Nucleotide Sequence-based Typing Research Articles

New real time and conventional RT-PCRs for updated molecular diagnosis of infectious bronchitis virus infection (IBV) in chickens in Egypt associated with frequent co-infections with avian influenza and Newcastle Disease viruses

In Egypt, currently two geographically restricted genotypes of the infectious bronchitis coronavirus (IBV) are circulating with detrimental effects for poultry industry. A sensitive real-time RT-PCR assay targeting the IBV nucleocapsid gene (N) was developed to screen clinical samples for presence of IBV. Conventional RT-PCRs amplifying hypervariable regions (HVRs 1–2 and 3) of the IBV S1 gene were developed and amplificates used for nucleotide sequence-based typing of IBV field strains in Egyptian chickens directly from clinical samples.

A Prospective Study of Enterovirus D68 in a Regional Uk Center

Aim: A prospective study was undertaken to establish the prevalence of enterovirus D68 (EV-D68) in the northwest of England. Patients & samples: In the period 24 October through 31 December 2014, respiratory samples were screened by a specific real-time PCR and EV-D68 was identified by nucleotide sequence-based typing. Results: Enterovirus was detected in 7.4% of samples and EV-D68 was confirmed in 32 cases. Phylogenetic analysis showed that EV-D68 strains corresponded to clades A and B. Clinical presentation of cases ranged from mild to severe respiratory infection. The majority of cases had comorbidities that might predispose to severe infection. Conclusion: EV-D68 was circulating in our region during late 2014 and has the potential to cause severe disease.

Characterization of swine leukocyte antigen alleles and haplotypes on a novel miniature pig line, Microminipig.

Microminipigs are extremely small-sized, novel miniature pigs that were recently developed for medical research. The inbred Microminipigs with defined swine leukocyte antigen (SLA) haplotypes are expected to be useful for allo- and xenotransplantation studies and also for association analyses between SLA haplotypes and immunological traits. To establish SLA-defined Microminipig lines, we characterized the polymorphic SLA alleles for three class I (SLA-1, SLA-2 and SLA-3) and two class II (SLA-DRB1 and SLA-DQB1) genes of 14 parental Microminipigs using a high-resolution nucleotide sequence-based typing method. Eleven class I and II haplotypes, including three recombinant haplotypes, were found in the offspring of the parental Microminipigs. Two class I and class II haplotypes, Hp-31.0 (SLA-1*1502-SLA-3*070102-SLA-2*1601) and Hp-0.37 (SLA-DRB1*0701-SLA-DQB1*0502), are novel and have not so far been reported in other pig breeds. Crossover regions were defined by the analysis of 22 microsatellite markers within the SLA class III region of three recombinant haplotypes. The SLA allele and haplotype information of Microminipigs in this study will be useful to establish SLA homozygous lines including three recombinants for transplantation and immunological studies.

Campylobacter sequence typing databases: applications and future prospects

Human campylobacteriosis, caused by the zoonotic bacteria Campylobacter jejuni and Campylobacter coli, remains a major cause of gastroenteritis worldwide. For many countries the implementation of effective interventions to reduce the burden of this disease is a high priority. Nucleotide sequence-based typing, including multilocus sequence typing (MLST) and antigen gene sequence typing (AGST), has provided unified, comprehensive, and portable Campylobacter isolate characterization, with curated databases of genotypes available (pubMLST.org/campylobacter). Analyses of large collections of isolates from various sources with these approaches have provided many insights into the epidemiology of these ubiquitous and diverse organisms. C. jejuni and C. coli populations are structured into clonal complexes, which reflect genealogy and are associated with specific phenotypes, e.g. the predisposition to infect particular animals, a property that has permitted the development of genetic means of attributing isolates from human disease to potential sources. This has identified retail meat, and especially chicken, as the likely cause of most human disease in many countries, although some human isolates have other likely origins. Such data have led directly to effective intervention studies and will be important in ongoing targeting of intervention strategies and the monitoring of their effectiveness. MLST and AGST data have also been employed in epidemiological investigations and studies of Campylobacter evolution and population biology. The sequence databases that have been established are compatible with the whole-genome sequencing (WGS) approaches likely to be implemented soon; indeed, the hierarchical approach adopted by MLST and AGST will be essential for the exploitation of WGS data.

Molecular typing methods for outbreak detection and surveillance of invasive disease caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review.

