Turbidimetric bioassays: A solution to antimicrobial activity detection in asymptomatic bacteriuria isolates against uropathogenic Escherichia coli.

Traditional bacteriocin screening methods often face limitations due to diffusion-related challenges in agar matrices, which can prevent the peptides from reaching their target organism. Turbidimetric techniques offer a solution to these issues, eliminating diffusion-related problems and providing an initial quantification of bacteriocin efficacy in producer organisms. This study involved screening the cell-free supernatant (CFS) from eight uncharacterized asymptomatic bacteriuria (ABU) isolates and Escherichia coli 83972 for antimicrobial activity against clinical uropathogenic E. coli (UPEC) strains using turbidimetric growth methods. ABU isolates exhibiting activity against five or more UPEC strains were further characterized (PUTS 37, PUTS 58, PUTS 59, S-07-4, and SK-106-1). The inhibition of the CFS by proteinase K suggested that the antimicrobial activity was proteinaceous in nature, potentially bacteriocins. The activity of E. coli PUTS 58 and SK-106-1 was enhanced in an artificial urine medium, with both inhibiting all eight UPECs. A putative microcin H47 operon was identified in E. coli SK-106-1, along with a previously identified microcin V and colicin E7 in E. coli PUTS 37 and PUTS 58, respectively. These findings indicate that ABU bacteriocin-producers could serve as viable prophylactics and therapeutics in the face of increasing antibiotic resistance among uropathogens.

Unravelling the genetic basis of Schizophrenia.

Neuronal development is a highly regulated mechanism that is central to organismal function in animals. In humans, disruptions to this process can lead to a range of neurodevelopmental phenotypes, including Schizophrenia (SCZ). SCZ has a significant genetic component, whereby an individual with an SCZ affected family member is eight times more likely to develop the disease than someone with no family history of SCZ. By examining a combination of genomic, transcriptomic and epigenomic datasets, large-scale 'omics' studies aim to delineate the relationship between genetic variation and abnormal cellular activity in the SCZ brain. Herein, we provide a brief overview of some of the key omics methods currently being used in SCZ research, including RNA-seq, the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and high-throughput chromosome conformation capture (3C) approaches (e.g., Hi-C), as well as single-cell/nuclei iterations of these methods. We also discuss how these techniques are being employed to further our understanding of the genetic basis of SCZ, and to identify associated molecular pathways, biomarkers, and candidate drug targets.

Detection of Genomic Imprinting for Carcass Traits in Cattle Using Imputed High-Density Genotype Data.

Genomic imprinting is an epigenetic phenomenon defined as the silencing of an allele, at least partially, at a given locus based on the sex of the transmitting parent. The objective of the present study was to detect the presence of SNP-phenotype imprinting associations for carcass weight (CW), carcass conformation (CC) and carcass fat (CF) in cattle. The data used comprised carcass data, along with imputed, high-density genotype data on 618,837 single nucleotide polymorphisms (SNPs) from 23,687 cattle; all animal genotypes were phased with respect to parent of origin. Based on the phased genotypes and a series of single-locus linear models, 24, 339, and 316 SNPs demonstrated imprinting associations with CW, CC, and CF, respectively. Regardless of the trait in question, no known imprinted gene was located within 0.5 Mb of the SNPs demonstrating imprinting associations in the present study. Since all imprinting associations detected herein were at novel loci, further investigation of these regions may be warranted. Nonetheless, knowledge of these associations might be useful for improving the accuracy of genomic evaluations for these traits, as well as mate allocations systems to exploit the effects of genomic imprinting.

The urinary microbiome and biological therapeutics: Novel therapies for urinary tract infections.

The discovery of microbial communities in the urinary tract (the urobiome) has fundamentally altered the previous doctrine regarding urine sterility and associated urinary disorders. Recent advances in culturing and culture-independent DNA sequencing technologies have characterised the resident microbial community in the urobiome, and has, in turn, demonstrated how community imbalances potentially contribute to infection and disease. As we enter a post-antibiotic era, the effectiveness of standard antimicrobial treatments against multi-drug resistant (MDR) uropathogens is vastly diminished. Preliminary research is accumulating surrounding microbiome-based therapies, and their potential as non-antibiotic therapeutics. In this context, the urobiome is significantly underexplored, and knowledge regarding the fundamental role of its constituents is lacking. Herein, we review the current state of the art concerning the urobiome; specifically, how it impacts health and disease states, in the context of urinary tract infections (UTIs). Furthermore, we discuss the development of novel biological therapeutics that may have the potential to provide significant advancements in UTI therapy, with a particular focus on bacterial interference, probiotics, antimicrobial peptides, bacteriocins, and bacteriophage.

