A comprehensive in vitro study on the performance of two different strategies to simplify adhesive bonding.

OBJECTIVE: The purpose of this study is to compare the bonding performance and mechanical properties of two different resin composite cements using simplified adhesive bonding strategies. MATERIALS AND METHODS: Shear bond strength of two resin composite cements (an adhesive cement: Panavia V5 [PV5] and a self-adhesive cement: RelyX Universal [RUV]) to human enamel, dentin, and a variety of restorative materials (microfilled composite, composite, polymer-infiltrated ceramic, feldspar ceramic, lithium disilicate and zirconia) was measured. Thermocycle aging was performed with selected material combinations. RESULTS: For both cements, the highest shear bond strength to dentin was achieved when using a primer (PV5: 18.0 ± 4.2 MPa, RUV: 18.2 ± 3.3 MPa). Additional etching of dentin reduced bond strength for RUV (12.5 ± 4.9 MPa). On enamel, PV5 achieved the highest bond strength when the primer was used (18.0 ± 3.1 MPa), while for RUV etching of enamel and priming provided best results (21.2 ± 6.6 MPa). Shear bond strength of RUV to restorative materials was superior to PV5. Bonding to resin-based materials was predominantly observed for RUV. CONCLUSIONS: While use of RUV with the selective-etch technique is slightly more labor intensive than PV5, RUV (with its universal primer) displayed a high-bonding potential to all tested restorative materials, especially to resin. CLINICAL SIGNIFICANCE: For a strong adhesion to the tooth substrate, PV5 (with its tooth primer) is to be preferred because etching with phosphoric acid is not required. However, when using a wide range of varying restorative materials, RUV with its universal primer seems to be an adequate option.

Development of genus-specific universal primers for the detection of flaviviruses.

BACKGROUND: Flaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods. METHODS: We developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates. RESULTS: The novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates. CONCLUSION: This PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology.

Universal Primers for Rapid Detection of Six Pospiviroids in Solanaceae Plants Using One-Step Reverse-Transcription PCR and Reverse-Transcription Loop-Mediated Isothermal Amplification.

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are listed as quarantine pathogens in many countries. Among them, columnea latent viroid, pepper chat fruit viroid, potato spindle tuber viroid, tomato apical stunt viroid, tomato chlorotic dwarf viroid, and tomato planta macho viroid are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step reverse transcription PCR (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from 1 fg to 10 ng, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR, but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate the ability to screen a large number of solanaceous plants and seeds intended for import and export.

Design of targeted primers based on 16S rRNA sequences in meta-transcriptomic datasets and identification of a novel taxonomic group in the Asgard archaea.

BACKGROUND: Amplification of small subunit (SSU) rRNA genes with universal primers is a common method used to assess microbial populations in various environmental samples. However, owing to limitations in coverage of these universal primers, some microorganisms remain unidentified. The present study aimed to establish a method for amplifying nearly full-length SSU rRNA gene sequences of previously unidentified prokaryotes, using newly designed targeted primers via primer evaluation in meta-transcriptomic datasets. METHODS: Primer binding regions of universal primer 8F/Arch21F for bacteria or archaea were used for primer evaluation of SSU rRNA sequences in meta-transcriptomic datasets. Furthermore, targeted forward primers were designed based on SSU rRNA reads from unclassified groups unmatched with the universal primer 8F/Arch21F, and these primers were used to amplify nearly full-length special SSU rRNA gene sequences along with universal reverse primer 1492R. Similarity and phylogenetic analysis were used to confirm their novel status. RESULTS: Using this method, we identified unclassified SSU rRNA sequences that were not matched with universal primer 8F and Arch21F. A new group within the Asgard superphylum was amplified by the newly designed specific primer based on these unclassified SSU rRNA sequences by using mudflat samples. CONCLUSION: We showed that using specific primers designed based on universal primer evaluation from meta-transcriptomic datasets, identification of novel taxonomic groups from a specific environment is possible.

