Looped primer and loop-de-loop method for detecting target nucleic acid

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 TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 989,140, ​​filed March 13, 2020. No. 6,313,525, filed on Oct. 13, 2003, which is hereby incorporated by reference in its entirety.

[0002] 2. Sequence Listing This application contains a sequence listing having XXX sequences, which were submitted via EFS-Web. The AS filed on XXX, is incorporated herein by reference in its entirety. The CII copy is named 48397WO_sequencelisting.txt , its size is XXX bytes. [Background technology]

[0003] 3.Background Methods for detecting target nucleic acids using nucleic acid sequence complementarity include traditional Southern hybridization. Since its creation, various improvements and modifications have been made to the present day. Nucleic acid amplification methods, such as polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid Sequence-based amplification (NASBA), rolling circle amplification (RCA) and loop-mediated isothermal amplification The establishment of loop-mediated amplification (LAMP) has made it possible to detect smaller amounts of target nucleic acid. These methods include medical diagnosis of infectious diseases, determining mutation genotypes, and single nucleotide polymorphisms (SNPs). and for use in 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 the gold standard of testing due to their high specificity and sensitivity. It was Dard.

[0004] However, amplification reactions and signal detection require precise procedures using controlled environments and expensive equipment. Current nucleic acid amplification methods are limited by the need for accurate measurements. are often cost-prohibitive for use in point-of-care situations. Additionally, this method has not been optimized for detection of multiplexed targets in a single patient sample. Detection of multiplexed targets can be achieved by signal multiplexing in a single-pot reaction (fluorescence spectrum multiplexing). ionization, electrochemical detector arrays), physical separation of multiple reactions into unique reaction vessels, or However, in the case of CLIA waiver testing, a single To simultaneously query a panel of nucleic acid targets using patient samples, users need only follow three steps: Therefore, complicated equipment or disposables are not required for the process. Physical separation of samples into separate chambers is not possible unless the samples are processed automatically. Spectral multiplexing with fluorescence quickly becomes impractical for the purpose of detecting the patterns of nucleic acid targets. Although it is possible to reduce the number of unique reactions required to target a channel, spectral multiplexing LAMP reactions require dramatic sacrifices in assay speed or signal strength, which This undermines the likelihood of successful application in POC trials.

[0005] This allows the target nucleic acid, especially multiplexed targets, to be detected with high sensitivity and specificity at low cost. There is a need to develop new methods that allow for easy amplification and detection. Summary of the Invention

[0006] 4. Overview The present disclosure provides a novel amplification method that allows easy detection of a target nucleic acid in a closed system. The method includes using a loop primer having a biosensor pair. This allows detection of small amounts of target nucleic acid with high specificity and sensitivity. The pair can be used, for example, to sterically tune the loop primer using fluorescent / quencher FRET technology. Loop-de-loop ("LDL") amplification of target sequences by detecting conformational changes Furthermore, the use of multiple biosensors allows the determination of Loop primers allow for the detection of multiplexed targets in a probe. LAMP, as well as any other nucleic acid amplification method that utilizes a strand-displacing polymerase. can be used in combination with

[0007] The applicants have demonstrated that the loop-de-loop amplification method can be used to detect the presence of inhibitory fluorescent probes, e.g., DARQ Previously known methods include (detection of amplification by emission of quenching) and OSD (single-step displacement) probes. With improved sensitivity and specificity, and faster turnaround time compared to previous methods. We demonstrated that the loop-delta fusion enables sequence-specific amplification of target nucleic acid molecules. The loop amplification method is different from QUASR (Quenching of Unincorporated Amplified Signal Reporter). This allows real-time detection of the amplified signal. However, a strong signal can be obtained, allowing the method to be performed with low-cost equipment.

[0008] Thus, the present invention provides a loop primer (e.g., a fluorophore-labeled primer) The present invention provides a method for detecting one or more target nucleic acids present in a sample using the method. The fluorophore-labeled primer is complementary to the target nucleic acid and is coupled to a biosensor, e.g. The 5' end or its vicinity of the primer sequence is not modified with a fluorophore or quencher molecule. and a fluorophore-labeled oligonucleotide having a continuous loop sequence internally labeled near The fluorophore-labeled primer is attached to the 5' end or Near the quencher or fluorophore, respectively, the In some embodiments, the fluorophore-labeled primers The fluorophore and quencher are attached at or near the 5' end of the added loop sequence. The fluorophore-labeled primer is labeled at an internal site having the following structure: and a first clamping sequence at the 3' end of the loop sequence (at the intersection with the This sequence may overlap with the unmodified primer sequence and may be It may be directly adjacent to the unmodified primer sequence or may be separated from the unmodified primer sequence. The sequences may be spaced apart by dNTPs, locked nucleic acids, or any other form of nucleic acid. It may include modifications or substitutions.

[0009] The melting temperature of the clamping sequence is preferably selected according to the uptake sequence using a strand-displacing polymerase. Approximately 10°C higher than the extension temperature of the assay but lower than or equal to the extension temperature of the assay or any amount higher.

[0010] If the melting temperature of the clamping sequence is lower than the extension temperature of the reaction, real-time detection can be achieved by: This method involves end point detection (cooling the reaction near or below the Tm of the clamping sequence). In this case, the loop primer is used at maximum strength (unmodified primer). Even when the primer is replaced 100% with a loop primer analog, the reaction is not inhibited. stomach.

[0011] If the melting temperature of the clamping sequence is equal to the extension temperature of the reaction, real-time detection is still possible. However, higher background may be observed until the reaction is cooled for endpoint determination. There may be some do-fluorescence.

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

[0013] Fluorophore-labeled primers are F when the primer is in a linear (extended) conformation. To inhibit RET, the 5'-terminus quencher or fluorophore is placed at an appropriate distance from the fluorophore. Separation of the internally conjugated fluorophore or quencher, resulting in the fluorescence The spacing sequence may be of any sequence and length. and can contain deoxyribonucleotides, locked nucleic acids, etc. Its length is It may be 25 nucleotides, but may be shorter or longer.

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

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

[0016] The looped fluorophore-labeled primers have further applications, e.g., nucleic acid capture, molecular To allow for barcoding, magnetic separation, column purification, and electrophoretic separation, The substrate may further comprise a sequence, molecule, purification tag, bead or other moiety comprising: Patterned probe capture oligonucleotides are used to capture the amplification products, which can produce fluorescent, colorimetric, luminescent, or other bands or zones.

[0017] Loop primers are often used to minimize costs or to increase sensitivity and specificity. To this end, the assay can be titrated to various degrees.

[0018] The loop primers described herein can be used with sensor molecules other than fluorophores, e.g., luminescence, color, change or other measurable signal when in close proximity or when moved far enough apart In some cases, a biosensor can be designed using the reporter molecules provided. For example, NanoLuc, Nanobit, NonoBRET can be used. .

[0019] When photoproteins are used, a decrease in signal can be an important indicator of a positive reaction. In some embodiments, a bioluminescent endpoint assay can be performed.

[0020] Amplification of the target sequence can be achieved using enzymes capable of strand displacement. Other reagents may also be used as required by the method.

[0021] The methods provided herein are directed to high background genomic DNA with high concentrations of non-target RNA or DNA. This allows for specific detection of fluorescent signals under standard conditions, thus eliminating false positives and non-specific amplification detection. Fluorescence detection can be used to detect amplicons that incorporate labeled primers and products. This allows for the specific detection of only those substances present in crude or unprocessed samples, e.g., genital tract Whether using swabs, feces, saliva, urine, blood, plant material, soil or environmental samples; Specific detection becomes possible.

[0022] In some embodiments, the methods involve the detection of two or more unique nucleic acid targets. In such a dual, triple or higher multiplex LAMP assay, The targets can be differentially labeled. For example, one target is labeled with FAM and the other with In some cases, detection has the potential to further distinguish true positives from false positives. To reduce the number of labeled primers required for sequencing, multiple labeled primers are used to detect a single nucleic acid target. In some cases, detection is performed using a single label, e.g. For example, using FAM, the identity of each target can be determined in real time or by ELISA, depending on the specific circumstances of the assay. Based on analysis of the endpoint signal (e.g., relative signal strength, time to result, etc.) In some cases, multiplexing can be achieved by performing reactions in physical reaction chambers. In some cases, multiplexing can be achieved within a single reaction chamber.

[0023] The method provided herein is advantageous compared to other real-time LAMP displacement probe techniques. This minimizes inhibition, making the method highly sensitive. Titration into the assay ensures that the complete reaction rate is The results demonstrate that at least 50% of the loop primer displacements are maintained. Titration to ensure full reaction velocity is maintained for at least 25% of the inner primer displacements. This demonstrates that

[0024] The loop primers provided herein can be used in combination with a strand-displacing polymerase, such as Geobacterium taurate. Geobacilus stearothermophilus (formerly Bacillus stearothermophilus) Polypeptides isolated or adapted from Bacillus stearothermophilus It can be used for loop-mediated amplification (LAMP) using ribosomal enzymes. The loop primers are forward inner primer, backward inner primer, Loop forward primer, loop backward primer, F3 primer and B3 It can be used together with other primers for LAMP, such as primers.

[0025] In some embodiments, the primers and optionally other reaction components are non-limiting. The dried ply is not dried, but may be dried using a process such as freeze drying. The mers can be included in diagnostic kits. In a preferred embodiment, the process of lyophilization does not affect the sensitivity of the LAMP primer set.

[0026] In one aspect, the present disclosure provides a loop for loop-de-loop amplification (LdL) of a target sequence. A primer having a first sensor molecule and a first clamping molecule in a 5' to 3' direction. spacing oligonucleotides e) and a second clamping oligonucleotide, , a first clamping oligonucleotide, a spacing oligonucleotide and a second The clamping oligonucleotide comprises a first clamping oligonucleotide and a second clamping oligonucleotide. A second clamping agent capable of forming a hairpin structure at a temperature lower than the melting temperature (Tm) a sensing oligonucleotide and a second sensor molecule, The sensor molecule is a first biosensor pair, and the second sensor molecule is a first binding site on the target sequence. a first primer sequence complementary to the site; and a loop primer comprising:

[0027] In some embodiments, the second clamping oligonucleotide is a first clamping oligonucleotide. The nucleic acid sequence is complementary to the nucleic acid sequence of the nucleic acid fragment.

[0028] In some embodiments, the first biosensor pair is an energy donor and receptor pair. In some embodiments, the first biosensor pair is a fluorescent sensor that detects fluorescence resonance energy transfer (FR Energy donors and acceptors for bioluminescence resonance energy transfer (BRET) or bioluminescence resonance energy transfer (BRET) In some embodiments, the first sensor molecule is a FRET fluorophore. and the second sensor molecule is a FRET quencher. In some embodiments, the first The sensor molecule is a FRET quencher and the second sensor molecule is a FRET fluorophore. In some embodiments, the first sensor molecule is a BRET energy donor. and the second sensor molecule is a BRET energy acceptor. The first sensor molecule is a BRET energy acceptor and the second sensor molecule is a BRET energy In some embodiments, the first sensor molecule and the second sensor molecule are A complex can be formed that produces a detectable optical signal.

