# US 2021/0172030 A1 · Method for detecting a tandem repeat

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2021/0172030 A1
- Document ID: US20210172030A1
- Status: Published application
- Kind: A1
- Application number: 16/973,414
- Filed: 2019-06-07
- Published: 2021-06-10
- Earliest claimed: 2018-06-08
- Assignees: Imperial College of Science, Technology and Medicine
- Applicants: Imperial College of Science, Technology and Medicine
- Inventors: Pantelis GEORGIOU; Ling-Shan YU; Kenny Malpartida CARDENAS; Matthew C. FISHER; Jesus Rodriguez MANZANO; Nicolas MOSER
- Web page: https://patentgrep.com/patent/US20210172030A1

## CPC

- C12Q 1/6895: for plants, fungi or algae
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6858: Allele-specific amplification
- C12Q 2600/156: Polymorphic or mutational markers
- C12R 2001/68: Aspergillus fumigatus
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

The present application relates to methods for detecting a tandem repeat in a nucleic acid sequence under isothermal conditions using primers.

## Claims

### Claim 1

A method for detecting a first allele of a single nucleotide polymorphism (SNP) in a nucleic acid sequence, wherein the method comprises:
(a) amplifying under isothermal conditions and stringent conditions a nucleic acid sequence from a sample, in a reaction mixture comprising
(i) the nucleic acid sequence,
(ii) a nucleic acid polymerase,
(iii) a nucleoside triphosphate mixture,
(iv) a forward inner primer targeting the first allele (FIPa1), comprising a F1c region which anneals to a F1 region of the nucleic acid sequence in the presence of the first allele and a F2 region which anneals to a F2c region of the nucleic acid sequence,
(v) a backward inner primer targeting the first allele (BIPa1), comprising a B1c region which anneals to a B1 region of the nucleic acid sequence in the presence of the first allele and a B2 region which anneals to a B2c region of the nucleic acid sequence,
(vi) a forward blocking primer targeting a second allele of the SNP (FBa2), comprising a B1c region which anneals to a B1 region of the nucleic acid sequence in the presence of the second allele, and
(vii) a backward blocking primer targeting the second allele (BBa2), comprising a F1c region which anneals to a F1 region of the nucleic acid sequence in the presence of the second allele,
wherein the F1 region of the nucleic acid sequence and the B1 region of the nucleic acid sequence both contain the SNP, and
(b) detecting an amplified nucleic acid sequence, wherein the detection of the amplified nucleic acid sequence indicates the presence of the first allele of the SNP.

### Claim 2 (depends on 1)

The method of claim 1, wherein, in the presence of the first allele, the annealing of the F1c region of FIPa1 and the B1c region of BIPa1 to the nucleic acid sequence is more energetically favorable than the annealing of FBa2 and BBa2 to the nucleic acid sequence.

### Claim 3 (depends on 1)

The method of claim 1, wherein the SNP is located proximal to the 5′ end of BBa2 and/or FBa2.

### Claim 4 (depends on 1)

The method of claim 1, wherein BBa2 is equal to or longer than the F1c region of FIPa1 and/or FBa2 is equal to or longer than the B1c region of BIPa1.

### Claim 5 (depends on 1)

The method of claim 1, wherein a concentration of BBa2 in the reaction mixture is higher than a concentration of FIPa1 and/or a concentration of FBa2 in the reaction mixture is higher than a concentration of BIPa1.

### Claim 6 (depends on 1)

The method of claim 1, wherein the reaction mixture further comprises a forward outer primer (F3) and/or a backward outer primer (B3).

### Claim 7 (depends on 1)

The method of claim 1, wherein the reaction mixture further comprises a forward loop primer (LF) and/or a backward loop primer (LB).

### Claim 8 (depends on 1)

A method for detecting drug resistance in an organism, the method comprising detecting the first allele of the SNP in the nucleic acid sequence according to claim 1, the method further comprising diagnosing drug resistance in the organism based on the detection of the amplified acid sequence indicating the presence of the first allele of the SNP.

