Desenvolvimento de sonda molecular para detecção do vírus da hepatite a em amostras ambientais de água

• Autor(a) principal: Santos, Matheus Ismerim Silva
• Data de Publicação: 2007
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFS
• Texto Completo: https://ri.ufs.br/handle/riufs/3727
• topic: Vírus da hepatite A; Sonda molecular; Monitoramento ambiental; Hepatitis A virus; Molecular probe; Environmental monitoring; CNPQ::CIENCIAS DA SAUDE::MEDICINA

Infection with hepatitis A virus (HAV) occurs worldwide and is the most common cause of acute viral hepatitis. The highest prevalence of this infection is seen where low standards of sanitation are adopted. Acquired primarily by the fecaloral route, HAV infection is easily disseminated, either by person-to-person contact or by ingestion of contaminated food or water. The HAV is a extremely steady non-enveloped particle, which comprises a single stranded plus-sense RNA with approximately 7,5 kb. The objective of this study was to develop, based into VP1 gene sequence, a DNA probe from the HAV genome by RTPCR to detect the virus. A 783 bp amplicon of HAV genome (Brazilian standard strain HAF-203), amplified by RT-PCR was labelled by coupling of alkaline phosphatase enzyme from Gene ImagesTM AlkPhos DirectTM System (Amersham). Specificity was determined by dot blot hybridization of RNA from standard strain HAF-203 and DNA of the own amplicon. To asses the sensitivity of detection hybridization was done in serially diluted up to 1 pg of purified viral RNA. Sewage water samples were artificially ontaminated with dilutions up to 10 PFU/mL of the virus. The particles were precipitated with amonium sulfate and the total RNA obtained by treatment with proteinase k and phenol:chloroform. Samples were transferred to a nylon membrane by dot blot and hybridized according to labelling system manufacturer s instructions. Dots were detected in spots containing 1 pg of purified RNA, just like the amplicon. The DNA probe did not hybridize to total DNA prepared from BoHV-1, XL2B DNA and Human total RNA. The probe hybridized to samples containing up to 100 PFU/mL of the virus. This results showed the probe specificity and sensitivity level is sufficient to detect the virus in environmental samples, making this technique able to be used in environmental molecular monitoring of the virus.