Species-specific DNA Fragments Research Articles

Rapid identification of goat (Capra hircus) and sheep (Ovis aries) species in raw meat using duplex PCR assay

Adulteration of high quality meat with their inferior/cheaper counterparts has become a common practice in the meat industry, which is not detectable by the naked eye or by eating. To circumvent this problem, molecular methods had been developed. The present study was carried out for detection of meat species with the use of cytochrome b gene variability by duplex PCR. Meat samples from goat, sheep and buffalo were utilized for molecular analysis. Genomic DNA was isolated from six samples of each species with some modifications. Mitochondrial cyotchrome b gene was amplified by conventional and duplex PCR using a common forward primer and species-specific reverse primer. PCR amplicons were resolved by agarose gel electrophoresis and for each species produce a characteristic band pattern in conventional and duplex PCR was obtained. The PCR products showed species-specific DNA fragments of 157 and 331 bps from goat and sheep respectively. The duplex PCR could detect upto 10 % of DNA of meat species. Thus, the duplex PCR was found to be a simple, reliable, sensitive and highly specific test for simultaneous detection of meat species.

Optimization of multiplex PCR for the identification of animal species using mitochondrial genes in sausages

Detection of species fraud in meat products is very important in order to protect consumers from undesirable adulteration, as well as for the economic, religious and health aspects. The most important reason for verification of the labeling statements is to detect fraudulent substitution of expensive meat components with other cheaper animals or mislabeling. The aim of this study was to develop a multiplex PCR that could be used in the simultaneous identification of multiple meat species. In this study, ten sausages with a minimum beef content of 55 %, from ten different manufacturing companies, and five samples of cow, chicken, goat, camel and donkey raw meats, for the purpose of positive control, were collected from food markets in Tehran, Iran. Total DNA was extracted from each sausage and the raw meats. Primers were selected in different regions of mitochondrial DNA (12S rRNA, cytochrome b and NADH dehydrogenase subunits 2) for identification of meat species. 12S rRNA and NADH dehydrogenase subunits 2 primers generated specific fragments of 183 and 145 bp length, for chicken and donkey, respectively. Three different specific primers were used for amplification of cytochrome b gene in goat, camel and cattle species and amplified species-specific DNA fragments of 157, 200 and 274 bp, respectively. The results proved that half of the specimens were contaminated with chicken meat, and this was greater than the proportion of beef stated on the label, while the other half only had chicken residuals, and no beef content. No contamination was found with goat, donkey or camel meats. These findings showed that molecular methods, such as multiplex PCR, is a potentially reliable, sensitive and accurate assay for the detection of adulterated meat species in mixed meat products.

Genetic Evidence Confirms the Presence of Pygmy Rabbits in Colorado

Abstract The pygmy rabbit Brachylagus idahoensis is a sagebrush-obligate species of conservation concern that occurs in the Great Basin and adjacent intermountain areas in the western United States. The species is not known to occur in Colorado, despite proximity to existing populations of pygmy rabbits in Wyoming. We provide the first documentation of the pygmy rabbit in Colorado. Fecal pellets diagnostic of pygmy rabbits were collected in the Vermillion Bluffs area of northwestern Colorado. Samples from 16 pellet clusters were collected for species identification via genetic analyses, and we were able to extract and amplify sufficient DNA from 7 of 16 pellet samples. All seven samples were identified as originating from pygmy rabbits based on a species-specific mitochondrial DNA fragment analysis test. To verify species identification, we also sequenced 225 base pairs of the mitochondrial DNA cytochrome b region from all seven pellet samples. Presence of pygmy rabbits was confirmed from three locations separated by 2.4–7.7 km and pellets represented both adult and juvenile rabbits. Based on the sparseness of burrows in the area, density of pygmy rabbits in the area likely is low; however, systematic surveys by trained observers are needed to delineate the range and density of this species in Colorado. Given the conservation concern for pygmy rabbits across their current range, the newly confirmed presence of this species suggests that assessment of their conservation status in Colorado is warranted.

Specific and Sensitive Detection of Venturia nashicola, the Scab Fungus of Asian Pears, by Nested PCR.