[0029] In some embodiments, the melting temperatures of the first and second clamping oligonucleotides (Tm) is greater than 60° C. In some embodiments, the first and second clamping options The melting temperature (Tm) of the oligonucleotide is greater than 65° C. The melting temperatures (Tm) of the first and second clamping oligonucleotides are above 70°C. In some embodiments, the melting temperatures ( In some embodiments, the first and second clamping oligurines have a Tm of greater than 80° C. The melting temperature (Tm) of the oligonucleotide is between 70 and 80° C. In some embodiments, The melting temperatures (Tm) of the first and second clamping oligonucleotides are from 72.5 to 77.5° C. In some embodiments, the first and second clamping oligonucleotides The melting temperature (Tm) of the oxidase is about 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 , 60 to 65°C.

[0030] In some embodiments, the first clamping oligonucleotide and the second clamping oligonucleotide In some embodiments, the nucleic acid sequence of the nucleic acid fragment is 3 to 10 nucleotides in length. , the first clamping oligonucleotide and the second clamping oligonucleotide In some embodiments, the first clamping oligo is 3 to 7 nucleotides in length. The nucleotide and the second clamping oligonucleotide are 6 nucleotides in length. In some embodiments, the spacing oligonucleotide is 5 to 35 nucleotides. In some embodiments, the spacing oligonucleotide is 10 to 20 In some embodiments, the spacing oligonucleotide is In some embodiments, the spacing oligonucleotide is 13 to 18 nucleotides in length. The fragment is 13 nucleotides in length. In some embodiments, the first clamping The oligonucleotide, the spacing oligonucleotide and the second clamping oligonucleotide The nucleotides are, in total, 15 to 35 nucleotides in length. A first clamping oligonucleotide, a spacing oligonucleotide and a second clamping oligonucleotide. The ramping oligonucleotides are, in total, 20 to 30 nucleotides in length. In some embodiments, a spacing oligonucleotide and a second clamping oligonucleotide are The nucleotides are, overall, 23 to 28 nucleotides in length.

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

[0032] In some embodiments, the loop primer comprises a first In some embodiments, the loop primer further comprises an additional oligonucleotide. a second additional interferometer between the first sensor molecule and the first clamping oligonucleotide; It further comprises an oligonucleotide.

[0033] In some embodiments, the first or second additional oligonucleotide comprises a barcode. It is an array.

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

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

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

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

[0038] In another aspect, the present disclosure provides a method for the detection of a target sequence comprising the loop primer provided herein. Provide a primer mix for loop-de-loop amplification in the column.

[0039] In some embodiments, the primer mixture comprises: (i) a forward inner primer (FIP), (ii) backward inner primer (BIP), (iii) forward The primer further comprises a forward primer (F3) and a backward primer (B3), and the primers are FIP, BI P, F3 and B3 bind to six different binding sites on the target sequence. In this embodiment, the primer mixture comprises: (i) a loop forward primer (LF); and (ii) The loop-backward primer (LB) is included, and LF and LB are two different primers on the target sequence. In some embodiments, FIP, BIP, F3, B3, LF or The LB binds to a first binding site on the target sequence. In some embodiments, the FIP , the amount of FIP and loop primer in the primer mixture that binds to the first binding site 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, the BIP binds to the first binding site and is present in the primer mix. The ratio of the amount of BIP to loop primer was 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21: In some embodiments, the LF is the first binding moiety. The ratio of the amount of LF and loop primer in the primer mixture was 1:1, 2: :1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1.52. In some embodiments, the LB binds to the first binding site and binds to the LB in the primer mixture. The ratio of primer amounts was 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 comprises an oligonucleotide of SEQ ID NO: 1, B3 comprises an oligonucleotide of the sequence The FIP comprises an oligonucleotide of SEQ ID NO: 3; BIP contains the oligonucleotide of SEQ ID NO:4, LF contains the oligonucleotide of SEQ ID NO:6 or LB comprises the oligonucleotide of SEQ ID NO:8.

[0041] In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO: 1 and B3 comprises the sequence The FIP comprises an 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. and LB comprises the oligonucleotide of SEQ ID NO:8.

[0042] In some embodiments, F3 comprises an oligonucleotide of SEQ ID NO: 9, B3 comprises an oligonucleotide of the sequence The FIP contains the oligonucleotide of SEQ ID NO:11. BIP contains the oligonucleotide of SEQ ID NO:12, and LF contains the oligonucleotide of SEQ ID NO:13. The LB comprises the oligonucleotide of SEQ ID NO:14.

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

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

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

[0046] In some embodiments, the primer mix further comprises a second loop primer. The second loop primer is connected to a third sensor molecule and a third clamping oligonucleotide. a second spacing oligonucleotide and a fourth clamping oligonucleotide. A third clamping oligonucleotide, a second spacing oligonucleotide, The third and fourth clamping oligonucleotides are It is possible to form a hairpin structure at a temperature lower than the melting temperature (Tm) of the oligonucleotide. a fourth clamping oligonucleotide, and a fourth sensor molecule, the second sensor molecule and the fourth sensor molecule are a second biosensor pair, and the second biosensor pair is a first A fourth sensor molecule, different from the first biosensor pair, and a first binding site on the second target sequence. and a second primer sequence complementary to the position.

[0047] In some embodiments, the third clamping oligonucleotide is a fourth clamping oligonucleotide. In some embodiments, the target sequence and the second oligonucleotide are complementary to each other. 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 comprises: (i) a second forward inner primer; (ii) second backward inner primer (SBIP), iii) a second forward primer (SF3) and (iv) a second backward primer and SFIP, SBIP, SF3, and SB3 further comprise a second target sequence. It binds to six different binding sites on the array.

[0049] In some embodiments, the primer mix comprises: (i) a second loop forward primer; and (ii) a second loop-backward primer (SLB). In contrast, SLF and SLB bind to two different binding sites on the second target sequence. In some embodiments, the primer mix further comprises a third loop primer, The loop primer comprises a fifth sensor molecule, a fifth clamping oligonucleotide, and The third is a spacing oligonucleotide, and the sixth is a clamping oligonucleotide. a fifth clamping oligonucleotide, a third spacing oligonucleotide, and the sixth clamping oligonucleotide, The sixth nucleotide can form a hairpin structure at a temperature lower than the melting temperature (Tm) of the leucine. a clamping oligonucleotide, and a sixth sensor molecule, and the sixth sensor molecule is a third biosensor pair, and the third biosensor pair is a first biosensor pair. a sixth sensor molecule different from the first biosensor pair and the second biosensor pair; and a third target sequence. and a second primer sequence complementary to the first binding site above. 5 clamping oligonucleotide 6 clamping oligonucleotide In some embodiments, the target sequence, the second target sequence, and the third target sequence are complementary to In some embodiments, the target sequence, the second target sequence, and the third target sequence are identical. In some embodiments, the primer mix comprises: (i) a third forward primer; (ii) the third backward inner primer (TB IP), (iii) the third forward primer (TF3) and (iv) the third backward primer (TF4). TFIP, TBIP, TF3 and TB3 are the first In some embodiments, the primers bind to six different binding sites on the three target sequences. The mixture contains (i) a third loop forward primer (TLF) and (ii) a third loop forward primer (TLF). and a primer for repeating the step backward (TLB), the TLF and the TLB being linked to a third target sequence. In some embodiments, the primer mixture binds to two different binding sites on the array. and a fourth loop primer. In some embodiments, the primer mix comprises , further comprising a fifth loop primer.

[0050] In yet another aspect, the present disclosure provides a method for the preparation of a loop primer or primer combination as provided herein. A dry primer mixture is provided which is obtained by freeze-drying the primer mixture.

[0051] In one aspect, the present disclosure provides a kit for loop-de-loop amplification of a target sequence, comprising: The loop primers, primer mixtures or dry primer mixtures provided herein In some embodiments, the kit further comprises a polymerase, The polymerase optionally comprises Bacillus stearothermophilus. In some embodiments, the kit includes dNTPs, MRNA, and a ribozyme. In some embodiments, the kit further comprises a reverse transcriptase. In some embodiments, the kit further comprises an RNase inhibitor. In some embodiments, the RNase inhibitor is a porcine or murine RNase inhibitor.

[0052] In another aspect, the present disclosure provides a method for detecting a target sequence in a sample, comprising providing the sample. (i) a primer, (ii) a mixture of primers, or (iii) a primer as described herein. A reconstituted primer mixture obtained by rehydrating the dry primer mixture described above. and a polymerase to the sample, thereby forming a reaction mixture; and incubating the mixture at 50 to 85° C. In some embodiments, the incubation is performed at 50 to 70° C. The incubation is carried out at 60-65° C. In some embodiments, the incubation In some embodiments, the polymerase is Bacillus subtilis. The polymerase is derived from Bacillus stearothermophilus. In some embodiments, the method further comprises detecting a signal from the reaction mixture. In some embodiments, the signal is a fluorescent signal. is performed during the incubation step. In some embodiments, the method further comprises: The method further comprises determining the presence or absence of a target sequence in the nucleic acid.

[0053] In some embodiments, the method further comprises the preceding step of preparing a sample. In some embodiments, preparing the sample comprises interacting an RNA molecule with a reverse transcriptase enzyme; In some embodiments, the sample comprises a step of: The step of preparing the RNA molecule comprises preheating the RNA molecule prior to or during interaction with the reverse transcriptase. In some embodiments, the reaction mixture further comprises adding an RNase inhibitor. In some embodiments, the RNase inhibitor is a porcine or murine RNA inhibitor. It is.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0077] The drawings depict various embodiments of the present invention for purposes of illustration only. Therefore, the structures shown herein may be modified without departing from the principles of the invention described herein. It will be readily understood that alternative embodiments of the structures and methods may be used.

[0078] 6. Detailed Description 6.1.Definition Unless otherwise defined, all technical and scientific terms used herein are defined by the principles of the present invention. As used herein, the term has the meaning commonly understood by a person skilled in the art to which the invention pertains. When used herein, the following terms have the meanings ascribed to them.

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

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

[0081] As used herein, the term "LOD" refers to the limit of detection. For example, LOD9 5 is the 95th percentile detection limit. This is the limit at which the assay will give a positive result 95% of the time. The concentration of target that is statistically expected to detect a therapeutic effect.

[0082] 6.2. Other Rules of Interpretation The ranges set forth herein are shorthand for all values ​​within the range, including the endpoints set forth. For example, the range 1 to 50 is understood to be 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 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 500, 510, 520, 530, 540, 550, 560, 570, do.

[0083] Unless otherwise indicated, a reference to a compound having one or more stereocenters refers to each of the All stereoisomers and combinations of stereoisomers are contemplated.

[0084] 6.3. Loop primer In one aspect, the present invention provides loop primers for loop-de-loop amplification. The loop primer is 5' to 3' A first sensor molecule; a first clamping oligonucleotide; a spacing oligonucleotide; A second clamping oligonucleotide, A first clamping oligonucleotide, a spacing oligonucleotide and a second The clamping oligonucleotide comprises a first clamping oligonucleotide and a second clamping oligonucleotide. Melting temperature (T m ) A second clamping agent capable of forming a hairpin structure at a lower temperature than the a binding oligonucleotide; A second sensor molecule, A second sensor, wherein the first sensor molecule and the second sensor molecule are a first biosensor pair. Molecules and a first primer sequence that is complementary to a first binding site on the target sequence; Includes.

