Multiplexed Genome Editing via an RNA Polymerase II Promoter-Driven sgRNA Array in the Diatom Phaeodactylum tricornutum: Insights Into the Role of StLDP

CRISPR/Cas9-mediated genome editing has been demonstrated in the model diatom P. tricornutum, yet the currently available genetic tools do not combine the various advantageous features into a single, easy-to-assemble, modular construct that would allow the multiplexed targeting and creation of marker-free genome-edited lines. In this report, we describe the construction of the first modular two-component transcriptional unit system expressing SpCas9 from a diatom episome, assembled using the Universal Loop plasmid kit for Golden Gate assembly. We compared the editing efficiency of two constructs with orthogonal promoter-terminator combinations targeting the StLDP gene, encoding the major lipid droplet protein of P. tricornutum. Multiplexed targeting of the StLDP gene was confirmed via PCR screening, and lines with homozygous deletions were isolated from primary exconjugants. An editing efficiency ranging from 6.7 to 13.8% was observed in the better performing construct. Selected gene-edited lines displayed growth impairment, altered morphology, and the formation of lipid droplets during nutrient-replete growth. Under nitrogen deprivation, oversized lipid droplets were observed; the recovery of cell proliferation and degradation of lipid droplets were impaired after nitrogen replenishment. The results are consistent with the key role played by StLDP in the regulation of lipid droplet size and lipid homeostasis.

General Taq PCR Master Mix -- CHEM 384/584 v2

SapphireAmp Fast PCR Master Mix contains a hot start PCR enzyme, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent as a 2X premix. SapphireAmp Fast PCR Master Mix is optimized for fast PCR and offers a rapid extension rate (10 sec. per kb). The inclusion of blue dye and a density reagent allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR. SapphireAmp Fast PCR Master Mix is ideal for fast colony PCR screening. Fast colony PCR amplification of a 5 kb insert can be completed in approximately 1 hr 15 min. Furthermore, it is possible to amplify fragments up to 6 kb from genomic DNA templates.

Optimization of multiplex PCR composition to screen for SARS-CoV-2 variants of concern

Background: The ongoing COVID-19 pandemic has led to the emergence of several variants of concern. To rapidly identify those variants, screening samples for whole-genome sequencing (WGS) prioritization could be performed. Objective: We optimized the polymerase chain reaction (PCR) screening method to identify the mutation in spike and ORF1a regions. Methods: We adopted primers targeting mutation in spike and ORF1a region from another study. We optimized the PCR screening method using kits readily available in Indonesia. Firstly, we compared N1 and N2 primers as internal positive control. We also compared GoTaq® 1-Step RT-qPCR System and Indonesia TFRIC-19 BioCOV-19 for the multiplex reaction. We used the optimized composition to screen SARS-CoV-2 positive samples from April – June 2021. Samples with spike and/or ORF1a target failure were subjected to whole genome sequencing (WGS). Results: The results demonstrated the N2 BioCOV-19 reaction as the optimized multiplex PCR composition for spike and ORF1a mutations screening. Whole-genome sequencing has shown that a sample with spike and ORF1a targets failure to be Alpha variant, while other samples with single target failure as non-variants of concern. Therefore, a multiplex RT-PCR composition has been optimized to detect mutation in spike and ORF1a regions. Conclusion: We have optimized a multiplex RT-PCR composition to detect mutation in spike and ORF1a regions.

Rnase A Enzyme Modification of Optimized SDS Protocol for DNA Extraction Suitable for Real-Time PCR Screening of GMOs

