Use of homoduplex ribosomal DNA spacer amplification products and heteroduplex cross-hybridization products in the identification of Salmonella serovars

Abstract

When the hypervariable 16S-23S intergenic spacer regions found in prokaryotic ribosomal DNA (rDNA) are amplified from conserved adjacent sequences, homoduplex double-stranded DNA structures and heteroduplex structures containing substantial regions of single-stranded DNA are generated. The electrophoretic separation of these structures results in product profile patterns, which may be organized into highly correlated pattern groups of ribosomal spacer and heteroduplex polymorphism (RS/HP) types. In a test panel of 380 Salmonella strains that were analyzed by this procedure, 36 unique RS/HP types were observed. Of the 28 serovars in the test group, 21 showed single characteristic RS/HP types. The remaining seven serovars each contained multiple RS/HP types, which were also unique to individual serovars. Formation of heteroduplex structures with a substantially reduced electrophoretic mobility was observed in 29 of the 36 RS/HP pattern types. Because the mobility of these heteroduplex structures is sensitive to intergenic spacer sequence composition, the presence of these structures adds an additional diagnostic feature that is extremely useful in the differentiation of Salmonella serovars. The RS/HP types show sufficient diversity to be useful in the identification of many commonly observed Salmonella serovars. This analytical procedure is simple to perform and is well suited to rapid and inexpensive screening of large numbers of Salmonella strains.

16S to 23S rRNA spacer fragment length polymorphism of Salmonella enterica at subspecies and serotype levels

“…The latter similarity was super®cial, and under non-denaturing electrophoresis conditions, separation of serotypes from different subspecies was obtained. The higher separation by non-denaturing conditions can be explained by presence of secondary structures of homoduplex and heteroduplex fragments, which change the electrophoresis pro®le compared with denaturing PAGE (Sambrook et al 1989;Jensen et al 1993;Jensen and Hubner 1996). This may further explain the presence of fragments of around 1 kbp length registered in the present as well as in previous investigations (Jensen et al 1993;Nastasi and Mammina 1995;Jensen and Hubner 1996;Lagatolla et al 1996) because nucleotide sequences of ITS have not been reported higher than 643 bs (Luz et al 1998) and ITS fragment lengths not higher than 645 bs (see Fig.…”

“…Typing of Salmonella by this approach has been reported to allow differentiation of serotypes (Jensen et al 1993;Jensen and Hubner 1996;Lagatolla et al 1996) and subtyping of serotypes (Jensen et al 1993;Nastasi and Mammina 1995;Jensen and Hubner 1996;Lagatolla et al 1996) by non-denaturing polyacrylamide or agarose gel electrophoresis. Both the primary ITS fragments and the mobility of secondary structures formed by base pairing are determined under the non-denaturing conditions (heteroduplexes) which contribute to the observed ITS fragment patterns.…”

Heteroduplex structures in 16S-23S rRNA intergenic transcribed spacer PCR products reveal ribosomal interoperonic polymorphisms within singleFrankiastrains

“…5). What was observed in Frankia is rather different from what occurred in Salmonella (Jensen and Hubner 1996;Baudart et al 2000) or in the Bacillus cereus group, where the main heteroduplex products are formed by two DNA strands that may differ by hundreds of nucleotides because of the presence ⁄ absence of one or more tRNA genes (Daffonchio et al 2000(Daffonchio et al , 2003. Hence, few nucleotides insertion ⁄ deletion in the ITS between different operons could be easily detected by heteroduplex assay in polyacrylamide or MDE gels.…”

Inactivation of Salmonella in liquid egg albumen by antimicrobial bottle coatings infused with allyl isothiocyanate, nisin and zinc oxide nanoparticles

“…Both of these isolates were previously used in FDA pasteurization studies and provided by Keith Lampel from the FDA (Shah et al 1991). The third isolate, Salmonella Oranienburg DD2229, was part of the USDA, FSIT culture collection and originally provided by Dupont (Wilmington, DE, USA) from their Salmonella library (Jensen and Hubner 1996). Salmonella Oranienburg was included based on its isolation from egg products and its inclusion in numerous published liquid egg pasteurization studies (Gibbons and Moore 1944;Schneider 1946;Solowey et al 1946;Winter et al 1946;Cantor and McFarlane 1949;Anellis et al 1954;Cotterill 1968;Cotterill and Glauert 1969, 1972McBee and Cotterill 1971;Cotterill et al 1973;Cunningham 1995;Jordan et al 2011).…”