Dataset Information

Development of a novel multiplex PCR assay to detect functional subtypes of KIR3DL1 alleles.

ABSTRACT: Among NK cell receptor-ligand partnerships, KIR3DL1 and HLA-Bw4 demonstrate the greatest diversity; permutations of their allelic combinations titrate NK reactivity. Balancing selection has maintained distinct subtypes of KIR3DL1 alleles in global populations, implying that each may provide unique fitness advantages and variably influence disease processes. Though approaches exist for determining HLA-B allotypes, practical methods for identifying KIR3DL1 alleles are lacking. We have developed a PCR-based approach that identifies functional subtypes of KIR3DL1 alleles; it is suitable for research and may have clinical application. Six allele subsets were identified based on expression characteristics of the eleven most common KIR3DL1 alleles represented in reported populations. The remaining 62 low-frequency alleles were distributed into these groups based on sequence homology to coding regions. Subtype-specific SNPs were found in exons 3, 4, and 7, and used as priming sites for five multiplex PCR reactions. Genomic DNA derived from 175 unrelated donors and 52 related individuals from 6 families demonstrated >99.5% concordance between sequence-based typing and our novel approach. Finally, PCR-based typing accurately predicted NK phenotypes obtained by flow cytometry after staining with DX9 and Z27 monoclonal antibodies. This novel approach facilitates high-throughput analysis of KIR3DL1 allotypes to enable a broader understanding of KIR3DL1 and HLA-Bw4 interaction in health and disease.

SUBMITTER: Boudreau JE

PROVIDER: S-EPMC4053526 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of a novel multiplex PCR assay to detect functional subtypes of KIR3DL1 alleles.

Boudreau Jeanette E JE Le Luduec Jean-Benoît JB Hsu Katharine C KC

PloS one 20140611 6

Among NK cell receptor-ligand partnerships, KIR3DL1 and HLA-Bw4 demonstrate the greatest diversity; permutations of their allelic combinations titrate NK reactivity. Balancing selection has maintained distinct subtypes of KIR3DL1 alleles in global populations, implying that each may provide unique fitness advantages and variably influence disease processes. Though approaches exist for determining HLA-B allotypes, practical methods for identifying KIR3DL1 alleles are lacking. We have developed a ...[more]

PMID: 24919192

Similar Datasets

Project description:Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenic Escherichia coli, are of considerable epidemiological, phylogenetic, and immunotherapeutic importance. However, to date, no method other than DNA sequencing has been generally available for their detection. In the present study, we developed and rigorously validated a novel PCR-based assay for the 11 recognized variants of papA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of the papA alleles among 75 E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence that papA sequences are highly conserved within each of the traditionally recognized F serotypes despite the diversity observed among F types. In certain strains, the assay detected serologically occult copies of papA, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional pap fragments. Among the urosepsis isolates, the assay revealed considerable segregation of papA alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer of papA alleles between lineages. Sequencing of papA from two strains that were papA positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel papA variants, one of which was actually more prevalent among the urosepsis isolates than were several of the known papA alleles. These findings provide novel insights into the papA alleles of extraintestinal pathogenic E. coli and indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection of papA alleles available to any laboratory with PCR capability.

Project description:BACKGROUND: Papillomaviruses (PVs) are a group of small, non-encapsulated, species-specific DNA viruses that have been detected in a variety of mammalian and avian species including humans, canines and felines. PVs cause lesions in the skin and mucous membranes of the host and after persistent infection, a subset of PVs can cause tumors such as cervical malignancies and head and neck squamous cell carcinoma in humans. PVs from several species have been isolated and their genomes have been sequenced, thereby increasing our understanding of the mechanism of viral oncogenesis and allowing for the development of molecular assays for the detection of PV infection. In humans, molecular testing for PV DNA is used to identify patients with persistent infections at risk for developing cervical cancer. In felids, PVs have been isolated and sequenced from oral papillomatous lesions of several wild species including bobcats, Asian lions and snow leopards. Since a number of wild felids are endangered, PV associated disease is a concern and there is a need for molecular tools that can be used to further study papillomavirus in these species. RESULTS: We used the sequence of the snow leopard papillomavirus UuPV1 to develop a PCR strategy to amplify viral DNA from samples obtained from captive animals. We designed primer pairs that flank the E6 and E7 viral oncogenes and amplify two DNA fragments encompassing these genes. We detected viral DNA for E6 and E7 in genomic DNA isolated from saliva, but not in paired blood samples from snow leopards. We verified the identity of these PCR products by restriction digest and DNA sequencing. The sequences of the PCR products were 100% identical to the published UuPV1 genome sequence. CONCLUSIONS: We developed a PCR assay to detect papillomavirus in snow leopards and amplified viral DNA encompassing the E6 and E7 oncogenes specifically in the saliva of animals. This assay could be utilized for the molecular investigation of papillomavirus in snow leopards using saliva, thereby allowing the detection of the virus in the anatomical site where oral papillomatous lesions develop during later stages of infection and disease development.

Dataset Information

Development of a novel multiplex PCR assay to detect functional subtypes of KIR3DL1 alleles.

Publications

Development of a novel multiplex PCR assay to detect functional subtypes of KIR3DL1 alleles.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets