High-throughput variant detection using a color-mixing strategy in real-time PCR reactions

Many diseases are related to multiple genetic alterations along a single gene. Probing for highly multiple (>10) variants in a single qPCR tube is not possible due to a limited number of fluorescence channels and one variant per channel, so many more tubes are needed. Here, we experimentally validate our novel color-mixing strategy that uses fluorescence combinations as digital color codes to probe multiple variants simultaneously. The color-mixing strategy relies on a simple intra-tube assay that can probe for 15 variants as part of an inter-tube assay that can probe for an exponentially increased number of variants. The color-mixing strategy is achieved using multiplex double-stranded toehold probes modified with fluorophores and quenchers; the probes are designed to be quenched or luminous after binding to wildtype or variant templates. We used the color-mixing strategy to probe for 21 pathogenic mutations in thalassemia and distinguishing between heterozygous and homozygous variants in 6 tubes, with a specificity of 99% and a sensitivity of 94%. To support tuberculosis diagnosis, we used the same strategy to simultaneously probe in Mycobacterium tuberculosis for rifampicin-resistance mutations occurring within one 81-bp region and one 48-bp region in rpoB gene, plus five isoniazid-resistance mutations in inhA and katG genes.

In vitrorumen fungi quantification in medium containing sodium hydroxide or formaldehyde treated sunflower meal using quantitative competitive PCR assay

The rumen anaerobic fungi are primary colonizers of fibrous plant materials that degrade lignin-containing plant cell walls. With the advancement of molecular enumeration methods, in particular 18S rDNA gene probing methods, researchers were able to monitor fungal species within the rumen (Stahlet al., 1998). Quantitative competitive PCR (QCPCR) techniques play an important role in nucleic acid quantification because of their significant lower cost of equipment and consumables (Franzet al., 2001). The aim of this study was to use the QC-PCR assay to evaluate the relative quantitative comparison of anaerobic fungi population in medium containing sunflower meal, either untreated or treated with formaldehyde (3 and 6 g/kg DM) or sodium hydroxide (40 g/kg DM).

Dissimilatory Arsenate Reduction with Sulfide as Electron Donor: Experiments with Mono Lake Water and Isolation of Strain MLMS-1, a Chemoautotrophic Arsenate Respirer

ABSTRACT Anoxic bottom water from Mono Lake, California, can biologically reduce added arsenate without any addition of electron donors. Of the possible in situ inorganic electron donors present, only sulfide was sufficiently abundant to drive this reaction. We tested the ability of sulfide to serve as an electron donor for arsenate reduction in experiments with lake water. Reduction of arsenate to arsenite occurred simultaneously with the removal of sulfide. No loss of sulfide occurred in controls without arsenate or in sterilized samples containing both arsenate and sulfide. The rate of arsenate reduction in lake water was dependent on the amount of available arsenate. We enriched for a bacterium that could achieve growth with sulfide and arsenate in a defined, mineral medium and purified it by serial dilution. The isolate, strain MLMS-1, is a gram-negative, motile curved rod that grows by oxidizing sulfide to sulfate while reducing arsenate to arsenite. Chemoautotrophy was confirmed by the incorporation of H14CO3 − into dark-incubated cells, but preliminary gene probing tests with primers for ribulose-1,5-biphosphate carboxylase/oxygenase did not yield PCR-amplified products. Alignment of 16S rRNA sequences indicated that strain MLMS-1 was in the δ-Proteobacteria, located near sulfate reducers like Desulfobulbus sp. (88 to 90% similarity) but more closely related (97%) to unidentified sequences amplified previously from Mono Lake. However, strain MLMS-1 does not grow with sulfate as its electron acceptor.

Vero cytotoxin-producingEscherichia coli, particularly serogroup O157, associated with human infections in England and Wales: 1992–4

SummaryInvestigations were performed by the Laboratory of Enteric Pathogens on Vero cytotoxin-producingEscherichia coli(VTEC) in England and Wales from 1992–4. Bacterial isolates, faeces and sera obtained from patients with diarrhoea, bloody diarrhoea and haemolytic uraemic syndrome were examined. Using serotyping, Vero cytotoxin gene probing and serodiagnostic tests forE. coliO157, evidence of infection was detected in 543, 434 and 491 individuals in 1992, 1993 and 1994 respectively; VTEC of serogroup O157 were isolated from 470, 385 and 411 cases. The O157 VTEC strains belonged to at least 19 different phage types (PT) although 84% belonged to PT2, PT49, PT8, PT1 or PT4. Antibodies toE. coliO157 lipopolysaccharide were detected in 13% of the cases. The average annual rate of infection with O157 VTEC was 0·83/100000 and 12% of the 1458 individuals with evidence of infection with VTEC orE. coliO157 developed haemolytic uraemic syndrome. There were at least 18 general outbreaks and many family outbreaks.