The fungus Venturia nashicola is the causal agent of scab on Asian pears. For the rapid and reliable identification as well as sensitive detection of V. nashicola, a PCR-based technique was developed. DNA fingerprints of three closely related species, V. nashicola, V. pirina, and V. inaequalis, were obtained by random amplified polymorphic DNA (RAPD) analysis. Two RAPD markers specific to V. nashicola were identified by PCR, after which two pairs of sequence characterized amplified region (SCAR) primers were designed from the nucleotide sequences of the markers. The SCAR primer pairs, designated as D12F/D12R and E11F/E11R, amplified 535-bp and 525-bp DNA fragments, respectively, only from genomic DNA of V. nashicola. The specificity of the primer sets was tested on strains representing three species of Venturia and 20 fungal plant pathogens. The nested PCR primer pair specific to V. nashicola was developed based on the sequence of the species-specific 525-bp DNA fragment amplified by primer set E11F/E11R. The internal primer pair Na11F/Na11R amplified a 235-bp fragment from V. nashicola, but not from any other fungal species tested. The nested PCR assay was sensitive enough to detect the specific fragment in 50 fg of V. nashicola DNA.

<I>Pseudococcus maritimus</I> (Hemiptera: Pseudococcidae) and <I>Parthenolecanium corni</I> (Hemiptera: Coccidae) Are Capable of Transmitting Grapevine Leafroll-Associated Virus 3 Between <I>Vitis</I> x <I>labruscana</I> and <I>Vitis vinifera</I>

The grape mealybug, Pseudococcus maritimus (Ehrhorn), and European fruit lecanium scale, Parthenolecanium corni (Bouché), are the predominant species of Coccoidea in Washington State vineyards. The grape mealybug has been established as a vector of Grapevine leafroll-associated virus 3 (GLRaV-3) between wine grape (Vitis vinifera L.) cultivars, elevating its pest status. The objective of this study was to determine if GLRaV-3 could be transmitted between Vitis x labruscana L. and V. vinifera by the grape mealybug and scale insects. Three transmission experiments were conducted with regard to direction; from V. vinifera to V. x labruscana L., from V. x labruscana L. to V. x labruscana L., and from V. x labruscana L. to V. vinifera. Each experiment was replicated 15 times for each vector species. Crawlers (first-instars) of each vector species were allowed 1-wk acquisition and inoculation access periods. The identities of viral and vector species were confirmed by reverse transcription-polymerase chain reaction, cloning, and sequencing of species-specific DNA fragments. GLRaV-3 was successfully transmitted by both species in all experiments, although Ps. maritimus was a more efficient vector under our experimental conditions. To the best of our knowledge, this study represents the first documented evidence of interspecific transmission of GLRaV-3 between two disparate Vitis species. It also highlights the potential role of V. x labruscana L. in the epidemiology of grapevine leafroll disease as a symptomless source of GLRaV-3 inoculum.

Molecular Detection of Vertebrates in Stream Water: A Demonstration Using Rocky Mountain Tailed Frogs and Idaho Giant Salamanders

Stream ecosystems harbor many secretive and imperiled species, and studies of vertebrates in these systems face the challenges of relatively low detection rates and high costs. Environmental DNA (eDNA) has recently been confirmed as a sensitive and efficient tool for documenting aquatic vertebrates in wetlands and in a large river and canal system. However, it was unclear whether this tool could be used to detect low-density vertebrates in fast-moving streams where shed cells may travel rapidly away from their source. To evaluate the potential utility of eDNA techniques in stream systems, we designed targeted primers to amplify a short, species-specific DNA fragment for two secretive stream amphibian species in the northwestern region of the United States (Rocky Mountain tailed frogs, Ascaphus montanus, and Idaho giant salamanders, Dicamptodon aterrimus). We tested three DNA extraction and five PCR protocols to determine whether we could detect eDNA of these species in filtered water samples from five streams with varying densities of these species in central Idaho, USA. We successfully amplified and sequenced the targeted DNA regions for both species from stream water filter samples. We detected Idaho giant salamanders in all samples and Rocky Mountain tailed frogs in four of five streams and found some indication that these species are more difficult to detect using eDNA in early spring than in early fall. While the sensitivity of this method across taxa remains to be determined, the use of eDNA could revolutionize surveys for rare and invasive stream species. With this study, the utility of eDNA techniques for detecting aquatic vertebrates has been demonstrated across the majority of freshwater systems, setting the stage for an innovative transformation in approaches for aquatic research.

