152 research outputs found
PathogenMip Assay: A Multiplex Pathogen Detection Assay
The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe
Experiences with array-based sequence capture; toward clinical applications
Although sequencing of a human genome gradually becomes an option, zooming in on the region of interest remains attractive and cost saving. We performed array-based sequence capture using 385K Roche NimbleGen, Inc. arrays to zoom in on the protein-coding and immediate intron-flanking sequences of 112 genes, potentially involved in mental retardation and congenital malformation. Captured material was sequenced using Illumina technology. A data analysis pipeline was built that detects sequence variants, positions them in relation to the gene, checks for presence in databases (eg, db single-nucleotide polymorphism (SNP)) and predicts the potential consequences at the level of RNA splicing and protein translation. In the samples analyzed, all known variants were reliably detected, including pathogenic variants from control cases and SNPs derived from array experiments. Although overall coverage varied considerably, it was reproducible per region and facilitated the detection of large deletions and duplications (copy number variations), including a partial deletion in the B3GALTL gene from a patient sample. For ultimate diagnostic application, overall results need to be improved. Future arrays should contain probes from both DNA strands, and to obtain a more even coverage, one could add fewer probes from densely and more probes from sparsely covered regions
Detection of virulence-associated genes of Pasteurella multocida isolated from cases of fowl cholera by multiplex-PCR
Divergent cerebrospinal fluid cytokine network induced by non-viral and different viral infections on the central nervous system
Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus
T-RECs: rapid and large-scale detection of recombination events among different evolutionary lineages of viral genomes
Risks associated with the use of live-attenuated vaccine poliovirus strains and the strategies for control and eradication of paralytic poliomyelitis
The Global Polio Eradication Initiative was launched in 1988 with the aim to eliminate paralytic poliomyelitis. Two effective vaccines are available: inactivated polio vaccine (IPV) and oral polio vaccine (OPV). Since 1964, OPV has been used instead of IPV in most countries due to several economic and biological advantages. However, in rare cases, the live-attenuated Sabin strains of OPV revert to neurovirulence and cause vaccine-associated paralytic poliomyelitis in vaccinees or lead to emergence of vaccine-derived poliovirus strains. Attenuating mutations and recombination events have been associated with the reversion of vaccine strains to neurovirulence. The substitution of OPV with an improved new-generation IPV and the availability of new specific drugs against polioviruses are considered as future strategies for outbreak control and the eradication of paralytic poliomyelitis worldwide
Amplification of Echoviruses genomic regions by different RT-PCR protocols - a comparative study
In the present report, the results of a comparative study in the
detection of all Echoviruses reference strains as well as of 38 clinical
isolates are presented. Using RT-PCR with already published primer pairs
(UG(52)-UC53, 292-222, 012-011 and EUG2a, 2b, 2c-EUC2) from the 5âUTR,
the VP1 region as well as a long genomic fragment including the VP1 3â
end, the entire coding sequence of 2A, 2B, and the 5â moiety of the
2C-coding region amplification was effective with all reference and
clinical Echovirus isolates with primer pair UG52-UC53 while with
292-222 and 012-011 were amplified 27/28 reference Echovirus strains and
all clinical isolates. As far as EUG2a,2b,2c-EUC2 is concerned, the
RT-PCR gave a positive result for 26/28 reference Echovirus strains and
34/38 clinical isolates. The sequence analysis of a large part of the
5âUTR has revealed that there is no correlation between 5âUTR identity
and the currently recognized human enterovirus species. It has been
suggested that part of VP1 coding sequence would correlate well with
serotype since a number of important neutralization epitopes, as well as
receptor recognition sequences, lie within the VP1 coding sequence.
Therefore, UG52-UC53 and 292-222 primer pairs seem to be the most
appropriate for Echovirus detection and, moreover, UG52-UC53 is useful
for the classification of enteroviruses into genetic clusters
(sub-groups) while 292-222 for the identification of enteroviruses by
amplicon sequencing. (C) 2004 Elsevier Ltd. All rights reserved
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