Cytochrome-b Gene Research Articles

Authentication of camel meat using species-specific PCR and PCR-RFLP.

In India and some of the African countries, one of the unconventional meats receiving the latest attention in meat adulteration is camel meat. So, the objective of this study was to develop a species-specific PCR based on mitochondrial cytochrome b (CYTB) gene and a PCR-RFLP assay of mitochondrial 12S rRNA to identify camel meat in suspected samples. Known sample of camel meat, samples suspected to be from illegally slaughtered camel and known samples of cattle, buffalo, sheep, goat, pork and chicken were used in the study. DNA were extracted from all samples following spin column method and PCR amplification were carried out using both CYTB and 12S rRNA gene primers. The CYTB gene amplification produced amplicon with a size of 435bp without any non-specific spurious amplification towards other species studied. Further, the 12S rRNA PCR products were analysed both by sequencing and by RFLP using enzyme AluI. On BLAST analysis the 448bp sequence obtained from suspected samples showed > 99% sequence homology to previously reported Camelus dromedaries (accession no: AM 9369251.1). On AluI digestion of the 448bp product from both known and suspected camel samples, a specific RFLP pattern with three distinct products of 90, 148 and 210bp size were evident, which were significantly different from the pattern of cattle, sheep, goat, chicken and buffalo. Further, after in-house validation, this cost effective and rapid method of camel meat identification is placed into practice for regular screening of vetero-legal samples in the lab.

Genetic variation of the Chilean endemic long-haired mouse Abrothrix longipilis (Rodentia, Supramyomorpha, Cricetidae) in a geographical and environmental context.

Quaternary climate and associated vegetational changes affected the fauna of the Chilean Mediterranean ecosystem. Here we studied the genetic variation of the long-haired mouse, Abrothrix longipilis, a sigmodontine rodent endemic to this area. Within an environmentally explicit context, we examined the geographic distribution of the genetic diversity and demographic history of the species based on sequences of the mitochondrial Cytochrome-b gene of 50 individuals from 13 localities and a large panel of single nucleotide polymorphisms of 17 individuals from 6 localities. The gene genealogy of A. longipilis revealed three intraspecific lineages that are allopatric and latitudinally segregated (northern, central, and southern lineages) with an estimated crown age for the whole species clade of 552.3 kyr B.P. A principal component analysis based on 336,596 SNP loci is in line with the information given by the the mitochondrial gene genealogy. Along its complete distributional range, A. longipilis showed patterns of isolation by distance and also isolation by environment. The general pattern of historical demography showed stability for most intraspecific lineages of A. longipilis. Northern and central lineages showed signals of historical demographic stability, while the southern lineage showed contrasting signals. In agreement with this, the niche models performed showed that in the northern range of A. longipilis, areas of high suitability for this species increased towards the present time; areas of central range would have remained relatively stable, while southern areas would have experienced more change through time. In summary, our study shows three distinct allopatric lineages of A. longipilis, each showing slightly different demographic history.

Assessing impact of dams on genetic diversity of native fish Mastacembelus armatus in river Yamuna using mitochondrial DNA cytochrome-b sequences as a molecular marker

Several dams have been constructed in Uttarakhand. In the present study an investigation on impact of dams on genetic diversity of native fish species Mastacembelus armatus at the Asan barrage on river Yamuna near Vikasnagar, Uttarakhand in India was done. Partial sequence of mitochondrial Cytochrome-b (Cyto-b) gene was used to determine the genetic variation in the population of Mastacembelus armatus. DNA was extracted from Mastacembelus armatus (n=33) samples, collected from river Yamuna and its tributary Asan from fin and fishes were released back in their habitat. Cytochrome c oxidase I (COI) was used to ascertain the species of fish along with morphometric characters. Analysis of 324 bp mtDNA fragment of Cyto-b revealed the presence of 06 haplotypes with nucleotide diversity, value ranged from 0.0172 to 0.0021 low pair wise Fst value was observed negative (-0.00125) when compared between Asan barrage and Kalsi site. No genetic subdivisions between the population were found after or before the dam sites. Tajima’s D value for river Asan, site A1 Mirzapur (before dam) was -0.1167 (a negative value). Negative Tajima D value can be indicative of recent selective sweep or population expansion after a recent bottleneck and linkage to a swept gene. Our data shows that fragmentation of habitat by dams does not have any impact on the genetic diversity of non-migrating Mastacembelus armatus fish species.

