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Brucellosis is considered a common bacterial zoonotic disease of high prevalence in countries of the Middle East and the Mediterranean region with economic and public health impact. The present study aimed to investigate the current situation of brucellosis in small ruminants reared in Médéa and Sidi Bel-Abbès provinces, north Algeria. To achieve this objective, 96 sera (77 sheep and 19 goat) and 57 milk (42 sheep and 15 goat) samples were collected from suspected infected animals and serologically analyzed by using ELISA. For isolation of Brucella spp., four placentas, two fetuses and forty-four milk samples were subjected to microbiological investigation. Whole genome sequencing (WGS) was used for genomic analysis of isolated Brucella species. The results of this study showed that anti- Brucella antibodies were detected in 46 (83.6%) and 52 (54.2%) milk and serum samples, respectively. However, among 27 cases where blood samples were negative, anti- Brucella antibodies were still detected in 19 of the corresponding milk samples, resulting in an overall discordance rate of 36.5%. Ten Brucella melitensis were isolated and identified from six sheep and four goats. Of these, eight originated from milk samples. The isolated strains were assigned to sequence type ST-11 using Multilocus sequence typing (MLST). Five isolates revealing high similarity (0–2 nucleotide differences) originated from different farms, indicating a close transmission link. However, two identical caprine isolates and three other isolates showed notable genotypic variation, in comparison. The highest base difference (449–462 nucleotides) was observed for an ovine isolate originating from Sidi Bel-Abbès. The phylogenetic analysis and clustering with the West Mediterranean lineage of B. melitensis revealed high genetic similarity of the investigated isolates with B. melitensis of human origin from North Africa and travel-associated 'European' cases, especially from Morroco, Tunisia, Sweden and Italy. The results of this study highlight brucellosis in small ruminants as a significant public health risk and will help to develop effective control strategies in Algeria. These findings provide specific evidence of this risk, with Brucella isolation from milk and by linking theses isolates to human cases in Algeria and abroad. The use of WGS-based analysis has revealed effective in tracing patterns of transmission, and can be recommended for tracking outbreaks at a high resolution. [ABSTRACT FROM AUTHOR]

The enteroinvasive bacterium Shigella flexneri is known as a highly host-adapted human pathogen. There had been no known other reservoirs reported until recently. Here 34 isolates obtained from animals (yaks, dairy cows and beef cattle) from 2016 to 2017 and 268 human S. flexneri isolates from China were sequenced to determine the relationships between animal and human isolates and infer the evolutionary history of animal-associated S. flexneri. The 18 animal isolates (15 yak and 3 beef cattle isolates) in PG1 were separated into 4 lineages, and the 16 animal isolates (1 yak, 5 beef cattle and 10 dairy cow isolates) in PG3 were clustered in 8 lineages. The most recent human isolates from China belonged to PG3 whereas Chinese isolates from the 1950s–1960s belonged to PG1. PG1 S. flexneri may has been transmitted to the yaks during PG1 circulation in the human population in China and has remained in the yak population since, while PG3 S. flexneri in animals were likely recent transmissions from the human population. Increased stability of the large virulence plasmid and acquisition of abundant antimicrobial resistance determinants may have enabled PG3 to expand globally and replaced PG1 in China. Our study confirms that animals may act as a reservoir for S. flexneri. Genomic analysis revealed the evolutionary history of multiple S. flexneri lineages in animals and humans in China. However, further studies are required to determine the public health threat of S. flexneri from animals.

Introduction: To get a comprehensive idea about the transmission and epidemiology of TB globally and locally, the use of molecular typing methods has become imperative not only for understanding genetic diversity but also the population structure of Mycobacterium tuberculosis complex (MTBC). We aimed to investigate the drug resistance pattern and genetic diversity of MTBC among previously treated patients with sputum smear-positive pulmonary tuberculosis in a South Indian population. Methodology: 104 patients with sputum smear positivity and who had previously undergone treatment were selected. Drug susceptibility testing, Spoligotyping, MIRU-VNTR, and SNP typing were performed. Results: Mono-resistance to isoniazid 16 (15.38%) was the highest among all drugs. Out of 104 isolates, 24 (23%) isolates were classified as MDR strains. The distributions of most common lineages were: EAI3-Ind-20 (19.23%), EAI5-13 (12.50%), Beijing-12 (11.54%), CAS1-Delhi-9 (8.65%), and 7 (6.73%) each of T-H37rv, Unknown and Orphan types. MIRU-VNTR-based analysis revealed that there are two major groups: CAS1-Delhi and Beijing groups. Out of 104 isolates, 82 belonged to well-defined lineages and 6 clusters, and the remaining 22 were singletons. SNP analysis showed no mutations associated with five sets of genes in 33 strains. Conclusions: The occurrence of 11.54% Beijing strains in South India is an important finding. High frequency of Isoniazid mono resistance noticed. Spoligotyping along with MIRU-VNTR and SNP typing is the best approach to the identification of strain lineages. No mutation with Antigen85C gene represents, can be used for vaccine candidates. [ABSTRACT FROM AUTHOR]

Plague is a typical natural focus disease that circulates in different ecology of vectors and reservoir hosts. We conducted genomic population and phylogenetic analyses of the Yersinia pestis collected from the 12 natural plague foci in China with more than 20 kinds of hosts and vectors. Different ecological landscapes with specific hosts, vectors, and habitat which shape various niches for Y. pestis. The phylogeographic diversity of Y. pestis in different kinds plague foci in China showed host niches adaptation. Most natural plague foci strains are region‐and focus‐specific, with one predominant subpopulation; but the isolates from the Qinghai–Tibet plateau harbor a higher genetic diversity than other foci. The Y. pestis from Marmota himalayana plague foci are defined as the ancestors of different populations at the root of the evolutionary tree, suggesting several different evolutionary paths to other foci. It has the largest pan‐genome and widest SNP distances with most accessory genes enriched in mobilome functions (prophages, transposons). Geological barriers play an important role in the maintenance of local Y. pestis species and block the introduction of non‐native strains. This study provides new insights into the control of plague outbreaks and epidemics, deepened the understanding of the evolutionary history of MHPF (M. himalayana plague focus) in China. The population structure and identify clades among different natural foci of China renewed the space cognition of the plague. [ABSTRACT FROM AUTHOR]

