Rotor gene 5plex hrm platform

Samples from bioreactor inoculated with MOI 0.1 and MOI 1.0 were submitted to qPCR analysis to compare the viral DNA quantification to OBs quantification. The OBs obtained from 3 and 7 dpi (MOI 0.1) or at 3, 7, 11 and 14 dpi (MOI 1.0) were dissolved in an alkaline solution and used to extract DNA with DNeasy Blood & Tissue (Qiagen), following the manufacturer’s instructions. The DNA was quantified with a low-DNA-mass ladder (Invitrogen) in 0.8% agarose gel electrophoresis. SfMNPV-6nd OBs obtained from larvae were extracted and serially diluted for absolute quantification. The qPCR for SfMNPV with specific primers for the sf32 gene was carried out in a Rotor gene 5plex HRM platform (Qiagen), according to [17 (link)].

Three different targets were analyzed for M. tuberculosis detection by real time PCR: IS6110 gene, 65 kDa Heat Shock Protein gene (hsp65 KDa), and the MPB64 protein encoding gene (MPB64) (Table 1). The reaction was performed on Rotor-Gene 5 plex/HRM platform (Qiagen, USA) by melting analyses (QuantiFast SYBR Green PCR (Qiagen, USA)) on 20 μL final volume. The limit of detection (LoD) was determined by a serial sevenfold dilutions prepared in ultrapure water (Sigma-Aldrich, Co. Ltd, Saint Louis, US).
The target specificity was carried out by testing the negative control group and CSF samples. The negative control group was made up by ATCC and known strains of bacteria, yeasts, and other mycobacteria (Table 2).