A revision of Neusticomys peruviensis (Rodentia: Cricetidae) with the description of a new subspecies

Abstract Neusticomys peruviensis is a poorly known sigmodontine rodent of the tribe Ichthyomyini, represented in collections by only five specimens collected in five localities from lowland forests of central and southern Peru. Recent expeditions in Llanchama, in northern Peru, north of the Río Amazonas, and near Allpahuayo Mishana Natural Reserve (Loreto, Peru), were successful in obtaining three specimens of Neusticomys. Based on morphological and meristic data, we found the population at Llanchama is distinct from the allopatric populations of N. peruviensis, and other species of Neusticomys. A species distribution model also shows the population at Llanchama is not highly predicted by the set of variables of the known localities of N. peruviensis. However, sequence data from the mitochondrial cytochrome-b gene indicate that genetic distinctiveness is low. Because intraspecific variability is important to understand evolutionary and biogeographic processes, and in concordance with the polytypic species concept, we interpret the population at Llanchama to represent a new subspecies of N. peruviensis that we describe in this paper.

Wild Boar-Specific PCR Assay and Sequence Analysis Based on Mitochondrial Cytochrome-B Gene for Halal Authentication Studies

Wild boar meat (WBM) is non-halal meat widely abused in Indonesia. The most common case is mixing beef with WBM either in raw or processed foods. Therefore, it is necessary to develop a detection method of WBM contamination. The objective of this study was to employ polymerase chain reaction (PCR) and sequence analysis using species specific primer (SSP) targeting on wild boar mitochondrial cytochrome-b (CYTBWB2-wb) gene for the identification of WBM in a meatball. The specificity of primer was tested, and the amplicon size was confirmed with conventional PCR and agarose electrophoresis. The base sequences were analyzed using GeneStudio software and subjected to BLAST using NCBI. CYTBWB2-wb primer was also used to test the reference meatballs made from beef and WBM using real-time PCR. The result showed that CYTBWB2-wb amplified wild boar Cyt-B mt-DNA gene specifically. The amplicon size was 194 base pair (bp) with a similarity of 93–98% toward gen Cyt-B mt-DNA of several wild boar types. The primer is able to detect WBM on the reference meatballs up to 0.1% wt/wt with efficiency value of 108.0% and coefficient of determination (R2) of 0.970. The CYTBWB2-wb primer proved to be specific and could be used as a standard method to identify the presence of WBM contamination in meatball products for halal authentication studies.

Investigation of ancient DNA to enhance natural history museum collections: misidentification of smooth-coated otter (Lutrogale perspicillata) specimens across multiple museums

Historical and modern natural museum collections are storehouses of extraordinary value for scientific research in a wide range of fields. Recent advances in molecular biotechnology (e.g., next generation genomics) have increased the range of collection material employable for DNA-based analyses to unprecedented levels. Nevertheless, the value of museum specimens strictly depends on reliability of data associated with them. We report on investigations of ancient DNA from specimens of smooth-coated otter (Lutrogale perspicillata, Mustelidae), the largest otter species living in Asia, in US and European mammal collections. Mitochondrial DNA Cytochrome-b gene sequencing proved that the studied specimens were not the expected taxon. Indeed, they actually belonged to three different species, namely the Asian small-clawed (Aonyx cinereus), Eurasian (Lutra lutra) and African clawless (Aonyx capensis) otters. This represents the first record of mustelid misidentification from museum collections. Detection of errors can be extremely difficult when based only on collectors’ notes and data. Hence, we warn scientists involved in otter research about potential challenges when dealing with museum specimens. We recommend curators pursue a multidisciplinary approach, including DNA analyses, to accurately catalogue the resources under their management and uphold the value of biodiversity information.

