Methods and compositions for characterizing drug resistant bacteria from formalin-fixed paraffin-embedded biological samples
Inventors
Zhou, Yi • Jung, Daniel • Rao, Kakuturu
Assignees
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Publication Number
US-10968489-B2
Publication Date
2021-04-06
Expiration Date
Abstract
The invention provides methods and compositions generally useful to the use of polymerase chain reaction (PCR) amplification of trace DNA sequences from formalin-fixed paraffin-embedded (FFPE) biopsy samples and specifically relevant to the identification of multi-drug resistant H. pylori in such biopsy samples.
Core Innovation
The disclosed subject matter relates to a method for detecting within a Helicobacter pylori sample mutations in a plurality of drug resistance genes. PCR primer pairs are identified for producing amplicons comprising regions of each gene containing one or more mutations, and primer pairs that interfere with amplicon generation by another primer pair are segregated into separate PCR primer pair pools.
Amplicons are generated from each separate PCR primer pair pool and target DNA isolated from the sample, then combined into a sample amplicon pool. A unique index sequence is added to generate an indexed sample amplicon pool, and optionally differentially indexed sample amplicon pools from different samples are further combined; all indexed sample amplicons are sequenced simultaneously.
Mutations within the indexed sequenced amplicons are identified by reference to corresponding wild-type gene sequences or SEQ ID Nos. 47-51, and a drug-resistant profile is determined based on the presence or absence of the identified mutations. The workflow is configured so that amplicons diagnostic for at least three different types of drug resistance genes are produced from two PCR reactions.
Claims Coverage
The disclosure contains two independent claims that share the same core inventive workflow: segregating interfering PCR primer pairs into separate pools, generating amplicons, indexing, and sequencing all indexed amplicons simultaneously, followed by mutation calling and drug-resistance profiling. The inventive features are the segregation/pooling strategy, the unique index sequence and simultaneous sequencing, mutation identification by reference to specified sequence references, and the claim-specific requirement that diagnostic amplicons for at least three different types of drug resistance genes are produced from two PCR reactions in one claim.
Segregated PCR primer pair pools to prevent primer interference
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.
Indexed sample amplicon pool for simultaneous sequencing
Combining all amplicons produced from each of the separate PCR primer pair pools and the target DNA into a sample amplicon pool, adding a unique index sequence to generate an indexed sample amplicon pool, optionally further combining differentially indexed sample amplicon pools from different samples, and sequencing all indexed sample amplicons simultaneously.
Mutation identification by reference to wild-type gene sequences
Identifying mutations within the indexed sequenced amplicons from a sample by reference to corresponding wild-type gene sequences.
Diagnostic amplicons for at least three different types of drug resistance genes from two PCR reactions
Amplicons diagnostic for at least three different types of drug resistance genes are produced from two PCR reactions.
Primer pool grouping into PCR primer pair pools 1-6 and patient drug-resistance profiling
Generating amplicons from DNA isolated from the patient-derived sample and from PCR primer pair pools 1 through 6, and determining the drug-resistant profile of H. pylori present in the patient-derived sample by the presence or absence of mutations identified.
Mutation identification by reference to SEQ ID Nos. 47-51
Identifying mutations within the sequenced indexed sample amplicons by reference to SEQ ID Nos. 47-51.
Across the independent claims, the patent covers methods that segregate interfering PCR primer pairs into separate pools, generate and combine amplicons into a uniquely indexed pool, sequence indexed amplicons simultaneously, and identify mutations based on predefined sequence references. One claim further specifies diagnostic amplicons for at least three different types of drug resistance genes from two PCR reactions, while the other specifies patient-derived drug-resistant profile determination by reference to SEQ ID Nos. 47-51.
Stated Advantages
Not explicitly described in patent.
Documented Applications
No documented applications found
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