Detection of species specific DNA fragments of tiger and leopard source materials by multiplex PCR method

Detection of species specific DNA fragments of tiger and leopard source materials by multiplex PCR method

Identifying commercially relevantEchinaceaspecies by AFLP molecular markers

The rising interest in medicinal plants has brought several species of the genus Echinacea to the attention of many scientists. Echinacea angustifolia, E. pallida, and E. purpurea are the most important for their immunological properties, well known and widely used by the native Americans. The three species are easily distinguishable on the basis of their morphological characteristics, but it would be difficult, if not impossible, to distinguish them in commercial preparations of ground, dry plant parts of E. purpurea (the most valuable species for chemotherapeutic properties) mixed with the other two species. Species-specific molecular markers could be useful to address this issue. In the present work, using fresh material collected from cultivated Echinacea spp., AFLP analysis was used to discriminate the three species and to detect species-specific DNA fragments. By using 14 primer combinations it was possible to detect a total of 994 fragments, of which 565 were polymorphic. Overall, 89 fragments were unique to E. purpurea, 32 to E. angustifolia, and 26 to E. pallida. E+CAC/M+AAT or E+CAC/M+AGC alone provided 13, 9, and 4 or 7, 5, and 5 specific fragments for E. purpurea, E. angustifolia, and E. pallida, respectively. A validation trial to confirm the results was carried out on bulked samples of 23 accessions covering most of the genetic diversity of the three species. The results are discussed in terms of practical applications in the field of popular medicine, detecting frauds, and implications for the genus Echinacea.

Generation of a Sequence Characterized Amplified Region Probe for Authentication of Mainland Serow (Capricornis sumatraensis)

Mainland serow is an endanged artiodactyl of southern Anhui province, China, that is often subject to poaching. To provide an easy, rapid and reliable marker for identification of bushmeat, skin and other tissues of the species, we developed a sequence characterized amplified region (SCAR) based on a species-specific random amplified polymorphic DNA (RAPD) marker. Initially, a 1012-bp species-specific DNA fragment of mainland serow was detected by a RAPD primer S1193. Then, a serow-specific primer pair (SCF/SCR) was designed according to the specific RAPD fragment, resulting in a 438-bp SCAR for the species. Finally, the reliability of the SCAR primers was tested by a common multiplex polymerase chain reaction using the combination of the SCAR and cyt b universal primers. The results that all mainland serow samples presented two target bands but the others failed to produce the SCAR indicated that the designed primers were highly diagnostic. Therefore, the SCAR probe developed in this study will be useful for quick authentication of mainland serow tissue samples for conservation biology and bushmeat regulation.

Simple Method to Accurately Differentiate Candida albicans Isolates Concurrently Using Polymorphic Patterns of PCR-Amplified, Species-Specific Nuclear and Mitochondrial Targets

We describe a simple method to accurately differentiate Candida albicans isolates by concurrent use of the restriction enzyme digestion patterns for PCR products, targeting two species-specific DNA regions originating from genetically different sources, the nuclear and mitochondrial genomes. The target sequence we used as the nuclear gene was derived from the PHO85 gene, a negative regulator of the PHO system, in which we found a restriction size polymorphism within the two alleles of PHO85 in the diploid genome of this fungus. The mitochondrial target was derived from EO3, a species-specific DNA fragment possessing a small size polymorphism among various clinical isolates. Our results should provide a new tool for molecular epidemiological surveys of patients suffering from candidiasis caused by C. albicans.

Unravelling below‐ground plant distributions: a real‐time polymerase chain reaction method for quantifying species proportions in mixed root samples

Knowledge on below-ground plant distributions is almost lacking to date, despite the fact that such information would be very valuable in understanding below-ground competition and species-specific interactions, processes that are expected to shape community structure. Methods available so far for below-ground species determination have drawbacks that we tried to challenge. Some methods make use of differences in the chemical composition between species, but this is highly variable upon environmental factors. DNA-based techniques - far less dependent on chemical composition - such as polymerase chain reaction on internal transcribed spacer (ITS) primers can so far only determine presence-absence of a species in a mixed root sample. Here, we present a quantitative DNA-based technique that allows investigation of relative species abundances in experimental mixed root samples. We used quantitative real-time polymerase chain reaction (PCR) on species-specific markers obtained from intersimple sequence repeat (ISSR) analyses in root samples. This molecular technique is novel in the field of root ecology and its development overcame three challenges: (i) determination of species-specific DNA fragments, (ii) development and optimization of the real time PCR protocol, (iii) designing a data treatment method based on a modified delta-delta-cycle threshold (CT) analysis. The method gained robustness from using relative DNA abundances in species mixtures rather than absolute concentration readings. This requires accurate multispecies reference series as a calibration. Test samples with different known biomass ratios of all species showed proof of concept of this method. The pro's and contra's of this method are discussed in the light of its contribution to advancing ecological research on below-ground plant-plant interactions.

