Perspective Chapter: Recent Progressions on the Colorimetric Diagnosis of SARS-CoV-2 by Loop-Mediated Isothermal Amplification (LAMP) Assay

A simple, fast, and accurate diagnosis of SARS-CoV-2 is of great importance for the patient’s isolation, treatment, and the control of the COVID-19 pandemic. Although RT-qPCR is accepted as the gold standard, studies to improve fast, simple, and more reliable diagnostic methods are continuing. Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a method that allows visual detection of SARS-CoV-2 without needing expensive fluorescence readers. However, the performance of the assay depends on some factors, such as selection of a target gene (i.e., N, RdRp, S, E, M), primer design, the dye used for visual observation—neutral red, calcein, cresol red, or phenol red—and the reaction conditions such as the buffer pH, reaction temperature, and enzyme concentration. In the last 2 years, plenty of research has been conducted to obtain the best performance. In this chapter, the recent progressions on colorimetric RT-LAMP assay for the diagnosis of SARS-CoV-2 are comprehensively elucidated

Genome of Wild Olive and the Evolution of Oil Biosynthesis

Here we present the genome sequence and annotation of the wild olive tree (Olea europaea var. sylvestris), called oleaster, which is considered an ancestor of cultivated olive trees. More than 50,000 protein-coding genes were predicted, a majority of which could be anchored to 23 pseudochromosomes obtained through a newly constructed genetic map. The oleaster genome contains signatures of two Oleaceae lineage-specific paleopolyploidy events, dated at similar to 28 and similar to 59 Mya. These events contributed to the expansion and neo-functionalization of genes and gene families that play important roles in oil biosynthesis. The functional divergence of oil biosynthesis pathway genes, such as FAD2, SACPD, EAR, and ACPTE, following duplication, has been responsible for the differential accumulation of oleic and linoleic acids produced in olive compared with sesame, a closely related oil crop. Duplicated oleaster FAD2 genes are regulated by an siRNA derived from a transposable element-rich region, leading to suppressed levels of FAD2 gene expression. Additionally, neofunctionalization of members of the SACPD gene family has led to increased expression of SACPD2,3, 5, and 7, consequently resulting in an increased desaturation of steric acid. Taken together, decreased FAD2 expression and increased SACPD expression likely explain the accumulation of exceptionally high levels of oleic acid in olive. The oleaster genome thus provides important insights into the evolution of oil biosynthesis and will be a valuable resource for oil crop genomics

Formulation of Manganese Zinc Spinel Ferrite (Mn0.5Zn0.5Fe2O4) Nanoparticles for the Growth Promotion of Plants

This research investigates the uptake and potential contribution of engineered manganese-zinc (MnZn) spinel ferrite nanoparticles (Mn0.5Zn0.5Fe2O4 NPs) to the growth performance of pumpkin (Cucurbita maxima L.). For this purpose, MnZn spinel ferrites were synthesized, and their structural, microstructural, and magnetic properties were determined. NPs (50, 100, 200, and 400 mg L-1) were applied to pumpkin seedlings in a hydroponic system for a week, and the root, stem, and leaf tissues were screened for NPs-uptake by using X-ray powder diffraction (XRD), vibrating sample magnetometry (VSM), and X-ray fluorescence (XRF). Besides, the effect of NPs treatments on some phenological parameters such as pigmentation, photosynthetic efficiency, and biomass was determined. The results showed that MnZn spinel ferrite treatment significantly increased Mn, Zn, and Fe content in the root, stem, and leaf. The Fe, Zn, and Mn content in the leaves increased by approximately 48, 67, and 20 times, respectively, when 400 mg L-1 was applied. Similarly, the magnitude of magnetization of root, stem, and leaf specimens confirmed the incorporation and translocation of magnetic NPs into plant tissues. Besides, the photosynthetic efficiency, pigmentation, and fresh weight were significantly enhanced, suggesting growth improvement by engineered NPs. NP concentrations for the most efficient plant growth were determined as 100 and 200 mg L-1. However, higher NP concentrations suppressed the growth due to the migration/translocation of excess NPs. These findings revealed the potential of engineered MnZn spinel ferrite NPs as nano-fertilizers to provide essential micronutrients, Mn, Zn, and Fe in this study. However, environmental concerns must be considered when using NPs at large scales.Deanship of Scientific Research (DSR) fund of Imam Abdulrahman Bin Faisal University (IAU) [2020-166-IRMC]The study is supported by the Deanship of Scientific Research (DSR) fund of Imam Abdulrahman Bin Faisal University (IAU) with the project no: 2020-166-IRMC

Development of loop-mediated isothermal amplification (LAMP) assays using five primers reduces the false-positive rate in COVID-19 diagnosis

