A Universal Primer Set for PCR Amplification of Nuclear Histone H4 Genes from All Animal Species

There are many PCR-based methods for animal species identification; however, their detection numbers are limited or could not identify unknown species. We set out to solve this problem by developing a universal primer PCR assay for simultaneous identification of eight animal species, including goat, sheep, deer, buffalo, cattle, yak, pig, and camel. In this assay, the variable lengths of mitochondrial DNA were amplified using a pair of universal primers. PCR amplifications yielded 760 bp, 737 bp, 537 bp, 486 bp, 481 bp, 464 bp, 429 bp, and 359 bp length fragments for goat, sheep, deer, buffalo, cattle, yak, pig, and camel, respectively. This primer pair had no cross-reaction with other common domestic animals and fish. The limit of detection varied from 0.01 to 0.05 ng of genomic DNA for eight animal species in a 20 µl PCR mixture. Each PCR product could be further digested into fragments with variable sizes and qualitative analysis by SspI restriction enzyme. This developed PCR-RFLP assay was sufficient to distinguish all targeted species. Compared with the previous published related methods, this approach is simple, with high throughput, fast processing rates, and more cost-effective for routine identification of meat in foodstuffs.

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Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Analysis of a Short Fragment of the Cytochrome b Gene for Identification of Flatfish Species

Journal of Food Protection ◽

10.4315/0362-028x-61.12.1684 ◽

1998 ◽

Vol 61 (12) ◽

pp. 1684-1685 ◽

Cited By ~ 15

Author(s):

ANA CÉSPEDES ◽

TERESA GARCÍA ◽

ESTHER CARRERA ◽

ISABEL GONZÁLEZ ◽

BERNABÉ SANZ ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Cytochrome B ◽

Pcr Amplification ◽

Cytochrome B Gene ◽

Polymorphism Analysis ◽

Chain Reaction ◽

Greenland Halibut ◽

Universal Primer ◽

Pcr Products ◽

Polymerase Chain

Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the specific identification of sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides). PCR amplification of the cytochrome b gene using a universal primer together with a primer specifically designed as a part of this study produced a 201-bp fragment in all species analyzed. Digestions of the PCR products with Sau3Al, BsmAl, Rsal, and Mn/l endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded specific profiles that enabled direct identification of each species analyzed.

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Extraction of high-quality genomic DNA and identification of different DNA barcoding markers for chickpea (Cicer arietinum L.)

Buletin Agroteknologi ◽

10.32663/ba.v1i1.1194 ◽

2020 ◽

Vol 1 (1) ◽

pp. 7

Author(s):

Umavathi Saraswathi ◽

Lakshmanan Mullainathan

Keyword(s):

Dna Barcoding ◽

Dna Extraction ◽

Genomic Dna ◽

Dna Isolation ◽

Genomic Library ◽

Pcr Amplification ◽

Slight Modification ◽

High Quality ◽

Universal Primer ◽

Genetic Studies

The genetic studies of individual plants, especially self-pollinated species like chickpea need to be evaluated at the DNA level with the help of molecular markers for identifying genetic variations among the plants. High-quality DNA extraction is a prerequisite for genetic studies. Extraction of intact genomic DNA with high – molecular mass is essential for the study of many molecular biology applications like Polymerase Chain Reaction, endonuclease restriction digestion, southern blot analysis, and also for the construction of a genomic library. Several plant DNA extraction methods are available, even though the DNA isolation methods that give good yield employing both quantity and quality is quite difficult especially for self-pollinated crops like a chickpea. This work was focused on developing a standard protocol for the extraction of genomic DNA and identifying different barcoding markers. The result revealed that the CTAB extraction method with slight modification in protocol had been optimized for DNA isolation. The purified DNA, which was isolated through the CTAB method, had excellent spectral qualities and is efficiently digested by a restriction endonuclease, and is found to be more suitable for long-fragment PCR amplification. DNA barcoding is considered as a promising tool because it provides a practical and standard identification of plants. The isolated DNA sample was processed with a classical DNA barcoding approach by amplifying and sequencing with a universal primer. According to the result, among the different barcoding markers studied, the RbcL and Mat K were found to given the best result for molecular species identification in chickpea.

