Isothermal LAMP Nucleic Acid Detection Method
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Solution Overview
Problem
Existing methods for determining nucleic acid sequences in animal samples are either highly sensitive and specific but slow or provide quick but unreliable results, lacking a balance between speed and reliability.
Innovation Solution
A method involving the addition of enzymes with RNA- and/or DNA-dependent DNA polymerase activity and at least five DNA primers, including forward and reverse complementary primers, to a sample, which is then incubated at a fixed temperature to detect the presence of a pre-determined nucleic acid sequence, specifically using a five-primer system without an F3 primer for efficient detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional nucleic acid detection methods are used, then sensitivity and specificity are improved, but detection time increases significantly
Solution Approach 1:
The patent changes the temperature parameter from cyclic variations (PCR) to a constant isothermal condition (65°C), enabling rapid amplification without the time-consuming heating and cooling cycles. This parameter change maintains detection reliability while reducing detection time to under 10 minutes.
Solution Approach 2:
The patent incorporates all necessary reaction components (enzymes, primers, dNTPs, buffers) into a pre-prepared master mix before adding the sample. This preliminary preparation eliminates setup time during detection and ensures optimal reaction conditions from the start, contributing to rapid and reliable results.
2Productivity
If rapid detection methods are used, then detection speed is improved, but result reliability decreases
Solution Approach 1:
The patent uses a specially designed primer set (FIP, BIP, LPF, LPB) as intermediaries that facilitate rapid isothermal amplification. These primers enable the Bst DNA polymerase to efficiently amplify target sequences at constant temperature, achieving both speed and reliability through their optimized sequence design and binding characteristics.
Solution Approach 2:
The patent employs a composite enzyme system combining Bst DNA polymerase with strand displacement activity and reverse transcriptase activity in a single reaction mixture. This composite enzymatic system enables simultaneous DNA amplification and RNA template conversion, achieving rapid and reliable detection of both DNA and RNA viral genomes.
3Measurement precision
If complex PCR protocols are used, then detection accuracy is improved, but procedural complexity increases
Solution Approach 1:
The patent extracts the essential function of nucleic acid amplification from the complex cyclic PCR protocol and implements it through a simplified isothermal LAMP reaction. By removing the need for temperature cycling and using a constant temperature incubation, the method maintains detection accuracy while dramatically reducing procedural complexity and equipment requirements.
Solution Approach 2:
The patent designs a universal isothermal amplification system that can detect both DNA and RNA viruses using the same reaction conditions and equipment. The Bst DNA polymerase with strand displacement activity and reverse transcriptase activity provides multi-functional capability, eliminating the need for separate protocols and enhancing procedural simplicity while maintaining high detection accuracy.
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 method achieves fast and sensitive detection of nucleic acid sequences, allowing for reliable identification of pathogens within ten minutes, improving the speed and reliability of nucleic acid detection in animal samples.
Implementation Method 1
adding one or more enzyme(s) providing activities of RNA- and/or DNA-dependent DNA polymerase activity
Implementation Method 2
adding one or more enzyme(s) providing activities of RNA- and/or DNA-dependent DNA polymerase activity and strand-displacement activity
Implementation Method 3
at least one DNA primer comprises a sequence hybridisable to the nucleic acid sequence and at least one DNA primer comprises a sequence hybridisable to the DNA sequence reverse-complementary to the nucleic acid sequence
Data Source
AI summary
The present invention relates to a method for determining presence of a pre-determined nucleic acid sequence in a sample, the method comprising the steps of adding one or more enzyme(s) providing activities of RNA- and/or DNA-dependent DNA polymerase activity and strand-displacement activity to the sample to be analysed for the presence of the pre-determined nucleic acid sequence; adding at least five DNA primers to the sample to be analysed for the presence of the pre-determined nucleic acid sequence, wherein at least one DNA primer comprises a sequence hybridisable to the nucleic acid sequence and at least one DNA primer comprises a sequence hybridisable to the DNA sequence reverse-complementary to the nucleic acid sequence; incubating the sample resulting at a fixed temperature; determining whether an elongated DNA sequence is present in the sample, wherein presence of the elongated DNA sequence in the sample is indicative of the presence of the pre-determined nucleic acid sequence in the sample, wherein the sample is obtained from an animal and wherein no F3 primer is used.