Abstract The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a cheaper and faster testing alternative for detecting SARS-CoV-2. However, a high false-positive rate due to misamplification is one of the major limitations. To overcome misamplifications, we developed colorimetric and fluorometric RT-LAMP assays using five LAMP primers, instead of six. The gold-standard RT-PCR technique verified the assays' performance. Compared to other primer sets with six primers (N, S, and RdRp), the E-ID1 primer set, including five primers, performed superbly on both colorimetric and fluorometric assays. The sensitivity of colorimetric and fluorometric assays was 89.5% and 92.2%, respectively, with a limit of detection of 20 copies/µL. The colorimetric RT-LAMP had a specificity of 97.2% and an accuracy of 94.5%, while the fluorometric RT-LAMP obtained 99% and 96.7%, respectively. No misamplification was evident even after 120 min, which is crucial for the success of this technique. These findings are important to support the use of RT-LAMP in the healthcare systems in fighting COVID-19

Impact of sonication time in nanoparticle synthesis on the nutrition and growth of wheat (Triticum aestivum L.) plant

Nanotechnology in agricultural applications is promising in improving plant nutrition and yield, pest control, and gene delivery. However, the method to synthesize nanoparticles or nanocomposites (NCs) can play a crucial role in determining the characteristics of NCs, such as size and morphology, which may be critical factors affecting plant nutrition and NCs` potential toxicity. This study elucidates the effect of sonication time in synthesizing NCs on its characteristics and plant use efficiency. For this purpose, a hard/soft nanocomposite (NC) (CoFe2O4/Ni0.8Cu0.1Zn0.1Fe2O4) was sonochemically synthesized at different sonication times (20 and 60 minutes) and comprehensively characterized. They were hydroponically applied to wheat seedlings (50, 100, 200, 400, and 800 mg/L). The physiological, morphological, and nutritional status of the seedlings were determined. The results showed that an increase in sonication time decreased the mean NC size: 26.7 nm (20 minutes) and 17.4 nm (60 minutes). Photosynthetic parameters, growth, and biomass were gradually reduced with the increasing NC concentrations, revealing their toxic effect. However, treating NCs at 60 min significantly improved the average root length, suggesting its beneficial role for plant growth at the germination stage. The content of elements in the composition of the NCs (Fe, Zn, Co, Ni, and Cu) was remarkably higher in the NC-treated roots compared to the untreated controls. In addition, 60 minutes of preparation showed better plant uptake than 20 minutes. This is the first study to evaluate the effect of sonication time in the preparation of NC on plant nutrition and their fate in plants

Effects of foliar iron oxide nanoparticles (Fe 3 O 4 ) application on photosynthetic parameters, distribution of mineral elements, magnetic behaviour, and photosynthetic genes in tomato ( Solanum lycopersicum var. cerasiforme) plants

This study aims to examine the effect of foliar magnetic iron oxide (Fe 3 O 4 ) nanoparticles (IONP) application on the physiology, photosynthetic parameters, magnetic character, and mineral element distribution of cherry tomatoes ( Solanum lycopersicum var. cerasiforme ). The IONP suspension (500 mg L -1 ) was sprayed once (S1), twice (S2), thrice (S3), and four times (S4) a week on seedlings. Upon 21 days of the treatments, photosynthetic parameters (chlorophyll, carotenoids, photosynthetic yield, electron transport rate) were elucidated. Inductivelycoupled plasma -optical emission spectrometer (ICP-OES) and vibrating sample magnetometer (VSM) were used to determine the mineral elements and abundance of magnetic power in the seedlings. In addition, the RTqPCR method was performed to quantify the expressions of photosystem-related ( PsaC , PsbP6 , and PsbQ ) and ferritin-coding ( Fer-1 and Fer-2 ) genes. Results revealed that the physiological and photosynthetic indices were improved upon S1 treatment. The optimal dosage of IONP spraying enhances chlorophyll, carotenoid, electron transport rate (ETR), and effective photochemical quantum yield of photosystem II (Y(II)) but substantially diminishes non -photochemical quenching (NPQ). However, frequent IONP applications (S2, S3, and S4) caused growth retardation and suppressed the photosynthetic parameters, suggesting a toxic effect of IONP in recurrent treatments. Fer-1 and Fer-2 expressions were strikingly increased by IONP applications, suggesting an attempt to neutralize the excess amount of Fe ions by ferritin. Nevertheless, frequent IONP treatment fluctuated the mineral distribution and caused growth inhibition. Although low -repeat foliar applications of IONP (S1 in this study) may help improve plant growth, consecutive applications (S2, S3, and S4) should be avoided.Deanship of Scientific Research (DSR) funds of Imam Abdulrahman Bin Faisal University (IAU) [2019-058-IRMC]The Deanship of Scientific Research (DSR) funds of Imam Abdulrahman Bin Faisal University (IAU) have supported this study under project numbers 2019-058-IRMC

Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2.

The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes (RdRP and E) and one human gene (RP) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2