Burkholderia (B.) mallei is a host-adapted equine pathogen that causes glanders, a re-emerging zoonotic disease, which is endemic in Pakistan and other developing countries and seriously impacts the global equine movement. Due to globalization, the geographical restriction of diseases vanishes and the lack of awareness of and experience with eradicated diseases in industrialized countries also promotes the re-introduction of infections in these regions. Owing to the high equine population, the Pakistani province Punjab is a potential hotspot where several glanders outbreaks have been seen over last two decades. For determining the genomic diversity of B. mallei in this and other equine-populated prefectures, the genomes of 19 B. mallei strains isolated between 1999 and 2020 in different locations were sequenced and their genotypes were determined. Particularly, for genetically highly homogenous pathogens like B. mallei genotyping techniques require a high discriminatory power for enabling differentiation on the strain level. Thus, core-genome single nucleotide polymorphism (cgSNP) analysis was applied for distinguishing the highly similar strains. Furthermore, a whole-genome sequence-based core genome multi locus sequence typing (cgMLST) scheme, specific to B. mallei, was developed and additionally applied to the data. It was found that B. mallei genotypes in Pakistan persisted over time and space and genotype clusters preferred connection with a time point rather than the place of isolation, probably due to frequent equine movement, which promotes the spread of glanders. The cgMLST approach proved to work in accord with SNP typing and may help to investigate future glanders outbreaks. [ABSTRACT FROM AUTHOR]

Burkholderia (B.) mallei is a host-adapted equine pathogen that causes glanders, a re-emerging zoonotic disease, which is endemic in Pakistan and other developing countries and seriously impacts the global equine movement. Due to globalization, the geographical restriction of diseases vanishes and the lack of awareness of and experience with eradicated diseases in industrialized countries also promotes the re-introduction of infections in these regions. Owing to the high equine population, the Pakistani province Punjab is a potential hotspot where several glanders outbreaks have been seen over last two decades. For determining the genomic diversity of B. mallei in this and other equine-populated prefectures, the genomes of 19 B. mallei strains isolated between 1999 and 2020 in different locations were sequenced and their genotypes were determined. Particularly, for genetically highly homogenous pathogens like B. mallei genotyping techniques require a high discriminatory power for enabling differentiation on the strain level. Thus, core-genome single nucleotide polymorphism (cgSNP) analysis was applied for distinguishing the highly similar strains. Furthermore, a whole-genome sequence-based core genome multi locus sequence typing (cgMLST) scheme, specific to B. mallei, was developed and additionally applied to the data. It was found that B. mallei genotypes in Pakistan persisted over time and space and genotype clusters preferred connection with a time point rather than the place of isolation, probably due to frequent equine movement, which promotes the spread of glanders. The cgMLST approach proved to work in accord with SNP typing and may help to investigate future glanders outbreaks.

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Abstract Background In 2007, a nationwide Salmonella Tennessee outbreak occurred via contaminated peanut butter. Here, we developed a single-nucleotide polymorphism (SNP)-typing method for S. Tennessee to determine the clonal subtypes of S. Tennessee that were associated with the peanut butter outbreak. Methods and results One seventy-six S. Tennessee isolates from various sources, including humans, animals, food, and the environment, were analyzed by using the SNP technique. Eighty-four representative SNP markers were selected by comparing the sequences of three representative S. Tennessee strains with different multi-locus sequence typing and variable number tandem repeats from our collection. The set of eighty-four SNP markers showed 100% typeability for the 176 strains, with the nucleotide diversity ranging from 0.011 to 0.107 (mean = 0.049 ± 0.018, median = 0.044) for each marker. Among the four clades and nine subtypes generated by the SNP typing, subtype 1, which comprised 142 S. Tennessee strains, was the most predominant. The dominance of single-strain clones in subtype 1 revealed that S. Tennessee is highly clonal regardless of outbreak-association, source, or period of isolation, suggesting the presence of an S. Tennessee strain prototype. Notably, a minimum 18 SNP set was able to determine clonal S. Tennessee strains with similar discrimination power, potentially allowing more rapid and economic strain genotyping for both outbreaks and sporadic cases. Conclusions The SNP-typing method described here might aid the investigation of the epidemiology and microevolution of pathogenic bacteria by discriminating between outbreak-related and sporadic clinical cases. In addition, this approach enables us to understand the population structure of the bacterial subtypes involved in the outbreak.

The use of whole genome sequencing (WGS) as a method for supporting outbreak investigations, studying Salmonella microbial populations and improving understanding of pathogenicity has been well-described (1–3). However, performing WGS on a discrete dataset does not pose the same challenges as implementing WGS as a routine, reference microbiology service for public health surveillance. Challenges include translating WGS data into a useable format for laboratory reporting, clinical case management, Salmonella surveillance, and outbreak investigation as well as meeting the requirement to communicate that information in an understandable and universal language for clinical and public health action. Public Health England have been routinely sequencing all referred presumptive Salmonella isolates since 2014 which has transformed our approach to reference microbiology and surveillance. Here we describe an overview of the integrated methods for cross-disciplinary working, describe the challenges and provide a perspective on how WGS has impacted the laboratory and surveillance processes in England and Wales.