The Association Between Genomic Heterozygosity and Carcass Merit in Cattle.

The objective of the present study was to quantify the association between both pedigree and genome-based measures of global heterozygosity and carcass traits, and to identify single nucleotide polymorphisms (SNPs) exhibiting non-additive associations with these traits. The carcass traits of interest were carcass weight (CW), carcass conformation (CC) and carcass fat (CF). To define the genome-based measures of heterozygosity, and to quantify the non-additive associations between SNPs and the carcass traits, imputed, high-density genotype data, comprising of 619,158 SNPs, from 27,213 cattle were used. The correlations between the pedigree-based heterosis coefficient and the three defined genomic measures of heterozygosity ranged from 0.18 to 0.76. The associations between the different measures of heterozygosity and the carcass traits were biologically small, with positive associations for CW and CC, and negative associations for CF. Furthermore, even after accounting for the pedigree-based heterosis coefficient of an animal, part of the remaining variability in some of the carcass traits could be captured by a genomic heterozygosity measure. This signifies that the inclusion of both a heterosis coefficient based on pedigree information and a genome-based measure of heterozygosity could be beneficial to limiting bias in predicting additive genetic merit. Finally, one SNP located on Bos taurus (BTA) chromosome number 5 demonstrated a non-additive association with CW. Furthermore, 182 SNPs (180 SNPs on BTA 2 and two SNPs on BTA 21) demonstrated a non-additive association with CC, while 231 SNPs located on BTA 2, 5, 11, 13, 14, 18, 19 and 21 demonstrated a non-additive association with CF. Results demonstrate that heterozygosity both at a global level and at the level of individual loci contribute little to the variability in carcass merit.

Low-Molecular-Weight Seaweed-Derived Polysaccharides Lead to Increased Faecal Bulk but Do Not Alter Human Gut Health Markers.

Seaweeds are potentially sustainable crops and are receiving significant interest because of their rich bioactive compound content; including fatty acids, polyphenols, carotenoids, and complex polysaccharides. However, there is little information on the in vivo effects on gut health of the polysaccharides and their low-molecular-weight derivatives. Herein, we describe the first investigation into the prebiotic potential of low-molecular-weight polysaccharides (LMWPs) derived from alginate and agar in order to validate their in vivo efficacy. We conducted a randomized; placebo-controlled trial testing the impact of alginate and agar LWMPs on faecal weight and other markers of gut health and on composition of gut microbiota. We show that these LMWPs led to significantly increased faecal bulk (20-30%). Analysis of gut microbiome composition by sequencing indicated no significant changes attributable to treatment at the phylum and family level, although FISH analysis showed an increase in Faecalibacterium prausnitzii in subjects consuming agar LMWP. Sequence analysis of gut bacteria corroborated with the FISH data, indicating that alginate and agar LWMPs do not alter human gut microbiome health markers. Crucially, our findings suggest an urgent need for robust and rigorous human in vivo testing-in particular, using refined seaweed extracts.

Phenotypic and genetic associations between feeding behavior and carcass merit in crossbred growing cattle.