Highly specific real-time quantification of diverse microRNAs in human samples using universal primer set frame.

In this study, one group of universal primer set frame, composed by one reverse transcription (RT) primer frame and a pair of quantitative real-time polymerase chain reaction (qRT-PCR) primer frames, was elaborately screened and designed by homebuilt software for sensitive and specific quantification of diverse miRNAs. The universal primer set frame can be applied for multiplex miRNAs detection by simply changing the RT-X part of RT primer frame and RP-Y part of qRT-PCR reverse primer frame based on target sequence. The maximum similarity of RT-Y, RT-Z and qRT-PCR forward primer to the human genome and human transcriptome is less than 76%, ensuring the high specificity in human sample detection. The high sensitivity and broad dynamic linear range of the developed approaches by using designed primer set frame were demonstrated on the in vitro detection of miR-21 and miR-155, with dynamic range of 10 fM to 10 nM and detection limit of 3.74 × 10-15 M and 5.81 × 10-15 M for miR-21 and miR-155, respectively. In particular, the developed assays also have high sequence specificity which could clearly discriminate a single base difference in miRNA sequence. The contents of miR-21 and miR-155 in tissue and serum samples have been successfully detected using the developed assays. Results indicated that miR-21 and miR-155 were elevated in cancer tissue and serum specimens than that of normal samples, implying the developed assays hold a great promise for further application in biomedical research and early clinical diagnosis. More importantly, the primer set frame can be universally used in any miRNA investigation.

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis.

Single universal primer multiplex ligation-dependent probe amplification with sequencing gel electrophoresis analysis.

In this study, a novel single universal primer multiplex ligation-dependent probe amplification (SUP-MLPA) technique that uses only one universal primer to perform multiplex polymerase chain reaction (PCR) was developed. Two reversely complementary common sequences were designed on the 5' or 3' end of the ligation probes (LPs), which allowed the ligation products to be amplified through only a single universal primer (SUP). SUP-MLPA products were analyzed on sequencing gel electrophoresis with extraordinary resolution. This method avoided the high expenses associated with capillary electrophoresis, which was the commonly used detection instrument. In comparison with conventional multiplex PCR, which suffers from low sensitivity, nonspecificity, and amplification disparity, SUP-MLPA had higher specificity and sensitivity and a low detection limit of 0.1 ng for detecting single crop species when screening the presence of genetically modified crops. We also studied the effect of different lengths of stuffer sequences on the probes for the first time. Through comparing the results of quantitative PCR, the LPs with different stuffer sequences did not affect the ligation efficiency, which further increased the multiplicity of this assay. The improved SUP-MLPA and sequencing gel electrophoresis method will be useful for food and animal feed identification, bacterial detection, and verification of genetic modification status of crops.

Universal primer multiplex PCR assay for detection and genotyping of porcine astroviruses.

Porcine astroviruses (PAstV) are members of the family Astroviridae, Mamastravirus genus and have been identified to have five genotypes (PAstV1-5). These viruses are highly prevalent in pigs and can cause enteric disease as well as neurological or respiratory symptoms depending on their genotypes. At present, the epidemiological impacts of some PAstV genotypes on pigs are largely unknown and hence continuously monitoring of these PAstVs may be needed. The purpose of this research was to develop an improved and efficient detection tool for PAstVs and to evaluate the developed method using clinical samples. Initially, a set of five chimeric primers (CP), each comprising genotype specific primer pairs with an identical universal adapter at the 5' end, and a universal primer (UP) that is identical to universal adapter sequence, were designed. With these tools in place, a novel multiplex PCR system with universal primer was established for the simultaneous detection of the five types of PAstV. This method can specifically detect PAstV genotypes, with a limit of detection (LOD) of 5 copies/µL for each genotype irrespective of single or mixed target template. Using this new assay, 273 pig fecal samples were investigated for further assay evaluation. Among all samples, the positive rate was 70.0% with PAstV4 in 56.8% of the samples, PAstV2 in 38.8%, PAstV1 in 16.8%, and PAstV5 in 11.0%. More than one PAstV in a sample were detected in 39.2% of the samples. The consistency rate between the novel multiplex PCR and singleplex PCRs was 96.4-100%. Given its rapidity, specificity and sensitivity, the novel multiplex PCR is a useful approach for demonstrating single or mixed genotype infections of PAstV.