A novel method of SNP typing in human mitochondrial DNA utilizing enzymatic labeling and electrochemical detection at disposable pencil graphite electrodes is described. The procedure is based on amplification of DNA stretches by cyclic primer extension (PEx) of SNP‐specific diagnostic primers in a mixture of biotinylated and natural nucleotides. The diagnostic primers are designed to recognize, by its 3'‐terminal nucleotide, the SNP‐site in target template. Under optimized conditions of the PEx reaction, efficient polymerase synthesis of biotin‐labeled strands takes place only in the case of full complementarity between the diagnostic primer and the target SNP site. There is also benefit from introducing many biotin molecules per extended DNA strand, resulting in another level of signal amplification. After adsorption of biotinylated PEx products at the electrode surface, streptavidin‐alkaline phosphatase conjugate was bound to the biotin tags, 1‐naphthol was enzymatically produced and electrochemically detected. Several critical steps and parameters of the assay, including termination of 3'‐OH ends of residual amplification primers, temperature for annealing of diagnostic primers, relative amount of biotinylated deoxynucleoside triphosphate in the PEx mixture and number of PEx cycles were optimized in this study to attain best SNP resolution, and reduction of time needed for the analysis. [ABSTRACT FROM AUTHOR]

Mycobacterium bovis , which belongs to the Mycobacterium tuberculosis complex, is a highly clonal pathogen. However, several lineages of M. bovis have been described worldwide and nine different clusters were identified in France. Targeted amplicon sequencing using next-generation sequencing technology of eighty-eight phylogenetically informative single nucleotide polymorphisms (SNPs) were used to infer the phylogenetic relationship of 630 strains of the National Reference Laboratory isolated between 1979 and 2018 from various animal species. This study allowed classifying 618 different genotypic profiles (combination of a spoligotype and 8 loci-MIRU-VNTR profiles) into the nine previously identified clusters. A global analysis of the entire collection of the National Reference Laboratory has made it possible to represent the evolution of clonal complexes and clusters in time and space for better assessing epidemiological changes of bovine tuberculosis in France. • A high-throughput targeted amplicon sequencing approach used to study bovine TB. • Nine M. bovis clusters found in France, including Eu1, Eu2 and Eu3 clonal complexes. • Two main lineages are responsible for almost all current French bTB outbreaks. • Changes in breeding practices led to the emergence of certain clones. [ABSTRACT FROM AUTHOR]

A new polymerase chain reaction (PCR) system using a low‐salt concentration buffer was developed, which is suitable for electrical PCR process monitoring. Using Taq polymerase in 10 mM of Tris‐HCl buffer without KCl, the PCR progressed and the target DNA PCR products were produced. As the ionic concentration in PCR buffer solution decreased, the quantity of PCR products decreased, but the PCR products could be detected down to a total ion concentration of 5 mM. Single nucleotide polymorphism typing was also successful under 10 mM of Tris‐HCl without KCl. Both alleles (G or T) were detected clearly. [ABSTRACT FROM AUTHOR]

Listeriosis remains among the most important bacterial illnesses, with a high associated mortality rate. Efforts to control listeriosis require detailed knowledge of the epidemiology of the disease itself, and its etiological bacterium, Listeria monocytogenes. In this study we provide an in-depth analysis of the epidemiology of 224 L. monocytogenes isolates from Australian clinical and non-clinical sources. Non-human sources included meat, dairy, seafood, fruit, and vegetables, along with animal and environmental isolates. Serotyping, Multi-Locus Sequence Typing, and analysis of inlA gene sequence were performed. Serogroups IIA, IIB, and IVB comprised 94% of all isolates, with IVB overrepresented among clinical isolates. Serogroup IIA was the most common among dairy and meat isolates. Lineage I isolates were most common among clinical isolates, and 52% of clinical isolates belonged to ST1. Overall 39 STs were identified in this study, with ST1 and ST3 containing the largest numbers of L. monocytogenes isolates. These STs comprised 40% of the total isolates (n = 90), and both harbored isolates from clinical and non-clinical sources. ST204 was the third most common ST. The high prevalence of this group among L. monocytogenes populations has not been reported outside Australia. Twenty-seven percent of the STs in this study contained exclusively clinical isolates. Analysis of the virulence protein InlA among isolates in this study identified a truncated form of the protein among isolates from ST121 and ST325. The ST325 group contained a previously unreported novel mutation leading to production of a 93 amino acid protein. This study provides insights in the population structure of L. monocytogenes isolated in Australia, which will contribute to public health knowledge relating to this important human pathogen. [ABSTRACT FROM AUTHOR]

Illegal trading of ivory is mainly responsible for the dramatic decline in elephant populations. Thailand is one of the largest laundering hotspots for African ivory, as the domestic Asian elephant ivory can be legally traded. So, to help combat ivory poaching and smuggling, an efficient method is needed to identify the elephant species from its ivory and ivory products. In this study, a mini-SNaPshot ® multiplex assay was developed and fully validated for the identification of confiscated ivory and low DNA template ivory products. Elephantid- and elephant species-specific mitochondrial single nucleotide polymorphisms (SNPs) were identified from 207 mammalian and 1705 elephant/mammoth cytochrome b sequence alignments. Seven informative SNPs were used for assay development. The assay unambiguously and accurately identified authentic elephant ivory and its species of origin on the basis of peak size and color observed in the haplotype profile. The assay was highly efficient for analysis of confiscated ivory and low-template ivory products with a 99.29% success rate ( N = 140). It was highly reproducible, exhibited no cross-reaction with eight other mammalian DNA; and had 100% identification accuracy. In addition, nested and direct PCR amplification were also compatible with the developed assay. This efficient assay should benefit wildlife forensic laboratories and aid in the prosecution of elephant-related crimes. [ABSTRACT FROM AUTHOR]

Highly pathogenic enterohemorrhagic Escherichia coli (EHEC) O157 cause a spectrum of clinical signs that include diarrhea, bloody diarrhea, and hemolytic uremic syndrome. The current evolutionary model of EHEC O157:H7/H– consists of a stepwise evolution scenario proceeding from O55:H7 to a node (hypothetical intermediate) that then branches into sorbitol-fermenting (SF) O157:H– and non-SF (NSF) O157:H7. To identify this hypothetical intermediate, we performed single nucleotide polymorphism analysis by sequencing of 92 randomly distributed backbone genomic regions of 40 O157:H7/H– isolates. Overall, 111 single nucleotide polymorphisms were identified in 75/92 partial open reading frames after sequencing 51,041 nt/strain. The EHEC O157:H7 strain LSU-61 from deer occupied an intermediate position between O55:H7 and both O157 branches (SF and NSF O157), complementing the stepwise evolutionary model of EHEC O157:H7/H–. The animal origin of this intermediate emphasizes the value of nonhuman reservoirs in the clarification of the evolution of human pathogens.

