive volunteers, the real-time loop-de-loop RT-LAMP assay was at least 370-fold higher sensitivity than required to detect both targets during the reaction.

[0073] [Figure 17] Figure 17 shows the real-time fluorescence signal obtained from loop-de-loop amplification of SARS-CoV-2 specific target sequences in nasal swabs acquired from negative subjects.

[0074] [Figure 18]Figure 18 shows the fluorescence signals obtained from multiple loop-de-loop amplification of SARS-CoV-2 target sequences and human target sequences, demonstrating the specificity of the loop-de-loop reaction. Since both the SARS-CoV-2 and human primer sets were modified for loop-de-loop using FAM-labeled primers, the double-positive control exhibits two amplification phenomena. RPPOS is positive for a respiratory pathogen panel (Exact Diagnostics LLC) containing genetic material from 22 non-target respiratory pathogens. PRNEG is a background matrix control of the RPPOS product without nucleic acids. This data demonstrates 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] Figure 19 shows the fluorescence signals obtained from loop-de-loop amplification of samples containing large amounts of C. trachomatis (Ct) (10,000 copy equivalents per reaction) and Neisseria gonorrhoeae (Ng) (10,000 copy equivalents per reaction). [Figure 19-2] (As stated above.)

[0076] [Figure 20-1] Figure 20 shows the fluorescence signals obtained from loop-de-loop amplification of negative controls—swab-only controls (the two panels on the left) or buffer-only controls (the two panels on the right). [Figure 20-2] (As stated above.) [Modes for carrying out the invention]

[0077] The drawings illustrate various embodiments of the present invention for illustrative purposes only. Those skilled in the art will understand the following description. Therefore, without departing from the principles of the present invention as described herein, the structure shown herein It will be readily apparent that alternative embodiments of the construction and method may be used.

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

[0079] As used herein, the term "biosensor pair" refers to a pair of sensor molecules that can generate a detectable signal upon a specific physical interaction between the two sensor molecules. For example, a biosensor pair can be a pair of donor and acceptor molecules used in Förster resonance energy transfer, such as fluorescence resonance energy transfer (FRET). In this case, a fluorescence 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. 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. 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. 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-mediated isothermal amplification" or "LdL amplification" refers to the amplification of a target nucleic acid using loop primers that can generate a fluorescence signal by the distance-dependent transfer of energy. the amplification of a target nucleic acid using loop primers that can generate a fluorescence signal by the distance-dependent transfer of energy.

[0081] As used herein, the term "LOD" refers to the limit of detection. For example, LOD9 5 is the limit of detection at the 95th percentile. This represents the limit of detection at which the assay is positive 95% of the time. This is the statistically predicted target concentration upon detection of the fruit.

[0082] 6.2. Other Interpretation Rules The ranges described herein are abbreviated representations of all values ​​within the range that include the specified endpoint. It is understood that there is a range from 1 to 50. For example, the range from 1 to 50 is 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, 3 6, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 It is understood that this includes any number, combination of numbers, or subrange from the group consisting of 50. ru.

[0083] Unless otherwise indicated, references to compounds having one or more stereocenters refer to each of them. This refers to stereoisomers 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 is applied from 5' to 3'. The first sensor molecule, The first clamping oligonucleotide, Spacing oligonucleotides and A second clamping oligonucleotide, First clamping oligonucleotide, spacing oligonucleotide and second The clamping oligonucleotides are the first and second clamping oligonucleotides Melting temperature (T m A second clamp that can form a hairpin structure at a lower temperature. ng oligonucleotides, A second sensor molecule, The first sensor molecule and the second sensor molecule constitute the first biosensor pair, the second sensor Molecules and, A first primer sequence complementary to the first binding site on the target sequence, Includes.

[0085] In some embodiments, the second clamping oligonucleotide is the first clamping oligonucleotide. It is complementary to the clamping oligonucleotide. In some embodiments, a second clamping Oligonucleotides can bind to the first clamping oligonucleotide. However, it is not completely complementary to the first clamping oligonucleotide.

[0086] The methods provided herein may utilize various biosensors known in the art. Yes, it is possible. For example, it can change color when very close or far enough away, or make it measurable. A pair of molecules that generate a signal (e.g., NanoLuc, based on luminescent proteins) Obit (NonoBRET technology) can be used.

[0087] In some embodiments, the first biosensor pair is a pair of an energy donor and a receptor. In some embodiments, the first biosensor pair measures the Förster resonance energy transfer. It is an energy donor and acceptor pair for movement. In some embodiments, the first bi The Osensor pair uses fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer. It is an energy donor-receptor pair for BRET. In some embodiments, the Sensor molecule 1 is a FRET fluorophore, and sensor molecule 2 is a FRET quencher It is a fret quencher. In some embodiments, the first sensor molecule is a fret quencher. , 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 5'Iowa Black (registered trademark) FQ, 5IABkFQ available from Integrated DNA technologies under the trade name. 5'Iowa Black (registered trademark) 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 any of Black Hole Quencher (registered trademark) (available from Biosearch Technologies), Iowa Black (registered trademark) quencher (available from Integrated DNA tech nologies), Zen (registered trademark) quencher (available from Integrated DNA Technologies), Onyx (registered trademark) quencher (available from Millipore-Sigma), or any of ATTO (registered trademark) quencher (available from ATTO-TEC GmbH).

[0089] In some embodiments, the FRET fluorophore is Int 6-FAM (Azid ​​​​Available from Integrated DNA Technologies under the name e). This is a capable i6-FAMK (FAM (Fluorescein) Azid). This form of FAM is, It can be attached to oligonucleotides using click chemistry. Inside this modification The version is bound to the oligo via a dT base. A dT nucleotide is attached to the modification position. It can be added. Alternatively, to avoid adding extra nucleotides, in the sequence The existing T nucleotides can be replaced with the necessary modifications. In some embodiments, Fluorophores include Cy3, Cy5, TAMRA, or Yakima Yellow. (Registered Trademark) (Available from Integrated DNA Technologies) )

[0090] In one embodiment, the loop primer is an internal quencher (e.g., Zen(registered trademark) ) or Onyx A (registered trademark)) and 5' fluorophores (e.g., Yakima Y Includes yellow (registered trademark) or HEX).

[0091] In some embodiments, the first sensor molecule and the second sensor molecule are detectable light signals It is possible to form a complex that generates numbers.

[0092] The first and second clamping oligonucleotides are complementary to each other, therefore It can be bound. First clamping oligonucleotide, spacing oligonucleotide The cleotide and the second clamping oligonucleotide are first and second clamping Melting temperature of oligonucleotides (T m ) allows for the formation of a hairpin structure at a lower temperature. Cut.

[0093] In some embodiments, the melting temperatures of the first and second clamping oligonucleotides (T m ) exceeds 60°C. In some embodiments, the first and second clamping Melting temperature of ligonucleotide (T m ) exceeds 65℃. In some embodiments, the Melting temperature of clamping oligonucleotides 1 and 2 (T m ) exceeds 70°C. In some embodiments, the melting temperatures of the first and second clamping oligonucleotides ( T m ) exceeds 80℃. In some embodiments, the first and second clamping olives Melting temperature of nucleotides (T m The temperature is 70 to 80°C. In some embodiments, , melting temperature (T) of the first and second clamping oligonucleotides m ) is from 72.5 The temperature is 77.5°C. In some embodiments, the first and second clamping oligonucleotides Melting temperature of Otide (T m The temperature is approximately 75°C. In some embodiments, the first and second The melting temperature (T) of the clamping oligonucleotide. m ) is below 60°C. In this embodiment, the melting temperature (T) of the first and second clamping oligonucleotides is m )teeth The temperature is between 60 and 65 degrees Celsius.

[0094] In some embodiments, the melting temperatures of the first and second clamping oligonucleotides (T m ) is 10°C higher than the extension temperature of assays using chain substitution polymerases. In that embodiment, the melting temperature is lower than or equal to the assay extension temperature, or It can be increased by any amount beyond that.

[0095] T m If the reaction is lower than the extension temperature, real-time detection is (T of the clamping sequence). m Nearby or T m (Cooling the reaction to below zero) can be replaced with endpoint detection, and full intensity Even when using loop-de-loop primers with 100% substitution, the reaction is inhibited. It's harmless.

