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The COVID-19 pandemic has forced the Bioinformatics course to switch from on-site teaching to remote learning. This shift has prompted a change in teaching methods and laboratory activities. Students need to have a basic understanding of DNA sequences and how to analyze them using custom scripts. To facilitate learning, we have modified the course to use Jupyter Notebook, which offers an alternative approach to writing custom scripts for basic DNA sequence analysis. This approach allows students to acquire the necessary skills while working remotely. It is a versatile and user-friendly platform that can be used to combine explanations, code, and results in a single document. This feature enables students to interact with the code and results, making the learning process more engaging and effective. Jupyter Notebook provides a hybrid approach to learning basic Python scripting and genomics that is effective for remote teaching and learning during the COVID-19 pandemic.
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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.
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Tarbinskiellus portentosus, commonly known as giant cricket one of the important edible cricket species. However, the genetic information of these species is still limited. Therefore, we have assembled and annotated the first mitochondrial genome of T. portentosus. The mitogenome is 15710 bp long and has GC content of 27.19%. The nucleotide composition is similar with other insect mitogenomes (A 40.6%; T 32.2%; C 17.3%; G 9.9%). The gene organization in the mitogenome of T. portentosus is identical to the mitogenome of other cricket species. The complete mitogenome of T. portentosus consisted 37 genes including 13 protein coding genes, 22 tRNA genes, and two rRNA genes. The newly assembled mitogenome will help molecular biology research on edible crickets. Since mitogenome genes are traditionally used for DNA barcoding and phylogenetic analysis, comparative analysis of T. portentosus mitogenome with other related cricket species will also aid researchers in developing universal primers for species identification toward food security. Apart from the main goal of providing full mitogenome of T. portentosus, paper also provides conceptual workflow based on de novo assembly and its correction for final mitogenome construction.
RESUMEN
Rapid and accurate species diagnosis accelerates performance in numerous biological fields and associated areas. However, morphology-based species taxonomy/identification might hinder study and lead to ambiguous results. DNA barcodes (Bar) has been employed extensively for plant species identification. Recently, CRISPR-cas system can be applied for diagnostic tool to detect pathogen's DNA based on the collateral activity of cas12a or cas13. Here, we developed barcode-coupled with cas12a assay, "Bar-cas12a" for species authentication using Phyllanthus amarus as a model. The gRNAs were designed from trnL region, namely gRNA-A and gRNA-B. As a result, gRNA-A was highly specific to P. amarus amplified by RPA in contrast to gRNA-B even in contaminated condition. Apart from the large variation of gRNA-A binding in DNA target, cas12a- specific PAM's gRNA-A as TTTN can be found only in P. amarus. PAM site may be recognized one of the potential regions for increasing specificity to authenticate species. In addition, the sensitivity of Bar-cas12a using both gRNAs gave the same detection limit at 0.8 fg and it was 1,000 times more sensitive compared to agarose gel electrophoresis. This approach displayed the accuracy degree of 90% for species authentication. Overall, Bar-cas12a using trnL-designed gRNA offer a highly specific, sensitive, speed, and simple approach for plant species authentication. Therefore, the current method serves as a promising tool for species determination which is likely to be implemented for onsite testing.