In growing cattle, the phenotypic and genetic relationships between feeding behavior and both live animal ultrasound measures and subsequent carcass merit are generally poorly characterized. The objective of the current study was to quantify the phenotypic and genetic associations between a plethora of feeding behavior traits with both pre-slaughter ultrasound traits and post-slaughter carcass credentials in crossbred Bos taurus cattle. Carcass data were available on 3,146 young bulls, steers, and heifers, of which 2,795 and 2,445 also had records for pre-slaughter ultrasound muscle depth and intramuscular fat percentage, respectively; a total of 1,548 steers and heifers had information on all of the feeding behavior, ultrasound, and carcass traits. Young bulls were fed concentrates, while steers and heifers were fed a total mixed ration. Feeding behavior traits were defined based on individual feed events or meal events (i.e., individual feed events grouped into meals). Animal linear mixed models were used to estimate (co)variance components. Phenotypic correlations between feeding behavior and both ultrasound and carcass traits were generally weak and not different from zero, although there were phenotypic correlations of 0.40, 0.26, and 0.37 between carcass weight and feeding rate, energy intake per feed event, and energy intake per meal, respectively. Genetically, cattle that had heavier carcass weights, better carcass conformation, or a higher dressing percentage fed for a shorter time per day (genetic correlations [±SE] of -0.46 ± 0.12, -0.39 ± 0.11, and -0.50 ± 0.10, respectively). Genetic correlations of 0.43 ± 0.12 and 0.68 ± 0.13 were estimated between dressing difference (i.e., differential between live weight pre-slaughter and carcass weight) and energy intake per feed event and energy intake per meal, respectively. Neither intramuscular fat percentage measured on live animals nor carcass fat score (i.e., a measure of subcutaneous fat cover of the carcass) was genetically associated with any of the feeding behavior traits. The genetic associations between some feeding behavior traits and both ultrasound and carcass traits herein suggested that indirect responses in the feeding behavior of growing cattle from selection for improved carcass merit would be expected. Such changes in feeding behavior patterns in cattle may be reduced by measuring and including feeding behavior in a multiple-trait selection index alongside carcass traits.

The urobiome, urinary tract infections, and the need for alternative therapeutics.

Improvements in bacterial culturing and DNA sequencing techniques have revealed a diverse, and hitherto unknown, urinary tract microbiome (urobiome). The potential role of this microbial community in contributing to health and disease, particularly in the context of urinary tract infections (UTIs) is of significant clinical importance. However, while several studies have confirmed the existence of a core urobiome, the role of its constituent microbes is not yet fully understood, particularly in the context of health and disease. Herein, we review the current state of the art, concluding that the urobiome represents an important component of the body's innate immune defences, and a potentially rich resource for the development of alternative treatment and control strategies for UTIs.

Genetic variability in the feeding behavior of crossbred growing cattle and associations with performance and feed efficiency.

The objectives of the present study were to estimate genetic parameters for several feeding behavior traits in growing cattle, as well as the genetic associations among and between feeding behavior and both performance and feed efficiency traits. An additional objective was to investigate the use of feeding behavior traits as predictors of genetic merit for feed intake. Feed intake and live-weight data on 6,088 growing cattle were used of which 4,672 had ultrasound data and 1,548 had feeding behavior data. Feeding behavior traits were defined based on individual feed events or meal events (where individual feed events were grouped into meals). Univariate and bivariate animal linear mixed models were used to estimate (co)variance components. Heritability estimates (± SE) for the feeding behavior traits ranged from 0.19 ± 0.08 for meals per day to 0.61 ± 0.10 for feeding time per day. The coefficient of genetic variation per trait varied from 5% for meals per day to 22% for the duration of each feed event. Genetically heavier cattle, those with a higher daily energy intake (MEI), or those that grew faster had a faster feeding rate, as well as a greater energy intake per feed event and per meal. Better daily feed efficiency (i.e., lower residual energy intake) was genetically associated with both a shorter feeding time per day and shorter meal time per day. In a validation population of 321 steers and heifers, the ability of estimated breeding values (EBV) for MEI to predict (adjusted) phenotypic MEI was demonstrated; EBVs for MEI were estimated using multi-trait models with different sets of predictor traits such as liveweight and/or feeding behaviors. The correlation (± SE) between phenotypic MEI and EBV for MEI marginally improved (P < 0.001) from 0.64 ± 0.03 to 0.68 ± 0.03 when feeding behavior phenotypes from the validation population were included in a genetic evaluation that already included phenotypic mid-test metabolic live-weight from the validation population. This is one of the largest studies demonstrating that significant exploitable genetic variation exists in the feeding behavior of young crossbred growing cattle; such feeding behavior traits are also genetically correlated with several performance and feed efficiency metrics. Nonetheless, there was only a marginal benefit to the inclusion of time-related feeding behavior phenotypes in a genetic evaluation for MEI to improve the precision of the EBVs for this trait.

Cryptosporidium spp. diagnosis and research in the 21st century.