Effects of Surface Treatment and Thermocycling on the Shear Bond Strength of Zirconia-Reinforced Lithium Silicate Ceramic.

PURPOSE: To investigate the effects of different surface treatments and thermocycling on shear bond strength (SBS) be-tween resin cement and zirconia-reinforced lithium-silicate (ZLS) ceramic. MATERIALS AND METHODS: 96 ZLS ceramic specimens were randomly allocated to four different surface treatment groups: etch and silane (ES), etch and universal primer (EUP), self-etching primer (SEP), and sandblasting and silane (SS). Stan-dardized composite cylinders were bonded to surface-treated ZLS ceramic, after which SBS was obtained either after 24-h water storage only or with an additional 5000 thermal cycles (TC), resulting in eight subgroups (n = 12). After evaluation of failure mode under a stereomicroscope, representative SEM images were acquired. To examine areal average surface roughness (Sa), additional ZLS specimens were prepared and randomly allocated to 3 groups: hydrofluoric acid etching, self-etching primer, and sandblasting (n = 10). Supplementary specimens were examined using field-emission scanning electron microscopy (FE-SEM) (n = 2) and atomic force microscopy (AFM) (n = 2) to investigate their surface topographies. RESULTS: ANOVA showed a statistically significant difference in SBS following different surface treatment protocols after 24-h water storage (p < 0.001). However, TC groups revealed no statistically significant difference in their SBS (p = 0.394). All surface treated groups were significantly affected by TC (p < 0.001), except for the SS group (p = 0.48). Sa was signifi-cantly influenced by the different surface treatment protocols (p < 0.001). CONCLUSION: The ability of self-etching primer to achieve comparable bond strength with a less technique-sensitive ap-proach makes it a favorable alternative to ES for the surface treatment of ZLS ceramics.

Shear Bond Strength of a Direct Resin Composite to CAD-CAM Composite Blocks: Relative Contribution of Micromechanical and Chemical Block Surface Treatment.

This study aims to compare the shear bond strength (SBS) of a direct resin composite to CAD-CAM resin composite blocks treated with different surface treatments: micromechanical, chemical or a combination of both. Eight CAD-CAM resin composite blocks, namely Brilliant Crios, Cerasmart 270, Vita Enamic, Grandio block, Katana Avencia, Lava Ultimate, Tetric CAD and Shofu Block HC were chosen. The micromechanical surface treatment protocols tested were hydrofluoric acid, polyacrylic acid or sandblasting, and the chemical one was a universal primer. These treated CAD-CAM blocks were tested to determine the SBS of a light-curing composite resin Z100 bonded to their surface. Two-way ANOVA followed by Tukey's post hoc test was used to investigate the difference in SBS. Failures were analyzed by Fisher's exact test. Bonding interfaces were examined by scanning electron microscopy. The micromechanical surface treatments give the highest SBS values: sandblasting appears to be the most efficient procedure for dispersed filler composite blocks, while hydrofluoric acid etching is preferable for polymer-infiltrated ceramic network (PICN) blocks. The use of universal primer does not improve SBS values on dispersed filler composite blocks. For PICN blocks, the use of universal primer significantly increases SBS values when combined with hydrofluoric acid etching.

Polymerase chain reaction-based snake origin tracing in commercial venom crystals by targeting the mitochondrial D-loop.