Escherichia coli O157:H7 has frequently been associated with foodborne infections and is considered an adulterant in raw non-intact beef in the U.S. Shiga toxin-producing E. coli (STEC) belonging to serogroups O26, O45, O103, O111, O121, and O145 (known as the “big six” non-O157) were estimated to cause > 70% of foodborne infections attributed to non-O157 serogroups in the U.S., as a result, these six serogroups have also been targeted by regulation in the U.S. The purpose of this study was to develop a rapid and high-throughput molecular method to group STEC isolates into seven clinically important serogroups (i.e., O157 and the “big six” non-O157 serogroups) targeted by regulation in the U.S. by interrogating single nucleotide polymorphisms (SNPs) in gnd . A collection of 195 STEC isolates, including isolates belonging to O157:H7 ( n = 18), O26 ( n = 21), O45 ( n = 19), O103 ( n = 24), O111 ( n = 24), O121 ( n = 23), O145 ( n = 21), and ten other STEC serogroups ( n = 45), was assembled and characterized by full gnd sequencing to identify informative SNPs for molecular serogrouping. A multiplex SNP typing assay was developed to interrogate twelve informative gnd SNPs by single base pair extension chemistry and used to characterize the STEC isolate collection assembled here. SNP types were assigned to each isolate by the assay and polymorphisms were confirmed with gnd sequence data. O-serogroup-specific SNP types were identified for each of the seven clinically important STEC serogroups, which allowed the differentiation of these seven STEC serogroups from other non-O157 STEC serogroups. Although serogroups of the “big six” non-O157 STEC and O157:H7 contained multiple SNP types per O-serogroup, there were no overlapping SNP types between serogroups. Our results demonstrate that molecular serogrouping of STEC isolates by interrogation of informative SNPs in gnd represents an alternative to traditional serogrouping by agglutination for rapid and high-throughput identification of clinically important STEC serogroups targeted by regulation for surveillance and epidemiological investigations. [ABSTRACT FROM AUTHOR]

Genetic variations such as single nucleotide polymorphism (SNP) and point mutations are important biomarkers to monitor disease prognosis and diagnosis. In this study, we developed a novel rotary microfluidic device which can perform multiplex SNP typing on the mutation sites of TP53 genes. The microdevice consists of three glass layers: a channel wafer, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a rotary plate in which twelve reaction chambers were fabricated. A series of sample injection, ligation-rolling circle amplification (L-RCA) reaction, and fluorescence detection of the resultant amplicons could be executed by rotating the top rotary plate, identifying five mutation points related with cancer prognosis. The use of the rotary plate eliminates the necessity of microvalves and micropumps to control the microfluidic flow in the channel, simplifying the chip design and chip operation for multiplex SNP detection. The proposed microdevice provides an advanced genetic analysis platform in terms of multiplexity, simplicity, and portability in the fields of biomedical diagnostics. [ABSTRACT FROM AUTHOR]

Leprosy is still a major health problem in India which has the highest number of cases. Multiple locus variable number of tandem repeat analysis (MLVA) and single nucleotide polymorphism (SNP) have been proposed as tools of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population from scale and duration were lacking for studying the transmission chain of leprosy. Seventy slit skin scrapings were collected from Purulia (West Bengal), Miraj (Maharashtra), Shahdara (Delhi), and Naini (UP) hospitals of The Leprosy Mission (TLM). SNP subtyping and MLVA on 10 VNTR loci were applied for the strain typing of Mycobacterium leprae . Along with the strain typing conventional epidemiological investigation was also performed to trace the transmission chain. In addition, phylogenetic analysis was done on variable number of tandem repeat (VNTR) data sets using sequence type analysis and recombinational tests (START) software. START software performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. These analyses include data summary, lineage assignment, and tests for recombination and selection. Diversity was observed in the cross-sectional survey of isolates obtained from 70 patients. Similarity in fingerprinting profiles observed in specimens of cases from the same family or neighborhood locations indicated a possible common source of infection. The data suggest that these VNTRs including subtyping of SNPs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci. The present study strongly indicates that multi-case families might constitute epidemic foci and the main source of M. leprae in villages, causing the predominant strain or cluster infection leading to the spread of leprosy in the community. [ABSTRACT FROM AUTHOR]

A single fluorophore based multi-color emission probing system has been developed in the current work. dA py was used as the fluorophore, which was incorporated at the end of folded oligonucleotide with increasing number. Depending on the number of dA py three different color emissions were observed, viz. blue, green and red. These interesting color patterns were further used for the SNP typing of DNA. Every folded oligonucleotide with different number of dA py s could discriminate the full matched target sequence from one base mismatched and two base mismatched sequences. Furthermore, this probing system has a simple structure and also exhibited large Stokes shifted signal for discriminating the target DNA. [ABSTRACT FROM AUTHOR]

