Improved Visual Detection of speB Gene in Streptococcus pyogenes Isolates by Real-time Loop-Mediated Isothermal Amplification Turbidimetry Method

Simon, Azi Simon; Subbiah, Suresh Kumar; Than, Leslie Thian Lung; Osman, Malina; Hamat, Rukman Awang

doi:10.5812/jjm.108540

Cited by 1 publication

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References 31 publications

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“…It is already known that LAMP design is performed by using the S. pyogenes speB gene and is used in clinical samples for rapid diagnosis [ 2 , 25 , 26 ]. In their study, Cao et al reported that the sensitivity was 71.21% [ 2 ].…”

Section: Discussionmentioning

confidence: 99%

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

Kirkoyun Uysal,

Eryildiz,

Demirci

2024

Micromachines

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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.

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