Technical improvement of the TBP (tubulin‐based polymorphism) method for plant species detection, based on capillary electrophoresis

Abstract

Nowadays, feed and food safety and traceability are of primary importance. Hence, a correct labeling of the different products is highly desirable in general, but mandatory for those people who are suffering from eating disorders and food allergies. Among the technologies that have been developed for feed and food analysis, the patented tubulin-based polymorphism (TBP) method emerges as an easy, versatile, and inexpensive diagnostic tool. Initially used to fingerprint different plant species and varieties, TBP was then successfully applied to trace species in mixtures of plant origin such as commercial feeds. TBP is a DNA-based molecular marker, that makes use of PCR for the selective amplification of plant β-tubulin introns. Amplified fragments are then separated by PAGE and visualized by silver staining. We have now developed an improved version of TBP. Based on capillary electrophoresis and fluorescence detection, it makes the method automatic, more sensible, reproducible, and faster. Compared to the classic TBP, this new version allows to obtain a better data resolution and an easier interpretation of the results, clearing the way to large-scale feed/food diagnostics.

The Tubulin-Based-Polymorphism Method Provides a Simple and Effective Alternative to the Genomic Profiling of Grape

“…The capillary electrophoresis TBP variant (CE-TBP) was used as the method of comparison. Performed under denaturing condition, CE-TBP provides more accurate molecular data then PAGE separation with reference to the actual sizes of the amplified fragments [ 53 ]. However, we could not use CE-TBP for marker analysis because this technique, in our hands, cannot provide a reliable resolution for fragments higher than 1200 bp in size, limit over which numerous alleles can still be detected.…”

“…Capillary Electrophoresis TBP (CE-TBP) was performed as described in Gavazzi et al [ 53 ] with the following modifications: 2 μl of FAM-labelled PCR product were mixed with 0,18 μl of GeneScan ™ 1200 LIZ ® Size Standard (Thermo Fisher Scientific) and 17,82 μl Hi-Di ™ Formamide (Thermo Fisher Scientific) to a final volume of 20 μl. After denaturation at 95°C for 5 minutes, the samples were loaded on the Applied Biosystems ® 3500 Genetic Analyser (Thermo Fisher Scientific).…”

A Journey Through Plant Cytoskeleton: Hot Spots in Signaling and Functioning

“…Gene organization and expression of main cytoskeletal proteins represent strong interest from opening possibilities to use this knowledge for different molecular genetic purposes. For example, length polymorphism of tubulin gene introns is proposed even to assess genetic diversity in plant species (Bardini et al, ; Breviario et al, ; Gavazzi et al, ). Rabokon et al () used this approach to differentiate between the accessions of Aegilops biuncialis Vis., a wild relative of wheat.…”

Capillary Electrophoresis Based on Nucleic Acid Detection as Used in Food Analysis

“…PCR‐CGE‐LIF was used to trace the origin of perch, and only 3 polymorphic microsatellite markers were adequate to reliably (>98%) show that a perch did or did not belong to the Swiss population, which demonstrated that this technique is a precise method of food authentication (Rolli and others ). Gavazzi and others () replaced polyacrylamide gel electrophoresis (PAGE) with CGE to identify plant species by TBP (tubulin‐based polymorphism)‐PCR‐CGE, which revealed that subjective errors caused by manually performing PAGE can be avoided; the use of CGE made the method more automatic, sensitive, reproducible, and rapid in comparison with TBP‐PCR‐PAGE. Hsieh and others () compared enzyme linked immune‐sorbent assay (ELISA), PCR‐RFLP‐slab‐gel electrophoresis (SGE), and PCR‐RFLP‐CE as methods of detecting undeclared animal byproducts in commercial canned canine foods, which revealed that CGE detected all 24 animal‐derived byproducts, whereas SGE and ELISA detected only 22/24 and 14/24 animal‐derived byproducts, respectively.…”

“…Third, the waste from these techniques may cause severe pollution. Fourth, these methods are limited by the difficulties of accurate size estimation, digital‐based data preparation, and data interpretation and transferability (Knetsch and others ), so the interpretation of the results may be subjective and error‐prone (Gavazzi and others ). These issues are increasingly important when considering future applications in food analysis, in which speed, safety, and effectiveness are compulsory for practical applications.…”