High-resolution melting system to perform multilocus sequence typing of Campylobacter jejuni

Multi-locus sequence typing (MLST) has emerged as the state-of-the-art method for resolving bacterial population genetics but it is expensive and time consuming. We evaluated the potential of high resolution melting (HRM) to identify known MLST alleles of Campylobacter jejuni at reduced cost and time. Each MLST locus was amplified in two or three sub fragments, which were analyzed by HRM. The approach was investigated using 47 C. jejuni isolates, previously characterized by classical MLST, representing isolates from diverse environmental, animal and clinical sources and including the six most prevalent sequence types (ST) and the most frequent alleles. HRM was then applied to a validation set of 84 additional C. jejuni isolates from chickens; 92% of the alleles were resolved in 35 hours of laboratory time and the cost of reagents per isolate was $20 compared with $100 for sequence-based typing. HRM has the potential to complement sequence-based methods for resolving SNPs and to facilitate a wide range of genotyping studies.     

A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms

A new MALDI-TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3'-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis.     

Development of an extended multilocus sequence typing for genotyping of Brucella isolates

By amplifying and sequencing longer sequences, an extended multi locus sequence typing (EMLST) theme was developed for Brucella. 61 isolates were genotyped by the EMLST with increased resolution. This strategy could be extended to other bacteria to improve MLST genotyping resolution without additional loci.     

An efficient procedure for genotyping single nucleotide polymorphisms

Analysis of single nucleotide polymorphisms (SNPs) has been and will be increasingly utilized in various genetic disciplines, particularly in studying genetic determinants of complex diseases. Such studies will be facilitated by rapid, simple, low cost and high throughput methodologies for SNP genotyping. One such method is reported here, named tetra-primer ARMS-PCR, which employs two primer pairs to amplify, respectively, the two different alleles of a SNP in a single PCR reaction. A computer program for designing primers was developed. Tetra-primer ARMS-PCR was combined with microplate array diagonal gel electrophoresis, gaining the advantage of high throughput for gel-based resolution of tetra-primer ARMS-PCR products. The technique was applied to analyse a number of SNPs and the results were completely consistent with those from an independent method, restriction fragment length polymorphism analysis.     

Accuracy of genotyping for single nucleotide polymorphisms by a microarray-based single nucleotide polymorphism typing method involving hybridization of short allele-specific oligonucleotides.

Advances in technologies for identifying genetic polymorphisms rapidly and accurately will dramatically accelerate the discovery of disease-related genes. Among a variety of newly described methods for rapid typing of single-nucleotide polymorphisms (SNPs), gene detection using DNA microarrays is gradually achieving widespread use. This method involves the use of short (11- to 13-mer) allele-specific oligonucleotides. This method allows simultaneous analysis of many SNPs in DNAs from a large number of individuals, in a single experiment. In this work, we evaluated the accuracy of a new microarray-based short allele-specific oligonucleotide (ASO) hybridization method. There is a 96-well formatted array on a single plate, in which up to 256 spots are included in each well. Fluorescent probes for our experiments were produced by multiplex PCR amplification often target SNP-containing regions. We genotyped 192 individuals across a panel of ten single base variations, which included an insertion/deletion polymorphism. For comparison, we genotyped the same individuals for the same SNPs by the method of single-base extension with fluorescence detection. The typing accuracies of the microarray-based PCR-ASO and single-base extension methods were calculated as 99.9% and 99.1%, respectively, on the basis of genotyping results determined by direct sequencing. We conclude that the microarray-based hybridization method using short ASO probes represents a potential breakthrough technology for typing large numbers of SNPs rapidly and efficiently.     

Development of a multilocus sequence typing tool for high-resolution genotyping of Enterocytozoon bieneusi

Thus far, genotyping of Enterocytozoon bieneusi has been based solely on DNA sequence analysis of the internal transcribed spacer (ITS) of the rRNA gene. Both host-adapted and zoonotic (human-pathogenic) genotypes of E. bieneusi have been identified. In this study, we searched for microsatellite and minisatellite sequences in the whole-genome sequence database of E. bieneusi isolate H348. Seven potential targets (MS1 to MS7) were identified. Testing of the seven targets by PCR using two human-pathogenic E. bieneusi genotypes (A and Peru10) led to the selection of four targets (MS1, MS3, MS4, and MS7). Further analysis of the four loci with an additional 24 specimens of both host-adapted and zoonotic E. bieneusi genotypes indicated that most host-adapted genotypes were not amplified by PCR targeting these loci. In contrast, 10 or 11 of the 13 specimens of the zoonotic genotypes were amplified by PCR at each locus. Altogether, 12, 8, 7, and 11 genotypes of were identified at MS1, MS3, MS4, and MS7, respectively. Phylogenetic analysis of the nucleotide sequences obtained produced a genetic relationship that was similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included most E. bieneusi genotypes in humans. Thus, a multilocus sequence typing tool was developed for high-resolution genotyping of E. bieneusi. Data obtained in the study should also have implications for understanding the taxonomy of Enterocytozoon spp., the public health significance of E. bieneusi in animals, and the sources of human E. bieneusi infections.     

