Summary of the conditions and results of each round of mutagenesis.

*<p>Note that in the 3rd round of mutagenesis there was a technical problem with the temperation, and cultures were also temperated during the night cycle, leading to many more cells dying than usual. A recovery at lower temperature was used to rescue surviving cells.</p

The Optimal Mutagen Dosage to Induce Point-Mutations in <em>Synechocystis</em> sp. PCC6803 and Its Application to Promote Temperature Tolerance

<div><p>Random mutagenesis is a useful tool to genetically modify organisms for various purposes, such as adaptation to cultivation conditions, the induction of tolerances, or increased yield of valuable substances. This is especially attractive for systems where it is not obvious which genes require modifications. Random mutagenesis has been extensively used to modify crop plants, but even with the renewed interest in microalgae and cyanobacteria for biofuel applications, there is relatively limited current research available on the application of random mutagenesis for these organisms, especially for cyanobacteria. In the presented work we characterized the lethality and rate of non-lethal point mutations for ultraviolet radiation and methyl methanesulphonate on the model cyanobacteria <em>Synechocystis</em> sp. PCC6803. Based on these results an optimal dosage of 10–50 J/m² for UV and either 0.1 or 1 v% for MMS was determined. A <em>Synechocystis</em> wildtype culture was then mutagenized and selected for increased temperature tolerance <em>in vivo</em>. During the second round of mutagenesis the viability of the culture was monitored on a cell by cell level from the treatment of the cells up to the growth at an increased temperature. After four distinct rounds of treatment (two with each mutagen) the temperature tolerance of the strain was effectively raised by about 2°C. Coupled with an appropriate <em>in vivo</em> screening, the described methods should be applicable to induce a variety of desirable characteristics in various strains. Coupling random mutagenesis with high-throughput screening methods would additionally allow to select for important characteristics for biofuel production, which do not yield a higher fitness and can not be selected for <em>in vivo</em>, such as fatty acid concentration. In a combined approach with full genome sequencing random mutagenesis could be used to determine suitable target-genes for more focused methods.</p> </div

Characterization of UV.

<p>Survival rate (green; triangle) and point mutation rate (blue; square) plotted over the administered dosage of UV irradiation. The counted colonies for the survival rate and the point mutation rate were normalized by setting the control to 100%.</p

Characterization of MMS.

<p>Survival rate (green; upward triangle: 5*10∧4 cells plated | downward triangle 5*10∧5 cells plated, counted colonies divided by 10) and point mutation rate (red; square) plotted over the administered dosage of MMS exposure. The counted colonies for the survival rate and the point mutation rate were normalized by setting the control to 100%.</p

Fig 2 -

Analysis of Ct/Cq values for (A) monkeypox virus PCR results and (B) for orthopoxvirus PCR results for different test systems. The grey boxes display all results for the respective sample, and the distributions of specific manufacturer-based collectives are illustrated as smaller, colored box plots in overlay with the total results. For all boxes, the whiskers stretch from the 1st quartile—1.5*(interquartile range) to the 3rd quartile + 1.5*(interquartile range).</p

Fig 1 -

Distribution of the qualitative PCR results for the four samples of the monkeypox EQA survey for A) monkeypox virus (MPXV) and B) orthopoxvirus (OPXV). Numbers in the columns represent the actual number of results for the corresponding category.</p

Sample properties.

BackgroundIn May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests.MethodsWe analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level.Results141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each.ConclusionThe EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted.</div

S4 Table -

Results of Levene’s test of equality of variance of the reported assay-specific Ct/Cq values per MPX positive sample for A) MPXV detection and B) OPXV detection. When equality of variance was not found (labeled in red colour), one collective (marked with *) was identified and excluded before rerun the test to be able to show the equality of the variances for the other collectives (labeled in green colour). (XLSX)</p

INSTAND Monkeypox EQA September 2022—all results.

This table contains the raw results of the EQA participants without any correction. Results that were excluded from evaluation are highlighted in orange. (XLSX)</p

Passing Bablok fit of laboratory-based Ct/Cq values (black dots) for all pairs of EQA samples, differing in concentration by a power of ten.

The Passing Bablok regression line (green) is shown with the lower and upper 95% confidence limits (red).</p