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A micro-method for the assay of polyadenylate-containing ribonucleic acid by gel electrophoresis.

ABSTRACT: A micro-method for the determination of the electrophoretic profile of the various poly(A)-containing RNA species in a RNA sample was developed. The method is simple to carry out and allows for great reproducibility. It combines the resolving power of electrophoresis in agarose with the specificity of binding of poly(A) to poly(U)-containing glass-fibre filters. It consists of the following steps. (1) The molecules in an RNA sample are first separated according to their molecular weight by electrophoresis in agarose, at low ionic strength. 2. The molecules thus seperated are then submitted to a second electrophoresis in 'binding buffer' in a direction perpendicular to the first one. In the course of this electrophoresis the poly(A)-containing RNA species are seperated from other RNA species as they bind to a poly(U)-containing glass-fibre filter which is placed across the electrophoresis path. The method was used to determine the electrophoretic profile of Rhynchosciara salivary-gland messenger RNA. It was found that the population of messenger RNA in the gland is dominated by forms moving as 18 and 158 S peaks, but there is also polydisperse RNA with slower mobility.

SUBMITTER: Lara FJ

PROVIDER: S-EPMC1172405 | biostudies-other | 1975 Dec

REPOSITORIES: biostudies-other

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Project description:African swine fever (ASF) is an acute, severe and hemorrhagic infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and wild boars. Since the outbreak of the disease in China in 2018, it has brought a great impact on China's pig industry. Classical swine fever (CSF) is an acute contact infectious disease of pigs caused by classical swine fever virus (CSFV) infection. Clinically, acute CSF usually shows persistent high fever, anorexia, extensive congestion and bleeding of the skin and mucosa, which are similar to ASF. It is of great significance to prevent, control and accurately detect ASF and CSF in pig farms. In this study, Recombinase aided amplification (RAA) technology combined with a nucleic acid test strip (RAA-strip) was established for simple and specific detection of ASFV/CSFV. The sensitivity and preliminary clinical application results showed that the RAA test strip established in this study could detect recombinant plasmids containing ASFV/CSFV gene fragments as low as 103 copies/µL. The minimum detection limits of virus DNA/cDNA were 10 and 12 pg respectively, and there was no cross-reaction with other porcine viruses. The specificity of the method was good. We used 37-42 clinical samples to evaluate the performance of our established method, and the positive concordance rates with conventional PCR were 94.1 and 57.1%, respectively. In addition, ASFV and CSFV double RAA agarose gel electrophoresis detection methods were established. The results showed that the method had good specificity. The detection limit of this method is 106 copies for ASFV p72 gene recombinant plasmid and 105 copies for CSFV NS5B Gene recombinant plasmid. The use of this method for clinical material detection was consistent with the PCR method. In summary, the developed method of RAA-strip assay for ASFV and CSFV realized the visual detection of pathogens, and the developed method of dual RAA agarose gel electrophoresis assay for ASFV and CSFV realized the simultaneous detection of two pathogens in one reaction, with good specificity, high sensitivity and rapid reaction rate, which was expected to be clinically feasible for the differential diagnosis of ASF and CSF provided technical support.

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A micro-method for the assay of polyadenylate-containing ribonucleic acid by gel electrophoresis.

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