RESUMO
ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.
Assuntos
Humanos , Peste/microbiologia , DNA Bacteriano/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Magnetismo/métodos , Yersinia pestis/isolamento & purificação , Yersinia pestis/classificação , Yersinia pestis/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/química , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Separação Imunomagnética , Primers do DNA/genética , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Magnetismo/instrumentaçãoRESUMO
Monoclonal antibody (mAb, NVRQS-DON) against deoxynivalenol (DON) was prepared. DON-Ag coated enzyme linked immunosorbent assay (ELISA) and DON-Ab coated ELISA were prepared by coating the DON-BSA and DON mAb. Quantitative DON calculation ranged from 50 to 4,000 ng/mL for DON-Ab coated ELISA and from 25 to 500 ng/mL for DON-Ag coated ELISA. 50% of inhibitory concentration values of DON, HT-2, 15-acetyl-DON, and nivalenol were 23.44, 22,545, 5,518 and 5,976 ng/mL based on the DON-Ab coated ELISA. Cross-reactivity levels of the mAb to HT-2, 15-acetyl-DON, and nivalenol were 0.1, 0.42, and 0.40%. The intra- and interassay precision coefficient variation (CV) were both <10%. In the mAb-coated ELISA, mean DON recovery rates in animal feed (0 to 1,000 microg/kg) ranged from 68.34 to 95.49% (CV; 4.10 to 13.38%). DON in a buffer solution (250, 500 and 1,000 ng/mL) was isolated using 300 microg of NVRQS-DON and 3 mg of magnetic nanoparticles (MNPs). The mean recovery rates of DON using this mAb-MNP system were 75.2, 96.9, and 88.1% in a buffer solution spiked with DON (250, 500, and 1,000 ng/mL). Conclusively we developed competitive ELISAs for detecting DON in animal feed and created a new tool for DON extraction using mAb-coupled MNPs.
Assuntos
Animais , Feminino , Camundongos , Ração Animal/análise , Anticorpos Antifúngicos/análise , Anticorpos Monoclonais/análise , Técnicas de Química Analítica/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Contaminação de Alimentos/análise , Fusarium/imunologia , Imidazóis/química , Magnetismo/métodos , Camundongos Endogâmicos BALB C , Micotoxinas/análise , Nanopartículas/química , Ovalbumina/química , Tricotecenos/análiseRESUMO
Nucleic acid amplification testing (NAT) was recently recommended by Brazilian legislation and has been implemented at some blood banks in the city of São Paulo, Brazil, in an attempt to reduce blood-born transmission of human immunodeficiency virus (HIV) and hepatitis C virus. OBJECTIVE: Manual magnetic particle-based extraction methods for HIV and HCV viral nucleic acids were evaluated in combination with detection by reverse transcriptase - polymerase chain reaction (RT-PCR) one-step. METHODS: Blood donor samples were collected from January 2010 to September 2010, and minipools of them were submitted to testing. ELISA was used for the analysis of anti-HCV/HIV antibodies. Detection and amplification of viral RNA was performed using real-time PCR. RESULTS: Out of 20.808 samples screened, 53 samples (29 for HCV and 24 for HIV) were confirmed as positive by serological and NAT methods. CONCLUSION: The manual magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donation for viremic donors to further increase the safety of blood products.
Assuntos
Humanos , HIV , Hepacivirus/isolamento & purificação , Magnetismo/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/sangue , Bancos de Sangue , Ensaio de Imunoadsorção Enzimática , HIV , Anticorpos Anti-HIV/sangue , Infecções por HIV/prevenção & controle , Hepacivirus/genética , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Hepatite C/prevenção & controle , Tamanho da Partícula , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The present experimental technique was utilized to record, 1 analyze, and correlate the electromyography [EMG] of human's triceps muscle [upper limb] under the effect of different successive MFS. The exposure time used for all fields employed was 15 minutes, which is the average duration recommended in magnetotherapy. It was evident that the PMF 50 G/50 Hz proved to be the most effective MF, it increases the muscle efficiency [A] by 21 percent if it was lonely applied. This parameter enhanced to 22 percent when double fields f[1]followed by f2 were applied. Then, it attained a maximum increase of 24 percent when triple fields F[1] followed by F[2], then F[3] were employed