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    Fabrication of a Microfluidic Test Device with a 3D Printer and Its Combination with the Loop Mediated Isothermal Amplification Method to Detect Streptococcus pyogenes
    (MDPI, 2024) Uysal, Hayriye Kirkoyun; Eryildiz, Meltem; Demirci, Mehmet
    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 x 10(2) 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 x 102-7.4 x 10(2) CFU/mL, respectively. Cohen's kappa (kappa) 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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    Helicobacter pylori-miRNA interaction in gastric cancer tissues: First prospective study from Turkey
    (Edizioni Int Srl, 2019) Demiryas, Suleyman; Kocazeybek, Bekir; Demirci, Mehmet; Caliskan, Reyhan; Kepil, Nuray; Uysal, Hayriye Kirkoyun; Dinc, Harika Oyku
    Helicobacter pylori (H. pylori) is involved in the etiology of gastric cancer (GC). miRNAs are short RNAs that regulate gene expression by marking mRNAs for degradation. miRNAs are involved in tumorigenesis, metastasis, and cell proliferation. We aimed to investigate the miRNA expression profiles of tissues from H. pylori Wand (-) GC patients. Forty GC patients, 20 H. pylori Wand 20 H. pylori (-), and a healthy control group were included. The miRNA expression levels were investigated by microarrays and quantitative RT-PCR. We detected 9 upregulated and 4 downregulated miRNAs by microarray. We selected 5 upregulated and 5 downregulated miRNAs for the quantitative RT-PCR assay. The relative fold changes of miRNAs in the cancerous tissue and non-tumor mucosa specimens of H. pylori (+) GC patients for hsa-miR-194 were 4.24- and 3.83-fold higher, respectively, whereas the hsa-miR-145 expression levels were downregulated 0.33-fold and 0.43-fold, respectively, in the same group. The presence of H. pylori significantly upregulated hsa-miR-194 and downregulated hsa-miR-145 expression levels in H. pylofi(+)GC cases, compared to H. pylori (-) GC cases. Regional differences in the virulence of H. pylori strains may also be involved in the up- or downregulation of miRNA expression levels.