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

[0086] The methods provided herein may utilize a variety of biosensors known in the art. For example, it can change color or be measurable when it is in close proximity or when it is far enough away. A pair of molecules that generate a unique signal (e.g., NanoLuc, based on photoproteins, NanoLuc ... obit, NonoBRET technology) can be used.

[0087] In some embodiments, the first biosensor pair is an energy donor and receptor pair. In some embodiments, the first biosensor pair is a Förster resonance energy transfer In some embodiments, the first biosensor is an energy donor and acceptor pair for the activation of the biosensor. The sensor pair is a pair of sensors that utilizes either fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BIT). In some embodiments, the first One sensor molecule is a FRET fluorophore and the second sensor molecule is a FRET quencher. In some embodiments, the first sensor molecule is a FRET quencher. and the second sensor molecule is a FRET fluorophore. In some embodiments, the first The sensor molecule is a BRET energy donor and the second sensor molecule is a BRET energy In some embodiments, the first sensor molecule is a BRET energy receptor. and the second sensor molecule is a BRET energy donor.

[0088] In some embodiments, the FRET quencher is 5'Iowa Black Integrated DNA technologies (trademark) FQ 5'Iowa Black® FQ is 5IABkFQ available from Broad absorbance spectrum ranging from 420 to 620 nm with peak absorbance at 31 nm This quencher is a FRET quencher with the following structure: It can be used with other fluorescent dyes that emit in the pink spectral range. In some embodiments, the quencher is a Black Hole Quencher®. (available from Biosearch Technologies), wa Black® Quencher (Integrated DNA tech available from Integrat Technologies), Zen® Quencher (available from Integrat Technologies, Inc.), ed DNA Technologies), Onyx® Quench Char (available from Millipore-Sigma) or ATTO (registered trademark) (Registered Trademark) Quencher (available from ATTO-TEC Global).

[0089] In some embodiments, the FRET fluorophore is Int 6-FAM (Azid e) available from Integrated DNA technologies i6-FAMK (FAM (fluorescein) azide) is a novel form of FAM. It can be attached to an oligonucleotide using click chemistry. The modified version is linked to the oligo via a dT base. Alternatively, to avoid adding extra nucleotides, In some embodiments, the existing T nucleotide can be replaced with the required modification. The fluorophore is Cy3, Cy5, TAMRA, or Yakima Yellow. (registered trademark) (available from Integrated DNA Technologies ).

[0090] In one embodiment, the loop primer comprises an internal quencher (e.g., Zen® ) or Onyx A®) and a 5' fluorophore (e.g., Yakima Y ellow (registered trademark) or HEX.

[0091] In some embodiments, the first sensor molecule and the second sensor molecule are A complex can be formed that produces a signal.

[0092] The first and second clamping oligonucleotides are complementary to each other and therefore The first clamping oligonucleotide, the spacing oligonucleotide, The nucleotide and the second clamping oligonucleotide are the first and second clamping oligonucleotides. Melting temperature (T m ) The hairpin structure can be formed at a lower temperature. Cut.

[0093] In some embodiments, the melting temperatures of the first and second clamping oligonucleotides (T m In some embodiments, the first and second clamping op . Melting temperature (T m ) is greater than 65° C. The melting temperatures (T m ) exceeds 70°C. In some embodiments, the melting temperatures ( T m ) is greater than 80° C. In some embodiments, the first and second clamping oli Melting temperature of oligonucleotide (T m ) is 70 to 80° C. In some embodiments , the melting temperatures (T m ) from 72.5 77.5° C. In some embodiments, the first and second clamping oligonucleotides The melting temperature of oxalate (T m ) is about 75° C. In some embodiments, the first and second Melting temperature (T m ) is less than 60°C. In this embodiment, the melting temperatures (T m )teeth , 60 to 65°C.

[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 the assay using the strand-displacing polymerase. In some embodiments, the melting temperature is lower than or equal to the extension temperature of the assay, or It can be any amount higher than that.

[0095] T m If is lower than the extension temperature of the reaction, real-time detection is possible at the T m Nearby or T m Cooling the reaction to less than 100% yields end point detection, allowing for full intensity detection. Even when loop-de-loop primers were used (100% substitution), the reaction was inhibited. There is no harm.

[0096] T m If t is equal to the extension temperature of the reaction, 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 is higher than the extension temperature of the reaction, real-time detection may be the basic mode of operation; Background fluorescence is minimal.

[0098] In some embodiments, the first clamping oligonucleotide and the second clamping oligonucleotide In some embodiments, the nucleic acid sequence of the nucleic acid fragment is 3 to 10 nucleotides in length. , the first clamping oligonucleotide and the second clamping oligonucleotide In some embodiments, the first clamping oligo is 3 to 7 nucleotides in length. The nucleotide and the second clamping oligonucleotide are 6 nucleotides in length. In an exemplary embodiment, a first clamping oligonucleotide and a second clamping oligonucleotide are The oligonucleotides have the same length.

[0099] In some embodiments, the spacing oligonucleotide is 5 to 35 nucleotides. In some embodiments, the spacing oligonucleotide is 10 to 2 In some embodiments, the spacing oligonucleotide is In some embodiments, the spacing oligonucleotide is 13 to 18 nucleotides in length. The nucleotide is 13 nucleotides long.

[0100] In some embodiments, the first clamping oligonucleotide, the spacing oligonucleotide The first and second clamping oligonucleotides, in total, are 15 to 35 nucleotides long. In some embodiments, the first clamping oligonucleotide is , the spacing oligonucleotide and the second clamping oligonucleotide are and is 20 to 30 nucleotides in length. In some embodiments, the first clamping The oligonucleotide, the spacing oligonucleotide and the second clamping oligonucleotide The nucleotides are, overall, 23 to 28 nucleotides in length.

[0101] The loop primer consists of (i) adenine, guanine, cytosine, thymine and uracil. (ii) a locked nucleic acid; (iii) a 2'O-methyl RNA base; (iv) phosphorothioated DNA bases, (v) phosphorothioated RNA bases, (v i) phosphorothioated 2'-O-methyl RNA bases, or (vii) combinations thereof. A first clamping oligonucleotide, a spacing oligonucleotide, The clamping oligonucleotide and the second clamping oligonucleotide are (i) adenine, guanine, cytosine (ii) a locked nucleic acid; and (iii) a nucleic acid base selected from 2-amino-3-phenylpropanediol, ... (iv) phosphorothioated DNA bases; (v) phosphorothioated (vi) phosphorothioated 2'-O-methyl RNA bases, or (vii) includes combinations thereof.

[0102] In some embodiments, the loop primer comprises a first In some embodiments, the loop primer further comprises an additional oligonucleotide. a second additional interferometer between the first sensor molecule and the first clamping oligonucleotide; In some embodiments, the first or second further oligonucleotide The oligonucleotide is a barcode sequence.

[0103] In some embodiments, the loop primer comprises a bar at the 5' end of the loop primer. The additional sequence may further comprise a coding sequence, a probe sequence or other sequence. The modifications may include:

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

[0105] In some embodiments, the target sequence is specific for Neisseria gonorrhoeae. In some embodiments, the target sequence is derived from porA or glnA. 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. In one embodiment, the target sequence is derived from tbc1d3. The sequence may be as follows:

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

[0108] In some embodiments, the primer mix includes one loop primer. In some embodiments, the primer mix includes two or more loop primers. When two or more loop primers are included, the primers in the mixture may be directed to a single target sequence or It is capable of binding to multiple target sequences. In some embodiments, multiple loop primers can be used. The mers are designed to detect target sequences from multiple sources. For example, a mixture may include: It contains multiple loop primers designed to detect target sequences from multiple pathogens. This can be done.

[0109] The primer mixture may further comprise additional primers for the amplification reaction. For example, the primer mixture may include: (i) a forward inner primer (FIP); ) backward inner primer (BIP), (iii) forward primer (F3 ) and a backward primer (B3), and B3 bind to six different binding sites on the target sequence. The primer mix consisted of (i) a loop-forward primer (LF) and (ii) a loop-back primer (LF). The primer further comprises a second word primer (LB), and LF and LB are two different primers on the target sequence. In some embodiments, one of the additional primers, e.g., F IP, BIP, F3, B3, LF or LB binds to the first binding site, i.e., It binds to the same binding site as the loop primer.

[0110] In some embodiments, the primer mix comprises one primer set. In some embodiments, the primer set comprises a loop-de-loop amplification sequence as provided herein. (i) a forward inner primer (FIP), (ii) a loop primer for width ) backward inner primer (BIP), (iii) forward primer (F3 ) and (iv) a backward primer (B3). The Immerset contains (i) a loop-forward primer (LF) and (ii) a loop-back primer (LF). It further contains the word primer (LB).

[0111] In some embodiments, the primer set comprises a loop-delta primer set as provided herein. (i) a loop primer for loop amplification and (ii) a forward inner primer (FIP) , (ii) backward inner primer (BIP), (iii) forward primer - (F3) and (iv) backward primer (B3) In some embodiments, the primer set comprises a loop primer, a BIP In some embodiments, the primer set comprises loop primers In some embodiments, the primer set comprises L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L14, L15, L16, L17, L23, L18, L19, L24, L19, L25, In some embodiments, the primers include FIP, BIP, and B3. The kit includes loop primer, FIP, BIP and F3.

[0112] In some embodiments, the primer set comprises a loop-delta primer set as provided herein. (i) a loop primer for loop amplification and (ii) a forward inner primer (FIP) , (ii) backward inner primer (BIP), (iii) forward primer - (F3), (iv) backward primer (B3), (v) loop forward primer (vi) loop backward primer (LB) In some embodiments, the primer set comprises a loop primer In some embodiments, the primer set includes: Examples of primers include loop primers, FIP, F3, B3, LF and LB. In this embodiment, the primer set is loop primer, FIP, BIP, B3, LF and LB. In some embodiments, the primer set comprises a loop primer, a FIP, a B In some embodiments, the primer set includes L1, L2, L3, L4, and L5. In some embodiments, the primers include FIP, BIP, F3, B3, and LF. The primer set includes loop primer, FIP, BIP, F3, B3 and LB.

[0113] In some embodiments, the primer mix comprises two primer sets. In some embodiments, the primer mix includes three primer sets. In an embodiment, the primer mix comprises 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 mixes amplify the same target sequence. In some embodiments, the primers include two or more primer sets for - The mixture contains two or more loop primers that bind to the same binding site on the same target sequence. Including Mar.

