# US 2020/0399685 A1 · Methods for detection of antibiotic resistant h.pylori

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

- Number: US 2020/0399685 A1
- Document ID: US20200399685A1
- Status: Published application
- Kind: A1
- Application number: 16/764,765
- Filed: 2018-11-16
- Published: 2020-12-24
- Earliest claimed: 2017-11-16
- Assignees: American Molecular Laboratories Inc.
- Applicants: American Molecular Laboratories, Inc.
- Inventors: Hongjun ZHANG; Yi ZHOU; Rajarao KAKUTURU
- Web page: https://patentgrep.com/patent/US20200399685A1

## CPC

- C12Q 1/689: for bacteria
- A61K 39/40: bacterial
- C12Q 1/68: involving nucleic acids
- C12Q 2600/156: Polymorphic or mutational markers
- 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
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present disclosure provides methods and materials for determining if antibiotic resistant H. pylori is present in a sample. The methods may comprise obtaining a threshold level of H. pylori DNA from the sample, amplifying a region of the H. pylori DNA to generate multiple copies of the region of the H. pylori DNA, sequencing the multiple copies of the region of the H. pylori DNA, comparing sequences of multiple copies of the region of the H. pylori DNA to a reference sequence, identifying the presence of a mutation in multiple copies of the region of the H. pylori DNA, and determining a number of the multiple copies of the region of the H. pylori DNA with the mutation, wherein antibiotic resistant H. pylori is present in the sample when the number of the multiple copies of the region of the H. pylori DNA with the mutation is above a predetermined amount.

## Claims

### Claim 1

A method for determining if antibiotic resistant H. pylori is present in a sample, the method comprising:
a) obtaining a threshold level of H. pylori DNA from the sample;
b) amplifying a region of the H. pylori DNA to generate multiple copies of the region of the H. pylori DNA;
c) sequencing the multiple copies of the region of the H. pylori DNA;
d) comparing sequences of the multiple copies of the region of the H. pylori DNA to a reference sequence;
e) identifying the presence of a mutation in the multiple copies of the region of the H. pylori DNA; and
f) determining a number of the multiple copies of the region of the H. pylori DNA with the mutation,
wherein antibiotic resistant H. pylori is present in the sample when the number of the multiple copies of the region of the H. pylori DNA with the mutation is above a predetermined amount.

### Claim 2 (depends on 1)

The method of claim 1, wherein the sample is a fecal sample.

### Claim 3 (depends on 2)

The method of claim 2, wherein the fecal sample is obtained by the method comprising:
a) exposing a first part of the fecal sample to an anti-H. pylori antibody;
b) separating H. pylori bound to the anti-H. pylori antibody from fecal material in the first part of the fecal sample;
c) extracting H. pylori DNA from the H. pylori separated from the first fecal material;
d) exposing a second part of the fecal sample to a DNA probe that binds to H. pylori DNA;
e) extracting the H. pylori DNA from the second part of the fecal sample; and
f) pooling the H. pylori DNA obtained from the first part of the fecal sample and the H. pylori DNA obtained from the second part of the fecal sample.

### Claim 4 (depends on 1)

The method of claim 1, wherein the mutation in the multiple copies of the region of the H. pylori DNA is detected by next generation sequencing.

### Claim 5 (depends on 1)

The method of claim 1 further comprising the step of providing one or more reference sequences of the amplified regions of H. pylori DNA.

### Claim 6 (depends on 1)

The method of claim 1, wherein the steps of amplifying and sequencing the one or more regions of H. pylori DNA comprise:
a) identifying PCR primer pairs suitable for producing amplicons comprising the one or more regions of the H. pylori DNA;
b) segregating PCR primer pairs comprising one or more primers that interfere with amplicon generation by another PCR primer pair into separate PCR primer pair pools, wherein each of the separate PCR primer pair pools contain a plurality of PCR primer pairs;
c) generating amplicons from each of the separate PCR primer pair pools and the H. pylori DNA; and
d) combining all amplicons produced from each of the separate PCR primer pair pools and the H. pylori DNA into a sample amplicon pool, adding a unique index sequence to the amplicons within the sample amplicon pool to generate an indexed sample amplicon pool, optionally further combining the indexed sample amplicon pool with one or more differentially indexed sample amplicon pools from different samples, and sequencing all indexed sample amplicons simultaneously.

