Silver nanoparticle production by the fungus Fusarium oxysporum: nanoparticle characterisation and analysis of antifungal activity against pathogenic yeasts

The microbial synthesis of nanoparticles is a green chemistry approach that combines nanotechnology and microbial biotechnology. The aim of this study was to obtain silver nanoparticles (SNPs) using aqueous extract from the filamentous fungus Fusarium oxysporum as an alternative to chemical procedures and to evaluate its antifungal activity. SNPs production increased in a concentration-dependent way up to 1 mM silver nitrate until 30 days of reaction. Monodispersed and spherical SNPs were predominantly produced. After 60 days, it was possible to observe degenerated SNPs with in additional needle morphology. The SNPs showed a high antifungal activity against Candida and Cryptococcus , with minimum inhibitory concentration values ≤ 1.68 µg/mL for both genera. Morphological alterations of Cryptococcus neoformans treated with SNPs were observed such as disruption of the cell wall and cytoplasmic membrane and lost of the cytoplasm content. This work revealed that SNPs can be easily produced by F. oxysporum aqueous extracts and may be a feasible, low-cost, environmentally friendly method for generating stable and uniformly sized SNPs. Finally, we have demonstrated that these SNPs are active against pathogenic fungi, such as Candida and Cryptococcus .     

Identification of genome-wide single nucleotide polymorphisms in allopolyploid crop Brassica napus

Background

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay.

Results

A total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%.

Conclusions

Despite the polyploid nature of B. napus, nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.     

A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2 ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis.     

Haplotyping using a combination of polymerase chain reaction–single-strand conformational polymorphism analysis and haplotype-specific PCR amplification

A single nucleotide polymorphism (SNP) may have an impact on phenotype, but it may also be influenced by multiple SNPs within a gene; hence, the haplotype or phase of multiple SNPs needs to be known. Various methods for haplotyping SNPs have been proposed, but a simple and cost-effective method is currently unavailable. Here we describe a haplotyping approach using two simple techniques: polymerase chain reaction–single-strand conformational polymorphism (PCR–SSCP) and haplotype-specific PCR. In this approach, individual regions of a gene are analyzed by PCR–SSCP to identify variation that defines sub-haplotypes, and then extended haplotypes are assembled from the sub-haplotypes either directly or with the additional use of haplotype-specific PCR amplification. We demonstrate the utility of this approach by haplotyping ovine FABP4 across two variable regions that contain seven SNPs and one indel. The simplicity of this approach makes it suitable for large-scale studies and/or diagnostic screening.     

Molecular haplotyping of tandem single nucleotide polymorphisms by allele-specific PCR

BARD1 Val507Met (1592A>G) is an interesting marker for association studies on cancer risk. However, studies are scarce in the literature, probably reflecting the methodological problem imposed by the fact that next to the 1592A>G stands the 1591C>T single nucleotide polymorphism (SNP). We have designed an allele-specific PCR method capable of molecular haplotyping tandem SNPs. In the tandem SNPs haplotyping assay (tSNPh), four reverse primers are designed to be perfect matches of each potential haplotype. The forward primer is labeled with a fluorochrome. PCR products are analyzed by capillary electrophoresis. Haplotyping is performed by size calling. To ascertain the accuracy and reproducibility of the assay, we measured the level of concordance with sequencing data in 124 samples. In vitro-generated templates have been used for further testing. We developed a novel and reliable assay that permits typing two SNPs directly adjacent to each other, avoiding mutual interferences. The method is amenable to automation and high throughput. We expect that this assay will contribute to clarifying the role of BARD1 in cancer susceptibility. In addition, we suggest that tandem SNPs are potentially interesting polymorphic markers in which molecular haplotyping can be performed easily.     

Ewing's sarcoma: Analysis of single nucleotide polymorphism in the EWS gene

We aimed to investigate single nucleotide polymorphisms (SNPs) in the EWS gene breaking region in order to analyze Ewing's sarcoma susceptibility. The SNPs were investigated in a healthy subject population and in Ewing's sarcoma patients from Southern Brazil. Genotyping was performed by TaqMan® assay for allelic discrimination using Real-Time PCR. The analysis of incidence of SNPs or different SNP-arrangements revealed a higher presence of homozygote TT-rs4820804 in Ewing's sarcoma patients (p = 0.02; Chi Square Test). About 300 bp from the rs4820804 SNP lies a palindromic hexamer (5′-GCTAGC-3′) and three nucleotides (GTC), which were previously identified to be in close vicinity of the breakpoint junction in both EWS and FLI1 genes. This DNA segment surrounding the rs4820804 SNP is likely to indicate a breakpoint region. If the T-rs4820804 allele predisposes a DNA fragment to breakage, homozygotes (TT-rs4820804) would have double the chance of having a chromosome break, increasing the chances for a translocation to occur. In conclusion, the TT-rs4820804 EWS genotype can be associated with Ewing's sarcoma and the SNP rs4820804 can be a candidate marker to understand Ewing's sarcoma susceptibility.     