As the number of genetically modified crops increases rapidly, their accurate detection is significant for labelling and safety assessment. Currently, real-time PCR is the “golden standard” method for GMO detection. Hence, extraction of high quality DNA represents a crucial step for accurate and efficient DNA amplification. For GMO presence evaluation in the extracted DNA from raw corn kernels and roasted soybean, we used real-time PCR method, in consistent with the ISO17025 accreditation standards. As for DNA extraction, modified basic SDS protocol by adding RNase A enzyme in different steps of the protocol, with different time and temperature of incubation was used. The results showed as most suitable, the protocol where 10 µl of RNase A enzyme was added together with the lysis buffer at 65 °C for 30 minutes. Data for DNA yield and purity for roasted soybean was 469.6±3.3 µg/ml with A260/280 absorbance ratio 1.78±0.01. Suitability of DNA extracts for GMO analysis was assessed by screening for the presence of 35S promotor and Tnos terminator. Diluted extracts in concentrations 10, 1, 0.1, 0.01 and 0.0027 ng/µl, were tested in six replicates. Positive signal of amplification (LOD) was detected in all concentrations for both genetic elements in both matrices. The LOQ for 35S and Tnos for both matrices was 0.1 ng, while for Tnos in raw corn kernels was 0.01 ng. This in-house developed DNA extraction method is simple and obtains high-quality DNA suitable for GMO screening of 35S promotor and Tnos terminator in both raw and processed matrices.

Molecular Detection and Characterization of Porcine Astrovirus on the basis of Partial ORF1b/ORF2 Region

The porcine astrovirus (PAstV) is distributed globally and exists as five distinct lineages (PAstV1-PAstV5). PAstV is considered one of the important pathogen associated with diarrhea among pigs. In the present study, the PAstV was detected in 13.4% (19/141) of fecal samples including 14.4% (16/111) diarrheic and 10% (3/30) non-diarrheic samples by RT-PCR based on partial ORF1b/ORF2 gene from Haryana, India. The results indicated that the weaning piglets were more susceptible to PAstV infection followed by suckling piglets. The phylogenetic analysis of the viral strains revealed the circulation ofPAstV4 (55.5%) and PAstV2 (44.4%) lineages with PAstV4 being the predominant lineage. To conclude, RT-PCR screening followed by sequencing of PAstV revealed high genetic diversity among the PAstV strains suggesting the wide range of heterogeneity and possible recombination events of viral strains in the state.

A Quality Improvement Project to Minimize COVID-19 Infections in Patients Receiving Haemodialysis and the Role of Routine Surveillance Using Nose and Throat Swabs for SARS-CoV-2 rRT-PCR and Serum Antibody Testing

<b><i>Background:</i></b> Patients receiving in-centre haemodialysis (ICHD) are highly vulnerable to COVID-19. <b><i>Objective:</i></b> We created a quality improvement (QI) project aimed to eliminate outbreaks of COVID-19 in haemodialysis units and evaluated the utility of surveillance rRT-PCR test and SARS-CoV-2 serum antibodies for prompt identification of patients infected with COVID-19. <b><i>Methods:</i></b> A multifaceted QI programme including a bundle of infection prevention control (IPC) measures was implemented across 5 ICHD units following the first wave of the pandemic in June 2020. Primary outcomes evaluated before and after QI implementation were incidence of outbreaks and severe COVID-19 illness defined as COVID-19-related death or hospitalization. Secondary outcomes included the proportion of patients identified in the pre-symptomatic/asymptomatic phase on surveillance rRT-PCR screening and the incidence and longevity of SARS-CoV-2 antibody response. <b><i>Results:</i></b> Following the implementation of the QI project, there were no further outbreaks. Pre- and post-implementation comparison showed a significant reduction in COVID-19-related mortality and hospitalization (26 vs. 13 events, respectively, <i>p</i> &#x3c; 0.001). Surveillance rRT-PCR screening identified 39 asymptomatic or pre-symptomatic cases out of a total of 59 rRT-PCR-positive patients (39/59, 66%). SARS-CoV-2 antibody levels were detected in 72/74 (97%) rRT-PCR-positive patients. Amongst rRT-PCR-positive patients diagnosed before August 2020, 96% had detectable antibodies until January 2021 (days from the rRT-PCR test to last antibody testing, 245–280). <b><i>Conclusions:</i></b> Systematic implementation of a bundle of IPC measures using QI methodology and surveillance rRT-PCR eliminated outbreaks in HD facilities. Most HD patients mount and sustain antibody response to COVID-19 for over 8 months.

