High-yield and facile genomic DNA extraction method on glass fiber strip for LAMP PCR based pathogenic bacteria detection
33rd International Symposium on Pharmaceutical and Biomedical Analysis, Ankara, Türkiye, 2 - 06 Temmuz 2023, ss.1-5, (Özet Bildiri)
- Yayın Türü: Bildiri / Özet Bildiri
- Basıldığı Şehir: Ankara
- Basıldığı Ülke: Türkiye
- Sayfa Sayıları: ss.1-5
- Gazi Üniversitesi Adresli: Evet
Özet
High-yield and facile DNA extraction from biological samples to detect pathogens is of great importance for Point of Care (POC) applications. Recently, glass fiber-based extraction method has become popular due to their ability to efficiently capture and retain DNA molecules. In our study, we demonstrated a simple DNA extraction process using glass fiber without centrifuge and any complex equipment reducing time required for sample processing and analysis for POC applications. The genomic DNA was isolated from S. aureus bacteria spiked reference blood sample using glass fiber strip and performed colorimetric LAMP PCR test. Glass fiber strip was fixed on glass slide. The blood samples spiked with Staphylococcus aureus bacteria culture was tested on the proposed material. The sample was incubated with lysis buffer and proteinase K solution. After drying pretreated glass fiber, the lysate was dropped on the strip and washed several times by ethanol solution to remove undesired molecules originated from biological samples on the strip, DNA was released from the fiber using elution buffer. Nanodrop UV absorbance spectrophotometer and colorimetric LAMP PCR measurements were carried out for the evaluation quantity and quality of DNA isolate. The higher (∼five times) DNA yield from glass fiber than the commercial spin column-based isolation kit was found via spectroscopic results. The limit of detection was determined as 10 cfu/ml of S.aureus bacteria in blood sample for colorimetric LAMP PCR. As a result of Enterococcus faecalis and Escherichia coli bacteria spiked blood samples for specifity evaluation of colorimetric LAMP, there wasn’t seen any cross reaction for that samples. Our work offers a simple, efficient and relatively inexpensive method based on glass fiber for isolating DNA and PCR based detection of pathogens.