Comparison between specific and multiplex reverse transcription-polymerase chain reaction for detection of hepatitis A virus, poliovirus and rotavirus in experimentally seeded oysters

Coelho, Clarimar José; Vinatea, Cecília Elena Barberena de; Heinert, A. P.; Simões, C. M. O.; Barardi, Célia Regina Monte

doi:10.1590/s0074-02762003000400006

Cited by 7 publications

(6 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A maioria dos manipuladores desconhece a possibilidade de serem portadores assintomáticos de microrganismos e utilizam práticas inadequadas de higiene. Além disso, o sururu é geralmente comercializado à temperatura ambiente, o que contribui para uma rápida proliferação de microrganismos (Delgado da Silva et al, 2002).…”

Section: Resultsunclassified

“…Porém, na prática, a aplicação desses conceitos é similar, com a definição de classes que restringem o aproveitamento dos moluscos com base em resultados de monitoramento de parâmetros microbiológicos em amostras dos bivalves provenientes das zonas de produção ou extração (Epagri, 2014). Na legislação brasileira é estabelecida que as coletas sejam realizadas de cinco amostras de moluscos obtidas em um mesmo dia e de diferentes pontos de coleta em uma mesma área de extração ou cultivo (Coelho et al, 2003), já na legislação europeia não é estabelecido um número de amostras a ser realizado no mesmo local e no mesmo dia (por padrão, sendo feita uma única amostra), porém considera-se a série histórica de até três anos de dados microbiológicos para a definição da classificação da zona de cultivo ou extração de moluscos (Coelho et al, 2003). Baseando-se na legislação europeia sugere-se que a estratégia de coleta seja mais segura do ponto de vista da saúde pública.…”

Section: Resultsunclassified

See 1 more Smart Citation

Qualidade microbiológica do sururu (Mytella charruana) comercializado por catadoras da Baía de Sepetiba, Rio de Janeiro, Brasil

Schmalz¹,

Calixto²,

Keller³

et al. 2018

Revista Brasileira De Ciência Veterinária

View full text Add to dashboard Cite

show abstract

Section: Resultsunclassified

Qualidade microbiológica do sururu (Mytella charruana) comercializado por catadoras da Baía de Sepetiba, Rio de Janeiro, Brasil

Schmalz¹,

Calixto²,

Keller³

et al. 2018

Revista Brasileira De Ciência Veterinária

View full text Add to dashboard Cite

show abstract

“…A multiplex reverse transcriptase polymerase chain reaction (RT-PCR) was also applied for the simultaneous detection of hepatitis A virus (HAV), poliovirus (PV) and simian rotavirus (RV-SA11), and compared with specific primers for each genome sequence. Three amplified DNA products representing HAV (192 bp), PV (394 bp) and RV (278 bp) were identified when positive controls were used [12] . A multiplex RT-PCR method was described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which could be used as an indicator of the effectiveness of the extraction and RT-PCR.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous detection of HBV and HCV by multiplex PCR normalization

Wang

Huard

Han

2004

WJG

View full text Add to dashboard Cite

AIM:To design and establish a method of multiplex PCR normalization for simultaneously detecting HBV and HCV. METHODS:Two pairs of primers with a 20 bp joint sequence were used to amplify the target genes of HBV and HCV by two rounds of amplification. After the two rounds of amplification all the products had the joint sequence. Then the joint sequence was used as primers to finish the last amplification. Finally multiplex PCR was normalized to a single PCR system to eliminate multiplex factor interference. Four kinds of nucleic acid extraction methods were compared and screened. A multiplex PCR normalization method was established and optimized by orthogonal design of 6 key factors. Then twenty serum samples were detected to evaluate the validity and authenticity of this method. RESULTS:The sensitivity, specificity, diagnostic index and efficiency were 83.3%, 70%, 153.3% and 72.2%, respectively for both HBsAg and anti-HCV positive patients, and were 78.6%, 80%, 258.6% and 79.2%, respectively for HBsAg positive patients, and were 75%, 90%, 165% and 83.3%, respectively for anti-HCV positive patients. CONCLUSION:The multiplex PCR normalization method shows a broad prospect in simultaneous amplification of multiple genes of different sources. It is practical, correct and authentic, and can be used to prevent and control HBV and HCV.Wang N, Gao XQ, Han JX. Simultaneous detection of HBV and HCV by multiplex PCR normalization.

show abstract

“…Positive controls consisted on the final oyster tissue extract inoculated with 6.0 x 10 5 PFU/mL. After virus inoculation or bioaccumulation, shells were opened at a hinge with an autoclaved oyster knife and the oyster extracts were prepared as described below (1,3,4,5,22,23).…”

Section: Bioaccumulation Assaysmentioning

confidence: 99%

“…The following method for virus extraction from oyster´s extracts was used for the subsequent RT-PCR and cell infection assays (1,3,4,22). Either for the bioaccumulation assay or for the directly inoculated oysters, the flesh of twelve oysters was transferred and homogenized with a shaft blender (Ultra-Turrax T-25 Ika®, France) at 24,000 rpm for 30s.…”

Section: Oyster Extract Preparationmentioning

confidence: 99%

Detection of poliovirus type 2 in oysters by using cell culture and RT-PCR

Vinatea¹,

Sincero²,

Simões³

et al. 2006

Braz. J. Microbiol.

View full text Add to dashboard Cite

Shellfish are readily contaminated with viruses present in water containing sewage due to the concentrating effect of filter feeding. Enteroviruses are generally used as a model for the detection of viruses from shellfish due to their public health significance. In the present work, oysters were placed in glass aquaria containing seawater plus unicellular algae. Two experiments were performed: 1) oysters bioaccumulating four different poliovirus type 2 concentrations: 5 x 10 4 , 2.5 x 10 4 , 5 x 10 3 and 5 x 10 2 PFU/mL during 20h; 2) oyster tissues directly inoculated with 6.0 x 10 5 and 1.0 x 10 5 PFU/mL. After viruses seeding, tissue samples were processed by an adsorption-elution-precipitation method. Positive controls were performed by seeding 6.0 x 10 5 PFU/ mL of poliovirus type 2 directly on the final oyster tissue extracts. Oyster extracts were assayed for viruses recovery by plaque assay, RT-PCR and integrated cell culture-PCR methodologies (ICC/PCR). The last one was based on the inoculation of the samples onto VERO cell monolayer followed by RT-PCR analysis of the infected cell fluid. In the first experiment (20h bioaccumulation) until 5 x 10 3 PFU were detected after 24 and 48h growth on VERO cells. Direct RT-PCR and ICC/PCR were able to detect 3 and 0.04 PFU of poliovirus, respectively, when bioaccumulation assay was used. When direct tissue virus seeding was performed, the plaque assays showed that polioviruses were recovered in all tested concentrations. Based on these results, it is possible to conclude that viable polioviruses can be detected in oysters after bioaccumulation and these techniques can be directly applied for monitoring virus contamination in environmental samples.

show abstract

Comparison between specific and multiplex reverse transcription-polymerase chain reaction for detection of hepatitis A virus, poliovirus and rotavirus in experimentally seeded oysters

Cited by 7 publications

References 19 publications

Qualidade microbiológica do sururu (Mytella charruana) comercializado por catadoras da Baía de Sepetiba, Rio de Janeiro, Brasil

Qualidade microbiológica do sururu (Mytella charruana) comercializado por catadoras da Baía de Sepetiba, Rio de Janeiro, Brasil

Simultaneous detection of HBV and HCV by multiplex PCR normalization

Detection of poliovirus type 2 in oysters by using cell culture and RT-PCR

Contact Info

Product

Resources

About