BR-102024018248-A2 - METHOD FOR EXTRACTING DNA FROM NASOPHARYNGEAL SWABS OF INDIVIDUALS INFECTED WITH SARS-CoV-2 AND OTHER RESPIRATORY PATHOGENS

BR102024018248A2BR 102024018248 A2BR102024018248 A2BR 102024018248A2BR-102024018248-A2

Abstract

This invention describes a method for extracting genomic DNA from nasopharyngeal swabs of individuals infected with SARS-CoV-2 (and other pathogens) using chelex®100 resin. The protocol details the treatment of nasopharyngeal swab samples with chelex®100, resulting in high-quality DNA for genetic analysis. Compared to traditional and commercial methods, the chelex®100 method offers lower cost, fewer steps, moderate execution time, higher final volume, higher DNA concentration and yield, and lower purity. DNA integrity is maintained in agarose gel, and amplification by PCR-RFLP and qRT-PCR is effective. The results indicate that the chelex®100 method is a viable and efficient alternative for DNA extraction, meeting the cost, time, and quality requirements for genetic studies related to COVID-19 and other diseases.

Inventors

RONALDO NASCIMENTO DE OLIVEIRA
RONALDO CELERINO DA SILVA
SUELEN CRISTINA DE LIMA
WENDELL PALOMA MARIA DOS SANTOS REIS
SEVERINO JEFFERSON RIBEIRO DA SILVA
LINDOMAR JOSE PENA

Assignees

UNIVERSIDADE FEDERAL RURAL DE PERNAMBUCO

Dates

Publication Date: 20260317
Application Date: 20240904

Claims (2)

1. METHOD FOR EXTRACTING DNA FROM NASOPHARYNGEAL SWABS OF INDIVIDUALS INFECTED WITH SARS-CoV-2 AND OTHER RESPIRATORY PATHOGENS"Characterized by having an isolation step with chelex®100 resin, and other conventional laboratory equipment.
2. METHOD FOR EXTRACTING DNA FROM NASOPHARYNGEAL SWABS OF INDIVIDUALS INFECTED WITH SARS-CoV-2 AND OTHER RESPIRATORY PATHOGENS" as per claim 1 Characterized by using chelex®100 resin for performing genomic and molecular analyses.

Description

Field of invention [001] The present invention describes a method for extracting DNA from nasopharyngeal swabs of individuals infected with SARS-CoV-2 and other respiratory pathogens using chelex®100 resin. Fundamentals of the invention [002] The SARS-CoV-2 pandemic, responsible for COVID-19, has presented unprecedented challenges on a global scale, especially in the field of public health. The virus is characterized by rapid spread and a wide spectrum of clinical manifestations, causing the death of thousands of people worldwide (Ng, O. T. et al. Lancet infect Dis, v.21, p.333-343, 2021). [003] In order to contain viral spread and safeguard human life, before the advent of effective vaccines, measures were adopted such as: social distancing, use of masks, hand hygiene and mass testing. Viral detection through amplification of nucleic acids extracted from nasopharyngeal swabs using RT-PCR is considered the gold standard for diagnosis (Kevadava, B. D. et al. Nat Mater, v.20, p.593-605,2021). However, the global spread of SARS-CoV-2 resulted in a shortage of diagnostic supplies, affecting not only the diagnosis for COVID-19, but for several other diseases. [004] The wide spectrum of clinical manifestations of COVID-19, ranging from asymptomatic to severe forms, frequently involves multiple organ failure due to immune imbalance caused by the pro-inflammatory cytokine storm (Rabaan, A. A. et al. Bosn J Basic Med Sci, v.23, p.37-52, 2022). Additionally, factors such as age, sex, and comorbidities, as well as the host's genetic background, can contribute to more critical disease outcomes (Initiative C-19 HG. Nature, v.600, p.472-477). [005] Studies of the genetic profile of individuals with COVID-19 (and other respiratory diseases) are needed for a better understanding of the genesis and development of the disease, as well as to explain the different clinical phenotypes of the disease. In such studies, the quality of the nucleic acids used in the analyses is an important factor to be considered, along with the design and characterization of the sample group (Gupta, N. J Cytol, v.36, p.116, 2019). [006] A good DNA extraction method must be safe, fast, and capable of generating a product of good quality in sufficient quantity for numerous analyses. Traditionally used techniques, such as organic extraction (phenol-chloroform), non-organic extraction (salting out and proteinase K treatment), adsorption-based methods and/or magnetic beads, although allowing consistent DNA isolation, differ in terms of quality, concentration, execution time, and costs. In-house methods generally have low cost but are laborious and time-consuming, in addition to using organic solvents harmful to human and environmental health (phenol, chloroform, etc.). On the other hand, commercial kits offer shorter extraction times and result in good quality DNA, but with high costs per sample, making their use unfeasible in countries with scarce research resources (Edwin, S.C.S. et al. Med J Malaysia. v.65, p.133-137, 2010). [007] DNA extraction using chelex®100 resin has emerged as a safe, economical, and sensitive method for extracting nucleic acids from a wide variety of samples, especially samples with low cell counts, without damaging the DNA (Walsh, P. S. et al. Biotechniques. v.54, p.506-513, 2013). In the context of COVID-19, the method can solve the lack of diagnostic supplies, reduce costs and execution time, and obtain DNA with sufficient quality and concentration for studies related to host genetics. Description of the invention [008] A method for extracting DNA from nasopharyngeal swab samples of individuals infected with SARS-CoV-2 was developed using chelex®100 resin. The method is applicable to other pathogens and also to non-human samples. For this, an aliquot of up to 250 μL of nasopharyngeal swab sample was added to 80 to 100 μL of chelex®100 resin, at a concentration of 5 g/mL, in a 0.5 mL transparent plastic microtube. The mixture was vortexed and incubated in a dry bath at a temperature of 56°C for up to 1 hour, and then at a temperature of 96°C for up to 30 minutes. After heating, the samples were centrifuged at a speed of 13000 rpm for 6 minutes. The supernatant was transferred to a new transparent plastic microtube with a volume of 1.5 mL. Up to 100 μL of ultrapure water was added to the supernatant for rehydration. The obtained genomic DNA was stored under refrigeration at a temperature of -20°C until use. [009] All genomic DNAs obtained by the chelex method were analyzed in relation to some criteria (execution time, costs, quality, concentration and amplification capacity by different methodologies) in comparison to a traditionally used in-house method (phenol-chloroform) and a commercial kit (Qiagem). [0010] The present invention is described by the following non-limiting examples, which are merely illustrative. Various modifications and variations of the embodiments are evident to those skilled in the art, without depart