[0115] The loop primer is mixed with additional primers in any ratio optimized for the amplification reaction. In some embodiments, the FIP binds to the first binding site and binds to the primer. - The ratio of the amount of FIP and loop primer in the mixture was 0:1, 1:1, 2:1, 3: In some embodiments, the ratio is 1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1. BIP binds to the first binding site, and the BIP and loop primers in the primer mix are The ratio of the amount of mer is 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, In some embodiments, LF binds to the first binding site and the protease activity is 8:1 or 9:1. The ratio of the amount of LF to loop primer in the primer mixture was 0:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1. In this state, the LB binds to the first binding site and binds to the LB and loop primers in the primer mixture. The ratio of the amount of mer is 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 mix comprises a primer specific for Neisseria gonorrhoeae. In some embodiments, F3 is designed to detect a target sequence that is different from the target sequence of SEQ ID NO: B1 contains the oligonucleotide of SEQ ID NO: 1, B2 contains the oligonucleotide of SEQ ID NO: 2, FIP contains the oligonucleotide of SEQ ID NO:3, and BIP contains the oligonucleotide of SEQ ID NO:4. LF comprises an oligonucleotide of SEQ ID NO:6, or LB comprises an oligonucleotide of SEQ ID NO:8. In one embodiment, F3 comprises an oligonucleotide of SEQ ID NO: 1, and B 3 comprises an oligonucleotide of SEQ ID NO: 2, and FIP comprises an oligonucleotide of SEQ ID NO: 3 wherein the BIP comprises an oligonucleotide of SEQ ID NO:4 and the LF comprises an oligonucleotide of SEQ ID NO:6. and LB comprises the oligonucleotide of SEQ ID NO: 8. In one embodiment, the loop primer is an oligonucleotide of SEQ ID NO:5 or SEQ ID NO:7. .

[0117] In some embodiments, the primer mixture is a primer for Chlamydia trachomatis. Some embodiments are designed to detect target sequences specific to A. trachomatis. In the example, F3 contains an oligonucleotide of SEQ ID NO:9, B3 contains an oligonucleotide of SEQ ID NO:10, The FIP comprises an oligonucleotide of SEQ ID NO: 11, the BIP comprises an oligonucleotide of SEQ ID NO: 12 oligonucleotides, LF comprises the oligonucleotide of SEQ ID NO: 13, and In one embodiment, LB comprises the oligonucleotide of SEQ ID NO: 14. In one embodiment, F3 comprises the oligonucleotide of SEQ ID NO: 9 B1 comprises an oligonucleotide of SEQ ID NO: 10, and B2 comprises an oligonucleotide of SEQ ID NO: 11, comprises an oligonucleotide of SEQ ID NO:11, and BIP comprises an 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. In some embodiments, the loop primer comprises an oligonucleotide having the sequence SEQ ID NO:1. 5 oligonucleotide.

[0118] In some embodiments, the primer mix comprises a primer sequence that is specific for Homo sapiens. In some embodiments, F3 is designed to detect the oligonucleotide sequence of SEQ ID NO: 16. B3 contains the oligonucleotide of SEQ ID NO: 17; FIP contains the oligonucleotide of SEQ ID NO: 1 8, BIP comprises an oligonucleotide of SEQ ID NO: 19, L F comprises an oligonucleotide of SEQ ID NO:20, or LB comprises an oligonucleotide of SEQ ID NO:21. In one embodiment, F3 comprises an oligonucleotide of SEQ ID NO: 16, and B3 comprises the oligonucleotide of SEQ ID NO:17, and FIP comprises the oligonucleotide of SEQ ID NO:18. BIP comprises an oligonucleotide of SEQ ID NO:19, and LF comprises an oligonucleotide of SEQ ID NO:20. and LB comprises the oligonucleotide of SEQ ID NO: 21. In some embodiments, the loop primer is an oligonucleotide of SEQ ID NO:22.

[0119] In some embodiments, the primer mixture is a primer for detecting a target sequence specific to a virus. In some embodiments, the virus is SARS-CoV-2. .

[0120] In some embodiments, the primer mixes provided herein comprise a primer mixture that is capable of binding to multiple target sequences. In some embodiments, the multiple target sequences are further combined for detection of different For example, the multiple target sequences are specific for different pathogens.

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

[0122] In some embodiments, the third clamping oligonucleotide is a fourth clamping oligonucleotide. In some embodiments, the third clamping oligonucleotide is complementary to the second clamping oligonucleotide. The oligonucleotide can be bound to a fourth clamping oligonucleotide. but is not perfectly complementary to the fourth clamping oligonucleotide.

[0123] In some embodiments, the primer mixture comprises: (i) a second forward inner primer; (ii) second backward inner primer (SBIP), iii) a second forward primer (SF3) and a second backward primer (S B3), wherein SFIP, SBIP, SF3 and SB3 are 6′-nucleotides on the second target sequence; In some embodiments, the primer mix comprises: (i) a primer mixture that binds to two different binding sites; (ii) a second loop-forward primer (SLF) and (iii) a second loop-backward primer (SLF). The SLF and SLB further comprise a primer (SLB), and the SLF and SLB bind to two different sequences on the second target sequence. It binds to the binding site.

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

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

[0126] In some embodiments, the primer mixture comprises: (i) a third forward inner primer; (ii) the third backward inner primer (TBIP), iii) a third forward primer (TF3) and a third backward primer (TF B3), wherein TFIP, TBIP, TF3 and TB3 bind to a 6′ amino acid sequence on a third target sequence. It binds to two different binding sites.

[0127] In some embodiments, the primer mix comprises: (i) a third loop forward primer; (ii) a third loop backword primer (TLB), LF and TLB bind to two different binding sites on a third target sequence.

[0128] In some embodiments, the primer mix contains two, three, four, five or six groups. The primer mixture contains two or more loop primers. In this case, each loop primer is a unique biotinylated molecule that provides a unique signal for detection. In some embodiments, each biosensor pair may include a fixed sensor for detection. In some embodiments, each biosensor pair provides a visual signal (e.g., color) that is representative of the sensor. , which contains unique dye molecules.

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

[0130] In some embodiments, the primer mixtures provided herein are lyophilized. The dry primer mixture may be any of the looper primers or primer mixtures described herein. In some embodiments, two or more loop plugs may be used. In some embodiments, the primer mixture containing the primer is lyophilized. The mixture is in the form of freeze-dried beads.

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

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

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

[0134] In some embodiments, the kit comprises multiple primer sets in a single container. In some embodiments, the kit comprises a plurality of primer sets, each of which Marset is packaged individually in a separate container.

[0135] In some embodiments, the kit further comprises a polymerase. In some embodiments, the polymerase is a strand-displacing DNA polymerase. The enzyme is produced by Bacillus stearothermophilus In some embodiments, the polymerase is a Bst 2.0 War nStart® 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. In some embodiments, the kit includes an RNase In some embodiments, the RNase inhibitor further comprises a porcine or murine RNase inhibitor. It is an RNase inhibitor.

[0136] In some embodiments, the kit further comprises reagents for an amplification reaction. In some embodiments, the reagents include dNTPs, MgSO4, and a buffer. In some embodiments, the buffer comprises a surfactant. In some embodiments, the composition comprises 4, 5, 6, 7, 8, 9, or 10% Tween-20. In some embodiments, the reagent comprises trehalose. In some embodiments, the reagent comprises sucrose. In some embodiments, the reagents include a polymer for stabilization. The method can be selected and optimized depending on the system.

[0137] In some embodiments, the kit includes dNTPs, MgSO4, buffer, loop deactivation, A mixture comprising one or more primer sets and a polymerase for loop amplification. In some embodiments, the kit comprises dNTPs, one or more primer sets for the The mixture includes an inhibitor.

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

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

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

[0141] In some embodiments, the kit is for diagnosing an infectious disease. The kit is suitable for detecting pathogenic infections, such as Chlamydia trachomatis. tis and Neisseria gonorrhoeae. In some embodiments, the The kit is used for the determination of single nucleotide polymorphisms (SNPs) and point mutations. In some embodiments, the kit is used for determining a mutant genotype. The kit is used to determine a mutant genotype associated with a drug resistance phenotype. For example, drug resistance markers, e.g., ceftriaxone / cefixime resistance markers, quinolones ciprofloxacin resistance marker, macrolide resistance marker (azithromycin ) can be detected.

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

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

[0144] In some embodiments, the method further comprises detecting a signal from the reaction mixture. In some embodiments, the method includes detecting a fluorescent signal. In some embodiments, the method includes detecting a change in color or turbidity. In some embodiments, the method includes detecting a non-visual signal. In some embodiments, the detecting step is performed during the incubation step. , after the incubation step is completed. In some embodiments, the signal is In some embodiments, the signal is recorded in real time and the ink is The incubation process is then completed and the analysis is performed.

[0145] In some embodiments, the method comprises preparing a sample for loop-de-loop amplification. In some embodiments, the step of preparing the sample further comprises the step of: Interacting with a transcriptase, thereby generating a sample containing DNA molecules. In this embodiment, the step of preparing the sample includes prepar- ing a sample containing RNA molecules prior to interaction with the reverse transcriptase. The method further comprises the step of preheating the sample or reaction mixture.

[0146] In some embodiments, the sample for loop-de-loop amplification is a purified polynucleotide. In some embodiments, the sample comprises purified RNA, purified DNA, total SAR, S-CoV-2 virus, whole human cells, saliva or nasal swabs, or nasal or nasopharyngeal specimens In some embodiments, the sample comprises genomic DNA, synthetic DNA, whole bacteria or Total human cells from vaginal swabs. In some embodiments, loop-de-loop amplification In some embodiments, the sample for loop-de-loop amplification is a crude sample. The sample is a purified sample.

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

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

[0149] In an exemplary embodiment, loop-mediated isothermal amplification ("LAMP") involves the loop amplification of a target nucleic acid. De-loop amplification is used in LAMP, which relies on the strand displacement activity of an enzyme known as polymerase. It is a method for isothermal DNA amplification that is dependent on the base specificity of the nucleotide bases in the DNA or It is added to the RNA strand to form a double-stranded nucleic acid with a complementary sequence. Polymers derived from the bacterium Geobacillus stearothermophilus Strand-displacing polymerases, such as Bst polymerase and its variants, polymerize the complementary strand. It displaces one strand of double-stranded DNA when it binds, so does not require thermal cycling.

[0150] The LAMP method was specifically designed to recognize six different regions of the target DNA sequence. Four different primers (F3, B3, inner forward primer or FIP, and Inner backward primers (BIPs) can be used to improve the rate of reaction. To improve the amplification efficiency, two additional "loop" primers may be added. The concentration of primers can vary, but typically is 100 mM for FIP and BIP primers. 0.6 μM for the forward and reverse loop primers (LF, LB), 0.8 μM for the parallel The primers F3 and B3 are set at 0.2 μM. In this embodiment, five primers can be utilized (two possible LAMP primers). The LAMP reaction utilizes a strand displacement reaction to induce a constant temperature The amplification and detection of the target requires the sample, primers, and a PCR product with strand displacement activity. The DNA polymerase, buffer, and substrate are incubated at a constant temperature. A typical mixture composition of LAMP is the following reagents: 20mM Tris-HCL, 10mM(NH4)2SO4, 50mM KCl, 8mM MgSO4, 0.1% Tween® 20, 1.4 mM dNTPs, 0.32 U / μL Bst polymerase, primers at the aforementioned concentrations, and water, pH 20°C. The pH is adjusted to 8.8 by pH adjustment. The reaction volume is typically 5 μL to 50 μL. The reaction time is optimized for the particular enzyme and primers used, and the reaction lasts for 5 to 60 minutes. LAMP has high sensitivity, specificity and efficiency.

