Sample Treatment with Trypsin for RT-LAMP COVID-19 Diagnosis.

The SARS-CoV-2 coronavirus is responsible for the COVID-19 pandemic resulting in a global health emergency. Given its rapid spread and high number of infected individuals, a diagnostic tool for a rapid, simple, and cost-effective detection was essential. In this work, we developed a COVID-19 diagnostic test, that incorporates a human internal control, based on the Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP). When working with synthetic SARS-CoV-2 RNA, the optimized RT-LAMP assay has a sensitivity of 10 viral copies and can be detected by fluorescence in less than 15 min or by the naked eye in 25 min using colorimetric RT-LAMP. To avoid the RNA extraction step, a pre-treatment of the sample was optimized. Subsequently, a validation was performed on 268 trypsin treated samples (including nasopharyngeal, buccal, and nasal exudates) and amplified with colorimetric RT-LAMP to evaluate its sensitivity and specificity in comparison with RT-qPCR of extracted samples. The validation results showed a sensitivity and specificity of 100% for samples with Ct ≤ 30. The rapid, simple, and inexpensive RT-LAMP SARS-CoV-2 extraction-free procedure developed may be an alternative test that could be applied for the detection of SARS-CoV-2 or adapted to detect other viruses present in saliva or nasopharyngeal samples with higher sensitivity and specificity of the antibody test.

EOSAL-CNV for Easy and Rapid Detection of CNVs by Fragment Analysis : EOSAL: A Fast and Reliable New Method for CNV Detection.

Copy number variations (CNVs) are a type of genetic variation involving from 50 base pairs (bps) to millions of bps and, in a general point of view, can include alterations of complete chromosomes. As CNVs mean the gain or loss of DNA sequences, their detection requires specific techniques and analysis. We have developed Easy One-Step Amplification and Labeling for CNV Detection (EOSAL-CNV) by fragment analysis in a DNA sequencer. The procedure is based on a single PCR reaction for amplification and labeling of all fragments included. The protocol includes specific primers for the amplification of the regions of interest with a tail in each of the primers (one for forward and another for the reverse primers) together with primers for tail amplification. One of the primers for tail amplification is labeled with a fluorophore, allowing the amplification and labeling in the same reaction. Combination of several tail pairs and labels allows the detection of DNA fragment by different fluorophores and increases the number of fragments that can be analyzed in one reaction. PCR products can be analyzed without any purification on a DNA sequencer for fragment detection and quantification. Finally, simple and easy calculations allow the detection of fragments with deletions or extra copies. The use of EOSAL-CNV allows simplifying and reducing costs in sample analysis for CNV detection.

Genetic variants in obesity-related genes and the risk of osteoporotic fracture. The Hortega Follow-up Study.

BACKGROUND: Osteoporosis and obesity are major public health problems that are closely correlated, as they share various features, including a genetic predisposition. A genetic correlation between obesity and osteoporosis due to the biological common pathways of bone and fat metabolism, which implies pleiotropic genes regulating has been described. The objective of our study was to analyse whether polymorphisms in obesity-related genes modify the risk of osteoporotic bone fracture. METHODS: We studied 575 subjects from the Hortega Study. The subjects were followed-up for 12-14 years. 202 subjects were overweight, 143 obese and 221 had bone fractures. The distribution of 39 genetic variants in 22 obesity-related genes were studied. RESULTS: The results showed a relationship between polymorphisms in the FTO and NEGR1 genes and the susceptibility to osteoporotic fracture. The variant genotype of the rs2568958 NEGR1 polymorphism and the rs6499649, rs3751812, and rs8044769 genetic variants in FTO were associated with susceptibility to bone fracture. In the best of our knowledge, this is the first time that these variants in NEGR1 and FTO genes have been associated with the susceptibility to osteoporotic bone fracture, supporting the hypothesis that the NEGR1 and FTO genes might be candidates for osteoporosis and bone fracture. CONCLUSIONS: In conclusion, this study associates obesity-related polymorphisms in the NEGR1 and FTO genes with osteoporotic bone fracture, reinforcing the hypothesis that obesity and bone metabolism are closely correlated genetically.