Isothermal HSV DNA Amplification via Restriction Enzyme Digestion
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Solution Overview
Problem
Current transcription-based amplification methods for diagnosing Herpes Simplex Virus (HSV) from double-stranded DNA are cumbersome and require multiple handling steps, including heat treatment and enzyme replenishment, making them inefficient compared to methods starting from single-stranded RNA.
Innovation Solution
A method using restriction enzyme digestion to create a defined 3′-end on DNA strands, followed by incubation with a promoter-primer and additional enzymes, allowing for isothermal amplification without the need for heat treatment, enabling efficient amplification and detection of HSV DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If transcription-based amplification is performed on double-stranded DNA using prior art methods, then amplification can be achieved, but multiple handling steps including heat treatment and enzyme replenishment are required, making the process cumbersome and inefficient
Solution Approach 1:
The method performs preliminary restriction enzyme digestion to create defined 3'-ends on DNA strands before the main amplification process. This preliminary action prepares the template in advance, allowing subsequent primers to bind efficiently without requiring heat treatment or enzyme replenishment during amplification, thereby reducing handling steps and improving operational ease
Solution Approach 2:
The invention introduces a restriction enzyme as an intermediary that processes the double-stranded DNA template before amplification. This intermediary enzyme creates the necessary structural conditions (defined 3'-ends) that enable the main amplification enzymes to work continuously without interruption, eliminating the need for heat treatment and enzyme replenishment steps
2Reliability
If prior art transcription-based amplification methods are used on double-stranded DNA, then amplification can occur, but the process requires heat treatment and enzyme replenishment, increasing handling time
Solution Approach 1:
The restriction enzyme digestion is performed as a preliminary step before amplification, creating defined 3'-ends that ensure reliable primer binding throughout the amplification process. This preliminary preparation eliminates the need for repeated heat treatment and enzyme replenishment, significantly reducing handling time while maintaining amplification reliability
Solution Approach 2:
The method establishes continuous useful action by creating a template structure that allows amplification enzymes to work continuously without interruption. The defined 3'-ends created by restriction digestion enable uninterrupted primer binding and extension, eliminating time-consuming intermediate steps while maintaining reliable amplification
3Measurement precision
If standard transcription-based amplification is performed on double-stranded DNA, then amplification can be achieved, but sensitivity and reaction kinetics are reduced compared to methods starting from single-stranded RNA
Solution Approach 1:
The restriction enzyme digestion performs preliminary structural optimization of the DNA template, creating defined 3'-ends that enhance primer binding efficiency and sensitivity. This preliminary action improves detection sensitivity to levels comparable with single-stranded RNA methods while maintaining the simplicity of working with double-stranded DNA templates
Solution Approach 2:
The method changes the structural parameters of the DNA template by creating defined 3'-ends through restriction digestion. This parameter change optimizes the template structure for subsequent amplification, improving sensitivity and reaction kinetics without requiring complex processing or conversion to single-stranded RNA
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 simplifies the amplification process, increases sensitivity, and reduces handling time, allowing for effective detection of HSV DNA, including both types 1 and 2, with improved reaction kinetics and sensitivity compared to prior art.
Implementation Method 1
one or more restriction enzymes capable of cleaving the double-stranded DNA at a selected restriction site, said restriction enzyme creating a defined 3′-end on one of the DNA strands
Implementation Method 2
a promoter-primer, said promoter-primer having a 5′-region comprising the sequence of a promoter recognized by a DNA-dependent RNA polymerase and a 3′-region hybridizing to the defined 3′-end of the DNA strand
Implementation Method 3
a DNA-dependent RNA polymerase and maintaining the thus created reaction mixture under the appropriate conditions for a sufficient amount of time for the amplification to take place
Data Source
AI summary
The present invention is related to a pair of oligonucleotide primers for the amplification of HSV nucleic acid comprising: a) an oligonucleotide, 10-50 nucleotides in length, preferably 10-35 nucleotides in length, comprising at least a fragment of 10 nucleotides of a sequence selected from the group consisting of: 5′-ACGTTCACCAAGCTGCTGCT-3′, or its complementary sequence and b) an oligonucleotide, 10-50 nucleotides in length, preferably 10-35 nucleotides in length, comprising at least a fragment of 10 nucleotides of a sequence selected from the group consisting of: 5′CCAGGGCCCTGGAGGTGCGG-3′, or its complementary sequence. The invention also relates to probes, method for amplifying an HSV DNA target, method of specific ou aspecific detection of HSV type 1 and 2 and test kit to do possible the detection of HSV. The present invention is especially useful in methods for practicing nucleic acid test.