Background & objectives: Genotyping has now become one of the major diagnostic means for almost all diseases. Among the advanced techniques that are used to study single nucleotide polymorphisms (SNPs), only a few are applicable for routine disease diagnosis. Their applicability mainly depends on three factors: cost, time, and accuracy. The primary objective of this study was to propose allele-specific real-time PCR as a rapid, low cost and simple genotyping method for routine diagnostics. Methods: Two SNPs, rs3014866 and rs2149356 were analysed using allele-specific real-time PCR. The polymerase chain reaction was carried out using RealQ PCR master mix containing SYBR Green DNA I dye followed by melt curve analysis. The results were validated by agarose gel electrophoresis and DNA sequencing. Results: The allelic discrimination and zygosity of the two SNPs were assessed by combined cycle threshold (Ct) and melting temperature (Tm) values. Variations in Ct and Tm values among the two alleles were observed in both rs3014866 (Ct: C allele - 24±1, T allele - 27±1; Tm: C allele - 82.5±0.3, T allele - 86.3±0.2) and rs2149356 (Ct: C allele - 24±1, A allele - 26±1; Tm: C allele - 79.4±0.2, A allele - 80.4±0.3). Based on the variations, homozygous and heterozygous alleles were detected. Agarose gel electrophoresis and DNA sequencing also confirmed the allelic variation and zygosity observed in real-time PCR. Interpretation & conclusions: In diagnostic settings where a large number of samples are analysed daily, allele-specific real-time PCR assay may serve as a simple, low cost and efficient method of genotyping. [ABSTRACT FROM AUTHOR]

The Ion Torrent™ HID SNP assay amplified 136 autosomal SNPs and 33 Y-chromosome markers in one PCR and the markers were subsequently typed using the Ion PGM™ second generation sequencing platform. A total of 51 of the autosomal SNPs were selected from the SNPforID panel that is routinely used in our ISO 17025 accredited laboratory. Concordance between the Ion Torrent™ HID SNP assay and the SNPforID assay was tested by typing 44 Iraqis twice with the Ion Torrent™ HID SNP assay. The same samples were previously typed with the SNPforID assay and the Y-chromosome haplogroups of the individuals were previously identified by typing 45 Y-chromosome SNPs. Full concordance between the assays were obtained except for the SNP genotypes of two SNPs. These SNPs were among the eight SNPs (rs2399332, rs1029047, rs10776839, rs4530059, rs8037429, rs430046, rs1031825 and rs1523537) with inconsistent allele balance among samples. These SNPs should be excluded from the panel. The optimal amount of DNA in the PCR seemed to be ?0.5 ng. Allele drop-outs were rare and only seen in experiments with <0.5 ng input DNA and with a coverage of <50 reads. No allele drop-in was observed. The great majority of the heterozygote allele balances were between 0.6 and 1.6, which is comparable to the heterozygote balances of STRs typed with PCR--CE. The number of reads with base calls that differed from the genotype call was typically less than five. This allowed detection of 1:100 mixtures with a high degree of certainty in experiments with a high total depth of coverage. In conclusion, the Ion PGM™ is a very promising platform for forensic genetics. However, the secondary sequence analysis software made wrong genotype calls from correctly sequenced alleles. These types of errors must be corrected before the platform can be used in case work. Furthermore, the sequence analysis software should be further developed and include quality settings for each SNP based on validation studies. [ABSTRACT FROM AUTHOR]

Chlorhexidine is one of the most widely used biocides in health and agricultural settings as well as in the modern food industry. It is a cationic biocide of the biguanide class. Details of its mechanism of action are largely unknown. The frequent use of chlorhexidine has been questioned recently, amidst concerns that an overuse of this compound may select for bacteria displaying an altered susceptibility to antimicrobials, including clinically important anti-bacterial agents. We generated a Salmonella enterica serovar Typhimurium isolate (ST24CHX) that exhibited a high-level tolerant phenotype to chlorhexidine, following several rounds of in vitro selection, using sub-lethal concentrations of the biocide. This mutant showed altered suceptibility to a panel of clinically important antimicrobial compounds. Here we describe a genomic, transcriptomic, proteomic, and phenotypic analysis of the chlorhexidine tolerant S. Typhimurium compared with its isogenic sensitive progenitor. Results from this study describe a chlorhexidine defense network that functions in both the reference chlorhexidine sensitive isolate and the tolerant mutant. The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur. In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism. These data show that sub-lethal concentrations of chlorhexidine induce distinct changes in exposed Salmonella, and our findings provide insights into the mechanisms of action and tolerance to this biocidal agent. [ABSTRACT FROM AUTHOR]

Background: Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains. Results: To validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal. Conclusion: Based on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings. [ABSTRACT FROM AUTHOR]

Background Coxiella burnetii causes Q fever in humans and Coxiellosis in animals; symptoms range from general malaise to fever, pneumonia, endocarditis and death. Livestock are a significant source of human infection as they shed C. burnetii cells in birth tissues, milk, urine and feces. Although prevalence of C. burnetii is high, few Q fever cases are reported in the U.S. and we have a limited understanding of their connectedness due to difficulties in genotyping. Here, we develop canonical SNP genotyping assays to evaluate spatial and temporal relationships among C. burnetii environmental samples and compare them across studies. Given the genotypic diversity of historical collections, we hypothesized that the current enzootic of Coxiellosis is caused by multiple circulating genotypes. We collected A) 23 milk samples from a single bovine herd, B) 134 commercial bovine and caprine milk samples from across the U.S., and C) 400 bovine and caprine samples from six milk processing plants over three years. Results We detected C. burnetii DNA in 96% of samples with no variance over time. We genotyped 88.5% of positive samples; bovine milk contained only a single genotype (ST20) and caprine milk was dominated by a second type (mostly ST8). Conclusions The high prevalence and lack of genotypic diversity is consistent with a model of rapid spread and persistence. The segregation of genotypes between host species is indicative of species-specific adaptations or dissemination barriers and may offer insights into the relative lack of human cases and characterizing genotypes. [ABSTRACT FROM AUTHOR]

