PCR Reaction Optimization for Whole Blood Optical Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The challenge in PCR methods is the generation of precipitates and turbidity in whole blood samples during DNA denaturation, which obstructs accurate optical detection of amplification products, requiring pre- and post-treatment processes that complicate the analysis and hinder real-time monitoring of amplification processes.
Innovation Solution
Setting the ratio of whole blood in the PCR reaction solution to 0.01-0.9% by volume, with optional addition of albumin, to suppress the formation of precipitates and turbidity, allowing for direct optical detection of amplification products without pre- or post-treatment, thereby enabling efficient amplification and analysis of target nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the ratio of whole blood sample in PCR reaction solution is increased to maintain PCR reaction activity, then PCR reaction efficiency is improved, but precipitate and turbidity generation increases blocking optical detection
Solution Approach 1:
The invention changes the concentration parameter of whole blood sample in PCR reaction solution to a specific range (0.01-10% by volume, preferably 0.05-5%). This parameter optimization balances two opposing requirements: maintaining sufficient PCR reaction activity while minimizing precipitate and turbidity generation that would block optical detection. Through systematic parameter adjustment, the invention resolves the contradiction between reaction efficiency and detection accuracy.
2Measurement precision
If pretreatment or aftertreatment purification processes are applied to remove precipitates, then optical detection accuracy is improved, but operation complexity and time consumption increase
Solution Approach 1:
The invention performs preliminary action by optimizing the whole blood sample ratio in the PCR reaction solution before the reaction begins. By pre-setting the sample concentration within the specific range of 0.01-10% by volume, the invention prevents excessive precipitate and turbidity formation during the PCR process itself, thereby eliminating the need for subsequent purification treatments and simplifying the overall workflow.
Solution Approach 2:
The invention converts the potentially harmful effect of whole blood components (which cause precipitates) into a beneficial outcome by optimizing their concentration. By controlling the whole blood sample ratio within the specific range, the invention ensures that sufficient genetic material is present for effective PCR amplification while the precipitate formation is limited to levels that do not significantly interfere with optical detection, thus turning a problem into a manageable parameter.
3Measurement precision
If pretreatment purification is applied to remove precipitate-causing substances, then optical detection accuracy is improved, but operation time and process complexity increase
Solution Approach 1:
The invention performs preliminary action by optimizing the whole blood sample ratio in the PCR reaction solution before the reaction begins. By pre-setting the sample concentration within the specific range of 0.01-10% by volume, the invention prevents excessive precipitate and turbidity formation during the PCR process itself, thereby eliminating the need for subsequent purification treatments and simplifying the overall workflow.
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 allows for accurate qualitative and quantitative analysis of amplification products and target nucleic acids using optical methods, enhancing amplification efficiency and eliminating the need for pre- and post-treatment processes, while maintaining PCR reaction integrity.
Implementation Method 1
annealing a primer to single-stranded DNA template; and extending the primer by DNA polymerase
Implementation Method 2
denaturing DNA (dissociation from double-stranded DNA into single-stranded DNA) by a heat treatment
Data Source
Figure 1~2
Figure 3
Figure 4~5
AI summary
A method of producing a PCR amplification product is provided that suppresses an effect of precipitate, turbidity, or the like derived from a whole blood sample on a detection in the detection of an amplified nucleic acid by an optical unit. The amplification product complementary to a target nucleic acid in the whole blood sample is produced by PCR in a condition where a ratio of the whole blood sample in a PCR reaction solution is in the range of 0.1 to 0.9% by volume or 0.01 to 1.8g/L in term of hemoglobin content. When the PCR is carried out with such conditions, even with an untreated whole blood sample, a monitoring of the amplification product by the optical unit can be done while suppressing the effect of the precipitate or the turbidity.