Point-of-care detection of respiratory tract infections using a novel PCR kit with gold nanoparticle-mediated dna polymerase

Abstract

Respiratory tract infections (RTIs) are common and can cause a wide range of illnesses, from mild colds to severe pneumonia, bronchitis, and other respiratory diseases. RTIs are a significant burden on public health, with high morbidity and mortality rates, particularly in vulnerable populations such as the elderly, children, and immunocompromised individuals. However, current diagnostic methods for respiratory tract infections (RTIs) are often time-consuming and require specialized equipment and trained personnel, resulting in long turnaround times and delays in treatment. In order to address this challenge, we developed a novel PCR kit for the detection of SARS-CoV-2, Influenza A virus, Influenza B virus, Respiratory Syncytial Virus (RSV) A/B, Human Adenovirus, Human Rhinovirus and Streptococcus pyogenes named Respiratory ID Panel 2 (RIDK-2), that uses gold nanoparticles to enhance DNA polymerase activity, thereby improving the sensitivity and specificity of RTI detection. In this study, we designed and optimized the gold nanoparticle-enhanced PCR kit and validated its analytical performance compared to same RT-qPCR kit produced without golden nanoparticles (AuNPs), including preliminary limit of detection (LoD), LoD, wet and in-silico inclusivity, wet and in-silico exclusivity, and stability studies. The kit demonstrated superior analytical performance, with high sensitivity and specificity for detecting target RTI pathogens mentioned above. The RIDK-2 has a turnaround time under 1 hour without the need of nucleic acid extraction with a LoD of 250 cp/mL for research samples prepared using reference materials and the kit showed no cross reactivity. The rapid turnaround time and accuracy of the gold nanoparticle-enhanced PCR kit make it a valuable tool for the timely diagnosis of RTIs, reducing the risk of intensive care unit admission and death. Furthermore, stability studies showed that the kit without gold nanoparticles could be stored at room temperature for up to 6 hours without significant loss of performance, while the gold nanoparticle-enhanced PCR kit was stable for up to 72 hours, making it suitable for use in clinics, including pediatric clinics, where specialized equipment and refrigeration may not be readily available. According to the analytical performance studies the kit's sensitivity and specificity was found as 99.74% and 99.61%. These findings suggest that the gold nanoparticle-enhanced PCR kit has the potential to significantly improve the management of RTIs, thereby enhancing patient outcomes and reducing healthcare costs.