Application of a commercial Salmonella real-time polymerase chain reaction (PCR) assay for the detection and quantitation of Salmonellaenterica in poultry ceca

Foodborne Salmonellosis is commonly associated with poultry and poultry products necessitating continued development of pre- and post-harvest food safety interventions and risk management strategies. Evaluating technologies and strategies is limited by availability of cost-effective, rapid laboratory methods. The objective of this work was to evaluate a commercial, qualitative PCR assay and its novel quantitative application to detect and enumerate Salmonella in poultry ceca as an analytical matrix. Ceca were collected at harvest, contents homogenized, and paired samples evaluated with Buffered Peptone Water (BPW) and BAX® MP + Supplement (MPS) pre-enrichment broths followed by PCR screening on BAX® System Q7 (PCR) and by isolation. Additional ceca were inoculated with Salmonella to develop a standard curve for the BAX® System SalQuant™ quantitative PCR application (QA), then estimates were obtained by the QA and Most Probable Number (MPN) methods. For pre-enrichment media, PCR outcomes performed equivalently to culture isolation for detecting Salmonella in ceca with 95.65% and 87.88% sensitivity and 82.00% and 100.00% specificity (P=0.074) for BPW and MPS, respectively. However, at the sample-level, BPW performed significantly worse (47.92%) than MPS (68.75%) for overall isolation of Salmonella (P&lt;0.0001). Post-standard curve development, the mean QA estimates obtained for the inoculated samples were 1.14 (95% CI; 0.62 - 1.66), 1.79 (95% CI; 1.50- 2.08), 2.91 (95% CI; 2.65 - 3.17) and 3.76 (95% CI; 3.26 - 4.25), respectively for each targeted inoculation of 1.0, 2.0, 3.0 and 4.0 Log10 CFU/mL and within or comparable to 95% confidence intervals of paired MPN estimates. These data demonstrate performance of MPS for the detection and isolation of Salmonella enterica from poultry ceca when screening with PCR and indicate the QA may be useful as an alternative tool to estimate Salmonella concentrations in ceca, which may support pre-harvest food safety activities.

One-step RT-PCR Ins214EPE assay for Omicron (B.1.1.529) variant detection v1

On 26 November 2021 WHO designated a new variant of concern B.1.1.529 named Omicron. This variant has a large number of mutations, some of which are concerning. Preliminary evidence suggests an increased risk of reinfection with this variant and reduced neutralization by convalescent and vaccinated sera, as compared to other VOCs. Implementation of the high-throughput rRT-PCR screening for Omicron is of great importance for monitoring the spread of this VOC in the population, especially in resource-limited countries lacking sufficient sequencing capacity. Omicron lineage B.1.1.529 (BA.1) has some indels that turned out to be a good target for its detection. In the current protocol, we use ins214EPE for this purpose. Here we describe the 1-step quantitative multiplex RT-qPCR assay consisting of the newly developed Ins214EPE detection set and widely used Hong Kong University N gene assay for SARS-CoV-2 detection (Chu et al., 2020). The assay was validated on the Omicron variant RNA kindly provided by the Pathogenic Microorganisms Variability Laboratory (Dr. Vladimir Guschin, Gamaleya Institute, Moscow, Russia) and RNA from the collection of Smorodintsev Research Institute of Influenza. Omicron RNA specimens were positive in the assay as expected. Negative controls were found negative. 10-fold serial dilutions of Omicron RNA were used to assess ins214EPE assay amplification efficiency. The amplification efficiency was 98,9% (R2 = 0,99). The developed rRT-PCR assay demonstrates high specificity. It was tested on 26 clinical samples (RNA extracted from oropharyngeal swabs) with previously characterized viruses belonging to 8 different SARS-CoV-2 lineages (including Delta B.1.617.2+AY.*) Specific signal was detected only in samples with SARS-CoV-2 Omicron lineage RNA (confirmed by whole-genome sequencing). Specificity was additionally tested on clinical samples positive for other respiratory viruses from the collection of Smorodintsev Research Institute of Influenza - influenza, parainfluenza, human seasonal coronaviruses (OC43, NL63, 229E, HKU1), hRSV, rhinoviruses, bocaviruses, metapneumovirus (33 in total) - with no false-positive results. Ins214EPE Cq 6x B.1.1.7 2x B.1.351 5x AT.1 6x B.1.617.2 4x AY.122 P.1 B.1.1.529 28,72 B.1.1.529 26,29 virus RP Cq SARS Cq Ins214 Cq RSV A 28,76 RSV A 30,56 RSV A 27,70 RSV B 31,49 RSV B 30,98 RSV B 32,33 NL63 32,20 NL63 30,42 NL63 24,95 Oc43 30,34 Oc43 30,69 Oc43 28,64 HKU1 30,06 HKU1 28,30 HKU1 30,73 229E 29,11 229E 32,52 229E 29,37 BoV 32,26 BoV 30,75 BoV 27,25 Rv 32,85 Rv 33,76 Rv 27,75 Piv1 28,63 Piv2 24,72 Piv3 27,01 Piv4 23,90 Adv 29,47 MPV 30,12 HIV A 29,13 HIV A 28,45 HIV A 28,16 39,06 c+ 34,15 26,61 28,44 Analytical sensitivity determination is underway. We consider developed assay to be useful in wide populational RT-PCR screening to assess the spread of Omicron variant.