Comparison of multilocus variable-number tandem-repeat analysis with multilocus sequence typing and pulsed-field gel electrophoresis for Enterococcus faecalis

Enterococcus faecalis represents recently an important etiological agent of health care-associated infections (HAIs) and there is a need for evaluation and comparison of typing methods available for this microorganism. We tested multilocus VNTR (variable-number tandem repeats) analysis (MLVA) on a well-characterized collection of 153 clinical isolates of E. faecalis, corresponding to 52 multilocus sequence types and 67 pulsed-field gel electrophoresis (PFGE) profiles. MLVA showed high discriminatory power, discerning 111 different types (diversity index equal 98.9%). The concordance MLVA/MLST and MLVA/PFGE was 0.95 and 0.74, respectively. High discriminatory power of MLVA indicates its utility for local epidemiology such as outbreak investigation, and for differentiation of clones defined by other methods.     

Multilocus sequence typing and antimicrobial resistance in Enterococcus faecium isolates from fresh produce

The purpose of the present study was to determine the relatedness of Enterococcus faecium isolates from fresh produce to E. faecium strains from other sources by using multi-locus sequence typing (MLST) and to determine the antimicrobial resistance of the isolates. MLST analysis of 22 E. faecium isolates from fresh produce revealed 7 different sequence types (ST 22, ST 26, ST 43, ST 46, ST 55, ST 94 and ST 296). Most isolates belonged to ST 296 (40.9 %), followed by ST 94 (27.3 %). All isolates were sensitive to vancomycin and to imipenem, and only one was resistant to ampicillin (MIC 32 mg/l). However, all were resistant to cefotaxime and ceftazidine. E. faecium isolates from fresh produce were inhibited by quaternary compounds (benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide), biguanides (chlorhexidine), polyguanides [poly-(hexamethylene guanidinium) hydrochloride], bisphenols (triclosan, hexachlorophene) and biocidal solutions of P3 oxonia and P3 topax 66. Didecyldimethylammonium bromide and triclosan were the least effective biocides in growth inhibition, while hexadecylpyridinium chloride was the most effective. Results from MLST typing and antibiotic resistance suggest that the studied E. faecium isolates from fresh produce are not related to the clinically-relevant clonal complex CC17.     

Multiplex single nucleotide polymorphisms genotyping using solid-phase single base extension on magnetic nanoparticles

To fulfill the increasing need for large-scale genetic research, we have developed a new solid-phase single base extension (SBE) protocol on magnetic nanoparticles (MNPs) for multiplex SNP detection using adapter polymerase chain reaction (PCR) products as templates. Extension primers were covalently immobilized on the MNPs, and allele-specific extension took place along the stretch of target DNA for one-color ddNTP incorporation. The MNPs with fluorophores were spotted on a glass slide to fabricate a “bead array” to discriminate their genotypes. Eight SNP loci of three DNA samples were interrogated, and the experiment demonstrated that it is an efficient method for large-scale SNP genotyping.     

A novel procedure for efficient genotyping of single nucleotide polymorphisms

Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of α-S-dNTPs and α-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5′-phosphodiesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the ‘GOOD Assay’ for SNP analysis.     

Comparison of two multilocus sequence based genotyping schemes for Leptospira species

Background

Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results.

Methods and Findings

A total of 48 isolates consisting of L. interrogans (n = 40) and L. kirschneri (n = 8) were typed by the 7 locus MLST scheme described by Thaipadungpanit et al., and the 6 locus genotyping scheme described by Ahmed et al., (termed 7L and 6L, respectively). Two L. interrogans isolates were not typed using 6L because of a deletion of three nucleotides in lipL32. The remaining 46 isolates were resolved into 21 sequence types (STs) by 7L, and 30 genotypes by 6L. Overall nucleotide diversity (based on concatenated sequence) was 3.6% and 2.3% for 7L and 6L, respectively. The D value (discriminatory ability) of 7L and 6L were comparable, i.e. 92.0 (95% CI 87.5–96.5) vs. 93.5 (95% CI 88.6–98.4). The dN/dS ratios calculated for each locus indicated that none were under positive selection. Neighbor joining trees were reconstructed based on the concatenated sequences for each scheme. Both trees showed two distinct groups corresponding to L. interrogans and L. kirschneri, and both identified two clones containing 10 and 7 clinical isolates, respectively. There were six instances in which 6L split single STs as defined by 7L into closely related clusters. We noted two discrepancies between the trees in which the genetic relatedness between two pairs of strains were more closely related by 7L than by 6L.

Conclusions

This genetic analysis indicates that the two schemes are comparable. We discuss their practical advantages and disadvantages.     