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Effect on sensitivity of PCR amplification of ribosomal DNA gene using universal primer with a mixture of yeast species

Journal of Food and Drug Analysis ◽

10.38212/2224-6614.2898 ◽

2020 ◽

Vol 6 (3) ◽

Author(s):

C.-C. Huang ◽

W.-C. Tsai ◽

H.-H. Wang

Keyword(s):

Ribosomal Dna ◽

Yeast Species ◽

Pcr Amplification ◽

Universal Primer

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Typing of Intimin Genes in Human and Animal Enterohemorrhagic and Enteropathogenic Escherichia coli: Characterization of a New Intimin Variant

Infection and Immunity ◽

10.1128/iai.68.1.64-71.2000 ◽

2000 ◽

Vol 68 (1) ◽

pp. 64-71 ◽

Cited By ~ 252

Author(s):

E. Oswald ◽

H. Schmidt ◽

S. Morabito ◽

H. Karch ◽

O. Marchès ◽

...

Keyword(s):

Escherichia Coli ◽

Animal Species ◽

Pcr Amplification ◽

Binding Activity ◽

Cell Binding ◽

Specific Primers ◽

E Coli ◽

Specific Manner ◽

Tight Association

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) produce the characteristic “attaching and effacing” (A/E) lesion of the brush border. Intimin, an outer membrane protein encoded by eae, is responsible for the tight association of both pathogens with the host cell. Severaleae have been cloned from different EPEC and EHEC strains isolated from humans and animals. These sequences are conserved in the N-terminal region but highly variable in the last C-terminal 280 amino acids (aa), where the cell binding activity is localized. Based on these considerations, we developed a panel of specific primers to investigate the eae heterogeneity of the variable 3′ region by using PCR amplification. We then investigated the distribution of the known intimin types in a large collection of EPEC and EHEC strains isolated from humans and different animal species. The existence of a yet-unknown family of intimin was suspected because several EHEC strains, isolated from human and cattle, did not react with any of the specific primer pairs, although these strains were eaepositive when primers amplifying the conserved 5′ end were used. We then cloned and sequenced the eae present in one of these strains (EHEC of serotype O103:H2) and subsequently designed a PCR primer that recognizes in a specific manner the variable 3′ region of this new intimin type. This intimin, referred to as “ɛ,” was present in human and bovine EHEC strains of serogroups O8, O11, O45, O103, O121, and O165. Intimin ɛ is the largest intimin cloned to date (948 aa) and shares the greatest overall sequence identity with intimin β, although analysis of the last C-terminal 280 aa suggests a greater similarity with intimins α and γ.

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8.Animal Species Identification through High Resolution Melting Real Time PCR (HRM) of the Mitochondrial 16S rRNA Gene

Annals of Animal Science ◽

10.1515/aoas-2015-0074 ◽

2016 ◽

Vol 16 (2) ◽

pp. 415-424 ◽

Cited By ~ 3

Author(s):

Pitchayanipa Klomtong ◽

Yupin Phasuk ◽

Monchai Duangjinda

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Species Identification ◽

Animal Species ◽

Restriction Enzymes ◽

Rrna Gene ◽

16S Rrna Gene Analysis ◽

Universal Primer ◽

Pcr Product ◽

Mitochondrial 16S Rrna Gene

Abstract Animal species identification has received growing attention, regarding genetic diversity and food traceability. The objective of this study is to apply a universal primer of part of the mitochondrial 16S rRNA gene analysis using the PCR-RFLP and HRM methods for identification of species origin in cattle, chicken, horse, sheep, pig, buffalo, and goat. PCR product size was 512 bp. The PCR product of 16S rRNA was digested with two restriction enzymes (BclI and MseI); sufficient to easily generate analyzable species-specific restriction profiles that could distinguish the unambiguity of all targeted species. The HRM method successfully identified all species by shape of melting temperature, and proved to be of higher resolution, and a more cost effective, alternative method compared with other identification techniques.

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Molecular tools for utilization of mitochondrial diversity in faba bean (Vicia faba)

Genetika ◽

10.2298/gensr1403745a ◽

2014 ◽

Vol 46 (3) ◽

pp. 745-762

Author(s):

Jelena Aleksic ◽

Jovanka Miljus-Djukic ◽

Zivko Jovanovic ◽

Branko Tomic ◽

Bojana Banovic

Keyword(s):

Vicia Faba ◽

Faba Bean ◽

Related Species ◽

In Silico ◽

Pcr Amplification ◽

Intraspecific Variability ◽

Pcr Primers ◽

Molecular Tools ◽

Universal Primer ◽

Centre Of Origin

We performed in silico PCR analyses utilizing complete mitochondrial (mtDNA) genome sequences of faba bean (Vicia faba) and two related species, Vigna angularis and Vigna radiata, currently available in GenBank, to infer whether 15 published universal primer pairs for amplification of all 14 cis-spliced introns in genes of NADH subunits (nad genes) are suitable for V. faba and related species. Then, we tested via PCR reactions whether seven out of 15 primer pairs would generate PCR products suitable for further manipulation in 16 genotypes of V. faba representing all botanical varieties of this species (major, minor, equina and subsp. paucijuga) of various levels of improvement (traditional and improved cultivars) originating from Europe, Africa, Asia and south America. We provide new PCR primers for amplification of nad1 intron 2/3 in V. faba, and demonstrate intraspecific variability in primary nucleotide sequences at this locus. Based on outcomes of both in silico predictions and PCR amplification, we report a set of PCR primers for amplification of five introns in nad genes that are promising molecular tools for future phylogeographic and other studies in this species for which unambiguous data on wild ancestors, centre of origin and domestication are lacking.

