Rapid, simple method of preparing rotaviral double-stranded ribonucleic acid for analysis by polyacrylamide gel electrophoresis

Abstract

A procedure for extracting rotaviral double-stranded ribonucleic acid (RNA) directly from fecal and intestinal specimens collected from calves and pigs is described. This procedure provides a rapid, simple, reproducible method of obtaining rotaviral double-stranded RNA preparations suitable for electrophoretic analysis in polyacrylamide-agarose composite gels. The rotaviral genome electrophoretic migration pattern produced by double-stranded RNA extracted directly from a specimen by this procedure was qualitatively identical to the electrophoretic migration pattern obtained with double-stranded RNA extracted from purified rotavirus derived from the same specimen. Direct extraction of specimens containing porcine rotavirus-like virus by this procedure gave preparations that had electrophoretic migration patterns similar, but not identical, to the characteristic electrophoretic migration pattern of the rotaviral genome. Sufficient rotaviral double-stranded RNA could be extracted from 6 ml of fecal or intestinal specimen by this procedure to permit 15 or more electrophoretic assays.

Genetic diversity of group A rotaviruses associated with repeated outbreaks of diarrhea in a farrow-to-finish farm: identification of a porcine rotavirus strain bearing a novel VP7 genotype, G26

“…The gels were stained using a Silver Stain Plus kit (Bio-Rad Laboratories, Hercules, CA, USA). GAR electropherotypes were determined by comparing the individual RNA migration patterns of genome segments on the gel [23].…”

“…The nucleotide sequences of the VP8* fragment of the VP4 genes (774 bp, corresponding to nucleotides 70 through 843 of the porcine P[7] OSU strain's VP4 gene) were successfully determined in 13 of 15 samples in which the VP7 gene sequences had been previously determined. BLAST search analysis and phylogenic analysis of these VP4 genes with those of 33 established P genotypes led to classification of the 13 GAR strains into three genotypes: P [23], P [7], and P[13]/ [22] (Table 2).…”

“…Eight P [23] strains were identified, with six strains detected at the first outbreak, one at the second, and one at the fourth ( Table 2). These strains had nucleotide identities of 99.7% to 100% compared to each other, 91.0% to the porcine GUB71 strain identified in Japan, and 85.6% to 89.7% to other porcine P [23] strains (Additional file 2, Table S2).…”

“…Based on the sequence and phylogenic analysis of GAR strains detected among the four outbreaks, at least five combinations of G and P genotypes (G/P combinations) were identified: G9P [23] at the first outbreak, G9P [13]/ [22] and G9P [23] at the second, G3P [7] at the third, and G26P [7], G9P [23], and G5P [13]/ [22] at the fourth ( Table 2).…”

“…Prior to the present study, at least 25 G genotypes and 33 P genotypes were identified in humans and animals [4,5]. Among them, at least 11 G genotypes (G1-G6, G8-G12) and 13 P genotypes (P [1], P[5]-P [8], P [11], P [13], P [19], P [13]/ [22], P [23], P [26], P [27], and P [32]) have been described in pigs [6][7][8][9][10][11].…”