High-throughput genotyping of single nucleotide polymorphisms using new biplex invader technology

The feasibility of large-scale genome-wide association studies of complex human disorders depends on the availability of accurate and efficient genotyping methods for single nucleotide polymorphisms (SNPs). We describe a new platform of the invader assay, a biplex assay, where both alleles are interrogated in a single reaction tube. The assay was evaluated on over 50 different SNPs, with over 20 SNPs genotyped in study cohorts of over 1500 individuals. We assessed the usefulness of the new platform in high-throughput genotyping and compared its accuracy to genotyping results obtained by the traditional monoplex invader assay, TaqMan genotyping and sequencing data. We present representative data for two SNPs in different genes (CD36 and protein tyrosine phosphatase 1β) from a study cohort comprising over 1500 individuals with high or low-normal blood pressure. In this high-throughput application, the biplex invader assay is very accurate, with an error rate of <0.3% and a failure rate of 1.64%. The set-up of the assay is highly automated, facilitating the processing of large numbers of samples simultaneously. We present new analysis tools for the assignment of genotypes that further improve genotyping success. The biplex invader assay with its automated set-up and analysis offers a new efficient high-throughput genotyping platform that is suitable for association studies in large study cohorts.     

Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay

Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of β-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the β-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.     

Confirming single nucleotide polymorphisms from expressed sequence tag datasets derived from three cattle cDNA libraries

Using the Phred/Phrap/Polyphred/Consed pipeline established in the National Livestock Research Institute of Korea, we predicted candidate coding single nucleotide polymorphisms (cSNPs) from 7,600 expressed sequence tags (ESTs) derived from three cDNA libraries (liver, M. longissimus dorsi, and intermuscular fat) of Hanwoo (Korean native cattle) steers. From the 7,600 ESTs, 829 contigs comprising more than two EST reads were assembled using the Phrap assembler. Based on the contig analysis, 201 candidate cSNPs were identified in 129 contigs, in which transitions (69%) outnumbered transversions (31%). To verify whether the predicted cSNPs are real, 17 SNPs involved in lipid and energy metabolism were selected from the ESTs. Twelve of these were confirmed to be real while five were identified as artifacts, possibly due to expressed sequence tag sequence error. Further analysis of the 12 verified cSNPs was performed using the program BLASTX. Five were identified as nonsynonymous cSNPs, five were synonymous cSNPs, and two SNPs were located in 3'-UTRs. Our data indicated that a relatively high SNP prediction rate (71%) from a large EST database could produce abundant cSNPs rapidly, which can be used as valuable genetic markers in cattle.     

The lactic acid enterococci Enterococcus faecium and Enterococcus durans: nucleotide sequence diversity in 16S rRNA genes

Among the strains used as starters for making sour milk products on the territory of the CIS, the bacteria Enterococcus faecium and Enterococcus durans are frequently found. In this work, we studied a new collection of lactic acid enterococci and also obtained more complete data on the nucleotide sequences of 16S rRNA genes in some strains studied earlier and found that most strains had certain distinctions in their 16S rRNA genes as compared with the E. durans and E. faecium genes available in the NCBI database. Based on these data, it is suggested that the strains of lactic acid enterococci represent new, earlier unknown taxa of enterococci that use milk as an ecological niche.     

Cost-effective procedures for genotyping of human FCN2 gene single nucleotide polymorphisms

L-ficolin (ficolin-2) is a complement-activating pattern-recognition lectin taking part in the innate immune response. Both its serum concentration and sugar binding capacity are influenced by single nucleotide polymorphisms (SNP) of the corresponding FCN2 gene. Cost-effective and simple procedures, based on polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism for an investigation of four FCN2 SNPs are proposed: ?64 A?>?C (rs7865453), ?4 A?>?G (rs17514136; both located in the promoter region), +6359 C?>?T (rs17549193), +6424 G?>?T (rs7851696; both in exon 8). Variant alleles of ?64 and +6424 (in strong linkage disequlibrium) are known to be associated with low L-ficolin level or activity. In contrast, variant alleles at positions ?4 and +6359 (also in strong linkage disequlibrium) correspond to higher values. Since several L-ficolin clinical associations have been reported, FCN2 genotyping seems to be a valuable tool for disease association studies.     

Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry

In the future, analysis of single nucleotide polymorphisms (SNPs) should become a powerful tool for many genetic applications in areas such as association studies, pharmacogenetics and traceability in the agro-alimentary sector. A number of technologies have been developed for high-throughput genotyping of SNPs. Here we present the simplified GOOD assay for SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI). The simplified GOOD assay is a single-tube, purification-free, three-step procedure consisting of PCR, primer extension and phosphodiesterase II digestion followed by mass spectrometric analysis. Due to the application of charge-tag technology, no sample purification is required prior to the otherwise very impurity-sensitive MALDI analysis. The use of methylphosphonate containing primers and ddNTPs or α-S-ddNTPs together with a novel DNA polymerase derived from Thermotoga maritima for primer extension allow the fluent preparation of negatively charge-tagged, allele-specific products. A key feature of this polymerase is its preference for ddNTPs and α-S-ddNTPs over dNTPs. The simplified GOOD assay was run with automatic liquid handling at the lowest manageable volumes, automatic data acquisition and interpretation. We applied this novel procedure to genotyping SNPs of candidate genes for hypertension and cardiovascular disease.