Methods for detection of antibiotic resistant H. pylori
Inventors
Zhang, Hongjun • Zhou, Yi • KAKUTURU, Rajarao
Assignees
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Publication Number
US-11708616-B2
Publication Date
2023-07-25
Expiration Date
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.
Core Innovation
The invention relates to methods to determine if antibiotic resistant Helicobacter pylori is present in a fecal sample. The method obtains a threshold level of H. pylori DNA from the fecal sample, amplifies a region of the H. pylori DNA to generate multiple copies, sequences the multiple copies, compares the sequences to a reference sequence, and identifies a mutation in the multiple copies of the region.
The method further determines a number of the multiple copies of the region of the H. pylori DNA with the mutation, where antibiotic resistant H. pylori is present in the fecal sample when the number of the multiple copies of the region of the H. pylori DNA with the mutation is above a predetermined amount. The obtaining of the H. pylori DNA includes exposing a first part of the fecal sample to an anti-H. pylori antibody, separating H. pylori bound to the anti-H. pylori antibody from fecal material, extracting H. pylori DNA, exposing a second part to a DNA probe that binds to H. pylori DNA, extracting the H. pylori DNA, and pooling DNA from the first and second parts.
In embodiments, mutation detection can be performed using next generation sequencing, and resistance-associated mutations are identified by comparison to reference or wild-type sequences. The disclosed approach can distinguish mutation-containing versus wild-type samples and detect mixtures of resistant strains based on mutation frequency. The method can use selected H. pylori genes including 23S rRNA, 16S rRNA, gyrA, rdxA, frxA, pbp1, and rpoB, and can define the threshold level of H. pylori DNA for analysis.
Claims Coverage
The independent claims cover three core areas: threshold-based determination of antibiotic resistant H. pylori in a fecal sample, pooled H. pylori DNA acquisition from a fecal sample using an anti-H. pylori antibody and a DNA probe, and treatment of an H. pylori infection based on the determination. Each area includes amplification, sequencing, comparison to reference sequence, mutation identification, and quantification of mutation-containing copies against a predetermined amount.
Threshold-based determination of antibiotic resistant H. pylori presence in fecal sample
Obtaining a threshold level of H. pylori DNA from the fecal sample, amplifying a region of the H. pylori DNA to generate multiple copies, sequencing the multiple copies, comparing sequences to a reference sequence, identifying a presence of a mutation in the 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 so that antibiotic resistant H. pylori is present when the number of the multiple copies with the mutation is above a predetermined amount.
Pooled H. pylori DNA acquisition from fecal sample using anti-H. pylori antibody and DNA probe
Obtaining H. pylori DNA from a fecal sample by exposing a first part to an anti-H. pylori antibody, separating H. pylori bound to the anti-H. pylori antibody from fecal material, extracting H. pylori DNA from the separated H. pylori, exposing a second part to a DNA probe that binds to H. pylori DNA, extracting the H. pylori DNA from the second part, and pooling the H. pylori DNA from the first and second parts.
Treatment of H. pylori infection based on mutation-quantified antibiotic resistance call from fecal DNA
Treating an H. pylori infection by obtaining a fecal sample from a subject, obtaining a threshold level of H. pylori DNA from the fecal sample, amplifying a region to generate multiple copies, sequencing the multiple copies, comparing sequences to one or more reference sequences, detecting a mutation in the multiple copies, determining a number of the multiple copies with the mutation such that antibiotic resistant H. pylori is present when the number is above a predetermined amount, and administering one or more antibiotics to which the H. pylori lacks resistance.
Across the independent claims, the inventive focus is pooled H. pylori DNA acquisition from fecal sample, amplification and sequencing of a region followed by comparison to reference sequence to identify mutations, and quantifying the number of mutation-containing sequence copies against a predetermined amount to call antibiotic resistant H. pylori presence, which is used to guide administering antibiotics lacking resistance.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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