# Fabrication of a Microfluidic Test Device with a 3D Printer and Its Combination with the Loop Mediated Isothermal Amplification Method to Detect Streptococcus pyogenes

## TL;DR

This paper describes a 3D-printed microfluidic device combined with LAMP to detect Streptococcus pyogenes, offering a rapid and cost-effective diagnostic tool.

## Contribution

The novelty is the integration of a 3D-printed microfluidic device with LAMP for rapid detection of S. pyogenes.

## Key findings

- The microfluidic device achieved 84–92% sensitivity and 90–94% specificity for S. pyogenes detection.
- LAMP reactions on the microfluidic device showed comparable performance to traditional microcentrifuge tube methods.
- The device's LOD was 1.5 × 10² CFU/mL, suitable for rapid pathogen detection in resource-limited settings.

## Abstract

New rapid, reliable, and cost-effective alternative systems are needed for the rapid diagnosis of Streptococcus pyogenes. The aim of this study was to fabricate a microfluidic test device to detect Streptococcus pyogenes by combining the Loop-mediated isothermal amplification method via a 3D printer. Microfluidic test devices were designed in CATIA V5 Release 16 software, and data were directly transferred to a 3D printer and produced using the FDM method with biocompatible PLA filament. The S. pyogenes ATCC 19615 and different ATCC strains was used. Following identification by classical culture methods, a 0.5 McFarland suspension was prepared from the colonies, and DNA isolation was performed from this liquid by a boiling method. S. pyogenes specific speB gene was used to desing LAMP primer sets in PrimerExplorer V5 software and tested on a microfluidic device. LAMP reactions were performed on microfluidic device and on a microcentrifuge tube separately. Both results were analyzed using the culture method as the standard method to diagnostic values. Melting curve analysis of the amplicons of the LAMP reactions performed on a LightCycler 480 system to detect amplification. Among the 50 positive and 100 negative samples, only four samples were found to be false negative by LAMP reaction in a microcentrifuge tube, while eight samples were found to be false negative by LAMP reaction on a microfluidic device. Six samples were found to be false positive by the LAMP reaction in the microcentrifuge tube, while ten samples were found to be false positive by the LAMP reaction on a microfluidic chip. The sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP reactions performed in the microcentrifuge tube and on the microfluidic device were 92–84%, 94–90%, 88.46–80.77%, and 95.92–91.84%, respectively. The limit of detection (LOD) was found to be the same as 1.5 × 102 CFU/mL and the limit of quantification (LOQ) values of the LAMP reactions were performed on the microcentrifuge tube and on the microfluidic device were 2.46 × 102–7.4 × 102 CFU/mL, respectively. Cohen’s kappa (κ) values of the LAMP reactions were performed on the microcentrifuge tube and on the microfluidic device were 0.620–0.705, respectively. In conclusion, our data showed that the LAMP method can be combined with microfluidic test device to detect S. pyogenes, this microfluidic device can be manufactured using 3D printers and results are close to gold standard methods. These devices can be combined with LAMP reactions to detect different pathogens where resources are limited and rapid results are required.