### Claim 9 (depends on 1)

A method for the diagnosis of an infectious disease or a drug resistant infection the method detecting the first allele of the SNP in the nucleic acid sequence according to claim 1, the method further comprising diagnosing an infectious disease or a drug resistant infection in a subject based on the detection of the amplified sequence indicating the presence of the first allele of the SNP.

### Claim 10 (depends on 9)

The method of claim 9, further comprising administering a drug to the subject to treat the infectious disease or the drug resistant infection in the subject.

### Claim 11 (depends on 1)

The method of claim 1, wherein the method is performed extemporaneously.

### Claim 12 (depends on 1)

The method of claim 1, wherein method steps (a) and (b) are a first reaction and the method further comprises a second reaction, the second reaction comprising:
(c) amplifying under isothermal conditions and stringent conditions a nucleic acid sequence from the sample, in a reaction mixture comprising
(i) the nucleic acid sequence,
(ii) a nucleic acid polymerase,
(iii) a nucleoside triphosphate mixture,
(iv) a forward inner primer targeting the second allele (FIPa2), comprising a F1c region which anneals to a F1 region of the nucleic acid sequence in the presence of the second allele and a F2 region which anneals to a F2c region of the nucleic acid sequence,
(v) a backward inner primer targeting the second allele (BIPa2), comprising a B1 c region which anneals to a B1 region of the nucleic acid sequence in the presence of the second allele and a B2 region which anneals to a B2c region of the nucleic acid sequence,
(vi) a forward blocking primer targeting a first allele of the SNP (FBa1), comprising a B1c region which anneals to a B1 region of the nucleic acid sequence in the presence of the first allele, and
(vii) a backward blocking primer targeting the first allele (BBa1), comprising a F1c region which anneals to a F1 region of the nucleic acid sequence in the presence of the first allele,
wherein the F1 region of the nucleic acid sequence and the B1 region of the nucleic acid sequence both contain the SNP,
(d) detecting an amplified nucleic acid sequence in the reaction mixture, wherein the detection of an amplified nucleic acid sequence indicates the presence of the second allele of the SNP.

### Claim 13 (depends on 12)

The method of claim 12 wherein, in the presence of the second allele, the annealing of the F1c region of FIPa2 and the Bic region of BIPa2 to the nucleic acid sequence is more energetically favourable than the annealing of FBa1 and BBa1 to the nucleic acid sequence.

### Claim 14 (depends on 12)

The method of claim 12, wherein:
(a) the SNP is located proximal to the 5′ end of BBa1 and/or FBa1;
(b) BBa1 is equal to or longer than the F1c region of FIPa2 and/or FBa1 is equal to or longer than the B 1 c region of BIPa2; and/or
(c) the concentration of BBa1 in the reaction mixture is higher than the concentration of FIPa2 and/or the concentration of FBa1 in the reaction mixture is higher than the concentration of BIPa2.

### Claim 15 (depends on 12)

The method of claim 12, wherein:
(a) the first reaction mixture and the second reaction mixture are the same reaction mixture, the detecting amplified nucleic acid sequences in the reaction mixture is by a single fluorescent channel and machine learning distinguishes the amplified nucleic acid sequence that indicates the presence of the first allele of the SNP from the amplified nucleic acid sequence that indicates the presence of the second allele of the SNP; or
(b) the first reaction mixture and the second reaction mixture are separate reaction mixtures.

### Claim 16 (depends on 1)

The method of claim 1, wherein
(a) the nucleic acid sequence is DNA, the nucleic acid polymerase is a DNA polymerase and the nucleoside triphosphate mixture comprises dNTPs, or
(b) the nucleic acid sequence containing the SNP is RNA, the method further comprises reverse transcription of the RNA to produce cDNA, the amplifying a nucleic acid sequence is amplifying the cDNA, the nucleic acid polymerase is a DNA polymerase and the nucleoside triphosphate mixture comprises dNTPs.

### Claim 17 (depends on 1)

The method of claim 1, wherein the sample is an environmental sample or a clinical sample.

