RESUMEN
Due to the huge demand for SARS-Cov-2 determination,alternatives to the standard qtPCRtestsare potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples.We isolated the naso-pharingealRNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5´-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks.The two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks. In conclusion, we describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 h. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR and increase the testing capacity.
Asunto(s)
Betacoronavirus/aislamiento & purificación , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/virología , Electroforesis Capilar/métodos , Neumonía Viral/virología , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Betacoronavirus/genética , COVID-19 , Prueba de COVID-19 , Infecciones por Coronavirus/diagnóstico , Cartilla de ADN/genética , ADN Complementario/genética , Genoma Viral , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Pandemias , Neumonía Viral/diagnóstico , SARS-CoV-2 , Sensibilidad y EspecificidadRESUMEN
Because broad genetic diversity has recently been detected in Torque teno sus viruses (TTSuV1 and TTSuVk2), the viral genome detection method needs to be improved to understand the prevalence of these viruses. Here, we established single PCR-based detection methods for the TTSuV1 and TTSuVk2a genomes with newly designed primer pairs and applied them to investigate the prevalence of TTSuV1 and TTSuVk2a in Japanese pig populations. The results revealed that 98.2% and 81.7% of the pig farms tested positive for the TTSuV1 and TTSuVk2a genomes, respectively, indicating that both TTSuV1 and TTSuVk2a are widespread in Japan.
Asunto(s)
Infecciones por Virus ADN/veterinaria , ADN Viral/aislamiento & purificación , Enfermedades de los Porcinos/virología , Torque teno virus/aislamiento & purificación , Animales , Infecciones por Virus ADN/virología , Genoma Viral , Japón/epidemiología , Reacción en Cadena de la Polimerasa , Prevalencia , Porcinos , Enfermedades de los Porcinos/epidemiologíaRESUMEN
INTRODUCTION: West Nile virus, a mosquito-borne viral zoonosis is responsible for human infections in Hungary. Laboratory diagnosis is based on serological tests, however the application of molecular methods has been appreciated. AIM: The aim of the study was to investigate blood, cerebrospinal-fluid and urine samples of acutely ill patients and to follow-up PCR positive cases to ascertain the length of virus excretion. METHOD: Clinical specimens were examined by indirect-immunofluorescent, haemagglutination-inhibition, two PCR tests and Sanger-sequencing. Virus isolation in case of two patients was successful. RESULTS: A follow-up study could be carried out in case of 5 patients. Viral nucleic acid was detectable in urine even for several weeks after symptom onset and viral RNA was present at higher concentration compared with other samples. CONCLUSIONS: PCR analysis of urine could provide useful epidemiological and diagnostic information. Therefore, it is recommended to collect urine samples in order to supplement the serological diagnosis. Orv Hetil. 2017; 158(20): 791-796.