The enigmatic Leiosaurae clade: Phylogeography, species delimitation, phylogeny and historical biogeography of its southernmost species

The clade Leiosaurae is composed of poorly-known species endemic to the southern region of South America. The difficulties of finding these lizards in the field, and their highly conserved morphology, have limited our taxonomic knowledge and understanding of their evolutionary histories. Here, we use data collected over 9 years to study the phylogenetic history, genetic diversity, and biogeographic history of almost all the southernmost species of Leiosaurae (except P. nigroigulus), including: Leiosaurus bellii, Diplolaemus darwinii, D. bibronii, D. sexcinctus and D. leopardinus. We use a fragment of the mitochondrial cytochrome-b gene to resolve general phylogeographic patterns, and add another mitochondrial gene and eight nuclear genes to perform species delimitation and phylogenetic analyses associated with divergence times. We found evidence for three putative new species-level taxa within L. bellii and five within Diplolaemus species, indicating high levels of geographic structure. We use a time-calibrated phylogeny to estimate ranges of ancestral distributions and to generate new hypotheses about their historical biogeography.

Non-native and hybrid in a changing environment: conservation perspectives for the last Italian red-legged partridge (Alectoris rufa) population with long natural history

The ever-increasing biotic homogenization - especially when associated with introgressive hybridization - raises concern for the reduction of the spatial component of diversity in wildlife worldwide. Nonetheless, there is a growing attention to the potential conservation value of hybridization in fastening the adaptive evolutionary responses to rapidly changing selective pressures. Under these premises, we investigated the genetic affinity of the red-legged partridge (Alectoris rufa) population inhabiting Elba Island (Italy) in the context of the overall species phylogeography and particularly of the nominate subspecies it is traditionally ascribed to. Although notoriously hybrid with the congeneric A. chukar, this island population is of undisputedly value because of its long natural history and self-sustainability. As such, its adaptive conservation management calls for a comprehensive knowledge including the assessment of its geographic origin. For this purpose, 110 fecal samples were collected across Elba, genotyped at their joint Cytochrome-b and Control Region genes (2,249 characters), and compared with 149 conspecifics from all over the species distribution range. We confirmed a widespread A. chukar mitochondrial DNA introgression in Elba partridges, whereas their expected formal assignment to the nominate subspecies from Italy and France was rejected, since these turned out to be closely related to conspecifics from the Iberian Peninsula. This counterintuitive result found support in a large variety of literary sources and compelling evidences from personal testimonies revealing recent intense management with farm-reared birds of Spanish origin. Although the nativeness of Elba partridges was disproved, we advise local authorities to keep warranting the ongoing conservation efforts - and especially restore the connectivity between the western and eastern island sub-populations - as this resource may still be conceived as the ultimate repository for part of the otherwise extinct Italian A. r. rufa genome. Indeed, the admixture with conspecifics from the Iberian Peninsula does not necessarily mean that the entirety of the native nuclear genome of Elba partridges has been wiped out. Furthermore, these latter represent an interesting case study in conservation biology to investigate the possible role played by introgressive hybridization in the adaptation to recent land use and vegetation cover changes associated with rural abandonment in an insular yet heavily anthropized context.

Taxonomic status of the nominal forms assigned to Necromys lactens (Rodentia, Cricetidae) as revealed by molecular and morphometric evidence