Development of a real time PCR Taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum

In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.     

High quality SNPs/Indels mining and characterization in ginger from ESTs data base

Ginger (Zingiber officinale Rosc.) is an important herb of the family Zingiberaceae. It is accepted as a universal cure for a multitude of diseases in Indian systems of medicine and its rhizomes are equally popular as a spice ingredient throughout Asia. SNPs, the definitive genetic markers, representing the finest resolution of a DNA sequence, are abundantly found in populations having a lower rate of mutation and are used for genomic analysis. The public ESTs sequences mostly lack quality files, making high quality SNPs detection more difficult since it is exclusively based on sequence comparisons. In the present study, current dbESTs of NCBI was mined and 38115 ginger ESTs sequences were obtained and assembled into contigs using CAP3 program. In this analysis, recent software tool QualitySNP was used to detect 11523 potential SNPs sites, 8810 high quality SNPs and 1008 indels polymorphisms with a frequency of 1.61 SNPs / 10 kbp. Of ESTs libraries generated from three ginger tissues together, rhizomes had a frequency of 0.32 SNPs and 0.03 indels per 10 kbp whereas the leaves had a frequency of 2.51 SNPs and 0.23 indels per 10 kbp and root is showing relative frequency of 0.76/10 kbp SNPs and 0.02/10 kbp indels. The present analysis provides additional information about the tissue wise presence of haplotypes (222), distribution of high quality exonic (2355) and intronic (6455) SNPs and information about singletons (7538) in addition to contigs transitions and transversions ratio (0.57). Among all tissue detected SNPs, transversions number is higher in comparison to the number of transitions. Quality SNPs detected in this work can be used as markers for further ginger genetic experiments.     

Determination of the CCR5∆32 frequency in Emiratis and Tunisians and the screening of the CCR5 gene for novel alleles in Emiratis

Background

The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5?32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5?32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians).

Methodology

The prevalence of CCR5 gene variants including CCR5?32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5?32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing.

Results

Among Emiratis, the allele frequency of the CCR5?32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5?32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans.

Conclusion

We conclude that the allele frequency of the most critical CCR5 polymorphism (?32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.     

Techniques de recherche des polymorphismes génétiques

Sequencing the human genome has allowed the discovery of millions of DNA sequence variants. Sequence variations in human DNA are mainly present asSingle Nucleotide Polymorphisms (SNPs); this common form of variation is found about once every 1,000 bases in the human genome and 1.8 million SNPs have now been identified and located. The accessibility of databases of SNPs opens the possibility of studying the influence of these polymorphisms on disease risks as well as on drug responses. Numerous approaches have been set up for the identification of SNPs. In this review we describe the main techniques used for the identification of these polymorphisms. They rely on two major consequences of sequence variations: the apparition or the disappearance of restriction enzyme sites or the alteration of DNA strand hybridization due to the presence of a mismatch. Southern blotting and restriction endonucleases have allowed the development of the technique ofrestriction fragment length polymorphisms (RFLPs), now performed on PCR products. Several other approaches such as denaturing high-performance liquid chromatography or real-time PCR can detect allele differences upon re-hybridization and heteroduplex formation. However, DNA sequencing remains the obligate step for the positive identification of known or unknown SNPs. At last, the development of high-throughput methods allows a large increase in the rate of discovery of SNPs likely.     