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Molecular identification of fungi associated with avocado (Persea americana Mill.) fruits

Agro-Science ◽

10.4314/as.v20i1.13 ◽

2021 ◽

Vol 20 (1) ◽

pp. 80-86

Author(s):

N.G. Iyanyi ◽

A.E. Ataga ◽

I.S. Rotimi ◽

I. Blessing

Keyword(s):

Pcr Amplification ◽

Fungal Species ◽

Persea Americana ◽

Molecular Techniques ◽

Control Measures ◽

Universal Primer ◽

Identification Of Fungi ◽

Pcr Products ◽

Fungal Organisms ◽

Health Complications

Avocado (Persea americana Mill.) is grown for its nutritious fruit. However, the quantity and quality of these fruits are threatened by some fungal organisms which can cause health complications when it is consumed by man. DNA extraction provides a unique tool for identification of organisms. This study was conducted to isolate and identify fungal species associated with avocado fruit using both morphological and molecular techniques. Fungal species were isolated from Persea americana purchased from Choba market, Port Harcourt, Rivers State, Nigeria using Potato Dextrose Agar (PDA) as a growth medium. The morphology of isolated fungi on PDA were cotton-like blackish grey spots, white villous colonies, greyish powdery spores and black spores for isolates 1 to 4 respectively. Extraction of DNA from fungal isolates was carried out using Zymo Fungal/Bacteria DNA Miniprep Kit. PCR amplification of the ITS1-2 regions of isolates was carried out using fungal universal primer pair; ITS4 and ITS5.PCR amplification of the ITS1-2 gene sequences yielded amplicons between 537-580 base pairs. PCR products were sequenced and the sequencing result after BLAST search revealed the identity of the four fungal species as follows: Lasiodiplodia theobromae, Fusarium proliferatum, Penicillium sp. and Aspergillus niger. This study will promote the knowledge of specific fungal species associated with Persea americanna and help plant pathologists to proffer preventive and control measures to enhance fruit protection and yield quality.

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

Genes ◽

10.3390/genes10030229 ◽

2019 ◽

Vol 10 (3) ◽

pp. 229 ◽

Cited By ~ 1

Author(s):

Laura Morello ◽

Luca Braglia ◽

Floriana Gavazzi ◽

Silvia Gianì ◽

Diego Breviario

Keyword(s):

Dna Sequencing ◽

Cell Biology ◽

Animal Species ◽

Analytical Procedure ◽

Dna Barcode ◽

Gene Families ◽

Genomic Profile ◽

Complex Matrix ◽

Universal Primer ◽

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.

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Combining a COI Mini-Barcode with Next-Generation Sequencing for Animal Origin Ingredients Identification in Processed Meat Product

Journal of Food Quality ◽

10.1155/2020/2907670 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Yanyi Pan ◽

Deyi Qiu ◽

Jian Chen ◽

Qiaoyun Yue

Keyword(s):

Next Generation Sequencing ◽

Animal Species ◽

Detection Efficiency ◽

Meat Product ◽

Animal Origin ◽

Processed Meat ◽

Next Generation ◽

Universal Primer ◽

Clone Sequencing ◽

Generation Sequencing

For revealing animal species in complex or adulterated processed meat product, we presented a method combining a novel cytochrome oxidase I (COI) mini-barcode with next-generation sequencing (NGS), which identifies various animal species (swine, bovine, Caprinae, and some of fish, shrimp, and poultry) accurately and efficiently in processed meat products. We designed a universal primer based on 140 sequences from 51 edible animal species. A mixture of 12 species raw meat samples were identified with the clone sequencing and also with a mini-barcode- (136 bp) combined NGS method, respectively. The mini-barcode of these 12 species was 100% identical to the target species sequence by Sanger sequencing. Compared to the clone sequencing method, the NGS method is superior in accuracy, sensitivity, and detection efficiency. Various edible animal species were identified in the species level both in the mixed samples and the 7 heavily processed food products. Moreover, some unlabeled species and dubious contamination were detected as well.

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