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.

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Introduction A global surge in SARS-CoV-2 cases is occurring due to the emergence of new disease variants, and requires continuous adjustment of public health measures. This study aims to continuously monitor and mitigate the impact of SARS-CoV-2 through genomic surveillance, to determine the emergence of variants and their impact on public health. Methods Data were collected from 50 full-genome sequences of SARS-CoV-2 isolates from Makassar, South Sulawesi, Indonesia. Mutation and phylogenetic analysis was performed of SARS-CoV-2 from Makassar, South Sulawesi, Indonesia. Results Phylogenetic analysis showed that two samples (4%) were of the B.1.319 lineage, while the others (96%) were of the B.1.466.2 lineage. Mutation analysis of the spike (S) protein region showed that the most common mutation was D614G (found in 100% of the sequenced isolates), followed by N439K (98%) and P681R (76%). Several mutations were also identified in other genomes with a high frequency, including P323L (nsp12), Q57H (ns3-orf3a), and T205I (nucleoprotein). Conclusion Our findings highlight the importance of continuous genomic surveillance to identify new viral mutations and variants with possible impacts on public health.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new virus responsible for the COVID-19 pandemic. The emergence of the new SARS-CoV-2 has been attributed to the possibility of evolutionary dynamics in the furin cleavage site (FCS) region. This study aimed to analyze the sequence of the FCS region in the spike protein of SARS-CoV-2 isolates that circulated in the Special Region of Yogyakarta and Central Java provinces in Indonesia. The RNA solution extracted from nasopharyngeal swab samples of confirmed COVID-19 patients were used and subjected to cDNA synthesis, PCR amplification, sequencing, and analysis of the FCS region. The sequence data from GISAID were also retrieved for further genome analysis. This study included 52 FCS region sequences. Several mutations were identified in the FCS region, i.e., D614G, Q675H, Q677H, S680P, and silent mutation in 235.57 C > T. The most important mutation in the FCS region is D614G. This finding indicated the G614 variant was circulating from May 2020 in those two provinces. Eventually, the G614 variant totally replaced the D614 variant from September 2020. All Indonesian SARS-CoV-2 isolates during this study and those deposited in GISAID showed the formation of five clade clusters from the FCS region, in which the D614 variant is in one specific cluster, and the G614 variant is dispersed into four clusters. The data indicated there is evolutionary advantage of the D614G mutation in the FCS region of the spike protein of SARS-CoV-2 circulating in the Special Region of Yogyakarta and Central Java provinces in Indonesia.

One of the most promising ways to produce high hydrogen yield is through dark fermentation by using dark fermentative bacteria due to the capability of these microbial agents to convert various organic compounds, particularly sugar, into hydrogen gas. In this study, three Gram-positive hydrogen-producing bacteria with a different character of colony on agar, namely as RP 009, RP 010, and RP 011, had been successfully isolated from Mount Pancar hot spring, West Java. All these isolates were able to produce hydrogen gas in all cheese whey concentration, consisting of cheese whey 30%, 60%, and 100%. RP 011 was the most favorable hydrogen producers in this study due to its high hydrogen productivity (4,400.625 ml biogas/L medium) as well as its ability to adapt and consecutively produce hydrogen even in the very high concentration of the organic compound. The best cheese whey concentration for hydrogen production in this study was 60%, considering the efficiency and effectiveness of the organic compound conversion into hydrogen gas. Ultimately, this study presented the potential of high hydrogen productivity of indigenous hot spring bacteria isolated from Mount Pancar hot spring in which had major potential for environmentally friendly bioenergy and biomass refineries.