A Recombinant Antigen-Based Elisa For The Simultaneous Differential Serodiagnosis Of Mycoplasma Gallisepticum, Mycoplasma Synoviae, And Mycoplasma Meleagridis Infections

We have previously identified species-specific DNA fragments, referred to as MS2/28 and Mm14, of Mycoplasma synoviae and Mycoplasma meleagridis, respectively. In the present study, we extended our analysis of the MS2/28 fragment that was found to encode a species-specific antigenic site, and we demonstrated the specificity of the Mycoplasma gallisepticum hemagglutinin protein encoded by pMGA1.2 (a member of the vlhA gene family). Then, we combined the Escherichia coli-expressed products of MS2/28, Mm14, and pMGA1.2, to develop a recombinant antigen-based enzyme-linked immunosorbent assay (recELISA), for the simultaneous and specific detection of antibodies to the three aforementioned major avian mycoplasma species. For comparative purposes, a novel in-house crude antigen capture ELISA (capELISA) was developed in parallel. In the latter protocol, the microtiter wells were enriched in species-specific antigens by capturing sonicated crude antigens on coated rabbit polyclonal antibodies that had been extensively adsorbed with the whole antigen of the heterologous species. With regard to rapid serum agglutination, both ELISA tests were highly specific, and they showed a significant correlation when field sera from naturally infected birds were tested. recELISA proved to be highly specific because absorbance values, with the heterologous species, were significantly lower (P < 0.001) than those obtained with capELISA. Given its cost-effectiveness and simplicity, the recombinant antigen-based ELISA seems to represent a valid tool for the specific screening of the three major avian mycoplasma species. recELISA will be particularly useful with regard to trade control because a large number of samples from various fields could be rapidly processed.

Genetic Characterization and Authentication of Embelia ribes using RAPD-PCR and SCAR Marker

Embelia ribes Burm. f. (Myrsinaceae) is one of the important plants used in Indian traditional medicine. RAPD-PCR analysis was performed to obtain species-specific DNA fragments. A band of 906 bp that was specific to Embelia ribes irrespective of the geographical source was obtained using the random decamer primer OPF 05. SCAR primers ER 1 (27 mer) and ER 2 (26 mer) were designed from the sequence of the RAPD marker which yielded an expected amplicon of 594 bp with the Embelia ribes DNA only. This methodology can be used for species identification of genuine Embelia ribes and to distinguish it from common substitutes and adulterants. BLAST: basic local alignment search tool ER 1: Embelia ribes forward primer ER 2: Embelia ribes reverse primer RAPD-PCR: random amplification of polymorphic DNA polymerase chain reaction SCAR: sequence characterized amplified region.

Development of species-specific primers for the identification of aphids in Taiwan

In aphids, an identification system based on adult morphology is difficult to apply to larvae or adults of a different morph because of their small size and extensive polymorphism. In this work, identification of aphids using random amplified polymorphic DNA (RAPD) fingerprinting to generate species-specific primers was studied. A total of 20 RAPD primers were screened on eleven members of Aphidinae, i.e., Acyrthosiphon pisum, Aphis craccivora, Aphis gossypii, Aphis rumicis, Cavariella salicicola, Indomegoura indica, Lipaphis erysimi, Myzus formosanus, Myzus hemerocallis, Myzus persicae, and Toxoptera odinae. DNA fragments potentially useful as species-specific markers were selected for six aphid species. A primers set, A02ApF/A02ApR, based on the nucleotide sequence of a specific 462 bp fragment obtained from Ac. pisum by RAPD-PCR using primer A02, amplified this fragment only from Ac. pisum genomic DNA, but not from that of other species. The specificity of the primers for M. persicae was further confirmed using individuals from several field populations. It is concluded that RAPD PCR-SCAR (sequence characterized amplified region) DNA assay is a useful method for the detection of species-specific DNA fragments in mixed populations, and that pertinent data could facilitate development of a realistic quarantine system for pea aphids.

Molecular and cytogenetic paternity testing of a male offspring of a hinny

An alleged male foal of a female mule, whose sire and grandparents were unknown, was identified for its pedigree. Parentage testing was conducted by comparing polymorphism of 12 microsatellite DNA sites and mitochondrial D-loop sequences of the male foal and the female mule. Both the sequence analysis of species-specific DNA fragments and a cytogenetic analysis were performed to identify the species of the foal and its parents. The results showed that the alleged female mule is actually a hinny, and the male foal, which possesses 62 chromosomes, qualifies as an offspring of the female hinny and a jack donkey.

