Methods and compositions for human papillomaviruses and sexually transmitted infections detection, identification and quantification
Inventors
Wang, Chunlin • MA, Zhihai • Gharizadeh, Baback
Assignees
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Publication Number
US-11149322-B2
Publication Date
2021-10-19
Expiration Date
Abstract
The present disclosure relates to compositions and methods for detection, identification and quantification of human papillomaviruses (HPV) and clinically significant sexually transmitted infections (STI) in a single amplification reaction. The disclosed method utilizes next-generation sequencing (NGS) to sequence amplified products. The present disclosure is also directed to kits containing primers specific to multiple HPV and STI targets.
Core Innovation
The invention relates to an NGS-based single-reaction multiplex assay for screening a sample from each of one or more subjects. Target-specific primers are hybridized with nucleic acid from each sample in the presence of barcoded universal primers to form a test reaction, including primers configured to bind to a target sequence specific to sexually transmitted infection. The barcoded universal primers include a universal priming portion at the 3′-end, a barcode portion in the middle, and a universal priming portion at the 5′-end.
Each test reaction is subjected to amplification conditions to generate amplicons, and at least a portion of the amplicons generated from each sample is subjected to bead cleanup to form enriched amplicons. The enriched amplicons formed from each sample are sequenced by next-generation sequencing. This workflow supports screening to detect, identify, and quantify multiple human papillomavirus types and multiple sexually transmitted infection species in a multiplex format.
The approach includes mapping-and-counting and read normalization using a human housekeeping gene, and it includes cross-contamination monitoring using an individual-specific polymorphic gene with a unique sequence. The assay design addresses minimizing primer-dimer and off-target artifacts using a primer compatibility score and parameter tuning, and it supports correct genotyping and quantification with a stated limit of detection of about 20 copies.
Claims Coverage
The independent claim defines a multiplex screening method with a defined barcoded universal primer architecture, a workflow of amplification, bead cleanup, and next-generation sequencing. Additional independent-claim refinements introduce pooling before sequencing, quantification by type and species, and cross-contamination monitoring using an individual-specific polymorphic gene.
Barcoded universal primer architecture for multiplex STI targeting
Each test reaction hybridizes a plurality of target-specific primers with nucleic acid from a sample in the presence of barcoded universal primers, wherein at least one target-specific primer is configured to bind to a target sequence specific to sexually transmitted infection, and wherein each target-specific primer comprises a specific sequence portion directed to a target nucleic acid sequence and a universal priming portion. Each barcoded universal primer comprises a universal priming portion at the 3′-end, a barcode portion in the middle, and a universal priming portion at the 5′-end.
Bead cleanup enrichment of amplified products before NGS
Amplification conditions generate amplicons from the test reaction, and at least a portion of the amplicons generated from each sample is subjected to bead cleanup to form enriched amplicons. The enriched amplicons formed from each sample are sequenced by next-generation sequencing.
Pooling enriched amplicons from each sample prior to sequencing
The method includes pooling enriched amplicons from each sample before sequencing.
Quantifying each type and species after sequencing
The method further includes quantifying each type and species in each sample after sequencing enriched amplicons.
Individual-specific polymorphic gene internal control for cross-contamination monitoring
The method uses a polymorphic gene with an individual-specific unique sequence as an internal control to monitor cross-contamination during the test.
Overall, the claim coverage centers on a multiplex screening workflow using target-specific primers with barcoded universal primers having defined 3′/5′ universal priming portions and a middle barcode, followed by amplification, bead cleanup to form enriched amplicons, and next-generation sequencing. Dependent refinements emphasize pooling before sequencing, quantification by type and species, and an internal control based on an individual-specific polymorphic gene for cross-contamination monitoring.
Stated Advantages
Minimizing primer-dimer and off-target artifacts via primer compatibility scoring and parameter tuning.
Correct genotyping and quantification with a stated limit of detection of about 20 copies.
Clinical validation with detection of additional genotypes/STIs in the evaluated samples.
Documented Applications
Screening samples from one or more subjects for multiple human papillomavirus types and multiple sexually transmitted infection species using an NGS-based multiplex assay.
Clinical validation on DNA samples including genotyping and quantification for HPV types and detection of additional genotypes/STIs.
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