[0096] T m If the reaction elongation temperature is equal to the reaction's extension temperature, real-time detection may still be feasible. However, higher background fluorescence may be present until the reaction is cooled for endpoint determination. .

[0097] T m If the reaction extension temperature is higher, real-time detection may be the basic operating mode. Background fluorescence is minimized.

[0098] In some embodiments, a first clamping oligonucleotide and a second clamping oligonucleotide The oligonucleotides are 3 to 10 nucleotides long. In some embodiments, The first clamping oligonucleotide and the second clamping oligonucleotide are , 3 to 7 nucleotides in length. In some embodiments, the first clamping oligo The nucleotide and the second clamping oligonucleotide are 6 nucleotides long. In a typical embodiment, a first clamping oligonucleotide and a second clamping oligonucleotide Ligonucleotides have the same length.

[0099] In some embodiments, the spacing oligonucleotides are 5 to 35 nucleotides. It is long. In some embodiments, the spacing oligonucleotides are 10 to 2 It has a length of 0 nucleotides. In some embodiments, the spacing oligonucleotide is , 13 to 18 nucleotides in length. In some embodiments, spacing oligonucleotides A creotide is 13 nucleotides long.

[0100] In some embodiments, a first clamping oligonucleotide, a spacing oligonucleotide The 5-nucleotide and the second clamping oligonucleotide consist of 15 to 35 nucleotides in total. It is the creotide length. In some embodiments, the first clamping oligonucleotide The spacing oligonucleotide and the second clamping oligonucleotide are, And it is 20 to 30 nucleotides long. In some embodiments, the first clamping Oligonucleotides, spacing oligonucleotides and second clamping oligonucleotides Cleotides are 23 to 28 nucleotides long in total.

[0101] The loop primers are (i) derived from adenine, guanine, cytosine, thymine, and uracil. Selected nucleic acid base, (ii) locked nucleic acid, (iii) 2'O-methyl RNA base, ( iv) phosphorothioate-modified DNA bases, (v) phosphorothioate-modified RNA bases, (v i) phosphorothioate-modified 2'-O-methylRNA bases, or (vii) combinations thereof May contain a first clamping oligonucleotide, a spacing oligonucleotide. The cytoside and the second clamping oligonucleotide are (i) adenine, guanine, and citose. (ii) nucleic acid base selected from , thymine and uracil, (ii) locked nucleic acid, (iii) 2 'O-methyl RNA base, (iv) phosphorothioate-modified DNA base, (v) phosphorothio (vi) phosphorothioate-modified RNA bases, or (vii) includes combinations of those.

[0102] In some embodiments, the loop primer has a first at the 5' end of the loop primer. Further comprising oligonucleotides. In some embodiments, loop primers. This involves a second further connection between the first sensor molecule and the first clamping oligonucleotide. Further comprising oligonucleotides. In some embodiments, a first or second further oligonucleotide. Ligonucleotides are barcode sequences.

[0103] In some embodiments, the loop primer has a bar at its 5' end. Further includes coding sequences, probe sequences, or other sequences. Further sequences may include nucleic acid bases or It can include modifications.

[0104] In some embodiments, the target sequence is specific to the pathogen genome. Morphologically, the target sequence is specific to Chlamydia trachomatis. In some embodiments, the target sequence is derived from orf8 or cds2. Specifically Specifically, the target binding site can have the sequence of sequence number 15.

[0105] In some embodiments, the target sequence is specific to Neisseria gonorrhoeae. In some embodiments, the target sequence is derived from porA or glnA. Specifically The target binding site may 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. Morphologically, the target sequence originates from tbc1d3. Specifically, the target binding site is sequence number It may have an arrangement of numbers 22.

[0107] 6.4. Primer mixture for loop-de-loop amplification In another embodiment, the present invention provides a primer mixture for loop-de-loop amplification. Provided. The primer mixture includes the loop primers 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. If two or more loop primers are included, the primers in the mixture target a single target sequence or It can bind to multiple target sequences. In some embodiments, multiple loop plies are used. MA is designed to detect target sequences from multiple sources. For example, a mixture, This includes multiple loop primers designed to detect target sequences from multiple pathogens. It is possible.

[0109] The primer mixture may further contain additional primers for amplification reactions. For example, the primer mixture is (i) forward inner primer (FIP), (ii ) Backward Inner Primer (BIP), (iii) Forward Primer (F3 ) and backward primer (B3) may further be included, FIP, BIP, F3 And B3 binds to six different binding sites on the target sequence. In some embodiments, The primer mixture consists of (i) loop forward primer (LF) and (ii) loop forward primer. The material further contains a keyword primer (LB), where LF and LB are two different target sequences. It binds to the binding site. In some embodiments, one of the additional primers, for example, F IP, BIP, F3, B3, LF, or LB are the first binding sites, i.e., on the target sequence. It binds to the same binding site as the loop primer.

[0110] In some embodiments, the primer mixture includes one set of primers. In some embodiments, the primer set is a loop-de-loop augmentation provided herein. (i) loop primer for width, (ii) forward inner primer (FIP), (ii) ) Backward Inner Primer (BIP), (iii) Forward Primer (F3 ) and (iv) backward primer (B3). In some embodiments, plastic The Immerset consists of (i) loop-forward primer (LF) and (ii) loop-back primer. Further includes word primer (LB).

[0111] In some embodiments, the primer set is provided herein by Loop de L Loop primers for loop amplification, and (i) forward inner primers (FIP) (ii) backward inner primer (BIP), (iii) forward primer - Three primers selected from (F3) and (iv) backward primer (B3) -Includes. In some embodiments, the primer set includes loop primers, BIP , including F3 and B3. In some embodiments, the primer set includes loop primers —Includes FIP, F3 and B3. In some embodiments, the primer set is Includes primer, FIP, BIP and B3. In some embodiments, primers The set includes loop primer, FIP, BIP, and F3.

[0112] In some embodiments, the primer set is provided herein by Loop de L Loop primers for loop amplification, and (i) forward inner primers (FIP) (ii) backward inner primer (BIP), (iii) forward primer - (F3), (iv) backward primer (B3), (v) loop forward primer Five primers selected from (LF) and (vi) loopback primers (LB) Includes primers. In some embodiments, the primer set includes loop primers. , including BIP, F3, B3, LF and LB. In some embodiments, primer set The components include loop primer, FIP, F3, B3, LF, and LB. Several implementations In this configuration, the primer set includes loop primer, FIP, BIP, B3, LF, and LB. Includes. In some embodiments, the primer set includes loop primer, FIP, B Includes IP, F3, LF, and LB. In some embodiments, the primer set is lu Includes primer, FIP, BIP, F3, B3 and LF. In some embodiments, The primer set includes loop primers, FIP, BIP, F3, B3, and LB.

[0113] In some embodiments, the primer mixture includes two primer sets. In some embodiments, the primer mixture includes three primer sets. In the embodiment, the primer mixture includes four or five primer sets.

[0114] In some embodiments, each primer set is used to amplify a unique target sequence. In some embodiments, the primer mixture amplifies the same target sequence. Includes two or more primer sets for. In some embodiments, the primer - The mixture contains two or more loop plies that bind to the same binding site on the same target sequence. Includes Mar.

[0115] The loop primer is mixed with additional primers in any ratio optimized for the amplification reaction. It is possible. In some embodiments, the FIP binds to the first binding site and the primer - The ratio of FIP to loop primer in the mixture is 0:1, 1:1, 2:1, 3: The ratios are 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 BIP and loop primer in the primer mixture. The ratios of the amounts of Ma are 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1. The ratio is 8:1 or 9:1. In some embodiments, LF is bonded to the first bonding site, The ratio 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. Several implementation forms In this state, LB binds to the first binding site, and LB and loop primer in the primer mixture The ratios of the amounts of Ma are 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1. It is 8:1 or 9:1.