[0151] LAMP is a novel method for the identification of 6 target sites (e.g., F3, B3, FIP, and BIP). Relying on at least four primers to amplify a specific DNA or RNA target (RNA The target must first be reverse transcribed into DNA). Loop primers (e.g., LF and L B) is included, a total of eight unique sites in the target nucleic acid are amplified by six primers. In various embodiments provided herein, out of a total of eight unique sites, One of the following can be recognized by the loop primer described herein. If present, an amplification reaction can occur resulting in large amounts of DNA.

[0152] The novel loop-de-loop method described herein is applicable to other isothermal amplification methods besides LAMP. To address the temperature cycle dependency of the polymerase chain reaction (PCR), A number of isothermal amplification methods have been developed. These methods can vary considerably, but they all They share some common characteristics. For example, DNA strands are not heat denatured, so all the Thermal methods rely on alternative approaches to allow primer binding and initiation of the amplification reaction. When the reaction is initiated, the polymerase is also still annealed to the sequence of interest. Isothermal methods are typically used to separate double-stranded DNA. The strand displacement activity of DNA polymerase is used. Polymerases with this ability include: In moderate temperature reactions (25-40°C), Klenow fragment (3'-5' exo-) , Bsu large fragment and phi29, and at higher temperatures (50-65°C) The reaction includes the large fragment of Bst DNA polymerase. For detection, a reverse transcriptase enzyme compatible with the reaction temperature is added to maintain the isothermal nature of the amplification. In addition to the strand displacement mechanism for separating sDNA, isothermal methods eliminate the requirement for initial denaturation for initiation. It may be necessary to design the enzyme or primers to avoid this.

[0153] As mentioned above, loop-mediated isothermal amplification (LAMP) amplifies 6–8 distinct regions of a target DNA. Uses 4-6 primers that recognize the region. Strand-displacing DNA polymerase initiates synthesis. The two primers then form a loop structure to facilitate subsequent amplification. Due to its speed, high sensitivity and extremely large amplification, LAMP is well suited for on-site diagnostics. The loop-de-loop primers may be either single or any combination or inner and / or outer can be used for the loop primer.

[0154] Strand displacement amplification (SDA) is a method using a strand-displacing DNA polymerase, typically Bst DNA polymerase. Reverse transcription by ligase, large fragment or Klenow fragment (3'-5'exo-) The primer is then subjected to a strand-restricting endonuclease or a nicking enzyme at the site contained therein. SDA starts at the nick created by one forward primer and one reverse primer. one bumping forward primer and one bumping reverse primer A reverse primer is required. The nicking site is regenerated at each polymerase displacement step. SDA is typically used in clinical diagnostics. Although 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 creation of restriction endonuclease enzymes to generate fluorescence. Either the forward or reverse SDA primer is paired with a fluorophore. For use in loop-de-loop assays that do not require a cleavage site location between the quencher pair The cleavage site can be adapted to the clamping sequence of the loop-de-loop primer. Next to the row, towards the 3' end of the primer, so that the restriction endonuclease Complete cleavage of the 5' end of the primer by a polymerase on the complementary strand prior to cleavage of the primer by Upon complete elongation, fluorescence is produced.

[0155] Helicase-dependent amplification (HDA) utilizes the double-stranded DNA unwinding activity of helicases. to separate the strands, allowing primer annealing and extension by a strand-displacing DNA polymerase. Similar to PCR, this system uses two primers: one forward primer and one HDA requires only one primer and one reverse primer. The primers in HDA are either loop-coupled or loop-coupled. Adapted for use in De-Loop assays for real-time closed-tube monitoring of reactions In HDA, the helicase enzyme mediates the loop-de-loop folding. The primer loop structure can be opened, which is stabilized by a single-stranded binding protein. This can then be converted into a double-stranded fluorescent amplicon by a DNA polymerase.

[0156] Nicking enzyme amplification reaction (NEAR) is a method to amplify DNA fragments by nicking at the nicks created by nicking enzymes. The method uses a strand-displacing DNA polymerase that initiates a gene transfer reaction to rapidly generate many short nucleic acids from a target sequence. The process is extremely rapid and sensitive, allowing detection of minute amounts of target within minutes. NEAR is commonly used for detecting pathogens in clinical and biosafety applications. Either the forward or reverse primer for NEAR is used. The first method generates real-time fluorescence via loop extension by a strand-displacing DNA polymerase. A loop-de-loop technique can be used to achieve this.

[0157] 6.7.How to use The loop-de-loop amplification methods provided herein detect target sequences from a variety of sources. For example, it can be used to extract viral genomes, bacterial genomes, ancient genomes, Bacterial genome, plant genome, animal genome, protist genome, prokaryotic genome or eukaryotic genome In some embodiments, the method can be used to detect target sequences specific to the genome. The method includes the step of detecting RNA (e.g., positive sense RNA, negative sense RNA) or DNA. In some embodiments, the method is used to detect synthetically produced A. The nucleic acid sequence is used to detect the target sequence.

[0158] In some embodiments, the loop-de-loop method is used to detect pathogen-specific DNA. In some embodiments, the pathogen is a virus, a bacterium, a fungus, a protozoan, or a parasite. In some embodiments, the loop detector is used to detect pathogens associated with STDs. In some embodiments, the pathogen is 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 one embodiment, the loop-de-loop method is used to determine the mutant genotype. In some embodiments, the loop-de-loop method comprises detecting a mutant gene associated with a drug resistance phenotype. For example, drug resistance markers, e.g., ceftriaxone / Cefixime resistance marker, quinolone (ciprofloxacin) resistance marker, macrolide resistance marker It is possible to detect the drug resistance marker (azithromycin).

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

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

[0162] In some embodiments, the loop-de-loop method is used to analyze or characterize a sample. In some embodiments, a loop-de-loop is used for sample application. For example, the loop-de-loop technique is used to identify human samples. Used.

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

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

[0165] Chlamydia trachomatis genomic DNA and Neisseria gonorrhoeae To separately detect C. gonorrhea genomic DNA, a LAMP reaction mixture was prepared. The reaction was prepared in a 10 μL volume and contained the following reagents: 20 mM Tris-HC1 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 (SEQ ID NOs: 1 to 4) with 8 μM LF and LB, and 0.2 μM F3 and B3 , 6, 8-14), 2.5 μM SYTO85 intercalating dye, and water. The target genomic DNA was prepared from stock solutions purchased from ATCC. The target DNA or DNA-containing Add 1 µL of the buffer containing no template (for the no template control) to the 9 µL solution mixture in each PCR tube. The temperature of the reaction was 65°C and the reaction was monitored by SYTO85 fluorescence. A real-time PCR machine was used to heat the reactions and measure fluorescence in real time. The reaction was carried out for 60 min. The data shown in Figure 2A indicates that the intercalating Chlamydia trachomatis monitored by dye ) (green) and Neisseria gonorrhoeae (blue) obtained from LAMP reactions. Representative curves of real-time fluorescence (in arbitrary units) on the vertical axis versus time on the horizontal axis are shown in Three levels of genomic DNA target were used: high (stock concentration), low (Ct = 10 -5 Dilutions, 10 Ng of stock DNA -6 dilution), and no template control (DNA (NTC)).

[0166] 6.8.2. Example 2: Chlamydia trachomatis (Ch) PCR amplification by loop-de-loop amplification Detection of Lamydia trachomatis Prepare the loop-de-loop LAMP reaction mixture to infect Chlamydia trachomatis (Chla The reaction mixture was prepared in a volume of 10 μL and consisted of the following: The following reagents were used: 20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 8 mM MgSO4, 0.1% Tween® 20, 1.4 mM M dNTP, 0.32U / μL Bst2.0 WarmStart® Poly 1.6 μM FIP and BIP, 0.4 μM LF and LF-LdL, 0.8 LB at 0.2 μM, primers (SEQ ID NOs: 9 to 15) with F3 and B3 at 0.2 μM, and The quantified target genomic DNA was diluted with water and adjusted to pH 8.8 at 20°C. The stock solution purchased from CC was diluted 10-fold or 2-fold (more precisely) with Tris-HCL buffer at pH 8.0. For finer resolution, the target DNA dilutions or DNA-free buffer (no template) were diluted (For the control) was added at 1 μL to the 9 μL solution mixture in each PCR tube, and assay Use up to 20 replicates per concentration over a multi-log concentration range to determine sensitivity The reaction temperature was 65 °C, and the F generated by the loop-de-loop primer was The reaction was monitored by AM fluorescence. The reaction was heated and fluorescence was measured in real time. The reaction was run for 60 min. Data shown in Figure 3 Chlamydia trachomatis against 10-fold dilutions of genomic DNA targets Real-time fluorescence (arbitrary units) obtained from loop-de-loop LAMP reactions of P. homatis A representative curve of 100 μm / s is shown versus time on the horizontal axis (each "cycle" represents 30 seconds). Sensitivity (limits of detection, 50% and 95% probability) was determined based on the end point determination of the assay. Estimated by ROBIT analysis. [Table 1]

[0167] 6.8.3. Example 3: Identification of Neisseria gonorrhoeae by Loop-de-Loop Amplification ) detection Prepare loop-de-loop LAMP reaction mixture to detect Neisseria gonorrhoeae Genomic DNA was detected. Reactions were prepared in 10 μL volumes and contained the following reagents: 20mM Tris-HCl, 10mM(NH4)2SO4, 50mM KCl, 8mM MgSO4, 0.1% Tween® 20, 1.4 mM dNTPs, 0.3 2U / μL Bst2.0 WarmStart® Polymerase, 1.6 μM FIP and BIP, 0.4 μM LF and LF-LdL, 0.8 μM LB, 0.2 μ A primer (SEQ ID NOs: 1 to 4, 6 to 8) having F3 and B3 of M, and water The pH was adjusted to 8.8 at 20° C. Quantitated target genomic DNA was purchased from ATCC. The stock solution was diluted 10-fold or 2-fold (for finer resolution) with Tris-HCL buffer at pH 8.0. Target DNA dilutions or DNA-free buffer (for no template controls) were diluted in 1 μL was added to the 9 μL solution mixture in each PCR tube to examine the sensitivity of the assay. A maximum of 20 replicates per concentration were used over a several log concentration range. The temperature was 65 °C, and the FAM fluorescence emitted by the loop-de-loop primer was used to detect the The reaction was monitored using a real-time PCR machine by heating the reaction and Fluorescence was measured in real time. The reaction was carried out for 60 min. Data shown in Figures 2B-2C Loop depletion assay of Neisseria gonorrhoeae against 10-fold dilutions of genomic DNA targets Representative real-time fluorescence (arbitrary units) versus time on the horizontal axis obtained from a loop-LAMP reaction. The curves shown in Figure 2A and Figure 2B show the signals obtained from the loop-de-loop assay. LAM was performed using SYTO85 dye without loop-de-loop primers, as The loop-de-loop assay is much more effective in the case of positive amplification. In Figure 2C, the reproducibility of the loop-de-loop assay as well as the Negligible background fluorescence and reduced late spurious amplification products in untyped controls were observed. The data shown in Figure 4 shows the results of the Neisseria gonorrhoeae assay against 10-fold dilutions of genomic DNA targets. Real-time fluorescence obtained from the loop-de-loop LAMP reaction of N. norrhoeae (any single A representative curve of the abscissa (position) versus the ordinate (time) (each "cycle" represents 30 seconds) is then shown. Assay sensitivity (limits of detection, 50% and 95% probability) was determined as P based on the endpoint determination of the assay. The estimate was based on 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 2. Preparing the Loop-de-Loop LAMP Reaction Mixture to Probe Homo sapiens Genomic DNA Reactions were prepared in 10 μL volumes and contained the following reagents: 20 mM Tris 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 A solution containing primers (SEQ ID NOs: 16 to 22) and water, maintained at 20°C and pH 8. The quantified target genomic DNA was diluted to pH 8.0 from the stock solution purchased from ATCC. The samples were diluted 10-fold or 2-fold (for finer resolution) with 10% Tris-HCL buffer. Add target DNA dilutions or DNA-free buffer (for no template controls) to each PCR tube. Add 1 μL of the solution to a 9 μL mixture and measure the sensitivity of the assay over a several log concentration range. A maximum of 20 replicates per concentration were used over the entire reaction time. Reactions are monitored by FAM fluorescence emitted by the loop-de-loop primer. A real-time PCR machine was used to heat the reactions and measure fluorescence in real time. Reactions were run for 60 minutes. The data shown in Figure 3 was obtained for a 10-fold dilution of genomic DNA target. Real-time fluorescence obtained from loop-de-loop LAMP reaction in Homo sapiens against A representative curve of (arbitrary units) versus time on the horizontal axis (each "cycle" represents 30 seconds) is shown. Assay sensitivity (limits of detection, 50% and 95% probability) is then determined for the endpoint determination of the assay. Based on this, estimates were made using PROBIT analysis. [Table 3]