Snake venom is a valuable raw material for numerous therapeutic formulations because of its life-saving pharmacological potential. However, due to their high price, fake "snake venoms" have captured a significant portion of the global market, and there is currently no reliable reported DNA-based method available for quickly distinguishing between fakes and originals. Therefore, in this study, a set of newly designed snake-specific universal primers targeting mitochondrial D-loop fragments were employed to detect snake origins in commercial venom crystals by only simplex polymerase chain reaction analysis. Under the optimal thermal cycling conditions, only the 145-149 bp snake-specific mitochondrial D-loop fragments from pure and mixed backgrounds were amplified by the newly designed primers. Specificity was achieved by confirming no DNA amplification occurred in the DNA admixture of ten different chordates, and universality by individual DNA amplification of nine different snakes. The primers that efficiently amplified the minimum mitochondrial DNA contained in a total of 10-2 ng in a 10.0 µl reaction were also successfully able to detect the snake origin in commercial cobra venom crystals. These findings suggest that the newly designed primers can be used to differentiate the original and fake commercial snake venom crystals in order to achieve the highest standards of snake venom-based medications through amplifying the snake-specific mitochondrial D-loop fragments.

Establishment of quantitative nested-PCR of Abelson interactor 1 transcript variant-11.

Abelson interactor 1 (ABI1), which presents 18 Transcript Variants (TSV), plays an important role in CRC metastasis. Different ABI1-TSVs play synergistic or antagonistic roles in the same pathophysiological events. ABI1 Transcript Variant-11 (ABI1-TSV-11) functionally promotes lymph node metastasis of left-sided colorectal cancer (LsCC) and is an independent molecular marker to evaluate the prognosis of patients with LsCC. However, there is still lack of a quick and accurate method to detect the expression of ABI-TSV-11, distinguishing ABI1-TSV-11 from other 17 TSVs. To establish a rapid method specific for ABI1-TSV-11detection, we developed a quantitative nested-PCR method composed of pre-amplification regular PCR using ABI1 universal primer pair and the followed Real Time (RT)-qPCR using ABI1-TSV-11 specific primer pair spanning exon-exon junction. ABI1-TSV-11-overexpressed SW480 and LoVo cell lines were used to verify the quantitative nested-PCR assay, and the sequencing data was used to evaluate the accuracy of ABI1-TSV-11 quantitative nested-PCR assay. The detection limit was 5.24×104 copies/ml. ABI1-TSV-11 quantitative nested-PCR provides a new technical means for the detection of ABI1-TSV-11.

A novel metabarcoding primer pair for environmental DNA analysis of Cephalopoda (Mollusca) targeting the nuclear 18S rRNA region.

Cephalopods are pivotal components of marine food webs, but biodiversity studies are hampered by challenges to sample these agile marine molluscs. Metabarcoding of environmental DNA (eDNA) is a potentially powerful technique to study oceanic cephalopod biodiversity and distribution but has not been applied thus far. We present a novel universal primer pair for metabarcoding cephalopods from eDNA, Ceph18S (Forward: 5'-CGC GGC GCT ACA TAT TAG AC-3', Reverse: 5'-GCA CTT AAC CGA CCG TCG AC-3'). The primer pair targets the hypervariable region V2 of the nuclear 18S rRNA gene and amplifies a relatively short target sequence of approximately 200 bp in order to allow the amplification of degraded DNA. In silico tests on a reference database and empirical tests on DNA extracts from cephalopod tissue estimate that 44-66% of cephalopod species, corresponding to about 310-460 species, can be amplified and identified with this primer pair. A multi-marker approach with the novel Ceph18S and two previously published cephalopod mitochondrial 16S rRNA primer sets targeting the same region (Jarman et al. 2006 Mol. Ecol. Notes. 6, 268-271; Peters et al. 2015 Mar. Ecol. 36, 1428-1439) is estimated to amplify and identify 89% of all cephalopod species, of which an estimated 19% can only be identified by Ceph18S. All sequences obtained with Ceph18S were submitted to GenBank, resulting in new 18S rRNA sequences for 13 cephalopod taxa.

