Blocking Primer Quantification for HIV Tropism
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Solution Overview
Problem
Current methods for determining HIV-1 tropism and quantifying HIV-1 RNA and proviral DNA are complex, costly, and have limitations in dynamic range and sensitivity, particularly for phenotypic assays and next-generation sequencing systems, which struggle with simultaneous genotyping and quantification.
Innovation Solution
A method that encodes quantitative information into a target sequence, allowing for simultaneous sequence identity and quantification through the use of hybridization domains and unique identifying features in primers, enabling efficient and accurate determination of HIV-1 tropism and viral load without the need for separate amplification steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If phenotypic assays are used to determine HIV-1 tropism, then comprehensive viral tropism determination is achieved, but the process becomes complex, costly, and time-consuming with high sensitivity thresholds
Solution Approach 1:
The invention extracts only the essential V3 loop sequence information needed for tropism determination from the complete envelope gene, eliminating the need for complex phenotypic assays. By focusing on the critical V3 loop region that determines co-receptor usage, the method achieves accurate tropism classification (R5, X4, or dual/mixed phenotype) through simplified genotypic analysis of this specific segment.
Solution Approach 2:
The invention introduces a blocking primer as an intermediary element that mediates between the quantification primer and the target V3 loop sequence. The blocking primer contains a 5' extension that hybridizes to the V3 loop while preventing quantification primer binding, enabling both sequence determination and quantification in a single assay system.
2Loss of information
If separate techniques are used for quantification and genotyping, then quantitative information and sequence identity are obtained, but the process requires multiple steps and increases labor and cost
Solution Approach 1:
The invention merges quantification and genotyping functions into a single integrated assay. The blocking primer design allows it to serve dual purposes: (1) its 5' extension hybridizes to the V3 loop for sequence identification, and (2) it blocks quantification primer binding to enable quantification. This eliminates the need for separate qPCR and sequencing procedures.
Solution Approach 2:
The blocking primer is designed with multi-functionality, serving both as a sequence-specific hybridization element (through its 5' extension matching V3 loop sequences) and as a quantification control element (by blocking quantification primer binding). This universal design enables a single primer to perform multiple functions in the assay.
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
This approach increases throughput and accuracy, reduces labor and costs, and provides cost-effective multiplexing capabilities, enabling precise quantification and genotyping within a single procedure.
Implementation Method 1
The quantification primer comprises a hybridization domain complementary to a region of the target and is of sufficient length to hybridize specifically with the target under reasonable conditions
Implementation Method 2
The blocking primer binds a portion of the amount of the target in the sample not bound by quantification primer, thereby blocking said portion from contact with any subsequent quantification primer comprising the same target-specific hybridization domain
Data Source
AI summary
The method includes obtaining a sample including a target nucleic acid, contacting the sample with a quantification primer, adding a quantity of blocking primer to the sample, and repeating the contacting and adding steps until the total quantity of blocking primer present in the sample meets or exceeds the amount of target present in the sample. Each repetition utilizes a quantification primer having a different unique identifying feature assigned to sequentially increasing quantification levels, and the quantity of blocking primer added at each repetition establishes numerical spacing between the quantification levels. The method further includes identifying the unique identifying feature present in any bound quantification primers that remain in the sample, where the presence of a unique identifying feature assigned to a particular quantification level indicates the approximate amount of the target present in the sample.