Abstract Necromys is a genus of sigmodontine rodent that inhabits grasslands and scrublands in South America. Eight extant species are recognized in the genus; one of these is Necromys lactens, which inhabits high-elevation grasslands in the Yungas from south-central Bolivia to northwestern Argentina. Morphological variation in N. lactens has been recognized by the description of three nominal forms. Geographically structured genetic diversity also has been observed, but a thorough revision of these nominal forms within an integrative framework has yet to be performed. We conducted a phylogeographic assessment based on an 801 base-pair fragment of the cytochrome-b gene that guided morphometric analyses (univariate and multivariate comparisons) of patterns of geographic variation in the species, and the distinction of its nominal forms. Haplotypes of N. lactens form a well-supported and geographically structured clade. Within it, there are two main clades; haplotypes from the northern range form a well-supported clade, sister and allopatric to a weakly supported southern clade, which includes variants collected at or near the type localities of three nominal forms. In turn, both main clades are composed by two allopatric subclades. Morphometric analyses indicated no differences in shape of the skull among the three nominal forms or between the recovered clades and subclades. Taking together all the available evidence, we consider N. lactens to be a monotypic species. Necromys es un género de roedor sigmodontino que habita los pastizales y arbustales de América del Sur. Se reconocen ocho especies actualmente en existencia en el género; una de ellas, Necromys lactens, habita pastizales de altura en las Yungas, desde el centro-sur de Bolivia hasta el noroeste de Argentina. Se ha reconocido variación morfológica en N. lactens con base en descripciones de tres formas nominales; también se ha observado diversidad genética geográficamente estructurada, pero una revisión exhaustiva de esas formas nominales dentro de un esquema integrativo aún no se ha llevado a cabo. Realizamos una evaluación filogeográfica basada en un fragmento de 801 pares de bases del gen citocromo b que orientó análisis morfométricos (comparaciones univariadas y multivariadas) respecto al patrón geográfico de variación de la especie y la distinción de sus formas nominales. Los haplotipos de N. lactens forman un clado bien apoyado y geográficamente estructurado. Dentro de este clado, los haplotipos del norte de su área de distribución forman un clado bien apoyado que es hermano y alopátrico con respecto de un clado austral débilmente apoyado, el cual incluye variantes colectadas en las localidades tipo de las tres formas nominales o sus cercanías. A su vez, ambos clados principales están compuestos por dos sub-clados alopátricos. Los análisis morfométricos no revelaron diferencias en la forma del cráneo entre las tres formas nominales ni entre los clados y sub-clados recuperados. Teniendo en cuenta toda la evidencia disponible, consideramos que N. lactens es una especie monotípica.

The Anatolian Diagonal and Paleoclimatic Changes Shaped the Phylogeography of Cynips quercus (Hymenoptera, Cynipidae)

Cynips quercus is a with widespread oak-gall wasp species distributed across the Western Palearctic from Europe, the northern Caucasus and North Africa to Iran. In this study, we used 175 asexual-generation individuals of C. quercus collected from 30 localities in Turkey to investigate the effects of topography and climatic factors on the phylogeographic structure of the species. Sequence analysis of the mitochondrial cytochrome-b gene (MT-CYB) and the nuclear ITS2 region supported a genetic break between the eastern and the western sides of the Anatolian Diagonal. The ITS2 results indicated that the populations underwent expansion, while in contrast the MT-CYB data implied a stable population structure for C. quercus. Molecular clock applied to the MT-CYB data suggested that the climatic and environmental changes during the Pliocene and Pleistocene had a profound effect on the phylogeography of the species.

Association of Phlebotomus guggisbergi with Leishmania major and Leishmania tropica in a complex transmission setting for cutaneous leishmaniasis in Gilgil, Nakuru county, Kenya.