The exact mode of transmission of leprosy is not clearly understood; however, many studies have demonstrated active transmission of leprosy around a source case. Families of five active leprosy cases and their household contacts were chosen from a high endemic area in Purulia. Fifty-two soil samples were also collected from different areas of their houses. DNA was extracted from slit-skin smears ( SSS) and soil samples and the Mycobacterium leprae-specific RLEP (129 bp) region was amplified using PCR. Molecular typing of M. leprae was performed for all RLEP PCR-positive samples by single nucleotide polymorphism ( SNP) typing and confirmation by DNA sequencing. SSS of these five patients and six out of the total 28 contacts were PCR positive for RLEP whereas 17 soil samples out of 52 showed the presence of M. leprae DNA. SNP typing of M. leprae from all RLEP PCR-positive subjects (patients and smear-positive contacts) and 10 soil samples showed the SNP type 1 genotype. M. leprae DNA from the five leprosy patients and the six contacts was further subtyped and the D subtype was noted in all patients and contacts, except for one contact where the C subtype was identified. Typing followed by subtyping of M. leprae clearly revealed that either the contacts were infected by the patients or both patients and contacts had the same source of infection. It also revealed that the type of M. leprae in the soil in the inhabited areas where patients resided was also of the same type as that found in patients. [ABSTRACT FROM AUTHOR]

Enterohemorrhagic Escherichia coli (EHEC) O26:H11/H− is the predominant non-O157 EHEC serotype among patients with diarrhea, bloody diarrhea, and hemolytic uremic syndrome (HUS) worldwide. To elucidate their phylogeny and association between their phylogenetic background and clinical outcome of the infection, we investigated 120 EHEC O26:H11/H− strains isolated between 1965 and 2012 from asymptomatic carriers and patients with diarrhea or HUS. Whole-genome shotgun sequencing (WGS) was applied to ten representative EHEC O26 isolates to determine single nucleotide polymorphism (SNP) localizations within a predefined set of core genes. A multiplex SNP assay, comprising a randomly distributed subset of 48 SNPs, was established to detect SNPs in 110 additional EHEC O26 strains. Within approximately 1 Mb of core genes, WGS resulted in 476 high-quality bi-allelic SNP localizations. Forty-eight of these were subsequently investigated in 110 EHEC O26 and four different SNP clonal complexes (SNP-CC) were identified. SNP-CC2 was significantly associated with the development of HUS. Within the subsequently established evolutionary model of EHEC O26, we dated the emergence of human EHEC O26 to approximately 19,700 years ago and demonstrated a recent evolution within humans into the 4 SNP-CCs over the past 1,650 years. WGS and subsequent SNP typing enabled us to gain new insights into the evolution of EHEC O26 suggesting a common theme in this EHEC group with analogies to EHEC O157. In addition, the SNP-CC analysis may help to assess a risk in infected individuals for the progression to HUS and to implement more specific infection control measures. [ABSTRACT FROM PUBLISHER]

Familial amyloidosis of the Finnish type (FAF) is an autosomal dominant form of systematic amyloidosis characterized by lattice corneal dystrophy, cranial neuropathy, and cutis laxa. Although FAF has been frequently found in the Finnish population, FAF is a considerably rare disorder in other regions. In this study, we examined the clinical characteristics as well as the haplotypes of six Japanese patients with FAF from five families. They showed the typical clinical presentations of FAF, but we found a broad range of ages at onset of neurological symptoms. All members had the c.654G>A mutation in GSN. To evaluate the disease haplotypes, high-density single-nucleotide polymorphism (SNP) arrays were used and disease-relevant haplotypes were reconstructed. Haplotype analysis in the four apparently unrelated families suggested a common founder haplotype. In a sporadic FAF patient, however, the haplotype was dissimilar to the founder haplotype. The present study demonstrated that a founder mutation in most of the Japanese families with FAF, except for a sporadic patient in whom a de novo mutation event was suggested as the origin of the mutation. [ABSTRACT FROM AUTHOR]

Capillary electrophoresis (CE) is a versatile and widely used analysis platform with application in diverse areas such as analytical chemistry, chiral separations, clinical, forensics, molecular biology, natural products, organic chemistry, and the pharmaceutical industry. Forensic applications of CE include fragment analysis, DNA sequencing, SNP typing, and analysis of gunshot residues, explosive residues, and drugs. Fragment analysis is a widely used method for short tandem repeat (STR) profiling for human identification (HID) due to the single-base resolution capability of CE. This approach circumvents the tedious and expensive approach of DNA sequencing for STR typing. The high sizing precision, ability to detect fluorescence emitted from multiple dyes, automated electrophoretic runs, and data collection software are key factors in the worldwide adoption of CE as the preferred platform for forensic DNA analysis. The most common CE systems used in forensic DNA analysis include the ABI PRISM® 310, 3100, 3100 Avant, 3130, 3130xl, 3500, and 3500xL Genetic Analyzers (GAs). The 3500 series GAs are developed with features useful for forensic scientists, including a normalization feature for analysis of the data designed to reduce the variation in peak height from instrument to instrument and injection to injection. Otherhardware and software features include improved temperature control, radio frequency identification (RFID) tags for monitoring instrument consumables, HID-focused software features, and security and maintenance. [ABSTRACT FROM AUTHOR]

Abstract: Leprosy is a disease caused by Mycobacterium leprae. Various modes of transmission have been suggested for this disease. Transmission and risk of the infection is perhaps related to presence of the infectious cases and is controlled by environmental factors. Evidence suggests that humidity may favor survival of M. leprae in the environment. Several reports show that non-human sources like ‘naturally’ infected armadillos or monkeys could act as reservoir for M. leprae. Inanimate objects or fomites like articles used by infectious patients may theoretically spread infection. However, it is only through detailed knowledge of the biodiversity and ecology that the importance of this mode of transmission can be fully assessed. Our study focuses here to decipher the role of environment in the transmission of the disease. Two hundred and seven soil samples were collected from a village in endemic area where active cases also resided at the time of sample collection. Slit skin smears were collected from 13 multibacillary (MB) leprosy patients and 12 household contacts of the patients suspected to be hidden cases. DNA and RNA of M. leprae were extracted and amplified using M. leprae specific primers. Seventy-one soil samples showed presence of M. leprae DNA whereas 16S rRNA could be detected in twenty-eight of these samples. Samples, both from the environment and the patients, exhibited the same genotype when tested by single nucleotide polymorphism (SNP) typing. Genotype of M. leprae found in the soil and the patients residing in the same area could help in understanding the transmission link in leprosy. [Copyright &y& Elsevier]

