Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

BACKGROUND: Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. METHODS: The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. CONCLUSIONS: The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. SIGNIFICANCE: The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food

Identification of GBV-D, a Novel GB-like Flavivirus from Old World Frugivorous Bats (Pteropus giganteus) in Bangladesh

Bats are reservoirs for a wide range of zoonotic agents including lyssa-, henipah-, SARS-like corona-, Marburg-, Ebola-, and astroviruses. In an effort to survey for the presence of other infectious agents, known and unknown, we screened sera from 16 Pteropus giganteus bats from Faridpur, Bangladesh, using high-throughput pyrosequencing. Sequence analyses indicated the presence of a previously undescribed virus that has approximately 50% identity at the amino acid level to GB virus A and C (GBV-A and -C). Viral nucleic acid was present in 5 of 98 sera (5%) from a single colony of free-ranging bats. Infection was not associated with evidence of hepatitis or hepatic dysfunction. Phylogenetic analysis indicates that this first GBV-like flavivirus reported in bats constitutes a distinct species within the Flaviviridae family and is ancestral to the GBV-A and -C virus clades