[0116] In some embodiments, the primer mixture is specifically for Neisseria gonorrhoeae. It is designed to detect heterogeneous target sequences. In some embodiments, F3 is the sequence number. B3 contains the oligonucleotide of sequence number 2, FIP BIP contains the oligonucleotide of sequence number 3, BIP contains the oligonucleotide of sequence number 4 LF contains the oligonucleotide of SEQ ID NO: 6, or LB contains the oligonucleotide of SEQ ID NO: 8. Contains a cleotide. In one embodiment, F3 contains the oligonucleotide of SEQ ID NO: 1, and B 3 contains the oligonucleotide of SEQ ID NO: 2, and FIP contains the oligonucleotide of SEQ ID NO: 3. BIP contains the oligonucleotide of SEQ ID NO: 4, and LF contains the oligonucleotide of SEQ ID NO: 6. Contains a cleotide, and LB contains the oligonucleotide of SEQ ID NO: 8. Several implementations Morphologically, the loop primer is the oligonucleotide of SEQ ID NO: 5 or SEQ ID NO: 7. .

[0117] In some embodiments, the primer mixture is used to kill Chlamydia trachomatis. Designed to detect target sequences specific to *A trachomatis*. Several embodiments So, F3 contains the oligonucleotide of SEQ ID NO: 9, and B3 contains the oligonucleotide of SEQ ID NO: 10. FIP contains leotide, FIP contains alkyl group of SEQ ID NO: 11, BIP contains SEQ ID NO: LF contains 12 oligonucleotides, and also contains the oligonucleotide of sequence number 13, LB contains the oligonucleotide of SEQ ID NO: 14. In one embodiment, F3 is SEQ ID NO: 9 It contains oligonucleotides, and B3 contains oligonucleotide of sequence number 10, FIP It contains the oligonucleotide of SEQ ID NO: 11, and BIP contains the oligonucleotide of SEQ ID NO: 12 It contains , LF contains oligonucleotide of sequence number 13, and LB contains oligonucleotide of sequence number 14 Contains oligonucleotides. In some embodiments, the loop primer is SEQ ID NO: 1 It is oligonucleotide number 5.

[0118] In some embodiments, the primer mixture targets a Homo sapiens-specific target sequence. Designed to detect. In some embodiments, F3 is the oligonucleotide of sequence number 16. B3 contains leotide, B3 contains alkyl group of SEQ ID NO: 17, FIP contains SEQ ID NO: 1 BIP contains the oligonucleotide of sequence number 19, L F contains oligonucleotide of SEQ ID NO: 20, or LB contains oligonucleotide of SEQ ID NO: 21. Contains ocide. In one embodiment, F3 contains the oligonucleotide of SEQ ID NO: 16, and B3 It contains the oligonucleotide of SEQ ID NO: 17, and FIP contains the oligonucleotide of SEQ ID NO: 18. It contains the oligonucleotide of sequence number 19, and LF contains the oligonucleotide of sequence number 20. It contains oligonucleotides, and LB contains oligonucleotide of sequence number 21. How many In that embodiment, the loop primer is the oligonucleotide of SEQ ID NO: 22.

[0119] In some embodiments, the primer mixture detects virus-specific target sequences. It is designed to do so. In some embodiments, the virus is SARS-CoV-2. .

[0120] In some embodiments, the primer mixture provided herein comprises multiple target sequences Further combinations are used for the detection of different target sequences. In some embodiments, multiple target sequences are used to detect different It is specific to a particular organism. For example, multiple target sequences are specific to different pathogens.

[0121] Therefore, in some embodiments, the primer mixture is a second loop primer The second loop primer further includes, The third sensor molecule, The third clamping oligonucleotide, The second spacing oligonucleotide, A fourth clamping oligonucleotide, Third clamping oligonucleotide, second spacing oligonucleotide and The fourth clamping oligonucleotide is the third and fourth clamping oligonucleotides. Melting temperature of T m A fourth type that can form a hairpin structure at a lower temperature. Lamping oligonucleotides and It is a fourth sensor molecule, The third and fourth sensor molecules constitute the second biosensor pair, and the second bio The sensor pair is different from the first biosensor pair, and is a fourth sensor molecule. A second primer sequence complementary to the first binding site on the second target sequence, Includes.

[0122] In some embodiments, the third clamping oligonucleotide is a fourth clamping oligonucleotide. It is complementary to the clamping oligonucleotide. In some embodiments, a third clamping The oligonucleotide can bind to the fourth clamping oligonucleotide. However, it is not entirely complementary to the fourth clamping oligonucleotide.

[0123] In some embodiments, the primer mixture is (i) a second forward inner primer Immer (SFIP), (ii) Second backward inner primer (SBIP), ( iii) Second forward primer (SF3) and second backward primer (S B3) further includes SFIP, SBIP, SF3, and SB3 on the second target sequence 6 It binds to different binding sites. In some embodiments, the primer mixture is (i) (ii) Second loop-forward primer (SLF) and (ii) second loop-back ward Further containing a lymer (SLB), SLF and SLB are two different structures on a second target sequence. It binds to the binding site.

[0124] In some embodiments, the primer mixture further comprises a third loop primer. The third loop primer is, The fifth sensor molecule, The fifth clamping oligonucleotide, A third spacing oligonucleotide and A sixth clamping oligonucleotide, The fifth clamping oligonucleotide, the third spacing oligonucleotide, and The sixth clamping oligonucleotide is the fifth and sixth clamping oligonucleotides. Melting temperature of T m A sixth type that can form a hairpin structure at a lower temperature. Lamping oligonucleotides and The sixth sensor molecule, The fifth and sixth sensor molecules constitute the third biosensor pair, and the third bio A sixth sensor pair whose sensor pair is different from the first biosensor pair and the second biosensor pair. Child, A second primer sequence complementary to the first binding site on the third target sequence, Includes.

[0125] In some embodiments, the fifth clamping oligonucleotide is the sixth clamping oligonucleotide. It is complementary to the clamping oligonucleotide. In some embodiments, a fifth clamping The oligonucleotide binds to the sixth clamping oligonucleotide, but the fifth clamping oligonucleotide The clamping oligonucleotide is not completely in phase with the sixth clamping oligonucleotide. Not complementary.

[0126] In some embodiments, the primer mixture is (i) a third forward inner primer (ii) Immer (TFIP), (ii) Third backward inner primer (TBIP), ( iii) A third forward primer (TF3) and a third backward primer (T B3) further includes TFIP, TBIP, TF3, and TB3 on the third target sequence. It binds to different binding sites.

[0127] In some embodiments, the primer mixture is (i) a third loop forward primer (ii) comprising a primer (TLF) and a third loopback primer (TLB), LF and TLB bind to two different binding sites on the third target sequence.

[0128] In some embodiments, the primer mixture contains two, three, four, five, or six Contains loop primers. The primer mixture contains two or more loop primers. In this case, each loop primer provides a unique signal for detection. It may include a pair of biosensors. In some embodiments, each pair of biosensors is fixed for detection. It provides a visual signal (e.g., color). In some embodiments, each biosensor pair is It contains unique pigment molecules.

[0129] In some embodiments, two or more loop primers are present in the primer mixture. This includes an identical pair of biosensors. In some embodiments, two in the primer mixture Alternatively, label the loop primers with FAM. In some embodiments, Two different loop primers in the mar mixture are labeled with FAM.

[0130] In some embodiments, the primer mixtures provided herein are freeze-dried. The dry primer mixture is a looper primer or primer mixture as described herein. It may include any of the following. In some embodiments, two or more loop plugs A primer mixture containing an primer is freeze-dried. In some embodiments, the primer The mixture is in the form of freeze-dried beads.

[0131] 6.5. Loop-de-loop amplification kit In another embodiment, a kit for loop-de-loop amplification is provided. This kit is This may include either a loop primer or a primer mixture provided in the specification. ru.

[0132] In some embodiments, the kit includes one primer set. In form, the primer set provided herein is for loop-de-loop amplification Loop primer, (i) Forward inner primer (FIP), (ii) Back loop (iii) Inner Primer (BIP), (iii) Forward Primer (F3) and Back Includes Quward primer (B3). In some embodiments, the primer set is ( i) Loop forward primer (LF) and (ii) Loop backward primer ( This also includes LB.

[0133] In some embodiments, the kit includes two primer sets. In terms of application, this kit includes three primer sets. In some embodiments, The kit includes four or five primer sets.

[0134] In some embodiments, the kit comprises multiple primer sets in a single container. Includes. In some embodiments, the kit includes multiple primer sets, and each primer The marsets are individually packaged in separate containers.