Development of an absolute quantification method for ribosomal RNA gene copy numbers per eukaryotic single cell by digital PCR.

Recent increase of Harmful Algal Blooms (HAB) causes world-wide ecological, economical, and health issues, and more attention is paid to frequent coastal monitoring for the early detection of HAB species to prevent or reduce such impacts. Use of molecular tools in addition to traditional microscopy-based observation has become one of the promising methodologies for coastal monitoring. However, as ribosomal RNA (rRNA) genes are commonly targeted in molecular studies, variability in the rRNA gene copy number within and between species must be considered to provide quantitative information in quantitative PCR (qPCR), digital PCR (dPCR), and metabarcoding analyses. Currently, this information is only available for a limited number of species. The present study utilized a dPCR technology to quantify copy numbers of rRNA genes per single cell in 16 phytoplankton species, the majority of which are toxin-producers, using a newly developed universal primer set accompanied by a labeled probe with a fluorophore and a double-quencher. In silico PCR using the newly developed primers allowed the detection of taxa from 8 supergroups, demonstrating universality and broad coverage of the primer set. Chelex buffer was found to be suitable for DNA extraction to obtain DNA fragments with suitable size to avoid underestimation of the copy numbers. The study successfully demonstrated the first comparison of absolute quantification of 18S rRNA copy numbers per cell from 16 phytoplankton species by the dPCR technology.

A Half-Day Genome Sequencing Protocol for Middle East Respiratory Syndrome Coronavirus.

Recent coronavirus (CoV) outbreaks, including that of Middle East respiratory syndrome (MERS), have presented a threat to public health worldwide. A primary concern in these outbreaks is the extent of mutations in the CoV, and the content of viral variation that can be determined only by whole genome sequencing (WGS). We aimed to develop a time efficient WGS protocol, using universal primers spanning the entire MERS-CoV genome. MERS and synthetic Neoromicia capensis bat CoV genomes were successfully amplified using our developed PCR primer set and sequenced with MinION. All experimental and analytical processes took 6 h to complete and were also applied to synthetic animal serum samples, wherein the MERS-CoV genome sequence was completely recovered. Results showed that the complete genome of MERS-CoV and related variants could be directly obtained from clinical samples within half a day. Consequently, this method will contribute to rapid MERS diagnosis, particularly in future CoV epidemics.

[Comparison of intestinal microbial community succession based on different universal primer sets].

The important role of intestinal microorganisms in human health has been widely confirmed. At present, most of the studies on intestinal microorganisms are based on amplification of the V3-V4 region of bacterial 16S rRNA gene, and little attention has been paid to archaea. In this study, a primer set which can amplify 16S rRNA gene of both bacteria and archaea at the same time was used. By comparing the community changes before and after probiotics intake, it showed that this primer set is suitable for analyzing the changes of human intestinal bacteria and archaea communities. The fecal samples of volunteers were collected, and the amplification and high-throughput sequencing were carried out by using bacterial primer set (B primer) and bacterial and archaeal universal primer (AB primer); several commonly used rRNA databases were used to determine the amplification ability of the primer set to bacteria and archaea. The results showed that AB primer could display the bacterial community amplified by B primer, and could obtain the sequence of common methanogenic archaea in intestinal tract. AB primer set can analyze the bacteria and archaea in the intestinal tract at the same time by only one amplification and sequencing, which can show the structure of intestinal microbial community more comprehensively, which is suitable for the research of intestinal microorganisms.

A rapid insect species identification system using mini-barcode pyrosequencing.