### Claim 18 (depends on 1)

The method of claim 1, wherein the nucleic acid is a nucleic acid from a pathogen, a nucleic acid from a host or a synthetic nucleic acid.

### Claim 19 (depends on 18)

The method of claim 18, wherein the SNP is selected from the group consisting of C580Y in the Plasmodium falciparum ketch 13 gene; F4461 in the Plasmodium falciparum ketch 13 gene; Y493H in the Plasmodium falciparum ketch 13 gene; R539T in the Plasmodium falciparum ketch 13 gene; I543T in the Plasmodium falciparum ketch 13 gene; or P553L in the Plasmodium falciparum ketch 13 gene.

### Claim 20 (depends on 19)

The method of claim 19, wherein:
(a) the SNP is C580Y in the Plasmodium falciparum ketch 13 gene and
(SEQ ID NO: 25)
(i) F1Pa1 comprises TACATAGCTGATGATCTTATGATCATCGTA
TGAAAGCATG,
(SEQ ID NO: 26)
(ii) B1Pa1 comprises ATGTTGCTTTTGATAATAAAATTTATGCT
AATAAGGCATATGGAAATTG,
(SEQ ID NO: 27)
(iii) FBa2 comprises TGTGTTGCTTTTGATAATAAAATTTATG,
(SEQ ID NO: 28)
(iv) BBa2 comprises CACACATAGCTGATGATCTAGGG,
(SEQ ID NO: 29)
(v) F1Pa2 comprises CACATAGCTGATGATCTTATGATCATCGTA
TGAAAGCATG,
(SEQ ID NO: 30)
(vi) B1Pa2 comprises GTGTTGCTTTTGATAATAAAATTTATGCT
AATAAGGCATATGGAAATTG,
(SEQ ID NO: 31)
(vii) FBa1 comprises GTATGTTGCTTTTGATAATAAAATTTATG
TCATTG,
(SEQ ID NO: 32)
(viii) BBa1 comprises ATACATAGCTGATGATCTAGGG,
(SEQ ID NO: 33)
(ix) F3 comprises CTATTATACCGAATGTAGAAGCA,
(SEQ ID NO: 34)
(x) B3 comprises TGCTCCTGAACTTCTAGC,
(SEQ ID NO: 35)
(xi) LF comprises T CAAAGGTGCCACCTCTACC,
and/or
(SEQ ID NO: 36)
(xii) LB comprises GGTGGAACTAATGGTGAGAGATTAA;
or
(b) the SNP is Y493H in the Plasmodium falciparum ketch 13 gene and
(SEQ ID NO: 37)
(i) F1IPa1 comprises GTAAGAAATTATTCAATACAGATTAGATA
TTAGTCAACAATGCTGG,
(SEQ ID NO: 38)
(ii) BIPa1 comprises C ACGTTTTTGGTGGTAAAAACGATCATA
CACCTCAGTT,
(SEQ ID NO: 39)
(iii) FBa2 comprises ATACGTTTTTGGTGGTAATAACTAT,
(SEQ ID NO: 40)
(iv) BBa2 comprises ATAAGAAATTATTCAATACAGCACTT,
(SEQ ID NO: 41)
(v) FI Pa2 may comprise ATAAGAAATTATTCAATACAGATTAG
ATATTAGTCAACAATGCTGG,
(SEQ ID NO: 42)
(vi) B1Pa2 may comprise TACGTTTTTGGTGGTAAAAACGATCA
TACACCTCAGTT,
(SEQ ID NO: 43)
(vii) FBa1 may comprise TACACGTTTTTGGTGGTAATAACTA
T,
(SEQ ID NO: 44)
(viii) BBa1 may comprise TGTAAGAAATTATTCAATACAGCAC
TT,
(SEQ ID NO: 45)
(ix) F3 comprises GGTGTAGAATATTTAAATTCGATGG,
(SEQ ID NO: 46)
(x) B3 comprises TTGAAACATACCATACATCT CTT,
(SEQ ID NO: 47)
(xi) LF comprises TGGTAGACATAGGTGTACACATACG,
and/or
(SEQ ID NO: 48)
(xii) LB comprises ATTATAAGGCTTTATTTGA;
or
(c) the SNP is R539T in the Plasmodium falciparum ketch 13 gene, and
(SEQ ID NO: 49)
(i) F1Pa1 may comprise GTACCATTTGACGTAACACTAAGAGAT
GTATGGTATGTTTCAAG,
(SEQ ID NO: 50)
(ii) B1Pa1 may comprise CAATTTATTGTATTGGGGGATCATAT
GCTTCTACATTCGGTAT,
(SEQ ID NO: 51)
(iii) FBa2 may comprise TAGAATTTATTGTATTGGGGGATAT,
(SEQ ID NO: 52)
(iv) BBa2 may comprise CTACCATTTGACGTAACACCAC,
(SEQ ID NO: 53)
(v) F1Pa2 may comprise CTACCATTTGACGTAACTAAGAGATGT
ATGGTATGTTTCAAG,
(SEQ ID NO: 54)
(vi) B1Pa2 may comprise AGAATTTATTGTATTGGGGGATCATA
TGCTTCTACATTCGGTAT,
(SEQ ID NO: 55)
(vii) FBa1 may comprise TACAATTTATTGTATTGGGGGATAT,
(SEQ ID NO: 56)
(viii) BBa1 may comprise TGTACCATTTGACGTAACACCAC,
(SEQ ID NO: 57)
(ix) F3 may comprise AACTGAGGTGTATGATCGTT,
(SEQ ID NO: 58)
(x) B3 may comprise ACCCATGCTTTCATACGA,
(SEQ ID NO: 59)
(xi) LF may comprise TTATTTCTTCTAG GTAT ATTT,
and/or
(SEQ ID NO: 60)
(xii) LB may comprise ATGATGGCTCTTCTATT.

