Establishing a Standardized DNA Extraction Method Using NaCl from Oral Mucosa Cells for Its Application in Imprinting Diseases Such as Prader-Willi and Angelman Syndromes: A Preliminary Investigation.

BACKGROUND: Diagnosing imprinting defects in neonates and young children presents challenges, often necessitating molecular analysis for a conclusive diagnosis. The isolation of genetic material from oral swabs becomes crucial, especially in settings where blood sample collection is impractical or for vulnerable populations like newborns, who possess limited blood volumes and are often too fragile for invasive procedures. Oral swab samples emerge as an excellent source of DNA, effectively overcoming obstacles associated with rare diseases. METHODS: In our study, we specifically addressed the determination of the quality and quantity of DNA extracted from oral swab samples using NaCl procedures. RESULTS: We compared these results with extractions performed using a commercial kit. Subsequently, the obtained material underwent MS-HRM analysis for loci associated with imprinting diseases such as Prader-Willi and Angelman syndromes. CONCLUSIONS: Our study emphasizes the significance of oral swab samples as a reliable source for obtaining DNA for MS-HRM analysis. NaCl extraction stands out as a practical and cost-effective method for genetic studies, contributing to a molecular diagnosis that proves particularly beneficial for patients facing delays in characterization, ultimately influencing their treatment.

PCR-based diagnosis is not always useful in the acute acquired toxoplasmosis in immunocompetent individuals.

Toxoplasmosis is the most prevalent zoonosis in the world and is associated with a large spectrum of diseases. Acute acquired toxoplasmosis (AAT) is considered a benign and self-limiting disease but severe postnatal infections have been reported, particularly in South America. Laboratory diagnosis is based on the detection of anti-Toxoplasma gondii IgM, IgG, and presence of low IgG avidity. However, these assays present limitations, and therefore, PCR has been suggested as an alternative diagnostic tool. In this study, we performed real-time and nested PCR in DNA blood samples from 59 individuals with AAT lasting less than 80 days. None of the patients had parasitic DNA detected by PCR, even in the more severe cases or when blood was collected early after disease onset. These negative results indicate that the parasitemia kinetics needs investigation to determine the best time for blood sampling, especially in immunocompetent individuals. Thus, we emphasize that a negative PCR result does not exclude recent T. gondii infection, and serological criteria are still decisive for the laboratory diagnosis of AAT.