The protozoan parasite Cryptosporidium has emerged as a leading cause of diarrhoeal illness worldwide, posing a significant threat to young children and immunocompromised patients. While endemic in the vast majority of developing countries, Cryptosporidium also has the potential to cause waterborne epidemics and large scale outbreaks in both developing and developed nations. Anthroponontic and zoonotic transmission routes are well defined, with the ingestion of faecally contaminated food and water supplies a common source of infection. Microscopy, the current diagnostic mainstay, is considered by many to be suboptimal. This has prompted a shift towards alternative diagnostic techniques in the advent of the molecular era. Molecular methods, particularly PCR, are gaining traction in a diagnostic capacity over microscopy in the diagnosis of cryptosporidiosis, given the laborious and often tedious nature of the latter. Until now, developments in the field of Cryptosporidium detection and research have been somewhat hampered by the intractable nature of this parasite. However, recent advances in the field have taken the tentative first steps towards bringing Cryptosporidium research into the 21st century. Herein, we provide a review of these advances.

Contribution of herd characteristics to best linear unbiased estimates of slaughter traits in beef cattle.

Genetic evaluations separate phenotypes into their contributing additive genetic effects and non-(additive) genetic effects, with the former termed best linear unbiased predictions, and the latter termed best linear unbiased estimates (BLUEs). For the purpose of the present study, genetic evaluations, along with phenotypic data from 4 137 376 animals, were used to generate herd, year of slaughter and sex contemporary group BLUEs for various slaughter-related traits. These slaughter traits included carcass weight (CW), carcass conformation (CC) and carcass fat (CF). For the 4 665 herds that were consistently slaughtering ≥10 animals/year between the years 2014 and 2018, inclusive, all relevant contemporary group BLUEs were collapsed into a single herd-year value; results herein relate to these herds. The within-year herd-year BLUE correlations between CW and CC, between CW and CF, and between CC and CF were 0.51, 0.10 and -0.04, respectively. The repeatability across years of the herd-year BLUEs for CW, CC and CF was 0.66, 0.59 and 0.50, respectively. Furthermore, when the herds were stratified, within year, on the percentile rank of their herd-year BLUEs, herds had the greatest probability of remaining in the same BLUE stratum from one year to the next. In addition, results from the present study determined that various herd characteristics are associated with differences in the herd BLUEs. Results from the present study could be used to advise beef producers on the most promising strategy to improve the carcass merit of their animals.

Development of a novel, high resolution melting analysis based genotyping method for Cryptosporidium parvum.

This study employed the post-real-time PCR application, high resolution melting (HRM) analysis, in order to differentiate between characterised clinical and reference Cryptosporidium parvum samples obtained from Cork University Hospital (Cork, Ireland) and the Cryptosporidium Reference Unit (Swansea, Wales). A sample set composed of 18 distinct C. parvum gp60-subtypes of the IIa gp60-subtype family (an allele family accounting for over 80% of all cryptosporidiosis cases in Ireland) was employed. HRM analysis-based interrogation of the gp60, MM5 and MS9-Mallon tandem repeat loci was found to completely differentiate between 10 of the 18 studied gp60-subtypes. The remaining eight gp60-subtypes were differentiated into three distinct groupings, with the designations within these groupings resolved to two to three potential gp60-subtypes. The current study aimed to develop a novel, reproducible, real-time PCR based multi-locus genotyping method to distinguish between C. parvum gp60-subtypes. These preliminary results support the further expansion of the multi-locus panel in order to increase the discriminatory capabilities of this novel method.

Genetic and non-genetic factors associated with health and vitality traits in beef calves.

Awareness and interest in calf health and wellbeing is intensifying, prompting change in the management and breeding decisions of producers and associated policy-makers. The objectives of the present study were to 1) quantify the risk factors associated with subjectively measured scores of vigor and birth size as well as diagnoses of scour and pneumonia in a large national dataset of beef calves, and 2) to estimate the contribution of genetic variance to such phenotypic measures. After edits, the data consisted of health and birth size data subjectively scored by producers on 88,207 calves born in 6,126 Irish beef herds. Vigor was recorded on a scale of 1 (very poor) to 5 (very good). Birth size was also scored on a scale of 1 (very small) to 5 (very large). Scour and pneumonia were both scored independently based on the suspected number of occurrence of each (0 = no occurrence, 1 = one occurrence, or 2 = more than one occurrence). On average, 14.7% of calves were recorded as having had at least one occurrence of scour within the first 5 mo of life, whereas 6.4% of calves were recorded as having had at least one occurrence of pneumonia within the first 5 mo of life. Relative to female calves, male calves had a worse vigor score and a suspected greater incidence of both scour and pneumonia. Relative to singletons, twins were, on average, smaller at birth, they had a worse vigor score, and they were more prone to scour. Calves born in the later periods of the calving season (i.e., late and very late) had a greater incidence of scour relative to calves in the herd born earlier in the calving season. Heritability estimates for vigor, birth size, and pneumonia were 0.12 (0.02), 0.33 (0.03), and 0.08 (0.02), respectively; no genetic variance was detected for scour. Breeding for vigorous calves that are less susceptible to pneumonia could provide producers with an additional strategy to ensure consumer concerns regarding food quality, safety, and calf wellbeing are being met.

