Magna pure lc 2.0 system

For each enrolled subject, a venous blood sample was collected on the date of entry. Blood samples were subjected to centrifugation at 4000 rpm for 10 min to separate serum and blood cells and stored at −78 °C prior to DNA extraction. Human genome DNA was done by using Magna Pure LC 2.0 system (Roche Applied Science, Mannheim, Germany). Six polymorphisms in TLRs including rs10759932, rs3804099, rs4696480, rs4986790, rs4986791 and rs5743708 were genotyped by employing Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry. All procedures were conducted strictly following the protocol as described by the manufacturer (Sequenom, San Diego CA, USA). A negative water control and reference DNA were employed as quality control measures during the genotyping assay. Moreover, approximately 5% of the samples were randomly selected and repeated for genotyping as duplicated controls. The genotyping call rate was 100% in accordance with the results generated from the platform.

Cell monolayers infected with HSV-1 were incubated with nHo-dimer (30 µM) or the vehicle during different times. Subsequently, the cells were harvested for further qPCR analysis. Nucleic acids were extracted from the harvested cells using the MagNA Pure LC 2.0 System (Roche Diagnostics GmbH, Penzberg, Germany), and qPCR was performed on the extracted DNA, following established protocols [15 (link)]. Briefly, specific HSV-1 sequences were amplified from 5 µL of extracted DNA using a 7500 Fast Real-Time PCR System and TaqMan^® Fast Universal PCR Master Mix (Thermo Fisher Scientific Inc., Waltham, MA, USA). To amplify a specific 81-nt sequence of the gene encoding the HSV-1 polymerase, we employed optimized primers and a FAM-labeled probe. The thermocycler settings for PCR amplification were as follows: initial denaturation at 95 °C for 20 s, followed by 45 cycles of denaturation at 95 °C for 3 s, and annealing/extension at 60 °C for 30 s. Quantification was accomplished by the simultaneous amplification of four HSV-plasmid-DNA standards of 5 × 10², 5 × 10³, 5 × 10⁴ and 5 × 10⁵ copies/mL.