Stress-Related Herpesvirus Reactivation in Badgers Can Result in Clostridium Proliferation

Clostridium perfringens is an important food-borne zoonotic pathogen and a member of the commensal gut microbiome of many mammals. Predisposing factors such as coinfection with other pathogens or diet change can, however, cause overgrowth and subsequent disease development. Here we investigated the occurrence of C. perfringens in a free-ranging badger population with up to 100% prevalence of herpesvirus infection. Herpesvirus reactivation is known to be associated with increased susceptibility bacterial infections. PCR screening of rectal swabs from 69 free-ranging badgers revealed 15.9% (11/69, 95% CI = 9.1–26.3%) prevalence of detectable C. perfringens (Type A) DNA in the digestive tracts of assymptomatic animals. The results of Fisher’s exact test revealed C. perfringens detection was not biased by age, sex and seasons. However, badgers with genital tract gammaherpesvirus (MusGHV-1) reactivation (p = 0.007) and infection with a specific MusGHV-1 genotype (p = 0.019) were more prone to of C. perfringens proliferation, indicating coinfection biased dynamics of intestinal C. perfringens. An inclusion pattern analysis further indicated that, causally, MusGHV-1 reactivation potentiated C. perfringens detection. Whether or not specific MusGHV-1 genotype infection or reactivation plays a role in C. perfringens overgrowth or disease development in badgers will require further investigation. Nevertheless, a postmortem examination of a single badger that died of fatal disease, likely associated with C. perfringens, revealed MusGHV-1 detection in the small intestine.

Rats exhibit age-related mosaic loss of chromosome Y

Mosaic loss of the Y chromosome (LOY) is the most frequent chromosomal aberration in aging men and is strongly correlated with mortality and disease. To date, studies of LOY have only been performed in humans, and so it is unclear whether LOY is a natural consequence of our relatively long lifespan or due to exposure to human-specific external stressors. Here, we explored whether LOY could be detected in rats. We applied a locus-specific PCR and target sequencing approach that we used as a proxy to estimate LOY in 339 samples covering eleven tissues from young and old individuals. We detected LOY in four tissues of older rats. To confirm the results from the PCR screening, we re-sequenced 60 full genomes from old rats, which revealed that the Y chromosome is the sole chromosome with low copy numbers. Finally, our results suggest that LOY is associated with other structural aberrations on the Y chromosome and possibly linked to the mosaic loss of the X chromosome. This is the first report, to our knowledge, demonstrating that the patterns of LOY observed in aging men are also present in a rodent, and conclude that LOY may be a natural process in placental mammals.