Herd solutions from genetic evaluations can be used as a tool to rescale the expected expression of genetic potential in cattle.

The objective of this study was to determine whether response to selection for carcass weight (CW), conformation (CC) and fat (CF), and the association between heterosis and carcass performance varied by herd environment in cattle. Following edits, carcass information was available for 4,616,761 cattle, of which the majority were some crossbred combination of the following breeds: Angus, Aubrac, Belgian Blue, Blonde d'Aquitaine, Charolais, Hereford, Holstein-Friesian, Jersey, Limousin, Saler, Shorthorn and Simmental. Herd environment was defined separately for each carcass trait using herd solutions outputted from carcass trait genetic evaluations. A total of 3,859 herds were stratified, for each trait, into one of five strata based on their corresponding percentile herd solution rank, with the response to selection and the effect of heterosis then estimated within each stratum. The response in CW and CC from selection on the respective estimated breeding values (EBV) increased between the lowest (0.71 kg and 0.89 CC score increase per unit increase in the respective EBV) and highest (0.99 kg and 1.25 CC score increase per unit increase in the respective EBV) corresponding herd stratum. The response in CF from selection on CF EBV, however, reduced between the lowest and highest CF herd stratum (respective increases of 0.93 and 0.83 CF scores per unit increase in CF EBV). In addition, the effect of a unit increase in heterosis coefficient on CW, CC and CF also varied by herd stratum. Furthermore, results (i.e. the area under relative operating characteristic curves) from the present study demonstrated that the response to selection and heterosis effects estimated for the different herd stratum can be used, along with EBVs and the herd solutions themselves, to improve the accuracy of phenotypic predictions. Results from the present study could help producers to make more informed breeding decisions that are bespoke to their herd.

A novel genotyping method for Cryptosporidium hominis.

Cryptosporidiosis remains the leading protozoan induced cause of diarrhoea-associated mortality worldwide. Cryptosporidium hominis, the anthroponotically transmitted species within the Cryptosporidium genus, contributes significantly to the global burden of infection, accounting for the majority of clinical cases in many countries. This study applied high resolution melting analysis, a post-real-time PCR application, to the differentiation of six globally prevalent C. hominisgp60-subtypes. This novel method targeted three microsatellite, tandem repeat containing genetic markers, gp60, the genetic marker upon which current Cryptosporidium subtype nomenclature is based, MSB, and MSE, by which to differentiate between C. hominis isolates. This multi-locus approach successfully differentiated between all six C. hominisgp60-subtypes studied, some of which, such as IbA10G2, are known to exhibit global ubiquity. Thus, this method has the potential to be universally employed as a sensitive, cost effective and highly reproducible means to rapidly differentiate between C. hominisgp60-subtypes. Such a method would be of particular utility in epidemiological studies and outbreak scenarios, providing cost effective, clinically accessible alternative to DNA sequencing. The success of this preliminary study also supports further analysis of an expanded C. hominisgp60-subtype range and the potential expansion of the multi-locus panel in order to improve the discriminatory power of this approach.

The roles of code in biology.

The way in which computer code is perceived and used in biological research has been a source of some controversy and confusion, and has resulted in sub-optimal outcomes related to reproducibility, scalability and productivity. We suggest that the confusion is due in part to a misunderstanding of the function of code when applied to the life sciences. Code has many roles, and in this paper we present a three-dimensional taxonomy to classify those roles and map them specifically to the life sciences. We identify a "sweet spot" in the taxonomy-a convergence where bioinformaticians should concentrate their efforts in order to derive the most value from the time they spend using code. We suggest the use of the "inverse Conway maneuver" to shape a research team so as to allow dedicated software engineers to interface with researchers working in this "sweet spot." We conclude that in order to address current issues in the use of software in life science research such as reproducibility and scalability, the field must reevaluate its relationship with software engineering, and adapt its research structures to overcome current issues in bioinformatics such as reproducibility, scalability and productivity.