### Claim 21 (depends on 1)

The method of claim 1, wherein the detecting is colorimetric detection, pH-based detection and/or detecting by one or more ion sensors.

### Claim 22 (depends on 21)

The method of claim 21, wherein the one or more ion sensors are one or more semiconductor-based ion sensors, optionally complementary metal-oxide-semiconductor (CMOS) based ion sensors.

### Claim 23

A kit comprising:
(i) a forward inner primer targeting a first allele (FIPa1), comprising a F1c region which anneals to a F1 region of a nucleic acid sequence in the presence of the first allele of a single nucleotide polymorphism (SNP) in a nucleic acid sequence and a F2 region which anneals to a F2c region of the nucleic acid sequence,
(ii) a backward inner primer targeting the first allele (B1Pa1), comprising a B1c region which anneals to a B1 region of the nucleic acid sequence in the presence of the first allele and a B2 region which anneals to a B2c region of the nucleic acid sequence,
(iii) a forward blocking primer targeting a second allele of the SNP (FBa2), comprising a B1c region which anneals to a B1 region of the nucleic acid sequence in the presence of the second allele, and
(iv) a backward blocking primer targeting the second allele (BBa2), comprising a F1c region which anneals to a F1 region of the nucleic acid sequence in the presence of the second allele,
wherein the F1 region of the nucleic acid sequence and the B1 region of the nucleic acid sequence both contain the SNP.

### Claim 24 (depends on 23)

The kit of claim 23, further comprising:
(v) a forward inner primer targeting the second allele (FIPa2), comprising a F1c region which anneals to a F1 region of the nucleic acid sequence in the presence of the second allele and a F2 region which anneals to a F2c region of the nucleic acid sequence,
(vi) a backward inner primer targeting the second allele (BIPa2), comprising a B1c region which anneals to a B1 region of the nucleic acid sequence in the presence of the second allele and a B2 region which anneals to a B2c region of the nucleic acid sequence,
(vii) a forward blocking primer targeting the first allele of the SNP (FBa1), comprising a B1c region which anneals to a B1 region of the nucleic acid sequence in the presence of the first allele, and
(viii) a backward blocking primer targeting the first allele (BBa1).

### Claim 25 (depends on 23)

A reaction mixture comprising the components of the kit according to claim 23, in a liquid medium.