BackgroundPhlebotomus (Larroussius) guggisbergi is among the confirmed vectors for cutaneous leishmaniasis (CL) transmission in Kenya. This scarring and stigmatizing form of leishmaniasis accounts for over one million annual cases worldwide. Most recent CL epidemics in Kenya have been reported in Gilgil, Nakuru County, where the disease has become a public health issue. However, little is known about the factors that drive its transmission. Here, we sought to determine the occurrence, distribution and host blood feeding preference of the vectors, and to identify Leishmania species and infection rates in sandflies using molecular techniques. This information could lead to a better understanding of the disease transmission and improvement of control strategies in the area.Methodology/ Principal findingsAn entomological survey of sandflies using CDC light traps was conducted for one week per month in April 2016, and in June and July 2017 from five villages of Gilgil, Nakuru county; Jaica, Sogonoi, Utut, Gitare and Njeru. Sandflies were identified to species level using morphological keys and further verified by PCR analysis of cytochrome c oxidase subunit I (COI) gene. Midguts of female sandflies found to harbour Leishmania were ruptured and the isolated parasites cultured in Novy-MacNeal-Nicolle (NNN) media overlaid with Schneider’s insect media to identify the species. Leishmania parasite screening and identification in 198 randomly selected Phlebotomus females and parasite cultures was done by PCR-RFLP analysis of ITS1 gene, nested kDNA-PCR and real-time PCR-HRM followed by sequencing. Bloodmeal source identification was done by real-time PCR-HRM of the vertebrate cytochrome-b gene. A total of 729 sandflies (males: n = 310; females: n = 419) were collected from Utut (36.6%), Jaica (24.3%), Sogonoi (34.4%), Njeru (4.5%), and Gitare (0.1%). These were found to consist of nine species: three Phlebotomus spp. and six Sergentomyia spp. Ph. guggisbergi was the most abundant species (75.4%, n = 550) followed by Ph. saevus sensu lato (11.3%, n = 82). Sandfly species distribution across the villages was found to be significantly different (p<0.001) with Jaica recording the highest diversity. The overall Leishmania infection rate in sandflies was estimated at 7.07% (14/198). Infection rates in Ph. guggisbergi and Ph. saevus s.l. were 9.09% (12/132) and 3.57% (2/56) respectively. L. tropica was found to be the predominant parasite in Gilgil with an overall infection rate of 6.91% (13/188) in Ph. guggisbergi (n = 11) and Ph. saevus s.l. (n = 2) sandflies. However, PCR analysis also revealed L. major infection in one Ph. guggisbergi specimen. Bloodmeal analysis in the 74 blood-fed sandflies disclosed a diverse range of vertebrate hosts in Ph. guggisbergi bloodmeals, while Ph. saevus s.l. fed mainly on humans.Conclusions/ SignificanceThe high infection rates of L. tropica and abundance of Ph. guggisbergi in this study confirms this sandfly as a vector of L. tropica in Kenya. Furthermore, isolation of live L. tropica parasites from Ph. saevus s.l. suggest that there are at least three potential vectors of this parasite species in Gilgil; Ph. guggisbergi, Ph. aculeatus and Ph. saevus s.l. Molecular identification of L. major infections in Ph. guggisbergi suggested this sandfly species as a potential permissive vector of L. major, which needs to be investigated further. Sandfly host preference analysis revealed the possibility of zoonotic transmissions of L. tropica in Gilgil since the main vector (Ph. guggisbergi) does not feed exclusively on humans but also other vertebrate species. Further investigations are needed to determine the potential role of these vertebrate species in L. tropica and L. major transmission in the area.

Quantitative morphological characters of the skull suggest that Akodon oenos (Rodentia, Cricetidae, Sigmodontinae) is not a junior synonym of A. spegazzinii

Abstract Akodon oenos, a nominal form with type locality in northern Mendoza province, Argentina, has an intricate taxonomic history. Recently, it was considered a junior synonym of Akodon spegazzinii on the basis of morphological similarities of one individual captured in southern Mendoza with specimens belonging to the type series of A. oenos, plus the genetic similarity of that single specimen with representatives of A. spegazzinii. We compared specimens of the Akodon boliviensis species group recently captured in Mendoza with the type series of A. oenos and series of A. spegazzinii from northwestern Argentina. We conducted an analysis of molecular markers (cytochrome-b gene) and quantitative morphological studies (comparisons of skull measurements). Our results showed that: (1) all sequenced specimens of the A. boliviensis group from Mendoza form a monophyletic clade closely related to A. spegazzinii; (2) contrary to previous hypothesis, specimens of Akodon from Mendoza are morphologically distinctive in size and shape of the skull, from A. spegazzinii; and (3) within the A. boliviensis species group, specimens from Mendoza are distinctive. We argue that the synonymy of A. oenos under A. spegazzinii must await further studies, including molecular markers other than mitochondrial and samples of DNA sequences from topotypical specimens of A. oenos.

