Polymerase Chain Reaction (PCR) is the most widely used
method for detection of infectious agents in clinical
samples by specific amplification of their nucleic acids.
However, PCR requires thermal cycling therefore an
instrument called thermal cycler which is only available in
molecular biology laboratories. Therefore PCR cannot be used
as a point of care diagnostic methods. In order to amplify
double stranded DNA the first step is the denaturation of
double strands by heating DNA to 95°C in the thermal cycler.
There is also loop mediated amplification technique (LAMP)
that can do DNA amplification without requiring thermal
cycling; however, it still requires a heating block since
isothermal amplification by LAMP is achieved at 65°C.
Our inspiration is to generate the path to the new
rapid, easy, low cost and accurate diagnostic tests.
As known, detection of infectious agents are diagnosed mostly by using PCR or rapid antigen-antibody interaction tests in clinical samples. But PCR is not practical since they require expensive laboratory instruments and experienced laboratory personnel. On the other hand, antigen tests are diagnostic tests that give results in a short time, however with less sensitivity due to lack of signal amplification ability like DNA or RNA templates.
Our target is to identify rapid easy point of care diagnostics without the need for any instrument and experienced laboratory personnel by combining antigen tests and nucleic acid amplification at room temperature which is REP Diagnostics. The inspiration behind our project is the need for an easy way to diagnose illnesses in order to help patients find treatment earlier.
Our method depends on cutting one strand of double stranded
DNA by a nicking enzyme and separation of strands from each
other by strand displacement activity of DNA polymerase.
For nucleic acid amplification, DNA strands are separated at
room temperature by using the nicking enzyme. When a nicking
enzyme breaks the sugar phosphate bond of one strand the
3’end of DNA functions like a primer where DNA polymerase
binds and starts elongating DNA on one strand. During this
DNA synthesis, the other strand is displaced separating the
two strands from each other. Amplification of double
stranded DNA is achieved by DNA synthesis on the displaced
strand using reverse primers. The new signal amplification
method can easily adapt to the nucleic acid sequence of
various infectious agents in clinical samples specifically.