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.

Herbal species authentication by melting fingerprint coupled with high resolution melting analysis (MF-HRM).

BACKGROUND: Herbal medicines have recently attracted increasing attention for use as food supplements with health benefits; however, species authentication can be difficult due to incomplete morphological characters. Here, a molecular tool was developed for the identification of species in the National List of Essential Medicinal Plants in Thailand. METHODS: The identification process used DNA fingerprints including start codon targeted (SCoT) and inter simple sequence repeat (ISSR) polymorphisms, coupled with high resolution melting (HRM), to produce melting fingerprint (MF)-HRM. RESULTS: Results indicated that MF-HRM, SCoT-HRM and ISSR-HRM could be used for DNA fingerprints as S34, S36, S9 and S8 of SCoT and UBC873, S25 and UBC841 of ISSR. The melting fingerprints obtained from S34 of SCoT exhibited the best primers for identification of herbal species with 87.5% accuracy and relatively high repeatability. The presence of intraspecific variation in a few species affected the shift of melting fingerprints within species. MF-HRM using S34 showed improved species prediction compared to DNA fingerprints. The concentration of DNA with 10 ng/µl was recommended to perform MF-HRM. MF-HRM enabled species authentication of herbal commercialized products at only 20% resulting from the low quality of DNA isolated, while admixture of multiple product species interfered with the MF process. CONCLUSION: Findings suggested that MF-HRM showed promise as a molecular tool for the authentication of species in commercial herbal products with high specificity, moderate repeatability and rapidity without prior sequence information. This information will greatly improve quality control and traceability during the manufacturing process.

Extraction of Heat-Stabilised Defatted Rice Bran Protein by Solid-State Fermentation Using Heterofermentative Microbes from Traditional Asian Starters.

Research background: Heat-stabilised defatted rice bran (HSDRB) is a primary by-product of rice bran oil extraction industry and a nutritious source of protein. However, despite the unique nutritional profile of rice bran protein, the protein-rich by-product, HSDRB is underutilised as a low-value animal feed. Research on protein extraction from HSDRB by enzymatic hydrolysis has attracted the attention of numerous scientists. However, a cost-effective extraction method is required to mitigate the high costs associated with the use of enzymes. Therefore, we have presented an alternative economical and natural approach for protein extraction from HSDRB by solid-state fermentation (SSF) with heterofermentative microbes. Experimental approach: SSF of HSDRB with two types of traditional Asian fermentation starters, namely loog-pang and koji, were evaluated for enzyme production and their efficacy in extracting proteins from HSDRB. For this purpose, HSDRB fermentation was carried out for 0, 12, 24, 48, 72 and 96 h followed by 24-hour hydrolysis to evaluate the extracted rice bran protein. In addition, microbiome diversity in the fermentation starters was also determined by metagenomic sequencing of 16S rRNA and internal transcribed spacer to identify bacteria and fungi, respectively. Results and conclusions: The microbial community in the fermentation starters showed the dominance of lactic acid bacteria (LAB) such as Bacillus subtilis in loog-pang and Streptococcus lutetiensis, Bacillus pumilus, Lactococcus cremoris, Lactococcus garvieae and Pediococcus pentosaceus in koji, while yeast species Saccharomycopsis fibuligera and Saccharomyces cerevisiae dominated the fungal diversity in loog-pang and koji starters, respectively. The results suggest that loog-pang and koji can produce cellulase, neutral and acid proteases during fermentation. Despite the discrepancy in their microbial diversity and the enzyme activity during SSF, both starters could effectively increase protein extraction from HSDRB. A positive relationship between the SSF duration and extracted protein was observed. During SSF with loog-pang and koji after 72 h followed by 24-hour hydrolysis, 65.66 and 66.67 % protein was extracted from HSDRB, respectively. The amino acid analysis of the protein hydrolysate produced by the non-fermented and fermented methods showed no difference and had an abundance of glutamic and aspartic acids, leucine, arginine, alanine and glycine amino acids, which accounted for approx. 58 % of the total amino acids. Novelty and scientific contribution: Loog-pang and koji (traditional Thai and Japanese fermentation starters, respectively) were found to be effective in extracting proteins from HSDRB by SSF although they are inexpensive microbial enzyme sources. Future research aimed at scaling up HSDRB protein extraction for usage in industrial applications can draw on our results.

Sensitive and rapid detection of Babesia species in dogs by recombinase polymerase amplification with lateral flow dipstick (RPA-LFD).

Canine babesiosis is a tick-borne disease caused by Babesia spp., which infects and destroys healthy erythrocytes, leading to mortality and morbidity in dogs. The diagnosis of babesiosis is tedious and time-consuming, especially in latent and chronic infections. Here, a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay was developed for rapid and accurate detection of Babesia spp. in canine blood specimens based on the 18S rRNA region. The RPA-LFD assay using rpaBab264 gave specificity to Babesia spp. in dogs (B. vogeli and B. gibsoni) without cross-amplification to other parasites (apicomplexans and non-apicomplexans), with detection limit of at least 22.5 copies/µl (0.1 fg/µl) at 40 °C for at least 10 min. The whole process of DNA amplification by RPA and readout by LFD did not exceed 30 min. To determine the performance of the RPA-LFD assay, a total of 30 clinical samples was examined and compared with conventional PCR (cPCR) and multiplex HRM (mHRM). Eight dogs (26.67%) were detected as positive by RPA-LFD, while seven and six were found positive by cPCR and mHRM, respectively. RPA-LFD and cPCR showed high agreement with Babesia spp. detection with kappa > 0.9. We confirmed that the dogs were infected by B. vogeli from sequences of positive PCR results. Our findings suggested that RPA-LFD using the rpaBab264 assay offered a rapid, accurate, cost-effective and simple method for Babesia spp. detection that is feasibly applicable to be rapid kit at a pet hospital or point-of-care testing.

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.

Can feline (Felis catus) flat and long bone morphometry predict sex or skull shape?

This study demonstrates sexual dimorphism in feline bones based on morphometric analysis of dried flat bones (scapula and os coxa) and long bones (humerus, radius, ulna, femur, tibia, and fibula) of 92 felines (50 male, 42 female). A total of 58 parameters (flat bones: scapula = 4 and os coxa = 7; long bones: humerus = 8, radius = 9, ulna = 10, femur = 9, tibia = 7, and fibula = 4) were measured using a digital vernier caliper. Twenty-three parameters were found to be significantly different between cats of different sexes and skull shapes. The correlation between the cephalic index and most parameters was negative. Analysis of bone morphometry enabled us to estimate both sex and skull shape with accuracy of up to 96 % and 71 %, respectively, through a stepwise logistic regression model and a stepwise discriminative analysis model. The stepwise logistic regression model was determined to be most suitable for classifying two categories of data and had higher prediction accuracy rate.