[0135] In some embodiments, the kit further comprises polymerase. In this case, polymerase is a strand-substitution DNA polymerase. In some embodiments, poly Melase is produced by Bacillus stearothermophilus. It is polymerase. In some embodiments, polymerase is Bst 2.0 War nStart(registered trademark) DNA Polymerase (available from NEB) In some embodiments, the kit further comprises other reaction enzymes, such as reverse transcriptase. In some embodiments, the reverse transcriptase is WarmStart® RTx reverse transcriptase. This is a RNase (available from NEB). In some embodiments, this kit is an RNase. The inhibitor further comprises an inhibitor in some embodiments. It is an RNase inhibitor.

[0136] In some embodiments, the kit further includes reagents for amplification reactions. In one embodiment, the reagent includes dNTPs, MgSO4, and a buffer. Several embodiments In this state, the buffer contains a surfactant. In some embodiments, the buffer is 1, 2, 3, Includes 4, 5, 6, 7, 8, 9, or 10% Tween-20. In some embodiments The reagent contains trehalose. In some embodiments, the reagent contains sucrose. In some embodiments, the reagent includes a polymer for stabilization. The amplification reagent is polymer It can be selected and optimized according to the requirements.

[0137] In some embodiments, this kit includes dNTPs, MgSO4, buffer, and loop de... A mixture containing one or more primer sets and polymerase for loop amplification. Includes. In some embodiments, this kit includes dNTP, loop-de-loop amplification. One or more primer sets, polymerase, reverse transcriptase, and RNase Contains a mixture including an inhibitor.

[0138] In some embodiments, this mixture is in liquid form. The mixture is in a dry form. In some embodiments, this mixture is freeze-dried into beads or It is formulated into pellets.

[0139] In some embodiments, the kit further includes apparatus for amplification reactions. In this embodiment, the kit includes a device for loop-mediated isothermal amplification.

[0140] In some embodiments, the kit further includes a reaction tube for carrying out amplification reactions. In some embodiments, the kit is used for filtering or purifying the sample before the amplification reaction. Includes further components.

[0141] In some embodiments, this kit is for the diagnosis of infectious diseases. This kit is for pathogenic infections, such as Chlamydia trachomatis. It is for the diagnosis of Neisseria gonorrhoeae and Neisseria tis. In some embodiments, The kit is used for determining single nucleotide polymorphisms (SNPs) and point mutations. Several implementations Morphologically, this kit is used for determining mutant genotypes. In some embodiments, This kit is used to determine mutant genotypes associated with drug resistance phenotypes. For example, drug resistance markers, such as ceftriaxone / cefixime resistance markers, quinol Ciprofloxacin resistance marker, macrolide resistance marker (azithromycin) ) can be detected.

[0142] 6.6. Loop-de-loop amplification method In another embodiment, a loop-de-loop amplification method is provided. This method is The process of preparing the sample, (i) Primers, primer mixtures, or dry primer mixtures provided herein (ii) a reconstituted primer mixture obtained by rehydrating the material, and (ii) polymer The process involves adding ze to the sample to generate a reaction mixture, The process involves incubating the reaction mixture at 50-85°C, It may include.

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

[0144] In some embodiments, the method further includes the step of detecting a signal from the reaction mixture. In some embodiments, the method includes the step of detecting a fluorescent signal. In some embodiments, this method includes a step of detecting a change in color or turbidity. This method includes the step of detecting a non-visual signal. In some embodiments, the step of detecting The process is carried out during the incubation process. In some embodiments, the detection process is This is done after the incubation process is complete. In some embodiments, the signal is real It is detected in time. In some embodiments, the signal is recorded in real time, and the ink It is analyzed after the incubation process is complete.

[0145] In some embodiments, this method prepares a sample for loop-de-loop amplification. The process further includes: In some embodiments, the sample preparation step involves inverting the RNA molecule. It is reacted with a deoxygenase to generate a sample containing DNA molecules. Several implementations In this state, the sample preparation process involves adding RNA molecules before interacting with reverse transcriptase. The process further includes preheating the sample or reaction mixture.

[0146] In some embodiments, the sample for loop-de-loop amplification is purified polynucleotides. Contains cytoplasmic molecules. In some embodiments, the sample is purified RNA, purified DNA, and total SAR. S-CoV-2 virus, whole human cells, saliva or nasal swab, or nasal or nasopharyngeal swab Includes a wab. In some embodiments, the sample is genomic DNA, synthetic DNA, whole bacteria or Contains whole human cells derived from vaginal swabs. In some embodiments, loop-de-loop amplification The sample used is a crude sample. In some embodiments, for loop-de-loop amplification The sample is a purified sample.

[0147] In some embodiments, two or more signals are detected. In some embodiments, Multiple fluorescent or other visual signals are detected. In some embodiments, multiple signals are detected. A number of target sequences are detected to determine whether they are present or absent. In some embodiments, Multiple signals are detected to determine the presence or absence of a single target sequence. In some embodiments, Multiple signals are detected to provide further sensitivity and specificity to this method.

[0148] Various amplification methods known in this field can be used to amplify the target sequence.

[0149] In a typical embodiment, loop-mediated isothermal amplification ("LAMP") is performed on the loop of a target nucleic acid. Used in de-loop amplification. LAMP is a chain substitution activity of the enzyme known as polymerase. This is a sex-dependent isothermal DNA amplification method that specifically amplifies nucleotide bases in DNA or It is added to the RNA chain to form a double-stranded nucleic acid with a complementary sequence. In isothermal amplification, geoba Polymer derived from the bacterium Geobacillus stearothermophilus Chain substitution polymerases, such as Bst polymerase and its variants, overlap the complementary chain. Since one strand of the double-stranded DNA is replaced during the fusion process, a thermal cycle is not required.

[0150] The LAMP method is specifically designed to recognize six different regions of a target DNA sequence. Four different primers (F3, B3, inner forward primer or FIP, and An inner backward primer (BIP) can be used. The reaction rate can be modified. To improve the situation, two additional "loop" primers may be added. The concentration of the primer may vary, but typically, for FIP and BIP primers, it is 1 0.6 μM, 0.8 μM for forward and reverse loop primers (LF, LB), and Furthermore, the F3 and B3 primers are set to 0.2 μM. Several LAMP methods In this embodiment, five primers can be used (two possible LAMP primers) (Using only one of the imers). The LAMP reaction utilizes chain substitution to operate at a constant temperature. The process proceeds at around 65°C. Target amplification and detection are performed using the sample, primers, and chain displacement activity. By incubating DNA polymerase, buffer, and substrate at a constant temperature... And it can be completed in one step. A typical LAMP mixture composition consists of the following reagents, namely, 20mM Tris-HCL, 10mM(NH4)2SO4, 50mM KCl, 8mM MgSO4, 0.1% Tween(registered trademark) 20, 1.4mM dNTP, 0.32 The mixture contains U / μL Bst polymerase, primer at the aforementioned concentrations, and water, with a pH of 20°C. The reaction is adjusted to 8.8. The reaction volume is typically 5 μL to 50 μL. The reaction time is optimized for the specific enzyme and primer used, and the reaction takes 5-60 minutes. It progresses. LAMP has high sensitivity, specificity, and efficiency.

[0151] LAMP recognizes six target sites (e.g., F3, B3, FIP, and BIP). At the very least, it relies on four primers to amplify a specific DNA or RNA target (RNA). The target first requires reverse transcription into DNA). Loop primers (e.g., LF and L) If B) is included, a total of eight unique sites in the target nucleic acid are targeted by the six primers. Recognized. In the various embodiments provided herein, of the eight unique parts in total One of these can be recognized by the loop primer described herein. If present, an amplification reaction can occur, potentially resulting in a large amount of DNA.

[0152] The novel loop-de-loop method described herein is suitable for other isothermal amplification methods besides LAMP. It can be used. To address the temperature cycle dependence of polymerase chain reaction (PCR), Number isothermal amplification methods have been developed. These methods can be quite different, but they all They share several common characteristics. For example, DNA strands are not denatured by heat, so all are equal. The warm method relies on alternative approaches to enable primer binding and the initiation of amplification reactions. Once the reaction begins, the polymerase is still annealed to the desired sequence. The strands must be replaced. Isothermal methods are typically used to separate double-stranded DNA. The strand displacement activity of the DNA polymerase is used. Polymerases possessing this ability include: In moderate temperature reactions (25-40°C), the Krenow fragment (3'-5'exo-) is produced. Examples include Bsu large fragments and Phi29, and are produced at higher temperatures (50-65°C). One example of the reaction is the large fragment of Bst DNA polymerase. To detect the reaction, a reverse transcriptase suitable for the reaction temperature is added to maintain isothermal amplification. In addition to the strand substitution mechanism for separating sDNA, isothermal methods meet the initial denaturation requirements for initiation. Designing enzymes or primers may be necessary to avoid this issue.