Invasive disease caused by the encapsulated bacteria Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae remains an important cause of morbidity and mortality worldwide, despite the introduction of successful conjugate polysaccharide vaccines that target disease-associated strains. In addition, resistance, or more accurately reduced susceptibility, to therapeutic antibiotics is spreading in populations of these organisms. There is therefore a continuing requirement for the surveillance of vaccine and non-vaccine antigens and antibiotic susceptibilities among isolates from invasive disease, which is only partially met by conventional methods. This need can be met with molecular and especially nucleotide sequence-based typing methods, which are fully developed in the case of N. meningitidis and which could be more widely deployed in clinical laboratories for S. pneumoniae and H. influenzae.

The secret life of the multilocus sequence type

Such are the challenges, and the potential, presented by complete genome sequences that the eventual erosion of the boundaries between biochemistry, ecology, bioinformatics, population biology, epidemiology and medical microbiology will perhaps be the most profound legacy of the genomics revolution. The development of nucleotide sequence-based typing schemes (multilocus sequence typing (MLST)) represents a similar synthesis, for this technique both matches the practical requirements for a highly portable standard for strain characterisation whilst also being firmly grounded in the population biology principles of multilocus enzyme electrophoresis (MLEE). Contrary to recent claims that population biology analyses of public health-oriented MLST data ‘obscures its utility in applied microbiology’ [Maiden MC. Multilocus sequence typing of bacteria. Annu Rev Microbiol 2006;60:561–88.], we argue that such an emphasis is essential for full interpretation of the data. Here we note a pertinent case in point; how a consideration of the rates of genetic recombination can help to explain why MLST data tend to correlate with virulence properties in some species ( Neisseria meningitidis) but not in others ( Staphylococcus aureus). We also discuss how the argument applies to the identification of recently emerged methicillin-resistant S. aureus (MRSA) clones using MLST. We conclude with a speculative rationale for promoting the ‘clonal complexes’ of S. aureus to species status.

Nucleotide sequence-based typing of meningococci directly from clinical samples.

The unpredictable characteristics of meningococcal disease (MD) make outbreaks complicated to monitor and consequently lead to high levels of public anxiety. Traditional molecular techniques have been utilized in order to understand better the epidemiology of MD, but some have disadvantages such as being highly specialized and labour-intensive, with low reproducibility. Some of these problems have been overcome by using multilocus sequence typing (MLST). This technique exploits the unambiguous nature and electronic portability of nucleotide sequencing data for the characterization of micro-organisms. The need for enhanced surveillance of MD after the introduction of serogroup C conjugate vaccines means that it is important to gain typing information from the infecting organism in the absence of a culture isolate. Here, the application of MLST for the laboratory confirmation and characterization of Neisseria meningitidis directly from clinical samples is described. This involved using a newly designed set of primers that were complementary to nucleotide sequences external to the existing MLST primers already in use for culture-based MLST of meningococci. This combination has produced a highly sensitive procedure to allow the efficient genotypic characterization of meningococci directly from clinical samples.

Nucleotide sequence-based typing of bacteria and the impact of automation.

DNA-based typing methods are increasingly important for the characterisation of bacteria. They are used to monitor the epidemiology of pathogens with public health significance and also to help understand the evolution and population biology of bacteria. However, these methods require accuracy and reproducibility and are often of a high-throughput nature. Laboratory automation is therefore the key to the successful implementation of such methods. This review describes the impact of automation on DNA-based typing methods, particularly multi-locus sequence typing (MLST), and the method components that can be automated.

Sequence typing confirms that Campylobacter jejuni strains associated with Guillain-Barré and Miller-Fisher syndromes are of diverse genetic lineage, serotype, and flagella type.

Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) are correlated with prior infection by Campylobacter jejuni in up to 40% of cases. Nucleotide sequence-based typing of 25 C. jejuni isolates associated with neuropathy permitted robust comparisons with equivalent data from approximately 800 C. jejuni isolates not associated with neuropathy. A total of 13 genetic lineages and 20 flaA short variable region nucleotide sequences were present among the 25 isolates. A minority of isolates (4 of 25) had the flaA short variable region nucleotide sequences that were previously proposed as a marker for GBS-associated isolates. These 4 isolates probably represented the Penner serotype 19 lineage, which has been proposed to have an association with GBS.