Evolving methods for single nucleotide polymorphism detection: Factor V Leiden mutation detection.

BACKGROUND: The many techniques used to diagnose the Factor V Leiden (FVL) mutation, the most common hereditary hypercoagulation disorder in Eurasians, and the most frequently requested genetic test reflect the evolving strategies in protein and DNA diagnosis. METHODS: Here, molecular methods to diagnose the FVL mutation are discussed. RESULTS: Protein-based detection assays include the conventional functional activated protein C resistance coagulation test and the recently reported antibody-mediated sensor detection; and DNA-based assays include approaches that use electrophoretic fractionation e.g., restriction fragment length polymorphism, denaturing gradient gel electrophoresis, and single-stranded conformational PCR analysis, DNA hybridization (e.g., microarrays), DNA polymerase-based assays, e.g., extension reactions, fluorescence polarization template-directed dye-terminator incorporation, PCR assays (e.g., amplification-refractory mutation system, melting curve analysis using real-time quantitative PCR, and helicase-dependent amplification), DNA sequencing (e.g., direct sequencing, pyrosequencing), cleavase-based Invader assay and ligase-based assays (e.g., oligonucleotide ligation assay and ligase-mediated rolling circle amplification). CONCLUSION: The method chosen by a laboratory to diagnose FVL not only depends on the available technical expertise and equipment, but also the type, variety, and extent of other genetic disorders being diagnosed.

qnrB, another plasmid-mediated gene for quinolone resistance.

A novel plasmid-mediated quinolone resistance gene, qnrB, has been discovered in a plasmid encoding the CTX-M-15 beta-lactamase from a Klebsiella pneumoniae strain isolated in South India. It has less than 40% amino acid identity with the original qnr (now qnrA) gene or with the recently described qnrS but, like them, codes for a protein belonging to the pentapeptide repeat family. Strains with qnrB demonstrated low-level resistance to all quinolones tested. The gene has been cloned in an expression vector attaching a polyhistidine tag, which facilitated purification to >or=95% homogeneity. As little as 5 pM of QnrB-His6 protected purified DNA gyrase against inhibition by 2 microg/ml (6 microM) ciprofloxacin. With a PCR assay qnrB has been detected in Citrobacter koseri, Enterobacter cloacae, and Escherichia coli isolates from the United States, linked to SHV-12 beta-lactamase and coding for a product differing in five amino acids from the Indian (now QnrB1) variety. The qnrB gene has been found near Orf1005 in some, but not all, plasmids and in association with open reading frames matching known chromosomal genes, suggesting that it too was acquired by plasmids from an as-yet-unknown bacterial source.

Role of efflux pumps and mutations in genes for topoisomerases II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains.

We have investigated the occurrence of mutations in topoisomerase II (DNA gyrase) subunit B(gyrB) and topoisomerase IV subunit E(parE) and the hyperexpression of genes for four efflux pump proteins in 20 previously described, fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa. Amino acid alterations were found in GyrB in five strains and in ParE in three strains with MIC of norfloxacin > or = 8 mg/L, and it is likely that some of the alterations contribute to the quinolone resistance exhibited by these strains. Seventeen of the 20 strains overproduced mRNA for one or more pump proteins (MexB, MexD, MexF, or MexY), which caused multidrug resistance phenotype in more than half of strains. Two strains were hypermutable and one of them was highly resistant, but the other strain was only moderately resistant.