The rapid emergence of Escherichia coli O157:H7 from an unknown strain in 1982 to the dominant hemorrhagic E. coli serotype in the United States and the cause of widespread outbreaks of human food-borne illness highlights a need to evaluate critically the extent to which genomic plasticity of this important enteric pathogen contributes to its pathogenic potential and its evolution as well as its adaptation in different ecological niches. Aimed at a better understanding of the evolution of the E. coli O157:H7 pathogenome, the present study presents the high-quality sequencing and comparative phylogenomic analysis of a comprehensive panel of 25 E. coli O157:H7 strains associated with three nearly simultaneous food-borne outbreaks of human disease in the United States. Here we present a population genetic analysis of more than 200 related strains recovered from patients, contaminated produce, and zoonotic sources. High-resolution phylogenomic approaches allow the dynamics of pathogenome evolution to be followed at a high level of phylogenetic accuracy and resolution. SNP discovery and study of genome architecture and prophage content identified numerous biomarkers to assess the extent of genetic diversity within a set of clinical and environmental strains. A total of 1,225 SNPs were identified in the present study and are now available for typing of the E. coli O157:H7 lineage. These data should prove useful for the development of a refined phylogenomic framework for forensic, diagnostic, and epidemiological studies to define better risk in response to novel and emerging E. coli O157:H7 resistance and virulence phenotypes. [ABSTRACT FROM AUTHOR]

Abstract: Short tandem repeat (STR) systems are the most powerful and widely used genetic marker systems in forensic DNA typing. Optimized amplification conditions and PCR reagents in combination with laser fluorescence based detection methods have increased the sensitivity and decreased the detection threshold down to approximately 100pg. The quality of human DNA from forensic samples can be influenced by environmental factors. These may cause different degrees of degradation which have a negative impact on the amplification process especially of STR systems with large amplicons. Therefore, methods which need only small amplicon sizes to detect DNA markers are a better choice for typing degraded DNA. Here we report investigations on different types of DNA markers and typing methods which should all be applicable for analysing degraded DNA. These are two commercially available mini STR kits and five SNP markers which were analysed with two self established assays, a 5′ nuclease assay and a minisequencing (SNaPshot) assay. The investigations comprised sensitivity studies, different types of stain material, as well as intact and degraded DNA. Results indicate that mini STRs are superior to standard STR typing methods, especially for typing old stain material with small amounts of degraded DNA. SNP typing based on the minisequencing (SNaPshot) assay achieved a better success rate in typing aged blood and saliva stains compared to standard STRs and SNP typing using the 5′ nuclease assay. [Copyright &y& Elsevier]

Abstract: Fast and accurate identification of Brucella suis at the biovar level is an important issue for public health laboratories because some of the biovars that infect suidae (boars and pigs) are pathogenic for humans while others are not. Since classical biovar typing methods are often time-consuming, hard to standardize and require high-level biosafety containment, methodological improvements are desirable. This article describes new single nucleotide polymorphism (SNP) signatures for the rapid identification and biovar characterization of B. suis. These SNPs were included together with previously described ones in real-time PCR assays applicable to low-biosafety conditions. Allelic profiles unique for each B. suis biovar were defined and the most relevant signatures were determined on a collection of 137 field strains of worldwide origin characterized previously. Biovars assigned with both present and classical methods were globally consistent except for some biovar 3 field strains which matched the allelic profile of biovar 1. [Copyright &y& Elsevier]

Abstract: A simple and non-expensive platform is critical to realize on-site SNP typing. In this study we typed an SNP existing at the 487th residue of human aldehyde dehydrogenase2 [wild: Glu (GAA); mutant: Lys (AAA)] using our unique isothermal DNA amplification method, ICAN and cycling probes. Both genotypes were identified by the naked eye using a non-expensive UV transilluminator as well as with real-time PCR apparatus or a fluorescence detector. Since ICAN does not need thermal cycling, a cost- and space-limiting factor when fabricating apparatus, the combination of ICAN and cycling probes will be able to realize affordable on-site SNP typing in the near future. [Copyright &y& Elsevier]

Abstract: 2- and 9-Anthracenecarboxamide labeled 2′-deoxyuridines were synthesized and their photophysical properties were examined. These oligonucleonucleotide probes are capable of detecting adenine base on a target DNA sequence. It was also found that 2-anthracene based oligonucleotide probe is more efficient than the corresponding 9-anthracene based oligonucleotide in the application for DNA chip based SNP detection, due to its longer emission wavelength and high fluorescence intensity. [Copyright &y& Elsevier]

Abstract: Association studies using SNPs provides one of the best tools that we have at the moment for looking for genes involved in physical traits. However the studies should be carefully designed from the very beginning in all the steps of the procedure: pre-genotyping, genotyping and the mathematical analysis of the results. If the actual knowledge is correctly applied in the design of the study the probability of being successful in finding an association can be considerably increased. Improved statistical analysis techniques are helping in the robustness of the findings. The current consensus from the literature indicates that this would be a good time to investigate complex or quantitative traits via dense SNP genotyping, and a number of studies have been published, providing potential models. The state of the art of candidate genes for pigmentation, stature and facial morphology is described. [Copyright &y& Elsevier]

Abstract: Fluorescence response upon hybridization of perylene labeled oligonucleotide probes depends on the microenvironment experienced by the perylene fluorophore. In mismatched duplex (Per U-C), enhanced fluorescence was observed while in matched duplex (Per U-A) fluorescence intensity decreased considerably. This observation will be a promising research effort in giving rise to a new powerful tool in detection of SNP. [Copyright &y& Elsevier]

Graft copolymers consisting of poly(N-isopropylacrylamide) and single-stranded DNA were found to form DNA-carrying nanoparticles above physiological temperature. Aggregation of the nanoparticles was induced at relatively high salt concentration by the hybridization of surface-anchored DNA with full match complementary DNA. In contrast, single-base mismatches at the distal end stabilized the colloidal dispersion so that the dispersion remained transparent. The phenomenon is applicable to SNP typing. [ABSTRACT FROM AUTHOR]

