Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based...

Area of Science:

• Molecular Biology
• Infectious Disease Diagnostics
• Biotechnology

Background:

• Real-time PCR is crucial for rapid infectious disease diagnosis.
• Validating the sensitivity of molecular diagnostic methods is essential for accurate results.
• Existing methods may have limitations in sensitivity and contamination control.

Purpose of the Study:

• To develop a recombinant plasmid-based standard for validating real-time PCR assay sensitivity.
• To compare the sensitivity of different molecular diagnostic methods using the developed standard.
• To introduce a method for simultaneous detection of target and exogenous sequences to prevent contamination.

Main Methods:

• Construction of chimeric plasmid DNA (cpDNA) containing pathogen genes and the Jonstrup assay target site.
• Validation of cpDNA as a standard for real-time PCR sensitivity testing.
• Evaluation of various real-time PCR methods, including VHS Garver and SARS-CoV-2 assays.
• Introduction of an additional probe attachment site for simultaneous detection.

Main Results:

• The developed standard demonstrated high sensitivity, detecting a single copy of the target gene, comparable to digital droplet PCR.
• Most real-time PCR methods showed high sensitivity, but VHS Garver and SARS-CoV-2 assays were tenfold less sensitive than the Jonstrup assay.
• The novel cpDNA design allowed simultaneous detection of target genes and exogenous sequences, preventing genetic contamination.

Conclusions:

• The recombinant plasmid-based standard effectively validates real-time PCR sensitivity for infectious disease diagnostics.
• The developed method improves diagnostic accuracy by enabling simultaneous detection and preventing contamination.
• This approach facilitates the development of more sensitive and reliable molecular diagnostic tools.