High resolution melting analysis as a novel DNA assay for meat speciation

Identification testing for meat species is of interest to regulatory bodies and cultural groups with special dietary requirements, e.g. Halal and Kosher foods. Current methodology for testing meat species is based on PCR assays; often followed by digestion with restriction enzymes to identify individual species [1,2,3]. These assays are time consuming and expensive to use as commercial test methods. The aim of this study was to determine the feasibility of using a universal PCR primer set and real-time PCR to amplify a single product from meat species DNA; followed by a more recent method for sequence verification – high resolution melting analysis (HRMA) - to identify and distinguish between the different meat species. Results across multiple assays show the methodology can qualitatively identify and distinguish different meat species in both single species and mixed species samples. There is also potential to develop the assay system to quantitatively analyse mixed samples

Effect of storage regime on the stability of DNA used as a calibration standard for real-time polymerase chain reaction

This article looks at storage factors influencing the stability of potential DNA calibration standards for use in quantitative polymerase chain reaction (PCR). Target sequences from the bacteria Campylobacter jejuni were cloned into a plasmid vector. Samples of these potential calibration standards were stored at +4, −20, and −80 °C as aqueous and lyophilized samples and were prepared as both single-use aliquots and multiple-use preparations. Results showed that the samples stored as single-use aqueous solutions at +4 °C and lyophilized samples stored at +4 and −20 °C were the most stable. Samples stored as frozen aqueous solutions at −20 °C were the least stable

Preliminary development and validation of a real-time reverse transcription PCR assay for the semi-quantification of viable Campylobacter jejuni in water samples

A TaqMan-based real-time reverse transcription PCR (RT-PCR) assay was developed for semi-quantification of viable Campylobacter jejuni in water samples. This preliminary assay is based on measuring the heat-shock induction of groEL messenger RNA (mRNA); the logic being that only viable cells can synthesise new mRNA. A 128-bp C. jejuni groEL DNA fragment was cloned and used as a positive control standard in TaqMan runs. The assay could detect as few as 13 genome equivalent copies of groEL plasmid DNA and 1–2 colony forming unit (CFU) of viable C. jejuni. A multi-step concentration technique was developed for collecting C. jejuni from large volumes of water samples. An average recovery of 20% (range: 8–47%) was obtained at spiking levels of 750–6,500 CFU per 10-litre of river water. Starting from concentration, the enumeration of viable C. jejuni in river water samples was achieved in approximately 12 hours. Quantification of viable C. jejuni in natural river water samples by this method showed similar trends to a culture-based double enrichment most probable number (MPN) -PCR method. There is potential to apply this fast, specific and sensitive semi-quantitative method to monitor a range of water samples for viable C. jejuni

Estrogenic pyrethroid pesticides regulate expression of estrogen receptor transcripts in mouse Sertoli cells differently from 17β–estradiol

Studies suggested that exposure to agricultural pesticides may affect male fertility. Pyrethroids are widely used pesticides due to their insecticidal potency and low mammalian toxicity. A recombinant yeast assay system incorporating the human agr-estrogen receptor was used to analyze the estrogenicity of a range of readily available pyrethroid pesticides. The commercial product Ripcord Plus showed estrogenic activity by this assay. To determine whether pyrethroid compounds might exert an effect on male fertility, mouse Sertoli cells were exposed in vitro to the endogenous estrogen, 17β-estradiol, and selected estrogenic pyrethroids. Following exposure, transcript levels of the agr- and β-estrogen receptors were assessed. Exposure of Sertoli cells to the pyrethroid compounds, both at high and at low published serum concentrations, affected the expression of the two estrogen receptors; however, the influence on estrogen receptor gene expression was different from the effect from exposure to 17β-estradiol. These results from our model systems suggest that (1) estrogenic pyrethroid pesticides affect the estrogen receptors, and therefore potentially the endocrine system, in a different manner from that of endogenous estrogen, and (2) should cells in the male testes be exposed to pyrethroid pesticides, male fertility may be affected through molecular mechanisms involving estrogen receptors

Deletion mapping of genetic regions associated with apomixis in Hieracium

Although apomixis has been quoted as a technology with the potential to deliver benefits similar in scale to those achieved with the Green Revolution, very little is currently known of the genetic mechanisms that control this trait in plants. To address this issue, we developed Hieracium, a genus of daisies native to Eurasia and North America, as a genetic model to study apomixis. In a molecular mapping study, we defined the number of genetic loci involved in apomixis, and we explored dominance and linkage relationships between these loci. To avoid difficulties often encountered with inheritance studies of apomicts, we based our mapping effort on the use of deletion mutagenesis, coupled with amplified fragment length polymorphism (AFLP) as a genomic fingerprinting tool. The results indicate that apomixis in Hieracium caespitosum is controlled at two principal loci, one of which regulates events associated with the avoidance of meiosis (apomeiosis) and the other, an unlinked locus that controls events associated with the avoidance of fertilization (parthenogenesis). AFLP bands identified as central to both loci were isolated, sequenced, and used to develop sequence-characterized amplified region (SCAR) markers. The validity of the AFLP markers was verified by using a segregating population generated by hybridization. The validity of the SCAR markers was verified by their pattern of presence/absence in specific mutants. The mutants, markers, and genetic data derived from this work are now being used to isolate genes controlling apomixis in this system

An accumulation of international reserves and external debt: Evidence from developing countries

The main analytical contribution of this paper is to analyze the cost of the decision to jointly hold reserves and sovereign debt. By analyzing the impact of holding reserves and sovereign debt on sovereign credit ratings will provide the evidence of the costs of holding reserves and debt with respect to credit risk. It is found that the positive effect of accumulation reserves that aims to improve sovereign ratings has been crowding-out by the negative effect of accumulation external debt which resulted in a net negative effect. As such, it is suggested that countries reduce their sovereign debt in order to maintain a good credit risk position while holds international reserves at the optimal level of 3.67 in a month of imports which is slightly higher than the conventional rule