Proposal of RT-PCReBased Mass Population Screening for Severe Acute Respiratory Syndrome Coronavirus 2 (Coronavirus Disease 2019)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing has lagged in many countries because of test kit shortages and analytical process bottlenecks. This study investigated the feasibility and accuracy of a sample pooling approach for wide-scale population screening for coronavirus disease 2019. A total of 940 nasopharyngeal swab samples (934 negative and 6 positive) previously tested for SARS-CoV-2 were deidentified and assigned random numbers for analysis, and 94 pools of 10 samples each were generated. Automated RNA extraction, followed by RT-PCR, was performed in a 96-well plate. Positive pools were identified, and the individual samples were reanalyzed. Of the 94 pools/wells, four were positive [Ct values: N (22.7 to 28.3), ORF1ab (23.3 to 27.2), and internal control (34.4 to 35.4)]. The 40 samples comprising the four pools were identified and reanalyzed individually; six samples were positive, with Ct values of N gene, ORF1ab, and internal control comparable to their respective wells. Additional experiments were performed on samples with high Ct values, and overall results showed 91.6% positive and 100% negative agreement compared with individual testing approach. Thus, 940 samples were tested in 148 reactions compared with 940 reactions in routine screening. The sample pooling strategy may help catch up with testing needs and minimal turnaround times and facilitate enormous savings on laboratory supplies, extraction, and PCR kits currently in short supply

Overexpression Of The Bacillus Thuringiensis (Bt) Cry2Aa2 Protein In Chloroplasts Confers Resistance To Plants Against Susceptible And Bt-Resistant Insects

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330-to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000-10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field

Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000–10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field

Energy storage and catalytic behaviour of cmWave assisted BZT and flexible electrospun BZT fibers for energy harvesting applications

Abstract High-performance lead-free Barium Zirconium Titanate (BZT) based ceramics have emerged as a potential candidate for applications in energy storage, catalysis for electro chemical energy conversion and energy harvesting devices as presented in this work. In the present study hybrid microwave sintered BZT are studied for dielectric, ferroelectric and phase transition properties. BZT ceramic exhibits tetragonal structure as confirmed by the Retvield refinement studies. XPS studies confirms the elemental composition of BZT and presence of Zr. Polarization versus electric field hysteresis loops confirms the ferroelectric behaviour of BZT ceramic. Encouragingly, the BZT showed a moderate energy storage efficiency of 30.7 % and relatively good electro chemical energy conversion (HER). Excellent catalytic activity observed for BZT electrode in acid medium with low Tafel slope 77 mV dec-1. Furthermore, electrospun nanofibers made of PVDF-HFP and BZT are used to make flexible piezoelectric nano generators (PENGs). FTIR studies show that the 16 wt% BZT composite ink exhibits a higher electroactive beta phase. The optimized open-circuit voltage and short circuit current of the flexible PENG exhibits 7Vpp and 750 nA under an applied force of 3N. Thus, flexible and self-powered BZT PENGs are alternative source of energy due to its reliability, affordability and environmental-friendly nature

SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance

Objectives: Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits. Methods: We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines. Results: Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 °C for 30-min and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60–180 copies/mL by absolute quantification. Furthermore, a five-sample-pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. Conclusion: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR)