Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a visual diagnostic platform for the detection of the emerging coronavirus SARS-CoV-2.

COVID-19, caused by the infection of SARS-CoV-2, has emerged as a rapidly spreading infection. The disease has now reached the level of a global pandemic and as a result a more rapid and simple detection method is imperative to curb the spread of the virus. We aimed to develop a visual diagnostic platform for SARS-CoV-2 based on colorimetric RT-LAMP with levels of sensitivity and specificity comparable to that of commercial qRT-PCR assays. In this work, the primers were designed to target a conserved region of the RNA-dependent RNA polymerase gene (RdRp). The assay was characterized for its sensitivity and specificity, and validated with clinical specimens collected in Thailand. The developed colorimetric RT-LAMP assay could amplify the target gene and enabled visual interpretation in 60 min at 65 °C. No cross-reactivity with six other common human respiratory viruses (influenza A virus subtypes H1 and H3, influenza B virus, respiratory syncytial virus types A and B, and human metapneumovirus) and five other human coronaviruses (MERS-CoV, HKU-1, OC43, 229E and NL63) was observed. The limit of detection was 25 copies per reaction when evaluated with contrived specimens. However, the detection rate at this concentration fell to 95.8% when the incubation time was reduced from 60 to 30 min. The diagnostic performance of the developed RT-LAMP assay was evaluated in 2120 clinical specimens and compared with the commercial qRT-PCR. The results revealed high sensitivity and specificity of 95.74% and 99.95%, respectively. The overall accuracy of the RT-LAMP assay was determined to be 99.86%. In summary, our results indicate that the developed colorimetric RT-LAMP provides a simple, sensitive and reliable approach for the detection of SARS-CoV-2 in clinical samples, implying its beneficial use as a diagnostic platform for COVID-19 screening.

Process optimization for an industrial-scale production of Diphtheria toxin by Corynebacterium diphtheriae PW8.

In this study, several parameters affecting the toxin production of Corynebacterium diphtheriae Parke Williams 8 (PW8) were investigated in detail. The comparison studies of amino acid profile in NZ Amine A-based medium (NZ medium) and beef digest-based medium (BD medium) suggested that an insufficient supply of amino acids was not responsible for low toxin yield observed in NZ medium. Supplementation of additional amino acids and growth promoting nutrient (in a form of yeast extract) into NZ medium enhanced only cell growth but not toxin production. Thus, BD medium was selected as the most suitable base medium for toxin production as it gave a significantly higher limit of flocculation (93 ± 0 Lf/ml) than NZ medium (46 ± 0 Lf/ml). Interestingly, a supplementation of 0.2% YE into BD medium resulted in a significant increase in growth as well as toxin production (235 ± 5 Lf/ml). In conclusion, consistently high toxin titer (174-239 Lf/ml) could be obtained from BD medium at a 5 L-scale production as long as 1) the protein content of BD medium was at least 24 g/L, 2) the iron content was below 0.15 ppm and 3) 0.2% YE was supplemented into the medium.

Improved docosahexaenoic acid production in Aurantiochytrium by glucose limited pH-auxostat fed-batch cultivation.

Fed-batch, pH auxostat cultivation of the docosahexaenoic acid (DHA)-producing microorganism Aurantiochytrium B072 was performed to obtain high cell density and record high productivity of both total fatty acid (TFA) and DHA. Using glucose feeding by carbon excess (C-excess) and by C-limitation at various feeding rates (70%, 50% or 20% of C-excess), high biomass density was obtained and DHA/TFA content (w/w) was improved from 30% to 37% with a 50% glucose feed rate when compared with C-excess. To understand the biochemistry behind these improvements, lipogenic enzyme assays and in silico metabolic flux calculations were used and revealed that enzyme activity and C-fluxes to TFA were reduced with C-limited feeding but that the carbon flux to the polyketide synthase pathway increased relative to the fatty acid synthase pathway. As a result, a new strategy to improve the DHA to TFA content while maintaining relatively high DHA productivity is proposed.