[0153] As described above, loop-mediated isothermal amplification (LAMP) involves 6 to 8 different regions of the target DNA. Use 4-6 primers that recognize the region. Strand substitution DNA polymerase initiates synthesis. The two primers then form a loop structure, which promotes subsequent amplification. LAMP is fast Because it is fast, highly sensitive, and has very large amplification, LAMP is well-suited for field diagnostics. Loop-de-loop primers are single or any combination or internal and / or It can be used as a loop primer.

[0154] Strand substitution amplification (SDA) is performed using strand substitution DNA polymerase, typically Bst DNA polymerase. Limerase, large fragment or klenow fragment (3'-5'exo-) The site contained in the primer is affected by chain-restricting endonucleases or nicking enzymes. It starts with a nick that is created as follows. SDA consists of one forward primer and one riva - Primer, and one bumping forward primer and one bumping primer A birth primer is required. Nicking sites are regenerated in each polymerase substitution step. This results in exponential amplification. SDA is typically used in clinical diagnosis. Existing fluorescence monitoring techniques exist for SDA (Nadeau et al., Real-Time, Se quence-specific detection of nucleic acids during strand displacement amplificat ion, 276m 2 177-187 (1999), the production of restriction endonuclease enzymes to generate fluorescence It depends on the application. Either forward or reverse SDA primer is applied to the fluorophore pair. For use in loop-de-loop techniques that do not require the position of the cutting site between the quencher pair. It can be adapted. The cut area is clamped with loop-de-loop primer. Located next to the column, toward the 3' end of the primer, and consequently, restriction endonuclease Before primer cleavage, complete cleavage of the 5' end of the primer by polymerase on the complementary chain. Fluorescence is generated during full extension.

[0155] Helicase-dependent amplification (HDA) utilizes the double-strand DNA unwinding activity of helicases. This separates the strands, enabling primer annealing and extension by strand-displacement DNA polymerase. This is done. Similar to PCR, this system uses two primers, i.e., one forward primer. Only a rimer and one reverse primer are required. HDA is a diagnostic device. It is used in and FDA-approved testing. Any primer in the HDA is loop- Adapted for use in the de-loop method, enabling real-time sealed tube monitoring of the reaction. It can be done in real time. In HDA, the helicase enzyme is used in loop de loop The lymer loop structure can be opened, which is stabilized by single-chain binding proteins. These can then be converted into double-stranded fluorescent amplicons by DNA polymerase.

[0156] Nicking enzyme amplification (NEAR) is an open reaction that uses the nicks produced by the Nicking enzyme. Using a strand substitution DNA polymerase, a large number of short nucleic acids are rapidly generated from a target sequence. This process is extremely fast and highly sensitive, allowing for the detection of even minute amounts of the target within minutes. NEAR can do this. NEAR is generally used in clinical and biosafety applications for the detection of pathogens. It is used as either a forward primer or a reverse primer for NEAR. This method generates real-time fluorescence via loop extension by strand substitution DNA polymerase. The loop-de-loop method can be used to achieve this.

[0157] 6.7.How to use The loop-de-loop amplification method provided herein involves detecting a target sequence from various sources. It can be used to extract, for example, viral genomes, bacterial genomes, ancient genomes. Bacterial genome, plant genome, animal genome, protist genome, prokaryotic genome, or eukaryotic genome It can be used to detect target sequences specific to NM. In some embodiments, This method uses RNA (e.g., positive sense RNA, negative sense RNA) or DN. Used to detect A. In some embodiments, this method is synthesized It is used to detect target sequences.

[0158] In some embodiments, the loop-de-loop method is used to detect pathogen-specific DNA. It is used. In some embodiments, the pathogen is a virus, bacteria, fungi, protozoa, or parasite. In some embodiments, loop-based detection of pathogens associated with STDs is used. • The loop method is used. In some embodiments, the pathogen is Chlamydia trachomatis. (Chlamydia trachomatis). In some embodiments, the pathogen is Neisseria gonorrhoeae. (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 this embodiment, the loop-de-loop method is used to determine the mutant genotype. In some embodiments, the loop-de-loop method identifies mutant genes associated with drug resistance phenotypes. Used for type determination. For example, drug resistance markers, e.g., ceftriaxone / Cefixime resistance marker, quinolone (ciprofloxacin) resistance marker, macrolase It can detect drug resistance markers (azithromycin).

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

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

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

[0163] The loop-de-loop method described herein can be used for the analysis of various samples. In some embodiments, blood, urine, semen, tissue, or saliva samples are analyzed. In some embodiments, the sample is taken from an animal or a human patient. In some embodiments, sperm The prepared sample is analyzed. In some embodiments, the crude sample is analyzed. In this embodiment, the sample consists of purified RNA, purified DNA, whole SARS-CoV-2 virus, and Includes whole nasal cells, saliva or nasal swab, or middle turbinate or nasopharyngeal swab. In one embodiment, the sample is genomic DNA, synthetic DNA, whole bacteria, or whole bacteria derived from a vaginal swab. Includes oocytes. [Examples]

[0164] 6.8. Examples The following examples are provided as illustrations, not as limitations. 6.8.1. Example 1: Chlamydia treatment using intercalate dye (SYTO) LAMP of Lacomatis (Chlamydia trachomatis) and Neisseria gonorrhoeae Issei

[0165] Chlamydia trachomatis genomic DNA and Neisseria gonorrhoeae (Niesser) To detect genomic DNA (Ia gonorrhea) separately, a LAMP reaction mixture was prepared. The reaction mixture was prepared in 10 μL volume and contained the following reagents: 20 mM Tris-HCl l, 10mM(NH4)2SO4, 50mM KCl, 8mM MgSO4, 0.1% Tween®20, 1.4mM dNTP, 0.32U / μL Bst2.0 WarmStart® polymerase, 1.6 μM FIP and BIP, 0. Primers containing 8 μM LF and LB, and 0.2 μM F3 and B3 (SEQ ID NOs: 1-4) Contains 6, 8-14), 2.5 μM SYTO85 intercalate dye, and water. The pH was adjusted to 8.8 at 20°C. The target genomic DNA was then mixed with the stock solution purchased from ATCC. The sample was diluted 10-fold with Tris-HCl buffer at pH 8.0. The sample contained target DNA or DNA. Add 1 μL of the uncoated buffer (for the uncoated control) to the 9 μL solution mixture in each PCR tube. μL was added. The reaction temperature was 65°C, and the reaction was monitored using SYTO85 fluorescence. We did this. Using a real-time PCR instrument, we heated the reaction product and measured the fluorescence in real time. The reaction was controlled. The reaction was carried out for 60 minutes. The data shown in Figure 2A represents the intercalating reaction. Chlamydia trachomatis monitored by dye Obtained from LAMP reactions for (green) and Neisseria gonorrhoeae (blue). The curves representing real-time fluorescence (in arbitrary units) on the vertical axis versus time on the horizontal axis are shown separately. This involves three levels of genomic DNA targeting: high (concentration of undiluted solution), and low (for Ct, 10% of the undiluted solution DNA). -5 For the dilution, Ng is 10 times the original DNA. -6 Diluted (DNA), and template-free control (DNA This shows the case for none (NTC).