[0169] 6.8.5. Example 5: Dry primer mix for loop-de-loop amplification Formulation into lyophilized reagents was performed in-house using a five-step lyophilization protocol. A loop-de-loop test kit designed to detect Neisseria gonorrhoeae The LAMP reaction mixture was prepared at 25 μL volume / tube and dispensed into each tube. The dry mixture contained the following reagents: 1.4 mM dNTPs, glycerol-free preparation. 0.32U / μL Bst 2.0 as a reagent WarmStart® polymer enzyme, 1.6 μM FIP and BIP, 0.4 μM LF and LF-LdL, 0.8 μM M LB, 0.2 μM F3 and B3 primers (SEQ ID NOs: 1 to 4, 6 to 8), The tubes contained 5% trehalose and water (up to 25 μL per reaction). The lids were removed for lyophilization. The tube strips were The samples were placed on metal shelves in a heated shelf freeze dryer unit, which is standard equipment in the industry. The dryer was programmed to run in 5 stages: Stage 1: Condenser ON, Vacuum OFF, Shelves and cool reagents to 41°F, 30 minutes. Stage 2: Condenser ON, vacuum OFF, shelves and reagents 2 Cool to 3F, 30 minutes. Stage 3: Condenser ON, vacuum OFF, cool shelves and reagents to -23F, 2 hours. Stage 4: Condenser ON, vacuum ON, shelves and reagents maintained at -23F, 10 hours. Stage 5: Condenser ON, vacuum ON, heat shelves and reagents to 77F, 5 hours. Once complete, the tubes were removed and capped to give the products shown in Figure 7. Lyophilization Assay The activity of was tested over a period of time after incubation in various environmental conditions. 8 shows a representative real-time loop-de-loop LAMP assay activity of the rehydration reaction. The rehydration protocol consisted of adding 24 μL of rehydration buffer to 1 μL of Neisseria gonorrhoeae. eae) consisted of adding the target genomic DNA to the dry reagents. The rehydration buffer was , 20mM Tris-HCL, 10mM(NH4)2SO4, 50mM KCl, 8m MgSO4, 0.1% Tween® 20 and water, pH 20 The temperature was adjusted to 8.8°C. Buffer was added to the tube, which was then resealed and vortexed or The reaction mixture was placed directly into the real-time qPCR machine without mixing. The reaction temperature was 65°C. The reaction was monitored by FAM fluorescence emitted by the loop-de-loop primer. A real-time PCR machine was used to heat the reaction and monitor the Fluorescence was measured. Reactions were run for 60 minutes. The data shown in Figure 8 shows the 100% genomic DNA target. Loop-de-loop LAMP reaction of Neisseria gonorrhoeae against 10-fold dilution Real-time fluorescence (arbitrary units) obtained from the experiment versus time on the horizontal axis (each "cycle" represents 30 seconds). Representative curves of the lyophilized loop-de-loop assay are shown. was found to be comparable in speed, sensitivity and specificity to the newly formulated assay. Furthermore, the magnitude of fluorescence is not affected by drying and rehydration, making the loop-de-loop method It has been shown that it is possible to provide 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, and one was used to amplify the ORF1ab gene. The other contains the POP7b primer set. Marset is a SARS-CoV-2 virus with a positive-sense single-stranded RNA genome. The POP7b primer set is specific for This primer set is specific for human RNA targets and therefore detects human R In both cases, loop-de-loop is used as a specific indicator of NA. In order to achieve this, one of the six component primers used in LAMP was modified to insert the seventh loop. The loop primer uses a pair of a fluorophore and a quencher. In this experiment, the sequence of the RNA target for each primer set was Moderately concentrated synthetic double-stranded DNA containing sequences on their positive-sense strands that correspond to the sequences The template was used as a target for LAMP reaction temperature optimization to minimize the reverse transcription step or to minimize the chance of encountering a rare target. The target DNA dilutions were then placed in a 384-well plate to minimize variability due to random noise. They were then incubated at various temperatures ranging from 55 to 70°C. The reaction was monitored by FAM fluorescence emitted by the loop-de-loop primer. A real-time PCR machine was used to heat the reaction and measure the fluorescence in real time. The light was measured. The reaction was carried out for 60 min. Two runs were performed over overlapping temperature ranges (first run The data are shown in Figures 9A and 9B. The results show that the primer set can be used at a wide range of temperatures to obtain sufficient signal. For example, the activity of POP7b was observed at temperatures between 57 and 70°C. Both the Immer and ORF1ab primer sets were within acceptable ranges. Optimal performance was achieved between ℃ and 68℃.

[0171] 6.8.7. Example 7: Multiplexed Detection of Both Purified Targets and Targets in Crude Samples Loop-de-loop reaction FIG. 14A, FIG. 14B, and FIG. 14C show ORF1ab and POP7b LdL, respectively. Loop-de-loop sequencing of SARS-CoV-2 and human target sequences using primer sets Two fluorescent signals obtained from the amplification are shown. SARS-CoV-2 ORF1ab (FAM ) and the signal obtained from the POP7b human internal control (Cy5). Sample (no template control) (Figure 14A), human nasal swab (Figure 14B), and heat-inactivated virus 3. Human nasal swabs (Figure 14C) combined with SARS-CoV-2 target sequences in the form of Two types of samples were used: human nasal swabs were self-collected from volunteers and no sample processing or nucleic acid analysis was performed. The swab was added directly to the reaction without extraction. The reaction mixture was stirred by twisting the swab for a few seconds. The SARS-CoV-2 target sequence was added to the SARS-CoV-2 positive reaction. The reaction was carried out as intact heat-inactivated virus (ATCC VR-1986HK) added to the The primers of ORF1ab LdL-FAM and POP7b LdL-Cy5 were introduced into the The primers were duplexed at a 1:1 ratio in the replication reaction volume. The assay utilized LdL primers at a ratio of 1:3 to unlabeled primer analogues (25% intensity). The reaction contained reverse transcriptase, strand-displacing polymerase, and RNase inhibitor. Using a real-time PCR device (Bio-Rad CFX-384®), The reaction was incubated at 55.6°C for 2.5 min while recording FAM and Cy5 fluorescence measurements. The reaction was incubated at 63.5°C for 60 minutes. As shown, replicates of the no-template control did not show any loop-de-loop fluorescent signal over the 60 min period. Reactions with COVID-19 negative nasal swab samples were evidenced by an increase in Cy5 fluorescence. As shown, we showed an amplification of the POP7b signal, whereas the ORF1ab signal remained flat. The samples spiked with heat-inactivated virus were negative for both RNAs in a single reaction vessel. The results showed that the loop-de-loop RT-LAM P enables single-tube spectral multiplexing of SARS-CoV-2 and human targets This shows that.

[0172] Multiple loop DNA sequencing of SARS-CoV-2 using ORF1ab LdL primer set The DeLoop test was as sensitive as the PCR test and did not require extraction. The reaction with the crude sample gave good results as shown in the table below. The 7b LdL primer set was used as an internal control. Intact heat-inactivated SA Serial dilutions of RS-CoV-2 virus (ATCC VR-1986HK) were The assay was then added to the de-looping reaction and monitored for real-time signal generation. The detection limit for a particular format of the test kit is LoD95=400cp / s Wab = 2.7 x 10 3 was estimated to be cp / mL. [Table 4]

[0173] A triplex loop-de-loop reaction was also tested in a single tube. It has the specificity of three targets. The assay showed differential amplification and maintained a fast time to result. The two loop-de-loop primer sets were used to detect SARS-CoV-2 virus. The two separate targets of the RNA were detected by Cy5-labeled human internal control loop determinants. A loop primer set detected a third RNA target. An internal Cy5 fluorophore The reaction was paired with a 5' Iowa Black® RQ quencher. Contains wabu eluate and spiked with heat-inactivated SARS-CoV-2.

[0174] For use with the loop-de-loop primer set for the POP7b human internal control, Additional loop primers were also tested. In one case, a second sensor molecule, the internal TAM The RA fluorophore was attached to the first sensor molecule, 5'Iowa Black® In another case, the first sensor molecule, 5'Yakima Yellow® (Epoch Biosciences) as the second sensor. The molecule, Zen™ (Integrated DNA Technology es) paired with a quencher. Regarding the placement of Yakima Yellow and Zen Three variations of the loop primer were made and tested. The first clamping oligonucleotide and the second clamping oligonucleotide are completely The spacing oligonucleotides were 13 bases long. In a second variation, the first clamping oligonucleotide had a length of 1:1 at its 5' end. The first clamping oligonucleotide is characterized by an additional base at the end, so that the first clamping oligonucleotide is 7 bases long. The first clamping oligonucleotide was 1 base long and the second clamping oligonucleotide was 6 bases long. Six complementary sequences were inserted between the clamping oligonucleotide and the second clamping oligonucleotide. The spacing oligonucleotide was 13 bases long. In the example, the first and second clamping oligonucleotides are both 7 bases long and have the sequence were perfectly complementary. The spacing oligonucleotide was 10 bases long.

[0175] These additional loop primers are used in the LAMP reaction, which is a step to amplify the target sequence. A sequence specific amplification signal is provided.