Metabolic networks and bioenergetics of Aurantiochytrium sp. B-072 during storage lipid formation

Baffled shake flask cultivation of Aurantiochytrium sp. B-072 was carried out at in a glucose-monosodium glutamate mineral medium at different C/N-ratios (30-165) with glucose fixed at 90 g/L. With increasing C/N-ratio, a modest increase in lipid content (60 to 73 % w/w) was observed whereas fat-free biomass decreased but overall biomass showed little variation. FA-profiles were not affected to a large extent by C/N-ratio and absolute docosahexaenoic (DHA)-levels fell in narrow range (5-6 g/L). However at C/N > 64 a rapid decrease in lipid synthetic rate and/or incomplete glucose utilization occurred. Glucose and FA-fluxes based on fat-free biomass peaked at a C/N ratio of 56. This condition was chosen for calculation of the redox balance (NAD(P)H) and energy (ATP) requirement and to estimate the in vivo P/O ratio during the main period of fatty acid biosynthesis. Several models with different routes for NADPH, acetyl-CoA formation and re-oxidation of OAA formed via ATP-citrate lyase were considered as these influence the redox- and energy balance. As an example, using a commonly shown scheme whereby NADPH is supplied by a cytosolic "transhydrogenase cycle" (pyruvate-OAA-malate-pyruvate) and OAA formed by ATP-citrate lyase is recycled via import into the mitochondria as malate, the calculated NADPH-requirement amounted to 5.5 with an ATP-demand of 10.5 mmol/(g fat-free biomass x h) and an in vivo P/O-ratio (not including non-growth associated maintenance) of 1.6. The lowest ATP requirement is found when acetyl-CoA would be transported directly from the mitochondria to the cytosol by carnitine acetyltransferase. Assay of some enzymes critical for NADPH supply indicates that activity of glucose-6-phosphate dehydrogenase, the first enzyme in the HMP pathway, is far insufficient for the required NADPH-flux and malic enzyme must be a major source. Activity of the latter (ca. 300 mU/mg protein) far exceeds that in oleaginous fungi and yeast.

Metabolic networks and bioenergetics of Aurantiochytrium sp. B-072 during storage lipid formation.

Baffled shake flask cultivation of Aurantiochytrium sp. B-072 was carried out at in a glucose-monosodium glutamate mineral medium at different C/N-ratios (30-165) with glucose fixed at 90 g/L. With increasing C/N-ratio, a modest increase in lipid content (60 to 73 % w/w) was observed whereas fat-free biomass decreased but overall biomass showed little variation. FA-profiles were not affected to a large extent by C/N-ratio and absolute docosahexaenoic (DHA)-levels fell in narrow range (5-6 g/L). However at C/N > 64 a rapid decrease in lipid synthetic rate and/or incomplete glucose utilization occurred. Glucose and FA-fluxes based on fat-free biomass peaked at a C/N ratio of 56. This condition was chosen for calculation of the redox balance (NAD(P)H) and energy (ATP) requirement and to estimate the in vivo P/O ratio during the main period of fatty acid biosynthesis. Several models with different routes for NADPH, acetyl-CoA formation and re-oxidation of OAA formed via ATP-citrate lyase were considered as these influence the redox- and energy balance. As an example, using a commonly shown scheme whereby NADPH is supplied by a cytosolic "transhydrogenase cycle" (pyruvate-OAA-malate-pyruvate) and OAA formed by ATP-citrate lyase is recycled via import into the mitochondria as malate, the calculated NADPH-requirement amounted to 5.5 with an ATP-demand of 10.5 mmol/(g fat-free biomass x h) and an in vivo P/O-ratio (not including non-growth associated maintenance) of 1.6. The lowest ATP requirement is found when acetyl-CoA would be transported directly from the mitochondria to the cytosol by carnitine acetyltransferase. Assay of some enzymes critical for NADPH supply indicates that activity of glucose-6-phosphate dehydrogenase, the first enzyme in the HMP pathway, is far insufficient for the required NADPH-flux and malic enzyme must be a major source. Activity of the latter (ca. 300 mU/mg protein) far exceeds that in oleaginous fungi and yeast.

Application of a mathematical model and Differential Evolution algorithm approach to optimization of bacteriocin production by Lactococcus lactis C7.

The effect of pH and temperature on cell growth and bacteriocin production in Lactococcus lactis C7 was investigated in order to optimize the production of bacteriocin. The study showed that the bacteriocin production was growth-associated, but declined after reaching the maximum titer. The decrease of bacteriocin was caused by a cell-bound protease. Maximum bacteriocin titer was obtained at pH 5.5 and at 22 degrees C. In order to obtain a global optimized solution for production of bacteriocin, the optimal temperature for bacteriocin production was further studied. Mathematical models were developed for cell growth, substrate consumption, lactic acid production and bacteriocin production. A Differential Evolution algorithm was used both to estimate the model parameters from the experimental data and to compute a temperature profile for maximizing the final bacteriocin titer and bacteriocin productivity. This simulation showed that maximum bacteriocin production was obtained at the optimal temperature profile, starting at 30 degrees C and terminating at 22 degrees C, which was validated by experiment. This temperature profile yielded 20% higher maximum bacteriocin productivity than that obtained at a constant temperature of 22 degrees C, although the total amount of bacteriocin obtained was slightly decreased.