[0166] 6.8.2. Example 2: Chlamydia trachomatis (Ch) amplified by loop-de-loop amplification Detection of Lamydia trachomatis Prepare a loop-de-loop LAMP reaction to test for Chlamydia trachomatis (Chla Mydia trachomatis genomic DNA was detected. The reaction mixture was prepared in 10 μL volume. The reagents below are 20 mM Tris-HCl, 10 mM (NH4)2SO4, and 50 mM KCl, 8 mM MgSO4, 0.1% Tween® 20, 1.4 mM M dNTP, 0.32U / μL Bst2.0 WarmStart(registered trademark) Melase, 1.6 μM FIP and BIP, 0.4 μM LF and LF-LdL, 0.8 Primers having μM LB, 0.2 μM F3 and B3 (SEQ ID NOs. 9-15), and The solution contained water and was adjusted to pH 8.8 at 20°C. The quantified target genomic DNA was then analyzed using AT. Dilute the stock solution purchased from CC 10-fold or 2-fold with pH 8.0 Tris-HCl buffer (more Diluted (for fine resolution). Target DNA dilution or DNA-free buffer (without template). (For the control case) Add 1 μL to the 9 μL solution mixture in each PCR tube and perform the assay. To investigate sensitivity, up to 20 copies per density were used across a range of several logarithmic density. The reaction temperature was 65°C, and F was emitted by the loop-de-loop primer. The reaction was monitored by AM fluorescence. A real-time PCR instrument was used to analyze the reaction products. The mixture was heated, and fluorescence was measured in real time. The reaction was run for 60 minutes. The data is shown in Figure 3. Chlamydia trachomatis (Chlamydia trac) is a 10-fold dilution of genomic DNA target. Real-time fluorescence obtained from the loop-de-loop LAMP reaction of homatis (arbitrary units) )A representative curve of time on the horizontal axis (each "cycle" represents 30 seconds) is shown. Next, The sensitivity (limit of detection, 50% and 95% probability) is determined based on the endpoint determination of the assay. This was estimated using ROBIT analysis. [Table 1]

[0167] 6.8.3. Example 3: Neisseria gonorrhoeae by loop-de-loop amplification ) Verification A LAMP reaction mixture was prepared to test for Neisseria gonorrhoeae. Genomic DNA was detected. The reaction was prepared in 10 μL volume and contained the following reagents: 20mM Tris-HCl, 10mM(NH4)2SO4, 50mM KCl, 8mM MgSO4, 0.1% Tween® 20, 1.4mM dNTP, 0.3 2U / μL Bst2.0 WarmStart® polymerase, 1.6μM FIP and BIP, 0.4 μM LF and LF-LdL, 0.8 μM LB, 0.2 μ Primers having M F3 and B3 (SEQ ID NOs: 1-4, 6-8), and water-containing The pH was adjusted to 8.8 at 20°C. The quantified target genomic DNA was purchased from ATCC. Diluting the stock solution 10-fold or 2-fold with Tris-HCl buffer at pH 8.0 (for finer resolution) (For this purpose) it was diluted. Target DNA dilution or DNA-free buffer (in the case of a template-less control) To examine the assay sensitivity, 1 μL was added to 9 μL of solution mixture in each PCR tube. To achieve this, a maximum of 20 copies per concentration were used across a range of several logarithmic concentrations. The reaction temperature The temperature is 65°C, and the FAM fluorescence emitted by the loop-de-loop primer is... The reaction was monitored. Using a real-time PCR instrument, the reaction product was heated and the reaction was monitored. Fluorescence was measured at 24-hour intervals. The reaction was run for 60 minutes. The data shown in Figures 2B to 2C are: Loop de genoplasm of Neisseria gonorrhoeae in response to a 10-fold dilution of genomic DNA target Real-time fluorescence (arbitrary units) obtained from loop LAMP reaction, with a representative time axis on the x-axis. The curve is shown. Figure 2B shows the signal obtained from the loop-de-loop assay, as shown in Figure 2A. As shown, LAM was performed using SYTO85 dye without loop-de-loop primers. Compare with the signal from the P assay. The loop-de-loop assay is much more effective in the case of positive amplification. It provides a large signal. Figure 2C shows the reproducibility of the loop-de-loop assay, as well as the casting Negligible background fluorescence and reduced late-stage pseudoamplification products were observed in the untyped control. The data shown in Figure 4 is for Neisseria gonorrhoeae (gonococcus gonorrhea) against a 10-fold dilution of genomic DNA target. Real-time fluorescence obtained from the loop-de-loop LAMP reaction of *Norrhoeae* (arbitrary single The graph shows a representative curve of the time on the horizontal axis (each "cycle" represents 30 seconds). Next, A Assay sensitivity (detection limit, 50% and 95% probability) is determined based on the endpoint determination of the assay. This was estimated from the results of serial dilution tests using ROBIT analysis. [Table 2]

[0168] 6.8.4. Example 4: Detection of Homo sapiens by loop-de-loop amplification A loop-de-loop LAMP reaction was prepared to examine Homo sapiens genomic DNA. The reaction was prepared in 10 μL volume and contained the following reagents: 20 mM Tri s-HCl, 10mM(NH4)2SO4, 50mM KCl, 8mM MgSO4, 0 .1% Tween®20, 1.4mM dNTP, 0.32U / μL Bs t2.0 WarmStart® polymerase, 1.6 μM FIP and BI P, 0.4 μM LF and LF-LdL, 0.8 μM LB, 0.2 μM F3 and B3 Primers having (SEQ ID NOs: 16-22), and a water-containing solution with a pH of 8 at 20°C. The pH was adjusted to 8. The quantified target genomic DNA was prepared using the stock solution purchased from ATCC at pH 8.0. Diluted 10-fold or 2-fold (for finer resolution) with Tris-HCl buffer. The target DNA dilution or DNA-free buffer (in the case of a template-less control) is placed in each PCR tube. Add 1 μL to 9 μL of solution mixture and examine the assay sensitivity within a few log concentration ranges. A maximum of 20 copies were used per concentration over the course of the experiment. The reaction temperature was 65°C. The reaction is monitored by FAM fluorescence emitted by the ov-de-loop primer. The reaction was heated using a real-time PCR instrument, and fluorescence was measured in real time. The reaction was carried out for 60 minutes. The data shown in Figure 3 are for a 10-fold dilution of the genomic DNA target. Real-time fluorescence obtained from the loop-de-loop LAMP reaction in Homo sapiens. This shows a representative curve of time on the horizontal axis (each "cycle" represents 30 seconds) in arbitrary units. Then, assay sensitivity (detection limit, 50% and 95% probability) is used to determine the endpoint of the assay. This was estimated using PROBIT analysis. [Table 3]

[0169] 6.8.5. Example 5: Dry primer mixture for loop-de-loop amplification Formulation into lyophilized reagents was carried out using an in-house lyophilization test with a five-step lyophilization protocol. The loop-de-loop was designed to detect Neisseria gonorrhoeae. The LAMP reaction mixture was prepared in 25 μL volumes per tube and dispensed into individual tubes. Freeze-dried. The dry mixture is prepared using the following reagents, namely 1.4 mM dNTPs, glycerol-free. WarmStart® polymer, 0.32 U / μL Bst 2.0, used as an agent. Lase, 1.6 μM FIP and BIP, 0.4 μM LF and LF-LdL, 0.8 μ Primers having M LB, 0.2 μM F3 and B3 (SEQ ID NOs: 1-4, 6-8), It contained 5% trehalose and water (up to 25 μL per reaction). The cap was removed for freeze-drying. The tube strip was used for pharmaceutical and biotechnology. It was placed on a metal shelf inside a heated shelf freeze dryer unit, which is standard equipment in industry. The dryer was programmed to run in 5 stages. Stage 1: Condenser ON, Vacuum OFF, Shelf Cool the reagents to 41°F for 30 minutes. Step 2: Turn the condenser ON, turn off the vacuum, and set the shelves and reagents to 2 Cool to 3F for 30 minutes. Stage 3: Turn on the condenser, turn off the vacuum, and cool the shelves and reagents to -23F. 2 hours. Stage 4: Turn on the condenser, turn on the vacuum, and maintain the shelves and reagents at -23°F for 10 hours. Stage 5: Turn on the condenser, turn on the vacuum, heat the shelves and reagents to 77°F for 5 hours. This process is complete. Once finished, the tube was removed, the cap was closed, and the product shown in Figure 7 was obtained. Lyophilization assay The activity of [the substance] was tested over a certain period after incubation under various environmental conditions. (Figure) Figure 8 shows the activity of a typical real-time loop-de-loop LAMP assay for rehydration reactions. The rehydration protocol involves adding 24 μL of rehydration buffer to 1 μL of Neisseria gonorrhoeae. The procedure involved adding the target genomic DNA to a dried reagent. The rehydration buffer was , 20mM Tris-HCL, 10mM(NH4)2SO4, 50mM KCl, 8m It consists of MgSO4, 0.1% Tween® 20, and water, with a pH of 20. The temperature was adjusted to 8.8°C. The buffer solution was added to the tube, then resealed, and vortexed or The reagents were placed directly into the real-time qPCR machine without any mixing. The reaction temperature was 65°C. The reaction is mediated by FAM fluorescence emitted by the loop-de-loop primer. We performed a real-time PCR test. Using a real-time PCR instrument, we heated the reaction product and performed the test in real time. Fluorescence was measured. The reaction was run for 60 minutes. The data shown in Figure 8 represents one of the genomic DNA targets. Is this a loop-de-loop LAMP reaction of Neisseria gonorrhoeae to a 0-fold dilution? The obtained real-time fluorescence (in arbitrary units) versus time on the horizontal axis (each "cycle" represents 30 seconds). The following shows a typical curve for the rate, sensitivity, and specificity of the freeze-dried loop-de-loop assay. It was found that the rate, sensitivity, and specificity were no different from those of the newly formulated assay. Furthermore, the fluorescence intensity is not affected by drying and rehydration, and the loop-de-loop method is effective. This demonstrates the possibility of providing a storage-stable in vitro diagnostic kit for pathogen detection.

