Diagnostic Host Gene Expression Analysis by Quantitative Reverse Transcription Loop-Mediated Isothermal Amplification...

Area of Science:

• Molecular Biology
• Infectious Disease Diagnostics
• Biomarker Discovery

Background:

• Accurate diagnosis of acute infections is crucial for appropriate antibiotic use and patient outcomes.
• Distinguishing bacterial from viral infections using host gene expression is challenging with current slow and expensive technologies.
• There is a need for rapid, cost-effective methods for host gene expression profiling in clinical settings.

Purpose of the Study:

• To develop an ultra-rapid test system for host gene expression profiling from blood.
• To enable discrimination between bacterial and viral etiologies using quantitative reverse transcription followed by loop-mediated isothermal amplification (qRT-LAMP).

Main Methods:

• Developed 10 messenger ribonucleic acid-specific assays using qRT-LAMP targeting 7 biomarkers for bacterial/viral discrimination and 3 housekeeping genes.
• Optimized qRT-LAMP for a 12-minute turnaround time without compromising specificity or precision.
• Validated the test system's accuracy against an orthogonal reference technology using blood samples from 57 patients.

Main Results:

• Achieved a Pearson correlation coefficient of 0.90 between bacterial/viral metascores generated by qRT-LAMP and the reference technology.
• Demonstrated the precision and specificity of the optimized qRT-LAMP assays.
• Confirmed the system's ability to generate accurate gene expression profiling data.

Conclusions:

• qRT-LAMP assays provide accurate gene expression profiling data for discriminating bacterial and viral infections.
• The developed system enables differentiation of etiologies using established biomarkers and a classification algorithm.
• This ultra-rapid test system has the potential to be integrated into clinical workflows for faster diagnosis.