Multiplex HPV Genotyping PCR for Self-Collected Cancer Screening Samples
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Solution Overview
Problem
Existing methods for detecting human papillomavirus (HPV) genotypes associated with cervical, oral, or anogenital cancer are inefficient, require medical intervention, are costly, and lack accuracy and automation.
Innovation Solution
A method involving multiplex real-time PCR followed by hybridization on a solid support, using specific probes and fluorophores to identify HPV genotypes, allowing for automated, accurate, and cost-effective detection and determination of HPV genotypes directly from self-collected samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing HPV detection methods are used, then detection can be performed, but the methods are inefficient, costly, and require medical intervention
Solution Approach 1:
The detection method is divided into distinct modular steps: sample collection by the subject, DNA extraction, multiplex real-time PCR amplification with genotype-specific probes, and automated analysis. This segmentation allows each step to be optimized independently and enables automated processing, significantly improving detection efficiency and reducing the time required compared to conventional methods that require continuous medical intervention.
Solution Approach 2:
The subject is empowered to collect their own biological sample (e.g., cervical swab) at home without requiring a doctor or medical personnel. This self-service approach eliminates the need for scheduled medical appointments and professional intervention for sample collection, thereby improving accessibility and reducing the overall time required for detection while maintaining sample quality.
2Reliability
If existing HPV detection methods are used, then detection can be performed, but the cost is high
Solution Approach 1:
The multiplex real-time PCR assay simultaneously detects multiple HPV genotypes (including high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and a human housekeeping gene in a single reaction using a panel of genotype-specific probes. This multi-functional approach replaces the need for multiple separate tests, reducing overall detection cost while maintaining high reliability through comprehensive genotype coverage and internal control.
Solution Approach 2:
The method employs real-time PCR with fluorescent probes that detect amplification products during the amplification process itself, rather than requiring post-PCR analysis. This parameter change in the detection timing and method reduces labor costs and increases throughput, thereby lowering the overall cost per test while maintaining or improving detection accuracy through automated fluorescent signal analysis.
3Extent of automation
If existing HPV detection methods are used, then detection can be performed, but automation is lacking and medical intervention is required
Solution Approach 1:
The method replaces manual visual inspection and interpretive analysis with automated fluorescent detection and computerized data analysis. The real-time PCR system automatically quantifies fluorescent signals from genotype-specific probes, and software algorithms automatically interpret results to determine which HPV genotypes are present. This substitution of mechanical and manual processes with automated electronic systems significantly increases the extent of automation while maintaining operational simplicity for the user.
4Measurement precision
If multiplex real-time PCR with hybridization is used, then sensitivity and specificity are improved, but reagent quantities and complexity increase
Solution Approach 1:
The method combines multiplex real-time PCR amplification with post-amplification hybridization to a solid support in an integrated workflow. Multiple genotype-specific probes are amplified simultaneously in the PCR reaction, then the amplification products are hybridized to a solid support containing complementary probes for each genotype. This merging of amplification and detection steps, along with the use of a solid support for organized probe presentation, improves measurement precision for genotype identification while managing assay complexity through systematic design.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables rapid, precise, and cost-effective detection and identification of HPV genotypes associated with cancer development, improving sensitivity and specificity through controlled hybridization temperatures.
Implementation Method 1
multiplex real-time PCR amplifies: (i) at least one first sequence comprised in said DNA, wherein each first sequence is a sequence of a marker of a genotype of the human papillomavirus
Implementation Method 2
amplification of each sequence is performed using a forward primer and a reverse primer
Implementation Method 3
a probe comprising a first fluorophore and a sequence, wherein said sequence is complementary to and specific for a sequence of a marker of the 16 genotype of the human papillomavirus
Implementation Method 4
subjecting the composition selected in step (a) to heating to at least 60 °C, and gradually cooling
Implementation Method 5
subjecting each amplified first sequence and second sequence of the composition selected in step (a) to hybridization on a solid support comprising probes attached thereto
Data Source
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AI summary
The invention relates to a method, uses, system and kit for detecting a human papillomavirus (HPV) genotype associated with development of cervical, oral or anogenital cancer in a subject and for determining the genotype or genotypes of HPV thus detected therein. Additionally, the present invention relates to uses of forward and/or reverse primers or amplicons generated by multiplex real-time PCR comprising a molecular tag for identifying marker of the genotype (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66) and/or (68) of the human papillomavirus.