Nonisotopic single‐strand conformation polymorphism analysis of sequence variability in ribosomal DNA expansion segments within the genus Trichinella (Nematoda: Adenophorea)

Abstract

A nonisotopic single-strand conformation polymorphism (SSCP) approach was employed to 'fingerprint' sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. In addition, phylogenetic analyses of the D3 sequence data set, employing three different tree-building algorithms, examined the relationships among all of them. These analyses showed strong support that the encapsulated species T. spiralis and T. nelsoni formed a group to the exclusion of the other encapsulated species T. britovi and its related genotypes Trichinella T8 and T9 and T. murrelli, and T. nativa and Trichinella T6, and strong support that T. nativa and Trichinella T6 grouped together. Also, these eight encapsulated members grouped to the exclusion of the nonencapsulated species T. papuae and T. zimbabwensis and the three representatives of T. pseudospiralis investigated. The findings showed that nonencapsulated species constitute a complex group which is distinct from the encapsulated species and supported the current hypothesis that encapsulated Trichinella group external to the nonencapsulated forms, in accordance with independent biological and biochemical data sets.

Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region

“…After PCR, individual amplicons were mixed with an equal volume of loading buffer (10 mM NaOH, 95 % formamide, 0.05 % of both bromophenol M. synoviae classification by SSCP and HRM curves blue and xylene cyanole) and the intensity of selected samples was verified on ethidium bromide-stained 2 % agarose gels using TBE (65 mM Tris/HCl, 27 mM boric acid, 1 mM EDTA, pH 9; Bio-Rad) as the buffer and WX174-HaeIII (Promega) as a size marker. After denaturation at 94 uC for 15 min and snap cooling on a freeze block (220 uC), the samples were subjected to SSCP analysis, as recently described (Gasser et al, 2004). In brief, samples (~12 ml each) were loaded into the wells of precast GMA S-12x13 gels (966261 mm; product no.…”

New pieces of the Trichinella puzzle

“…Maximum likelihood (ML), parsimony and/or NJ methods were subsequently used to study Trichinella phylogeny focusing on the D3 domain of the nuclear ribosomal DNA (Gasser et al, 2004). Strong bootstrap support was observed for monophyly between T. spiralis and T. nelsoni, and between T. nativa and Trichinella T6.…”

Differentiation ofEntamoeba histolytica fromEntamoeba dispar by PCR-coupled nonisotopic SSCP analysis

“…Subsequently, individual samples (12 mL) were denatured at 947C for 15 min, snap cooled on a freeze-block (2207C) and then loaded immediately into the wells of precast GMA ™ S-50 gels (96 mm6261 mm; product no. 3548, Elchrom Scientific) and subjected to electrophoresis for 5 h at 110 V and 7.47C (constant) in a horizontal SEA2000 ™ apparatus (Elchrom Scientific) connected to a MultiTemp III (Pharmacia) cooling system [18]). After electrophoresis, gels were stained for 15 min with SYBR Gold ™ (0.5 mg/mL), destained in water for the same time and then photographed (using 667 film, Polaroid) upon UV transillumination.…”