The potential of mitochondrial DNA markers and polymerase chain reaction-restriction fragment length polymorphism for domestic and wild species identification

Poaching is increasingly presenting challenge to conservational authorities in Africa. Accurate and reliable methods for the identification of poached wildlife meat when morphological features are missing, has been lacking in Africa. We describe a molecular based approach that has a potential of serving as a tool for game and domestic meat identification in Africa. A mitochondrial (mt246) marker and Rsa1 restriction enzyme were used in the PCR-RFLP species identification of game and domestic meat. Species-specific reference DNA fragment patterns were obtained using fresh meat from ten major wild herbivores, representing the highly targeted wild meat species in Tanzania and four domesticated animal species. With the exception of the zebra, all species produced unique monomorphic RFLP patterns that were species specific. These reference fragment patterns enabled identification of about 75% of unknown meat samples, demonstrating the ability of the technique in discriminating between and among wild and domestic species. The results provide preliminary promising fingerprints which need further validation for future use for the control of the up-surging bush meat trade in the continent.

Tick-Borne Rickettsial Pathogens in Ticks and Small Mammals in Korea

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.

Species and gender differentiation between and among domestic and wild animals using mitochondrial and sex-linked DNA markers

In many African countries accurate and reliable identification of poached wildlife products like carcasses or meat presents a big problem when morphological characters such as skin hair or bones are missing. We describe a molecular based approach that has a potential of serving as a forensic tool in game meat identification in Africa. A mitochondial DNA marker (mt700) and one restriction enzyme, Rsa1 were used in the PCR-RFLP species identification of game meat obtained from two National Parks in Tanzania. Species-specific reference DNA fragment patterns were obtained using fresh meat from ten wildlife and four domesticated species. All species except the zebra, produced unique monomorphic RFLP patterns. Collectively, these patterns demonstrate the potential ability of genetic techniques for discriminating between and among wildlife and domestic species. The reference PCR-RFLP fragments enabled species identification of about 79% of unknown meat samples. In addition, sex was also assigned to all of the samples following successful amplification of gender-specific, SRY and ZFY/X, chromosomal domains. Although the present study has been conducted on a limited range both in numbers and genetic diversity of wildlife species present in Africa, the results demonstrate the potential usefulness of the DNA approach in wildlife forensics in the continent.

Polymerase Chain Reaction Method to Detect Canis Materials by Amplification of Species-Specific DNA Fragment

Rapid identification of mammal materials in feeding stuffs and food is essential for effective control of a potential source of pathogens, such as those that cause bovine spongiform encephalopathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a canis-specific mitochondrial DNA sequence in foodstuffs and food. The amplified canis-specific PCR product was a 213 base pair band from the D-loop DNA fragment of mitochondria, a high copy gene which should improve the possibility of amplifying template molecules of adequate size among the degraded DNA fragments brought about by heat denaturation. The specificity of this method was confirmed by 8 canis blood DNA samples (from different breeds of dog) and 9 noncanis animal blood DNA samples (bovine, sheep, porcine, chicken, fish, donkey, rabbit, deer, horse). This method was able to detect the presence of canis material in foodstuffs and in food mixtures even when the concentration of canis-derived meat was reduced to 0.05%. Furthermore, it did not appear to be affected by prolonged heat treatment. This method was developed for detection of canis materials in feeding stuffs, and occasionally for medical jurisprudence detection of canis-derived materials.

Molecular epidemiological study for tick-borne disease (Ehrlichia and Anaplasma spp.) surveillance at selected U.S. military training sites/installations in Korea.

Vector-borne diseases are a potential public health threat to U.S. Forces Korea (USFK). Ehrlichia and Anaplasma spp., transmitted by ticks, are only two of several diseases that may affect military readiness and operations. Rodents were collected at selected U.S. military installations and training sites in the Republic of Korea. DNA was extracted from spleen tissues and assayed by PCR methods for Ehrlichia and Anaplasma species. From rodents and mustelids collected during 1999 and 2000, a total of 196 Apodemus agrarius (striped field mouse), 2 Mustela sibirica (weasel), and 1 Cricetulus triton nestor (Korean greater long-tailed hamster) were assayed for Ehrlichia and Anaplasma species-specific DNA fragments. Rodent surveillance indicated a very high prevalence of Ehrlichia and Anaplasma spp. at selected training sites. Ehrlichia/Anaplasma DNA were identified from spleen tissue from 157 Apodemus agrarius, 1 Mustela sibirica, and 1 Cricetulus riton nestor. Species-specific DNA fragments of E. canis (45), E. ewingii (16), A. phagocytophila (5), and A. platys (62) were amplified by PCR techniques. Seventy-one striped field mice had single infections, while 24 had mixed infections of 2 (17 specimens), 3 (7 specimens), or 4 (1 specimen) pathogens. The striped field mouse plays a role as a reservoir for latent infections of various Ehrlichia or Anaplasma species.