Polymorphisms involving the Pneumocystis jirovecii-related genes in AIDS patients in eastern China

ObjectiveTo investigate the genetic polymorphisms of mitochondrial large ribosomal subunit (mtLSU)-rRNA, dihydrofolate reductase (DHFR), dihydropteroate synthase (DHPS), cytochrome b (CYB), and superoxide dismutase (SOD) genes and its correlation with clinical outcomes of Pneumocystis jirovecii pneumonia in acquired immune deficiency(AIDS) patients. MethodsEighty AIDS patients with P. jirovecii pneumonia that were admitted to our hospital from 2016 to 2018 were included in this study. Their demographic information and clinical data were collected, as well as corresponding saliva specimens for PCR and sequencing of mtLSU-rRNA, DHFR, DHPS, CYB, and SOD genes to analyze genetic polymorphisms, different polymorphic combinations, and clinical outcomes. ResultsOf the 80 saliva specimens, mtLSU-rRNA was successfully amplified and sequenced in 30 cases; CYB was successfully amplified and sequenced in 26 cases; and SOD, DHFR, and DHPS were successfully amplified and sequenced in 18 cases. These results indicate that The mtLSU-rRNA, CYB, and SOD genes were highly polymorphic. mt85T and CYB1 were the variants dominantly detected at the mtLSU-rRNA and CYB loci, respectively. The SOD1 and SOD2 variants (each in 50% of the cases) were detected at the SOD locus. Among the 18 cases that were successfully amplified and sequenced for DHFR and DHPS, three DHFR nonsense mutations and no DHPS mutation were observed. The mt85C, CYB1, SOD1, and DHFR312T genes harbored common polymorphisms (n = 4; 22.22%) and the mt85T, CYB1, SOD1, DHFR312T genes were associated with poor clinical outcomes. ConclusionThe types of genetic polymorphisms and polymorphic combinations of mtLSU-rRNA, DHFR, DHPS, CYB, and SOD in P. jirovecii were related to the clinical outcomes of patients with P. jirovecii pneumonia in Zhejiang Province, China.

Development of a PCR-based assay for detection of Chinese mink tissue in meat products based on the mitochondrial DNA cytochrome-b gene

Species authentication of meat product origins has become an important subject for ensuring the health of consumers. Based on the cytochrome b gene, we developed a PCR-based assay kit for identification of Chinese mink tissues from 10 animal species in meat products, and to evaluate its quality indices including specificity, stability, sensitivity, and repeatability. Kits were made up of DNA extraction and PCR amplification systems based on species-specific, and universal primers. The reference meat mixtures and commercial samples were extracted by the kit and PCR technique was performed to identify the species of mink authenticity. The kit was effective after 20 repeated freeze–thaw cycles and it could be stored at –20 °C for 1 year. The sensitivity showed that a concentration as low as 0.1 ng/μL still can amplify the target band. The specificity test confirmed that the kit was 100% specific. The kit proved to be effective, stable, and reliable for extraction of efficient contents of the genomic DNA and routine analysis of Chinese mink source composition from meat products.

Preliminary report on the genetic structure of Glyphoglossus molossus (Anura: Microhylidae) from the Khorat Plateau, north-eastern Thailand

ABSTRACTGlyphoglossus molossus, is one of the most economically important amphibians in the Khorat Plateau, north-eastern Thailand. It is categorised as a near-threatened species by IUCN due to over-exploitation. Here, we examined the genetic structure of G. molossus using partial mitochondrial cytochrome-b gene sequences from specimens collected at 11 localities. The sequence analysis revealed the presence of 15 haplotypes, 5 of which are shared by 2 or more populations, and 10 haplotypes that are confined to a single population. The relatively low haplotype (h) and nucleotide (π) diversities suggest that the Phu Phan Mountain Range is not high enough to act as an effective dispersal barrier between the Sakon Nakhon and Khorat Basins. However, AMOVA and phylogenetic analyses based on Maximum likelihood (ML) and Bayesian inference (BI) strongly supported the presence of two genetically divergent clades, Sakon Nakhon Basin and the northern part of the Khorat Basin (clade A) and the southern part of the Khorat Basin (clade B). These two lineages are separated by substantial geographical distance, which has putatively resulted in a reduction of gene flow. Further studies, with more extensive genetic sampling, especially from throughout the species range will aid in better interpreting our results.

