Integration of IFAST-based nucleic acid extraction and LAMP for on-chip rapid detection of Agroathelia rolfsii in soil.

Agroathelia rolfsii (A. rolfsii) is a fungal infection and poses a significant threat to over 500 plant species worldwide. It can reduce crop yields drastically resulting in substantial economic losses. While conventional detection methods like PCR offer high sensitivity and specificity, they require specialized and expensive equipment, limiting their applicability in resource-limited settings and in the field. Herein, we present an integrated workflow with nucleic acid extraction and isothermal amplification in a lab-on-a-chip cartridge based on immiscible filtration assisted by surface tension (IFAST) to detect A. rolfsii fungi in soil for point-of-need application. Our approach enabled both DNA extraction of A. rolfsii from soil and subsequent colorimetric loop-mediated isothermal amplification (LAMP) to be completed on a single chip, termed IFAST-LAMP. LAMP primers targeting ITS region of A. rolfsii were newly designed and tested. Two DNA extraction methods based on silica paramagnetic particles (PMPs) and three LAMP assays were compared. The best-performing assay was selected for on-chip extraction and detection of A. rolfsii from soil samples inoculated with concentrations of 3.75, 0.375 and 0.0375 mg fresh weight per 100-g soil (%FW). The full on-chip workflow was achieved within a 1-h turnaround time. The platform was capable of detecting as low as 3.75 %FW at 2 days after inoculation and down to 0.0375 %FW at 3 days after inoculation. The IFAST-LAMP could be suitable for field-applicability for A. rolfsii detection in low-resource settings.

Optimization and application of loop-mediated isothermal amplification technique for sex identification in red-whiskered bulbul (Pycnonotus jocosus).

The red-whiskered bulbul (Pycnonotus jocosus) is a popular avian species in Thailand and many other countries. The red-whiskered bulbul has a high economic value, but breeding is challenging since sex identification is difficult. The PCR method is now used for sex identification. However, PCR amplification and post-PCR analysis necessitate the use of a laboratory equipped with specialized scientific instruments, which is inconvenient for field operations. This research describes a method for amplification of DNA samples using the loop-mediated isothermal amplification (LAMP) approach, which is a molecular biology methodology for isothermal amplification that is extremely sensitive, fast, and easy for post-LAMP product visualization. Herein, total of 23 blood samples were collected and DNA was extracted. Two sets of LAMP primers were designed for CHD-Z and CHD-W genes. The colorimetric assay was used to investigate the best conditions for LAMP reactions and post-LAMP product visualization. LAMP reactions for sex identification were compared to traditional PCR in terms of sensitivity and specificity. LAMP reactions were found to be 10-fold more sensitive than PCR at 1 ng of DNA. When compared to electrophoresis analysis, the visualization with colorimetric assay using GelRed® and SYTO™ 9 was 100% accurate. The optimal LAMP condition tested simple DNA extracted from bird feathers using the HotSHOT technique. The result showed that the optimal condition could distinguish the sex of red-whiskered bulbuls totally and accurately. A powerful method for red-whiskered bulbul sex identification is demonstrated in this study, which can be used in field studies because it is quick and easy to perform, has high sensitivity, and does not require advanced scientific equipment.

Water Treatment for Fish Aquaculture System by Biochar-Supplemented Planting Panel System.

Rice husk biochars were prepared by carbonization at 400-600°C. The products were analyzed by FTIR, SEM-EDS, BET, and approximate analysis in order to find final products with the best properties and the lowest carbonization temperature. It has been found that the biochar prepared at 500°C, which has 37.86 ± 0.11% yield, 341.0776 m2/g of BET surface area, and 0.136639 cm3/g of micropore volume, is suitable for use as a root supplement in the aquaponic system. The aquaponic systems consist of aquaculture and a hydroponic system with and without biochar supplement. The control experiment consists of an aquaculture and planting panel with biochar supplement disconnected from each other. Tilapia and Chinese morning glory were used for growth studies. The water quality from all aquaculture ponds has also been analyzed at an interval of 10 days for 47 days. The results showed that the growth rates of Tilapia and Chinese morning glory in the aquaponic system with biochar were clearly higher than in the control experiment, which is in accordance with the water quality in each aquaculture pond. However, the growth rates of Tilapia (23.5 g/body vs. 22.7 g/body) and morning glory (3.907 g/stem vs. 2.609 g/stem) in supplemented biochar system tend to be higher than the nonsupplemented biochar system. It has been shown that rice husk biochar can help in treating water in the aquaponic system by increasing the amount of dissolved oxygen in the aquaculture water and conversion of toxic compounds to those beneficial for plant growth.