[0176] 6.8.8. Example 8: SARS-CoV in human samples by loop-de-loop amplification -2 detection Prepare loop-de-loop LAMP reaction mixture to isolate S from unprocessed human saliva. ARS-CoV-2 was detected. Lyophilized enzyme, dNTP, and oligonucleotide primers The immersion mixture was rehydrated with a 10% volume / volume mixture of human saliva in a pH-buffered salt solution. Reactions were prepared in PCR tubes by adding 100 µL of lyophilized primer mix to the PCR tubes. It contained primer sets for SARS-CoV-2 and human internal control RNA sequences. After rehydration with the liquid sample, the reaction is incubated at the preheated temperature for a specified period of time to allow for viral lysis. lysis, RNase inhibition, and reverse transcription, followed by higher efficiency for LAMP DNA amplification. Custom equipment was used to obtain temperature-controlled data and real-time Time fluorescence data was collected.

[0177] Heat-inactivated SARS-CoV-2 was spiked into pools of fresh saliva collected from anonymous donors. Three-fold serial dilutions of saliva were prepared. 20 samples were amplified using the loop-de-loop amplification method. Samples were tested using the mobile application, visual inspection, or real-time curve inspection. The readouts are summarized below. These results indicate that the LoD is approximately 2,500 cp / mL. This shows that: [Table 5]

[0178] Self-collected nasal swabs were obtained from volunteer subjects and reversed by twisting 10 times. Figure 16 shows that the IgG-positive individuals were later confirmed as COVID-positive by PCR. 4 shows amplification results from nasal swabs obtained from symptomatic volunteers with confirmed SARS-CoV-2. The results from the positive and negative control samples are also shown. These results were obtained with the samples (1× swab). (B) was 365 times higher than the concentration required to detect a positive sample in the loop-de-loop assay. The LoD is estimated to be about 2,500 cp / mL. Therefore, the specific sample is about 9.1 × 10 5 SARS-CoV-2 virus RN in cp / mL It was assumed to contain A.

[0179] FIG. 17 shows the amplification results obtained from nasal swabs obtained from negative volunteers. The patient was detected as negative by both loop-de-loop reaction and PCR testing.

[0180] The reaction mixture for detecting SARS-CoV-2 was prepared by combining human genome sequences in a 1:1 ratio. The results of the multiplexed amplification are shown in FIG. The results show specific and sensitive detection of two target sequences without cross-reactivity.

[0181] 6.8.9. Example 9: Isolation of Chlamydia trachomatis in human samples by loop-de-loop amplification Detection of Chlamydia trachomatis and Neisseria gonorrhoeae Three vaginal swabs (BD BBL Culture Swabs, sourced from unique individual donors, 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, The fluid was eluted in 294 μL of rehydration buffer (431 μL per swab). After 10 min of fluid loss, 1095 μL of pooled vaginal swab eluate was obtained. Fluid recovery was 85%. The swab eluates were used for loop-de-loop LAMP (one for Ct and one for One for Ng and one for human process control) were used to make injection molded prototypes containing lyophilized reaction mixtures. The solution was pipetted into a disposable container.

[0182] Each reaction was rehydrated with: · 18 μL swab eluate (swab swirled in rehydration buffer); 1 μL of total Ct pathogen suspended in rehydration buffer 1 μL of total Ng pathogen suspended in rehydration buffer The Ct and Ng pathogen samples were placed in separate disposable reaction chambers. Thus, for example, the Ct assay detects all IgG and human targets and IgG present in the swab sample. The objective of the study was to detect Ct in the simultaneous presence of various bacterial environments.

[0183] The amplification results are shown in Figures 19 to 22. Figure 19 shows the results of a large amount of Ct (10,000 per reaction). copy equivalents) and a large amount of Ng (10,000 copy equivalents per reaction). Figure 20 shows the fluorescence signal obtained from the negative control - swab only control (left two panels) ) or buffer only controls (right two panels). There was no amplification of Ct or Ng in the control, but the human genomic sequence was amplified as expected. There was no amplification of Ct or Ng in the solution-only control. Amplification of Homo sapiens was observed in one buffer. In the reaction with only buffer (Test 20), the signal was detected later. , this is likely a spurious amplification.

[0184] These results demonstrate that the assay has a Ct of approximately 100 copies per reaction and These results show that the antibody was sensitive enough to detect more than approximately 1,000 copies of Ng. 7. Arrays [Table 6]

[0185] 8. INCORPORATION BY REFERENCE All publications, patents, patent applications, and other references cited in this application are hereby expressly incorporated by reference in their entirety as if each was individually incorporated by reference. Any publications, patents, patent applications or other documents are incorporated by reference for all purposes. This document is hereby incorporated by reference in its entirety for all purposes to the same extent as if each individual INCORPORATED INTO THE SPECIFICATION.

[0186] 9. Equivalents The present disclosure provides, inter alia, compositions of cannabinoids and associated compositions. In addition, the cannabinoid composition and the associated composition are administered to treat neurodegenerative diseases. Although various specific embodiments have been illustrated and described, the above specification Various modifications may be made without departing from the spirit and scope of the present invention. It will be understood that many variations will become apparent to those of skill in the art upon review of this specification. It will become clear.

Claims

1. A loop primer for loop-de-loop amplification (LdL) of a target sequence, comprising: From 'to 3', A first sensor molecule; a first clamping oligonucleotide; a spacing oligonucleotide; A second clamping oligonucleotide, The first clamping oligonucleotide, the spacing oligonucleotide, and and the second clamping oligonucleotide, The melting temperature (T ｍ ) can form hairpin structures at lower temperatures a second clamping oligonucleotide; and A second sensor molecule, The first sensor molecule and the second sensor molecule are a first biosensor pair. and a sensor molecule of a first primer sequence complementary to a first binding site on the target sequence; A loop primer comprising:

2. The second clamping oligonucleotide is The loop primer of claim 1 which is complementary to the oxidase.

3. 3. The method according to claim 1, wherein the first biosensor pair is an energy donor and receptor pair. The loop primer according to claim 1.

4. The first biosensor pair is a pair of sensors that uses fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRE).

3. A pair of an energy donor and an energy acceptor for BRET. The loop primer described.

5. The first sensor molecule is a FRET fluorophore and the second sensor molecule is a FRET fluorophore. The loop primer of claim 3 which is an ET quencher.

6. The first sensor molecule is a FRET quencher and the second sensor molecule is a FRE The loop primer of claim 3, wherein the T fluorophore is a T fluorophore.

7. The first sensor molecule is a BRET energy donor and the second sensor molecule is a B The loop primer of claim 3 which is a RET energy acceptor.

8. The first sensor molecule is a BRET energy acceptor and the second sensor molecule is a B The loop primer of claim 3 which is a RET energy donor.

9. The first sensor molecule and the second sensor molecule form a composite that generates a detectable optical signal. The loop primer of claim 1 , which is capable of forming a body.

10. The melting temperatures (T ｍ ) exceeds 60°C The loop primer according to any one of claims 1 to 9.

11. The melting temperatures (T ｍ ) exceeds 65°C The loop primer according to claim 10.

12. The melting temperatures (T ｍ ) exceeds 70°C The loop primer according to claim 11 .

13. The melting temperatures (T ｍ ) exceeds 80°C The loop primer according to claim 12.

14. The melting temperatures (T ｍ ) from 70 to 8 The loop primer of claim 12, wherein the temperature is 0°C.

15. The melting temperatures (T ｍ ) is 72.5 The loop primer according to claim 13, wherein the temperature is from 77.5°C to 77.5°C.

16. The melting temperatures (T ｍ ) at about 75°C The loop primer according to claim 14 .

17. The melting temperatures (T ｍ ) is less than 60°C The loop primer according to any one of claims 1 to 9,

18. The melting temperatures (T ｍ ) from 60 to 6 The loop primer according to any one of claims 1 to 9, wherein the temperature is 5°C.

19. The first clamping oligonucleotide and the second clamping oligonucleotide The loop according to any one of claims 1 to 18, wherein the nucleotide is 3 to 10 nucleotides in length. P-primer.

20. The first clamping oligonucleotide and the second clamping oligonucleotide 20. The loop primer of claim 19, wherein the nucleotide is 3 to 7 nucleotides in length.

21. The first clamping oligonucleotide and the second clamping oligonucleotide 21. The loop primer of claim 20, wherein the nucleotide is 6 nucleotides in length.

22. 1 , wherein the spacing oligonucleotide is 5 to 35 nucleotides in length.

22. A loop primer according to any one of claims 1 to 21.

23. The spacing oligonucleotide is 10 to 20 nucleotides in length.

23. A loop primer according to 22.

24. The spacing oligonucleotide is 13 to 18 nucleotides in length.

24. A loop primer according to 23.

25. 25. The method of claim 24, wherein the spacing oligonucleotide is 13 nucleotides in length. The loop primer is shown.

26. the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide, in total, is 15 to 35 nucleotides. The loop primer according to any one of claims 1 to 25,

27. the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide, in total, is 20 to 30 nucleotides long.

27. The loop primer of claim 26,

28. the first clamping oligonucleotide, the spacing oligonucleotide, and the second clamping oligonucleotide, in total, is 23 to 28 nucleotides long.

28. The loop primer of claim 27,

29. The first clamping oligonucleotide, the spacing oligonucleotide, and and the second clamping oligonucleotide is selected from the group consisting of (i) adenine, guanine, and cytosine. , thymine and uracil; (ii) a locked nucleic acid; (iii) a 2' (iv) phosphorothioated DNA bases; (v) phosphorothioated (vi) phosphorothioated 2'-O-methyl RNA bases, or (vii) The loop according to any one of claims 1 to 28, including combinations thereof. Rhymer.

30. further comprising a first additional oligonucleotide at the 5' end of said loop primer; A loop primer according to any one of claims 1 to 29.

31. A second further intervening region is provided between the first sensor molecule and the first clamping oligonucleotide. The loop according to any one of claims 1 to 30, further comprising an oligonucleotide consisting of Primer.

32. 31. The method of claim 30, wherein the first or second additional oligonucleotide is a barcode sequence. Or the loop primer according to 31.

33. The method according to any one of claims 1 to 32, wherein the target sequence is specific to a pathogen genome. Loop primer.

34. 34. The loop of claim 33, wherein the target sequence is specific for Chlamydia trachomatis. Primer.

35. 35. The loop primer of claim 34, wherein the target sequence is derived from orf8 or cds2. -

36. 35. The loop primer of claim 34, comprising the oligonucleotide of SEQ ID NO:

15.

37. 34. The loop primer of claim 33, wherein the target sequence is specific for Neisseria gonorrhoeae.

38. 38. The loop primer of claim 37, wherein the target sequence is derived from porA or glnA. Mah.

39. The loop fragment according to claim 37, comprising an oligonucleotide of SEQ ID NO: 5 or SEQ ID NO:

7. Rhymer.

40. 34. The loop primer of claim 33, wherein the target sequence is specific to a virus.

41. The loop primer of claim 40, wherein the virus is SARS-CoV-2.

42. The method according to any one of claims 1 to 30, wherein the target sequence is specific to Homo sapiens. Loop primer.

43. The loop primer of claim 42, wherein the target sequence is derived from tbc1d3.

44. 43. The loop primer of claim 42, comprising the oligonucleotide of SEQ ID NO:

22.

45. A primer mixture for loop-de-loop amplification of a target sequence, comprising: A primer mixture comprising the loop primer according to any one of claims 4.