### Claim 7 (depends on 6)

The method of claim 6, further comprising the step of identifying mutations within the indexed sequence amplicons from a sample by reference to corresponding wild type gene sequences.

### Claim 8 (depends on 1)

The method of claim 1, wherein the region of H. pylori DNA is one or more H. pylori gene selected from the group comprising: 23S rRNA, gyrA, rdxA, frxA, pbp1, 16S rRNA, and rpoB.

### Claim 9 (depends on 1)

The method of claim 1, wherein the mutation in the multiple copies of the region of the H. pylori DNA is selected from the group comprising: A2143G and A2142G mutations in 23S rRNA; A272G Asp91Gly and G271A Asp91Asn in gyrA; pGlu194, G352A, pCys87, pR41Rfs in rdxA; and T926C and C927A in 16A rRNA.

### Claim 10 (depends on 1)

The method of claim 1, wherein the threshold level of H. pylori DNA is DNA from at least 10 H. pylori genomes.

### Claim 11 (depends on 1)

The method of claim 1, wherein the threshold level of H. pylori DNA is DNA from at least 50 fragments of H. pylori DNA.

### Claim 12 (depends on 1)

The method of claim 1, wherein the threshold level of H. pylori DNA is DNA from between 50 and 100 fragments of H. pylori DNA.

### Claim 13 (depends on 1)

The method of claim 1, wherein the sample is a dental plaque, gastric juice, or a gastric biopsy.

### Claim 14 (depends on 1)

The method of claim 1, wherein the sample is formalin fixed or formalin-fixed and paraffin embedded (FFPE).

### Claim 15 (depends on 1)

The method of claim 1, wherein the antibiotic resistant H. pylori is resistant to one or more of the following: macrolides, metronidazole, quinolones, rifamycins, amoxicillin, and tetracycline.

### Claim 16

A method for obtaining H. pylori DNA from a fecal sample, the method comprising:
a) exposing a first part of the fecal sample to an anti-H. pylori antibody;
b) separating H. pylori bound to the anti-H. pylori antibody from fecal material in the first part of the fecal sample;
c) extracting H. pylori DNA from the H. pylori separated from the first fecal material;
d) exposing a second part of the fecal sample to a DNA probe that binds to H. pylori DNA;
e) extracting the H. pylori DNA from the second part of the fecal sample; and
f) pooling the H. pylori DNA obtained from the first part of the fecal sample and the H. pylori DNA obtained from the second part of the fecal sample.

### Claim 17 (depends on 16)

The method of claim 16, further comprising the step of homogenizing the fecal sample.

### Claim 18 (depends on 16)

The method of claim 16, wherein the anti-H. pylori antibody is labeled.

### Claim 19 (depends on 18)

The method of claim 18, wherein the anti-H. pylori antibody is labeled with biotin.

### Claim 20

A method for treating H. pylori infection in a subject, the method comprising:
a) obtaining a sample from the subject;
b) obtaining a threshold level of H. pylori DNA from the sample;
c) amplifying a region of the H. pylori DNA to generate multiple copies of the region of the H. pylori DNA;
d) sequencing the multiple copies of the region of the H. pylori DNA;
e) comparing sequences of the multiple copies of the region of the H. pylori DNA to one or more reference sequences;
f) detecting a mutation in the multiple copies of the region of H. pylori DNA;
g) determining a number of the multiple copies of the region of the H. pylori DNA with the mutation, wherein antibiotic resistant H. pylori is present in the sample when the number of the multiple copies of the region of the H. pylori DNA with the mutation is above a predetermined amount; and
h) administering to the subject one or more antibiotics to which the H. pylori lacks resistance.