[0170] 6.8.6. Example 6: Temperature for Loop-de-Loop Amplification The loop-de-loop LAMP reaction mixture was prepared as described above, with one side being ORF1ab. One includes a primer set, and the other includes a POP7b primer set. ORF1ab primer Marset is a SARS-CoV-2 virus with a positive sense single-stranded RNA genome. It is specific to [specific target]. The POP7b primer set does not exist naturally as a DNA template. This primer set is specific to human RNA targets, and therefore, this primer set targets human R in the sample. It is useful as a specific indicator of NA. In both cases, loop-de-loop is used. Then, modify one of the six constituent primers used in LAMP to create the seventh loop. Primers were prepared. Loop primers utilize a pair of fluorophores and quenchers. This generated an observable signal. In this experiment, the distribution of RNA targets in each primer set Moderately high concentrations of synthetic double-stranded DNA containing sequences on their positive sense strands corresponding to the columns. The template is used as a target for optimizing the LAMP reaction temperature, and the reverse transfer process or dilute target is subjected to Variations due to encountered stochastic noise were minimized. Target DNA diluent was prepared in 384 wells. They were added to the mixture in the container. They were incubated at various temperatures ranging from 55 to 70°C. The reaction is monitored by FAM fluorescence emitted by the loop-de-loop primer. Taring was performed. Using a real-time PCR instrument, the reaction was heated and the fireflies were observed in real time. Light was measured. The reaction was run for 60 minutes. Two experiments were conducted over overlapping temperature ranges (the first experiment). The first and second tests were conducted, and the data is shown in Figures 9A and 9B. This figure shows the detection This shows the time required to obtain a sufficient signal. This result indicates that the primer set is suitable for a wide range of temperatures. This indicates that it is active over a range of temperatures. For example, at approximately 57-70°C, POP7b plastic Both the primer set and the ORF1ab primer set were within acceptable limits. 60 Optimal performance was achieved between ℃ and 68℃.

[0171] 6.8.7. Example 7: Multiplexing for detecting both purified target and target in crude sample Loop-de-loop reaction Figures 14A, 14B, and 14C show ORF1ab and POP7b LdL, respectively. Loop-de-loop testing of SARS-CoV-2 and human target sequences using primer sets. The two fluorescence signals obtained from amplification are shown. SARS-CoV-2 ORF1ab(FAM) The signals obtained from ) and POP7b human internal control (Cy5) are shown. Control without target sequence. Sample (unformed control) (Figure 14A), human nasal swab (Figure 14B), and thermally inactivated virus. Three human nasal swabs (Figure 14C) combined with SARS-CoV-2 target sequences in the form of swabs. Various types of samples were used. Human nasal swabs were self-collected from volunteers and sampled or nucleic acid-based. The reaction was mixed by twisting the swab for a few seconds without extraction. The SARS-CoV-2 target sequence was eluted into a substance. It reacts as an intact, heat-inactivated virus (ATCC VR-1986HK) that is added. It was introduced into the device. ORF1ab LdL-FAM and POP7b LdL-Cy5 The merset was double-chained in a 1:1 ratio at the replication reaction volume. Both primer sets This involves using LdL primer in a 1:3 ratio to unlabeled primer analog (25% strength). The reaction mixture contained reverse transcriptase, chain substitution polymerase, and an RNase inhibitor. Using a real-time PCR instrument (Bio-Rad CFX-384®) While recording the fluorescence measurements of FAM and Cy5, add the reaction mixture at 55.6°C for 2.5 minutes. The mixture was incubated, then incubated at 63.5°C for 60 minutes. As expected... However, the untemplated control replica did not show a loop-de-loop fluorescence signal over 60 minutes. The reaction, including COVID-19 negative nasal swab samples, was indicated by an increase in Cy5 fluorescence. As revealed, the POP7b signal was amplified, but the ORF1ab signal remained flat. The result was (negative). The sample to which the heat-inactivated virus was added tested in both RNs in a single reaction vessel. Spectral dual detection of target A was demonstrated. This result was obtained using loop-de-loop RT-LAM. P enables single-tube spectral multiplexing of SARS-CoV-2 and human targets. This indicates.

[0172] Multiple loops of SARS-CoV-2 using the ORF1ab LdL primer set. The de Loop test was as sensitive as the PCR test and did not require extraction. This is because the reaction with the crude sample yielded favorable results, as shown in the table below. POP A 7b LdL primer set was used as an internal control. Intact thermally inactivated SA Double-loop serial dilutions of RS-CoV-2 virus (ATCC VR-1986HK) The assay was performed by adding the substance to the de Loop reaction and monitoring the real-time signal generation. The detection limit for a specific format of the test kit is LoD95 = 400 cp / s Wab = 2.7 × 10 3 It was estimated to be cp / mL. [Table 4]

[0173] The tripled loop-de-loop reaction was also tested in a single tube. It was a characteristic of the three targets. It exhibited heterogeneous amplification and maintained a fast time to results. Labeled with FAM fluorophores. Using two loop-de-loop primer sets, SARS-CoV-2 virus Two distinct targets of the rus RNA were detected. The human internal control loop was labeled with Cy5. • The loop primer set detected a third RNA target. Internal Cy5 fluorophore This was paired with the 5' Iowa Black (registered trademark) RQ Quencher. The reaction product was coarse nasal stool. The sample contained a vasectomy solution and was supplemented with heat-inactivated SARS-CoV-2.

[0174] Regarding the use of the POP7b human internal control loop-de-loop primer set, Additional loop primers were also tested. In some cases, the internal TAM was used as a second sensor molecule. The RA fluorophore is the first sensor molecule, 5'Iowa Black (registered trademark). It was paired with the FQ quencher. In another case, the first sensor molecule was 5'Yakima Yellow (registered trademark) (Epoch Biosciences) is the second sensor The molecule is called Internal Zen (trademark) (Integrated DNA Technology). es) Paired with Quencher. Regarding the placement of Yakima Yellow and Zen. Three variations of the loop primer were prepared and tested. In the first variation, Clamping oligonucleotide 1 and clamping oligonucleotide 2 are completely They were complementary and each had a length of 6 bases. The spacing oligonucleotide had 13 bases. It was long. In the second modification, the first clamping oligonucleotide has its 5' end. Characterized by an additional base at the end, the first clamping oligonucleotide is 7 bases. The first clamping oligonucleotide was 6 bases long. Six complementary bonds are formed between the clamping oligonucleotide and the second clamping oligonucleotide. A base was present. The spacing oligonucleotide was 13 bases long. Third variation So, both the first and second clamping oligonucleotides are 7 nucleotides long, and the sequence They were perfectly complementary. The spacing oligonucleotide was 10 base pairs long.

[0175] These additional loop primers are used in the LAMP reaction. This reaction targets the target It provides a specific amplified signal for the column.