Low genetic diversity in the vulnerable Goitred Gazelle, Gazella subgutturosa (Cetartiodactyla: Bovidae), in Iran: potential genetic consequence of recent population declines

The Goitered Gazelle, Gazella subgutturosa, is the most widespread gazelle species in the Middle East and central Asia inhabiting desert and semi-desert habitats. Today it is threatened and its geographic range and population size have experienced significant decline in the last decades. In Iran, the remnant populations are confined to fragmented habitats. We aimed to characterise genetic diversity and phylogenetic status of the populations of Goitered Gazelle in Central Iran and to evaluate the potential effect of a historic population bottleneck on the genetic variation of today’s population. We used noninvasive sampling to uncover structure and level of genetic variation in a fragment of the cytochrome-b gene from 170 samples. Genealogical analyses were performed using HKY+I model and phylogenetic trees reconstructed using Bayesian inference and maximum likelihood. We found extremely low levels of genetic variation, with altogether only five haplotypes in samples from different populations. Overall haplotype diversity was 0.081 and nucleotide diversity 0.0003. The mean observed mismatch between any two sequences was 0.093 with the largest peak for small numbers. The mismatch distribution fit the model of population expansion and suggested that gazelles had experienced a sudden expansion. An unrooted median-joining network analysis of mtDNA haplotypes showed a star-like structure which few mutations steps separating the haplotypes from other regions. Our findings strengthen the urgency of preserving the species’ genetic diversity to prevent local extinction.

Thermal adaptation of Japanese sand lance (Ammodytes personatus) in different ocean currents revealed by the cytochrome-b gene

To assess the possible thermal selection on mitochondrial coding genes in cold water species, we explored the population structure of Japanese sand lance (Ammodytes personatus) and performed selection tests on the mitochondrial cytochrome-b gene of species, using 174 individuals from eight different sea temperature populations in different ocean currents. Two distinct haplotype lineages were present in this species, and the lineage frequencies varied with changes in sea temperature. The selection tests showed that A. personatus was subject to purifying selection. Populations living in cold climates had a considerably smaller non-synonymous substitution rate/synonymous substitution rate (dN/dS) than those in temperate areas. The efficiency of the electron transfer chain system may be affected by amino acid changes at codons 353 and 371. These findings provide new evidence that temperature may affect the contemporary distribution of mitochondrial DNA clade frequencies in A. personatus.

Application of real-time polymerase chain reaction using species specific primer targeting on mitochondrial cytochrome-b gene for analysis of pork in meatball products.

Objective:This study aimed to design specific primers derived from mitochondrial cytb of Sus Scrofa (1F1R primer) used in the pork meatball analysis using real time polymerase chain reaction (RT-PCR) method.Materials and Methods:Such designed primers were validated and these included specificity of primer, linearity, and sensitivity of the method as well as the repeatability test. The primers were specifically affirmed in the fresh tissue of chickens, cows, pigs, and goats. The linearity and sensitivity of the method was conducted by measuring the amplification curve from a series of dilution (0, 1, 1, 10, 100, 1,000, and 10,000 pg/μl of DNA) extracted from 100% pork meatball formulation. The repeatability test was conducted by determining the cycle threshold (Ct) values of RT-PCR amplification from 100% pork meatball formulation as many as six times.Results:Primer of 1F1R (forward: 5′-ACG CGA TAT AAG CAG GTA AA-3′; reverse: 5′-CTG CTT TCG TAG CAC GTA TT-3′) was specific in analyzing the presence of pork in meatball formulation at 47.1°C, which was optimum annealing temperature. The DNA identification was able to use the primers by RT-PCR with 1 pg as the limit of detection, efficiency value was 242.58%, and the coefficient of determination value (R2) was 0.956. The coefficient of variance was 4.13%. The developed method was also fruitfully applied to analyze commercial meatballs.Conclusion:RT-PCR method using specific primers targeting on mitochondrial gene (1F1R primer) could be used as the standard method for identification of pork in food samples intended for halal authentication studies.