Ranking microbiome variance in inflammatory bowel disease: a large longitudinal intercontinental study.

OBJECTIVE: The microbiome contributes to the pathogenesis of inflammatory bowel disease (IBD) but the relative contribution of different lifestyle and environmental factors to the compositional variability of the gut microbiota is unclear. DESIGN: Here, we rank the size effect of disease activity, medications, diet and geographic location of the faecal microbiota composition (16S rRNA gene sequencing) in patients with Crohn's disease (CD; n=303), ulcerative colitis (UC; n = 228) and controls (n=161), followed longitudinally (at three time points with 16 weeks intervals). RESULTS: Reduced microbiota diversity but increased variability was confirmed in CD and UC compared with controls. Significant compositional differences between diseases, particularly CD, and controls were evident. Longitudinal analyses revealed reduced temporal microbiota stability in IBD, particularly in patients with changes in disease activity. Machine learning separated disease from controls, and active from inactive disease, when consecutive time points were modelled. Geographic location accounted for most of the microbiota variance, second to the presence or absence of CD, followed by history of surgical resection, alcohol consumption and UC diagnosis, medications and diet with most (90.3%) of the compositional variance stochastic or unexplained. CONCLUSION: The popular concept of precision medicine and rational design of any therapeutic manipulation of the microbiota will have to contend not only with the heterogeneity of the host response, but also with widely differing lifestyles and with much variance still unaccounted for.

COVID-19: mask efficacy is dependent on both fabric and fit.

Aim: Face masks are an important addition to our arsenal in the fight against COVID-19. The aim of this study is to present a novel method of measuring mask performance which can simultaneously assess both fabric penetration and leakage due to poor fit. Materials & methods: A synthetic aerosol is introduced into the lung of a medical dummy. A conical laser sheet surrounds the face of the dummy where it illuminates the aerosol emitted during a simulated breath. The system is demonstrated with five mask types. Conclusions: The curved laser sheet highlights both penetration through the mask fabric and leakage around the edges of the mask. A large variation in both material penetration and leakage was observed.

A genomic analysis of osmotolerance in Staphylococcus aureus.

A key phenotypic characteristic of the Gram-positive bacterial pathogen, Staphylococcus aureus, is its ability to grow in low aw environments. A homology transfer based approach, using the well characterised osmotic stress response systems of Bacillus subtilis and Escherichia coli, was used to identify putative osmotolerance loci in Staphylococcus aureus ST772-MRSA-V. A total of 17 distinct putative hyper and hypo-osmotic stress response systems, comprising 78 genes, were identified. The ST772-MRSA-V genome exhibits significant degeneracy in terms of the osmotic stress response; with three copies of opuD, two copies each of nhaK and mrp/mnh, and five copies of opp. Furthermore, regulation of osmotolerance in ST772-MRSA-V appears to be mediated at the transcriptional, translational, and post-translational levels.

Development of novel methodology for the molecular differentiation of Cryptosporidium parvum gp60 subtypes via high resolution melting analysis.

Cryptosporidium species subtypes are generally identified via DNA sequencing of the gp60 gene tandem repeat motif region. Due to the immunogenic nature of its glycoprotein products, gp60 is subject to host selective pressures, genetic recombination and evolutionary processes that drive extensive polymorphism at this locus. The elucidation of the polymorphic nature of this gene has led to the current mainstay in Cryptosporidium subtyping nomenclature. This study aimed to develop a real-time polymerase chain reaction based method utilising a post-PCR application, high resolution melting (HRM) analysis, in conjunction with the abovementioned gp60 nomenclature system, in order to differentiate between Cryptosporidium parvum gp60 subtypes. Subtype differentiation is based on the difference between the melting temperatures of individual subtypes conferred by variations in the polymorphic region of gp60. • Nested gp60 primers were designed to amplify a target region of <200 base pairs for effective HRM analysis • This method presents a rapid, sensitive, cost effective alternative to conventional sequencing. • This method is highly flexible and may be applied to other loci in order to facilitate multi-locus analysis and improve the discriminative abilities of the method.