[0176] 6.8.8. Example 8: SARS-CoV in human samples by loop-de-loop amplification Detection of -2 Loop-de-loop LAMP reaction mixture was prepared using untreated human saliva. ARS-CoV-2 was detected. Lyophilized enzyme, dNTP, and oligonucleotide plastic Rehydrate the immer mixture with a 10% volume / volume mixture of human saliva in a pH buffer solution. The reaction was prepared in a PCR tube by the following method. The lyophilized primer mixture was It contained primer sets for SARS-CoV-2 and human internal control RNA sequences. After rehydrating with the liquid sample, the reaction mixture is incubated at the preheating temperature for a predetermined period to dissolve the virus. It promotes resolution, RNase inhibition, and reverse transcription, and then higher LAMP DNA amplification. The mixture was incubated at the reaction temperature. Custom equipment was used to obtain temperature control data and real-time data. Time fluorescence data was collected.

[0177] Heat-inactivated SARS-CoV-2 was added to a pool of fresh saliva collected from anonymous donors. Three-fold serial dilutions of saliva were prepared. Twenty samples were collected using the loop-de-loop amplification method. The samples were tested using mobile applications, visual estimation, or real-time curve analysis. The readout is summarized below. These results indicate that the LoD is approximately 2,500 cp / mL. This indicates that. [Table 5]

[0178] Nasal swabs collected by volunteers are obtained by twisting them 10 times. It was added directly to the reaction mixture. Figure 16 shows that it was later positive for COVID by PCR testing. The amplification results obtained from nasal swabs acquired from symptomatic volunteers who were confirmed to have the condition are shown. The results obtained from positive / negative control samples are also shown. These results are based on the sample (1×swallow). (B) is 365 times higher than the concentration required to detect a positive sample in the loop-de-loop assay. This indicates that it has been doubled in concentration. The LoD is estimated to be approximately 2,500 cp / mL. Therefore, a specific sample is approximately 9.1 × 10 5 SARS-CoV-2 virus RN at cp / mL It was presumed to contain A.

[0179] Figure 17 shows the amplification results obtained from nasal swabs acquired from negative volunteers. The patient tested negative by both the loop-de-loop test and the PCR test.

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

[0181] 6.8.9. Example 9: Chlamydia thalamus in human samples by loop-de-loop amplification Detection of Chlamydia trachomatis and Neisseria gonorrhoeae Using a 30-second, 1Hz rotation method (Panpradist et al., 2016), three vaginal swabs (BD) were used. BBL Culture Swabs, supplied by unique individual donors Lee B I purchased 1 polyurethane foam swab from iosolutions (MO). It eluted into 294 μL of rehydration buffer (431 μL per swab). A small amount of fluid was absorbed by the swab. After loss, 1095 μL of pooled vaginal swab eluate was obtained. The fluid recovery rate was 85%. It was %. This swab eluate was used for loop-de-loop LAMP (one for Ct, one for Injection molding prototype containing lyophilized reaction mixture (one for Ng, one for human process control) I piped it into a disposable container.

[0182] Each reactant was rehydrated as follows. • 18 μL of 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 The Ct and Ng pathogen samples were placed in separate disposable reaction chambers. Therefore, for example, the Ct assay detects Ng and human targets as well as the substances present in the swab sample. I was tasked with detecting Ct in the simultaneous presence of various bacterial environments.

[0183] The amplification results are shown in Figures 19 to 22. Figure 19 shows the amplification of a large amount of Ct (10,000 per reaction). A sample containing copy equivalents and a large amount of Ng (10,000 copy equivalents per reaction) The resulting fluorescence signals are shown. Figure 20 shows the negative control - swab-only control (the two panels on the left). The signals are shown from a control with ) or buffer solution only (the two panels on the right). In the diffractometer, there was no amplification of Ct or Ng, but the human genome sequence was amplified as expected. Buffer In the liquid-only control, there was no amplification of Ct or Ng. Amplification in Homo sapiens was observed in one of the weaker strains. In the reaction with only the volcanic fluid (Test 20), it was detected with a delay, but considering the delay in the signal... This is likely a false amplification.

[0184] These results suggest that this assay produces approximately 100 copies of Ct per reaction and per reaction This indicates that it was sensitive enough to detect over 1,000 copies of Ng. 7. Array [Table 6]

[0185] 8. Incorporation by reference All publications, patents, patent applications and other documents cited in this application are treated as if each one were an individual. Publications, patents, patent applications, or other documents are incorporated by reference for all purposes. Just as it is shown individually, the whole is by reference for all purposes. It will be included in the specifications.

[0186] 9. Equal parts This disclosure provides, in particular, cannabinoid compositions and associated compositions. Furthermore, by administering cannabinoid compositions and associated compositions, neurodegenerative diseases can be treated. The present specification provides a method for doing so. Various specific embodiments have been illustrated and described, but the above specification is This invention is not limited. Various modifications may be made without departing from the spirit and scope of the present invention. It will be understood that this is possible. Many variations will be apparent to those skilled in the art upon examination of this specification. It will become softer.

Claims

1. A primer mixture for loop-de-loop amplification of a target sequence, (i) From 5' towards 3', (a) The first sensor molecule and (b) The first clamping oligonucleotide and (c) Spacing oligonucleotides that are not complementary to the target sequence, (d) A second clamping oligonucleotide, (e) The second sensor molecule, (f) A first primer sequence complementary to the first binding site on the target sequence, A loop primer comprising, The first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide can form a hairpin structure at a temperature lower than the melting temperature (Tm) of the first and second clamping oligonucleotides. The first sensor molecule and the second sensor molecule constitute the first biosensor pair. The loop primer is configured to be amplified by a chain substitution polymerase when it binds to the target sequence. (ii) Forward Inner Primer (FIP), (iii) Backward Inner Primer (BIP), (iv) Forward primer (F3), (v) Backward primer (B3), (vi) Loop forward primer (LF) and (vii) Loopback primer (LB) A primer mixture comprising the FIP, BIP, F3, and B3 binding to six different binding sites on the target sequence, and at least one of the FIP, BIP, F3, B3, LF, and LB binding to the first binding site.

2. The second loop primer further comprises, (a) A third sensor molecule, (b) A third clamping oligonucleotide, (c) A second spacing oligonucleotide and (d) A fourth clamping oligonucleotide, (e) The fourth sensor molecule, (f) A second primer sequence complementary to the first binding site on the second target sequence, Includes, The third clamping oligonucleotide, the second spacing oligonucleotide, and the fourth clamping oligonucleotide can form a hairpin structure at a temperature lower than the melting temperature (Tm) of the third and fourth clamping oligonucleotides. The primer mixture according to claim 1, wherein the third sensor molecule and the fourth sensor molecule constitute a second biosensor pair, and the second biosensor pair is different from the first biosensor pair.

3. The primer mixture according to claim 2, wherein the first target sequence and the second target sequence are different.

4. The primer mixture according to claim 2 or 3, 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. The primer mixture according to any one of claims 2 to 4, 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. The third loop primer further comprises, (a) The fifth sensor molecule, (b) A fifth clamping oligonucleotide, (c) A third spacing oligonucleotide and (d) A sixth clamping oligonucleotide, (e) The sixth sensor molecule, (f) A third primer sequence complementary to the first binding site on the third target sequence, Includes, The fifth clamping oligonucleotide, the third spacing oligonucleotide, and the sixth clamping oligonucleotide can form a hairpin structure at a temperature lower than the melting temperature (Tm) of the fifth and sixth clamping oligonucleotides. The primer mixture according to any one of claims 2 to 5, wherein the fifth sensor molecule and the sixth sensor molecule constitute a third biosensor pair, and the third biosensor pair is different from the first biosensor pair and the second biosensor pair.

7. A kit for loop-de-loop amplification of a target sequence, A kit comprising a primer mixture according to any one of claims 1 to 6 and a polymerase, wherein the polymerase is a chain-substituted polymerase.

8. The kit according to claim 7, further comprising reverse transcriptase.

9. A method for detecting a target sequence in a sample, (1) The step of preparing the sample, (2) (i) A step of adding the primer mixture according to any one of claims 1 to 6 and polymerase to the sample to generate a reaction mixture, (3) A step of incubating the reaction mixture at 50 to 85°C, Methods that include...

10. The method according to claim 9, further comprising the step of detecting a fluorescent signal from the reaction mixture.

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