Abstract: Recently we reported that gold nanoparticles (GNPs) with fully matched duplexes on their surfaces are selectively deposited onto walls of poly(dimethylsiloxane) (PDMS) microchannels at high salt concentrations. In this study, the surface plasmon resonance (SPR) imaging technique was applied to monitor this phenomenon for improvement of detection sensitivity and elucidation of the phenomenon. The microchip was fabricated by bonding a surface-patterned PDMS plate and a gold thin film-deposited glass substrate. Probe oligonucleotide-modified GNPs were hybridized with target oligonucleotides to make fully matched or single-base-mismatched duplexes. The hybridized GNP solution was mixed with an NaCl solution in a Y-shaped microchannel. The deposition of the GNPs onto the gold sensor surface was detected by SPR imaging. Discrimination of the targets was possible with limit of detection of 32nM (19fmol) without temperature control in 5min. Detailed analysis indicated that a seed layer of GNPs was initially adsorbed onto the sensor surface regardless of the target sequence. Therefore, in combination with a portable SPR device, the proposed method is promising for point-of-care testing of single-nucleotide polymorphsims. [Copyright &y& Elsevier]

Abstract: Genomic alternations, including dosage and allelic imbalance, constitute a major basis of neoplastic and other genetic disorders. Using oligonucleotide genotyping microarrays, here we report the development and usage of an algorithm, called genome imbalance map (GIM) algorithm, for allelic as well as total gene dosage analysis. Using the GIM algorithm, global genome imbalance status at over 100,000 loci was simultaneously analyzed with unprecedented accuracy and allelic discrimination. [Copyright &y& Elsevier]

The study demonstrates an advanced method—reverse transcription-hairpin-probe-polymerase chain reaction (RT-Hpro-PCR)—to specifically and efficiently identify miRNAs that differ even by one base. The technique developed here could help uncover the unprecedented opportunity to understand the normal biological processes of miRNAs and their roles in the development of disease. [Display omitted] • MicroRNAs (miRNAs) in the same family have similar sequences and are hard to identify. • Reverse transcription-hairpin-probe-polymerase chain reaction (RT-Hpro-PCR) reported. • RT-Hpro-PCR uses an RT primer with a DNA tag to detect miRNAs with similar sequences. • RT-Hpro-PCR can precisely detect miRNAs with similar sequences. • This technique may help explore the roles of miRNAs in several biological processes. MicroRNAs (miRNAs) belonging to the same family have similar sequences and are difficult to identify. Herein, we report the reverse transcription-hairpin-probe-polymerase chain reaction (RT-Hpro-PCR) technique, which utilises a reverse transcription (RT) primer containing a 5′-end deoxyribonucleic acid (DNA) tag, to detect miRNAs with similar sequences. This strategy follows a two-step RT-PCR method using 6–7-mer RT-primers with a ~ 10-mer tag sequence at the 5′-end and a probe with a hairpin structure (Hpro), including two C-bulges, attached. The findings demonstrate that the specificity of RT could be increased by shortening the complementary part of the RT primer containing a different base, wherein the PCR could successfully progress with the use of 5′-end DNA tag because of an increase in the length of the hybridised tagged primer. This study shows the potential of RT-Hpro-PCR to precisely detect miRNAs with similar sequences, which could help explore the roles of miRNAs in several biological processes. [ABSTRACT FROM AUTHOR]

Abstract: It is common in forensic casework to encounter highly degraded DNA samples from a variety of sources. In this category bone and teeth samples are often the principal source of evidential material for criminal investigations or identification of long-deceased individuals. In these circumstances standard STRs are prone to fail due to their long amplicon sizes (since DNA becomes progressively more fragmented as it degrades). To successfully resolve such cases alternative markers can be used and until recently the only other tool available was mitochondrial DNA, which despite being more resistant to degradation, is much less informative. A rapidly developing approach to analyzing degraded DNA is the typing of loci from short-amplicon PCR products based on markers such as mini-STRs and autosomal SNPs. We have performed an analysis of several cases with naturally degraded DNA using established STRs plus mini-STRs and autosomal SNPs in order to make an objective comparison of the performance of each method using challenging DNA. The main aim was to establish the benefits and drawbacks of each marker set to help the practitioner choose the DNA analysis method most suited to the circumstances of each case. [Copyright &y& Elsevier]

Abstract: The NanoChip® Molecular Biology Workstation using electronic microarrays is an approach for rapid and high throughput analysis of SNPs. This instrument is fully automated and uses a microchip for electronic addressing of capture probes to specific array sites followed by electronic hybridisation of the single stranded PCR products, and passive hybridisation of fluorescently labelled reporter probes. Discrimination is achieved by applying thermal stringency to denature the mismatched reporters. 48 SNP assays have been designed using the ‘capture down’ assay which applies a thermal ‘touch down’ strategy to obtain the best reporter probe discrimination. [Copyright &y& Elsevier]

Genetic markers located on the Y chromosome are of increasing importance in human identity testing. In an effort to evaluate the forensic utility of Y chromosome single nucleotide polymorphism (SNP) markers, we constructed several novel multiplex allele-specific primer extension (ASPE) assays and utilized a new commercial allele-specific hybridization (ASH) multiplex kit to examine 50 Y-SNP markers in 229 males from two US Caucasian and African American populations. The novel ASPE assays covered 18 Y-SNP markers in three multiplex reactions while a commercial ASH kit was used to type 42 Y-SNPs plus amelogenin for sex-typing purposes. There were 10 overlapping loci between the ASPE and ASH methods permitting an evaluation of concordance on over 2000 allele calls. The 50 Y-SNP markers examined in this study define 45 of the 159 possible Y Chromosome Consortium (YCC) haplogroups. Only 18 different haplogroups were observed in our samples. [Copyright &y& Elsevier]