Genomic Structure of the Cyanobacterium Synechocystis sp. PCC 6803 Strain GT-S

Synechocystis sp. PCC 6803 is the most popular cyanobacterial strain, serving as a standard in the research fields of photosynthesis, stress response, metabolism and so on. A glucose-tolerant (GT) derivative of this strain was used for genome sequencing at Kazusa DNA Research Institute in 1996, which established a hallmark in the study of cyanobacteria. However, apparent differences in sequences deviating from the database have been noticed among different strain stocks. For this reason, we analysed the genomic sequence of another GT strain (GT-S) by 454 and partial Sanger sequencing. We found 22 putative single nucleotide polymorphisms (SNPs) in comparison to the published sequence of the Kazusa strain. However, Sanger sequencing of 36 direct PCR products of the Kazusa strains stored in small aliquots resulted in their identity with the GT-S sequence at 21 of the 22 sites, excluding the possibility of their being SNPs. In addition, we were able to combine five split open reading frames present in the database sequence, and to remove the C-terminus of an ORF. Aside from these, two of the Insertion Sequence elements were not present in the GT-S strain. We have thus become able to provide an accurate genomic sequence of Synechocystis sp. PCC 6803 for future studies on this important cyanobacterial strain.     

Genomic regions involved in yield potential detected by genome-wide association analysis in Japanese high-yielding rice cultivars

Background

High-yielding cultivars of rice (Oryza sativa L.) have been developed in Japan from crosses between overseas indica and domestic japonica cultivars. Recently, next-generation sequencing technology and high-throughput genotyping systems have shown many single-nucleotide polymorphisms (SNPs) that are proving useful for detailed analysis of genome composition. These SNPs can be used in genome-wide association studies to detect candidate genome regions associated with economically important traits. In this study, we used a custom SNP set to identify introgressed chromosomal regions in a set of high-yielding Japanese rice cultivars, and we performed an association study to identify genome regions associated with yield.

Results

An informative set of 1152 SNPs was established by screening 14 high-yielding or primary ancestral cultivars for 5760 validated SNPs. Analysis of the population structure of high-yielding cultivars showed three genome types: japonica-type, indica-type and a mixture of the two. SNP allele frequencies showed several regions derived predominantly from one of the two parental genome types. Distinct regions skewed for the presence of parental alleles were observed on chromosomes 1, 2, 7, 8, 11 and 12 (indica) and on chromosomes 1, 2 and 6 (japonica). A possible relationship between these introgressed regions and six yield traits (blast susceptibility, heading date, length of unhusked seeds, number of panicles, surface area of unhusked seeds and 1000-grain weight) was detected in eight genome regions dominated by alleles of one parental origin. Two of these regions were near Ghd7, a heading date locus, and Pi-ta, a blast resistance locus. The allele types (i.e., japonica or indica) of significant SNPs coincided with those previously reported for candidate genes Ghd7 and Pi-ta.

Conclusions

Introgression breeding is an established strategy for the accumulation of QTLs and genes controlling high yield. Our custom SNP set is an effective tool for the identification of introgressed genome regions from a particular genetic background. This study demonstrates that changes in genome structure occurred during artificial selection for high yield, and provides information on several genomic regions associated with yield performance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-346) contains supplementary material, which is available to authorized users.     

Homogenisation of cystic fibrosis sputum by sonication--an essential step for Aspergillus PCR

The importance of Aspergillus as a lung pathogen in cystic fibrosis (CF) is becoming increasingly recognised. However, fungal culture of CF sputum is unreliable and there is no consensus for identifying phenotypes beyond ABPA that may benefit from antifungal therapy. There are no published studies using real-time PCR to detect Aspergillus in CF sputum. The major barrier to sensitive detection of Aspergillus using PCR is sputum homogenisation. This study aimed to optimise sputum homogenisation utilising sonication to improve Aspergillus DNA extraction. Sonication amplitude and duration that enabled sputum homogenisation but ensured preservation of DNA integrity were first determined. 160 sputum samples were collected from CF patients. 49 of the sputum samples were split, one half was used for standard culture and the other half was homogenised with NALC-NaOH before undergoing DNA extraction. The subsequent 111 samples were homogenised with dithiothreitol plus sonication prior to culture and DNA extraction. Real-time PCR targeting a portion of the 18S rDNA of Aspergillus was performed on all DNA extractions. In the 49 samples with no sonication 8 (16%) were culture positive but only 4 of these were PCR positive. However, PCR was positive in 11 culture negative samples. PCR after sonication showed a significant improvement in sensitivity: 33 (30%) were culture and PCR positive, 48 (43%) were culture negative, but PCR positive (p < 0.0001) and 30 (27%) were culture and PCR negative. The combination of dithiothreitol and sonication to homogenise sputum increases PCR yield, with PCR being substantially more sensitive than culture.     

Molecular Approaches to Taenia asiatica

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.