46. (i) forward inner primer (FIP), (ii) backward inner primer (iii) forward primer (F3) and backward primer (F4). (B3), wherein the FIP, the BIP, the F3 and the B3 are 46. ​​The primer mix of claim 45, which binds to six different binding sites on the sequence.

47. (i) a loop forward primer (LF) and (ii) a loop backward primer (LB), wherein the LF and the LB are capable of binding to two different binding sites on the target sequence.

47. The primer mixture of claim 46 which binds to the site.

48. The FIP, the BIP, the F3, the B3, the LF, or the LB is The primer according to any one of claims 45 to 47, which binds to the first binding site on the array. Mer mixture.

49. The FIP binds to the first binding site and is coupled to the FIP in the primer mixture. The ratio of the amount of the loop primers is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 49. The primer mixture of claim 48, wherein the ratio of the primers to the primers is 1:1, 8:1, or 9:

1.

50. The BIP binds to the first binding site and is coupled to the BIP in the primer mixture. The ratio of the amount of the loop primers is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:

49. The primer mixture of claim 48, wherein the ratio is 1, 8:1 or 9:

1.

51. The LF binds to the first binding site, and the LF and the LF in the primer mixture are The ratio of the amount of primer to the amount of primer is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 49. The primer mixture of claim 48, which is 8:1 or 9:

1.

52. The LB binds to the first binding site, and the LB and the LU in the primer mixture are The ratio of the amount of primer to the amount of primer is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 49. The primer mixture of claim 48, which is 8:1 or 9:

1.

53. The F3 comprises an oligonucleotide of SEQ ID NO: 1, and the B3 comprises an oligonucleotide of SEQ ID NO:

2. the FIP comprises an oligonucleotide of SEQ ID NO:3; the BIP comprises an oligonucleotide of SEQ ID NO:4; The LF comprises an oligonucleotide of SEQ ID NO: 4, and the LF comprises an oligonucleotide of SEQ ID NO:

6. or the LB comprises an oligonucleotide of SEQ ID NO:

8. A primer mixture according to any one of claims 1 to 4.

54. The F3 comprises an oligonucleotide of SEQ ID NO: 1, and the B3 comprises an oligonucleotide of SEQ ID NO:

2. said FIP comprising an oligonucleotide of SEQ ID NO:3; said BIP comprising an oligonucleotide of SEQ ID NO:4; The oligonucleotide of SEQ ID NO: 4, and the LF of SEQ ID NO: 6 and the LB comprises an oligonucleotide of SEQ ID NO:

8. A primer mixture according to any one of claims 1 to 4.

55. The F3 comprises an oligonucleotide of SEQ ID NO: 9, and the B3 comprises an oligonucleotide of SEQ ID NO:

10. The BI comprises a nucleotide sequence of SEQ ID NO:

11. P comprises an oligonucleotide of SEQ ID NO: 12, said LF comprises an oligonucleotide of SEQ ID NO: 13 or the LB comprises an oligonucleotide of SEQ ID NO:

14.

53. A primer mixture according to any one of claims 52.

56. The F3 comprises an oligonucleotide of SEQ ID NO: 9, and the B3 comprises an oligonucleotide of SEQ ID NO:

10. nucleotides, the FIP comprises an oligonucleotide of SEQ ID NO: 11, and the BI P comprises an oligonucleotide of SEQ ID NO: 12 and said LF comprises an oligonucleotide of SEQ ID NO: 13 and the LB comprises an oligonucleotide of SEQ ID NO:

14.

53. A primer mixture according to any one of claims 52.

57. The F3 comprises an oligonucleotide of SEQ ID NO: 16, and the B3 comprises an oligonucleotide of SEQ ID NO:

17. The FIP comprises an oligonucleotide of SEQ ID NO:

18. The IP comprises an oligonucleotide of SEQ ID NO: 19, and the LF comprises an oligonucleotide of SEQ ID NO:

20. or the LB comprises an oligonucleotide of SEQ ID NO:

21.

53. A primer mixture according to any one of claims 1 to 52.

58. The F3 comprises an oligonucleotide of SEQ ID NO: 16, and the B3 comprises an oligonucleotide of SEQ ID NO:

17. the FIP comprises an oligonucleotide of SEQ ID NO: 18, the B The IP comprises an oligonucleotide of SEQ ID NO: 19, and the LF comprises an oligonucleotide of SEQ ID NO:

20. and said LB comprises an oligonucleotide of SEQ ID NO:

21.

53. A primer mixture according to any one of claims 1 to 52.

59. and a second loop primer, the second loop primer comprising: a third sensor molecule; and a third clamping oligonucleotide; and a second spacing oligonucleotide; and a fourth clamping oligonucleotide, The third clamping oligonucleotide, the second spacing oligonucleotide and the fourth clamping oligonucleotide is the third and fourth clamping oligonucleotide. The melting temperature (T ｍ ) hairpin structures can be formed at lower temperatures. a fourth clamping oligonucleotide, A fourth sensor molecule, the third sensor molecule and the fourth sensor molecule are a second biosensor pair, a fourth sensor molecule, wherein a second biosensor pair is different from the first biosensor pair; a second primer sequence complementary to the first binding site on the second target sequence; The primer mixture according to any one of claims 45 to 58, comprising:

60. The third clamping oligonucleotide is 60. The primer mixture of claim 59, which is complementary to the nucleotide sequence of

61. 61. The primer of claim 59 or 60, wherein the target sequence and the second target sequence are identical. Mer mixture.

62. 61. The primer of claim 59 or 60, wherein the target sequence and the second target sequence are different. mixture.

63. (i) a second forward inner primer (SFIP); (ii) a second backward inner primer (SFIP); (iii) a second forward primer (SF3) and (iv) a second backward primer (SB3), The SBIP, the SF3 and the SB3 bind to six different binding sites on the second target sequence. The primer mixture according to any one of claims 59 to 62, which binds to the above-mentioned positions.

64. (i) a second loop-forward primer (SLF) and (ii) a second loop-back primer The method further comprises the steps of: The method according to any one of claims 59 to 60, wherein the target sequence is bound to two different binding sites. Primer mixture.

65. and a third loop primer, the third loop primer comprising: A fifth sensor molecule; and a fifth clamping oligonucleotide; and a third spacing oligonucleotide; and a sixth clamping oligonucleotide, The fifth clamping oligonucleotide, the third spacing oligonucleotide the fifth and sixth clamping oligonucleotides are The melting temperature (T ｍ ) hairpin structures can be formed at lower temperatures. a sixth clamping oligonucleotide, A sixth sensor molecule, the fifth sensor molecule and the sixth sensor molecule are a third biosensor pair, The third biosensor pair is different from the first biosensor pair and the second biosensor pair. , a sixth sensor molecule; and a second primer sequence complementary to the first binding site on a third target sequence; The primer mixture according to any one of claims 45 to 64, comprising:

66. The fifth clamping oligonucleotide is 66. The primer mixture of claim 65, which is complementary to the nucleotide sequence of

67. 66. The method of claim 65, wherein the target sequence, the second target sequence and the third target sequence are identical. Or a primer mixture according to claim 66.

68. 66. The method of claim 65, wherein the target sequence, the second target sequence and the third target sequence are different.

67. The primer mixture according to claim 66.

69. (i) a third forward inner primer (TFIP); (ii) a third backward inner primer (TFIP); (iii) a third forward primer (TF3) and (iv) a third backward primer (TB3), The TBIP, the TF3, and the TB3 bind to six different binding sites on the third target sequence. The primer mixture according to any one of claims 65 to 68, which binds to the binding site.

70. (i) a third loop forward primer (TLF) and (ii) a third loop back The method further comprises the steps of: The method according to any one of claims 65 to 69, wherein the target sequence is bound to two different binding sites. Primer mixture.

71. The plasmid of any one of claims 65 to 70, further comprising a fourth loop primer. immer mixture.

72. 72. The primer mix of claim 71, further comprising a fifth loop primer.

73. The loop primer according to any one of claims 1 to 44 or any one of claims 45 to 72. A dried primer obtained by freeze-drying the primer mixture according to any one of claims 1 to 4. mixture.

74. 1. A kit for loop-de-loop amplification of a target sequence, comprising: The loop primer according to any one of claims 1 to 44, any one of claims 45 to 72 or a dry primer mixture according to claim 73. ,kit.

75. and further comprising a polymerase, said polymerase optionally being selected from Bacillus stearosa 75. The kit of claim 74, wherein the polymerase is a M. mophilus polymerase.

76. dNTPs, MgSO ４ and a buffer solution. Kit included.

77. 77. The kit of any one of claims 74 to 76, further comprising a reverse transcriptase.

78. The kit of any one of claims 74 to 77, further comprising an RNase inhibitor.

79. 79. The method of claim 78, wherein the RNase inhibitor is a porcine or murine RNase inhibitor. Kit included.

80. 1. A method for detecting a target sequence in a sample, comprising: providing a sample; (i) a primer according to any one of claims 1 to 44, (ii) a primer according to any one of claims 45 to 72 or (iii) The drying plate according to claim 73 The reconstituted primer mixture was obtained by rehydrating the primer mixture, and the polymerase adding a zeta to the sample, thereby forming a reaction mixture; incubating the reaction mixture at 50-85° C.; A method comprising:

81. 81. The method of claim 80, wherein the incubation is carried out at 50-70°C.

82. 82. The method of claim 81, wherein the incubation is carried out at 60-65°C.

83. 83. The method of claim 82, wherein the incubation is carried out at 62-65°C.

84. 9. The method of claim 8, wherein the polymerase is a Bacillus stearothermophilus polymerase.

84. The method of any one of claims 0 to 83.

85. Any of claims 80 to 84, further comprising the step of detecting a signal from the reaction mixture.

13. The method according to claim 1.

86. 86. The method of claim 85, wherein the signal is a fluorescent signal.

87. 8. The method of claim 85, wherein the detecting step is performed during the incubation step.

7. The method according to any one of claims 6 to 6.

88. The method according to claim 80, further comprising determining the presence or absence of the target sequence in the sample.

87. The method of any one of claims 87.

89. 89. The method according to any one of claims 80 to 88, further comprising the preceding step of preparing the sample. Method of posting.

90. The step of preparing the sample includes allowing a reverse transcriptase to interact with an RNA molecule, 90. The method of claim 89, thereby generating the sample comprising DNA molecules.

91. The step of preparing the sample comprises allowing the sample to interact with the reverse transcriptase before or after the reverse transcriptase.

91. The method of claim 90, further comprising the step of pre-heating the RNA molecule during

92. 92. The method of claim 80, wherein the reaction mixture further comprises an RNase inhibitor. The method described.

93. 93. The method of claim 92, wherein the RNase inhibitor is a porcine or murine RNA inhibitor. method.

94. The sample may be purified RNA, purified DNA, whole SARS-CoV-2 virus, whole human cells, , saliva or a nasal swab, or a nasal or nasopharyngeal swab. The method according to any one of claims 1 to 5.

95. The sample contains genomic DNA, synthetic DNA, total bacteria, or total human cells from a vaginal swab. The method according to any one of claims 80 to 93.

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