The employment of q-PCR using specific primer targeting on mitochondrial cytochrome-b gene for identification of wild boar meat in meatball samples.

Objective:The objective of this study was to employ real-time or quantitative polymerase chain reaction (q-PCR) using novel species specific primer (SSP) targeting on mitochondrial cytochrome-b of wild boar species (CYTBWB2-wb) gene for the identification of non-halal meat of wild boar meat (WBM) in meatball products.Materials and Methods:The novel SSP of CYTBWB2-wb was designed by our group using PRIMERQUEST and NCBI software. DNA was extracted using propanol-chloroform-isoamyl alcohol method. The designed SSP was further subjected for validation protocols using DNA isolated from fresh meat and from meatball, which include specificity test, determination of efficiency, limit of detection and repeatability, and application of developed method for analysis of commercially meatball samplesResults:The results showed that CYTBWB2-wb was specific to wild boar species against other animal species with optimized annealing temperature of 59°C. The efficiency of q-PCR obtained was 91.9% which is acceptable according to the Codex Allimentarius Commission (2010). DNA, with as low as 5 pg/μl, could be detected using q-PCR with primer of CYTBWB2-wb. The developed method was also used for DNA analysis extracted from meatball samples commercially available.Conclusion:q-PCR using CYTBWB2-wb primers targeting on mitochondrial cytochrome-b gene (forward: CGG TTC CCT CTT AGG CAT TT; Reverse: GGA TGA ACA GGC AGA TGA AGA) can be fruitfully used for the analysis of WBM in commercial meatball samples.

Laboratory challenges of Plasmodium species identification in Aceh Province, Indonesia, a malaria elimination setting with newly discovered P. knowlesi.

The discovery of the life-threatening zoonotic infection Plasmodium knowlesi has added to the challenges of prompt and accurate malaria diagnosis and surveillance. In this study from Aceh Province, Indonesia, a malaria elimination setting where P. knowlesi endemicity was not previously known, we report the laboratory investigation and difficulties encountered when using molecular detection methods for quality assurance of microscopically identified clinical cases. From 2014 to 2015, 20 (49%) P. falciparum, 16 (39%) P. vivax, 3 (7%) P. malariae, and 2 (5%) indeterminate species were identified by microscopy from four sentinel health facilities. At a provincial-level reference laboratory, loop-mediated isothermal amplification (LAMP), a field-friendly molecular method, was performed and confirmed Plasmodium in all samples though further species-identification was limited by the unavailability of non-falciparum species-specific testing with the platform used. At a national reference laboratory, several molecular methods including nested PCR (nPCR) targeting the 18 small sub-unit (18S) ribosomal RNA, nPCR targeting the cytochrome-b (cytb) gene, a P. knowlesi-specific nPCR, and finally sequencing, were necessary to ultimately classify the samples as: 19 (46%) P. knowlesi, 8 (20%) P. falciparum, 14 (34%) P. vivax. Microscopy was unable to identify or mis-classified up to 56% of confirmed cases, including all cases of P. knowlesi. With the nPCR methods targeting the four human-only species, P. knowlesi was missed (18S rRNA method) or showed cross-reactivity for P. vivax (cytb method). To facilitate diagnosis and management of potentially fatal P. knowlesi infection and surveillance for elimination of human-only malaria in Indonesia and other affected settings, new detection methods are needed for testing at the point-of-care and in local reference laboratories.