BACKGROUND: Rapid and accurate species identification is not only important for biodiversity studies and pest quarantine and management, but in some cases may also influence the results of international trade negotiations. In this study, we developed a rapid species identification system for insects. RESULTS: A universal DNA mini-barcode primer pair was designed to target ∼ 120 bp of the mitochondrial 16S rDNA gene. This primer set can amplify the targeted region from all 300 species of 26 insect orders tested as well as other classes of Arthropoda. Although we found no within-species variation in this region, it provided enough information to separate closely related species or species complexes, in particular Thrips spp. and Bemisia spp. By combining a quick DNA extraction method with pyrosequencing, we were able to generate DNA sequences and complete species identification within 5 h. CONCLUSION: Mini-barcode pyrosequencing of 16S rDNA coupled with the GenBank database provides a rapid, accurate, and efficient species identification system. This system is therefore useful for biodiversity discovery, forensic identification, and quarantine control and management. © 2019 Society of Chemical Industry.

Multiplex pyrosequencing quantitative detection combined with universal primer-multiplex-PCR for genetically modified organisms.

A multiplex pyrosequencing quantitative detection technique combined with universal primer-multiplex-PCR (UP-M-PCR) was established. In this study, a pyrosequencing results analysis software was first self-compiled, which realized the DNA sequences degeneration, and converted the pyrosequencing results and base composition of the target sequences into mathematic relations. Five calculation models were put forward based on the actual situation, which adjusted the values smaller than zero or the detection limit. By applying this method, samples containing five genetically modified (GM) lines mixed in random ratio were quantified, it showed that the quantification was very close to the actual value, and the detection sensitivity was as low as 1.47% of a single component, which satisfied most labeling policies. This novel method is realized without fluorescent group labeling, hence the number of targets is not limited by factors inherent in method or equipment, and is proven to be a reliable tool for the quantitative detection.

A 'turn-on' ultra-sensitive multiplex real-time fluorescent quantitative biosensor mediated by a universal primer and probe for the detection of genetically modified organisms.

Among the existing multiplex genetically modified organism (GMO) detection methods, significant problems are highlighted, including amplification asymmetry of different targets, and the low detection throughput, which limits their capacity to meet the requirements of high-throughput analysis. To mitigate these challenges, a 'turn-on' ultra-sensitive multiplex real-time fluorescent quantitative biosensor is developed. In this system, the multiplex ligation-dependent amplification (MLPA), universal primer and universal probe are innovatively combined, which can enhanced the amplification specificity, overcome asymmetric amplification and guarantee the homogeneity of amplification efficiency simultaneously. Furthermore, both single and multiplex detection results can be output by the fluorescent group labeled on universal TaqMan probes for different targets in real-time. After optimization, the quantitative detection limit was 5 pg. In conclusion, this strategy could serve as an important tool for GMO detection in processed and commercially available products, even in the fields that require reliable and sensitive detection of DNA targets.

Tubulin-Based DNA Barcode: Principle and Applications to Complex Food Matrices.

The DNA polymorphism diffusely present in the introns of the members of the Eukaryotic beta-tubulin gene families, can be conveniently used to establish a DNA barcoding method, named tubulin-based polymorphism (TBP), that can reliably assign specific genomic fingerprintings to any plant or/and animal species. Similarly, many plant varieties can also be barcoded by TBP. The method is based on a simple cell biology concept that finds a conveniently exploitable molecular basis. It does not depend on DNA sequencing as the most classically established DNA barcode strategies. Successful applications, diversified for the different target sequences or experimental purposes, have been reported in many different plant species and, of late, a new a version applicable to animal species, including fishes, has been developed. Also, the TBP method is currently used for the genetic authentication of plant material and derived food products. Due to the use of a couple of universal primer pairs, specific for plant and animal organisms, respectively, it is effective in metabarcoding a complex matrix allowing an easy and rapid recognition of the different species present in a mixture. A simple, dedicated database made up by the genomic profile of reference materials is also part of the analytical procedure. Here we will provide some example of the TBP application and will discuss its features and uses in comparison with the DNA sequencing-based methods.