Objectives
Monitoring the spread of the G614 in specific locations is critical as this variant is highly transmissible and can trigger the emergence of other mutations. Therefore, a rapid and accurate method that can reliably detect the D614G mutation will be beneficial. This study aims to analyze the potential use of the two-step Reverse Transcriptase
quantitative polymerase chain reaction - high resolution melting
analysis (RT-qPCR-HRM) to detect a specific mutation in the SARS-CoV-2 genome.
Methods
Six SARS-CoV-2 RNA samples were synthesized into cDNA and analyzed with the qPCR-HRM method in order to detect the D614G mutation in Spike protein of SARS-CoV-2. The primers are designed to target the specific Spike region containing the D614G mutation. The qPCR-HRM analysis was conducted simultaneously, and the identification of the SARS-CoV-2 variant was confirmed by conventional PCR and Sanger sequencing methods.
Results
The results showed that the melting temperature (T
m
) of the D614 variant was 79.39 ± 0.03°C, which was slightly lower than the T
m
of the G614 variant (79.62 ± 0.015°C). The results of the HRM analysis, visualized by the normalized melting curve and the difference curve were able to discriminate the D614 and G614 variant samples. All samples were identified as G614 variants by qPCR-HRM assay, which was subsequently confirmed by Sanger sequencing.
Conclusions
This study demonstrated a sensitive method that can identify the D614G mutation by a simple two-step RT-qPCR-HRM assay procedure analysis, which can be useful for active surveillance of the transmission of a specific mutation.
Abstract:Microalgae use photosynthesis to convert solar energy into chemical energy, such as lipid and they can be a replacement for oil-based fuels. They are among the fastest growing plants in the world, and about 50% of their weight is oil. This lipid oil can be used to make biodiesel. Unfortunately, there are only some of potential strains isolated from Indonesia and most of the biodiesel productions are usually using a single strain. Then, although they are rich of oils, their biomass productivity is still low. Salinity treatment can be used to increase their biomass as well as their lipid content. Therefore, the research aim was to study the effect of salinity on the growth, dry weight and lipid content of mixed microalgae isolated from Glagah, Yogyakarta. The mixed microalgae were cultured in 3NBBM medium with different salinities or types of water (sea water, brackish water, and fresh water). The cultures were incubated at light intensity 3,000 lux under dark:light exposure of 12:12 h for 7 days. The number of cells was counted every 24 h with a Haemocytometer, and the biomass was calculated based on the dry weight. The lipid content was measured on days 0, 3, and 7 using NR (Nile Red) staining, and then the amount of lipid was analyzed using a fluorescence microscope and measured with CellProfiler 2.0 software. The highest dry weight and lipid content were found in seawater medium, they accounted for 3.42 mg/mL and 13.58% at day 7, respectively. Whereas, the highest number of cells was found in freshwater medium, this was 9.8 × 10 6 cells/mL.
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