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Real-time polymerase chain reaction (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, that is, amplification inhibition. Humic substances (HS) are well-known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, that is, quench the fluorescence signal of double-stranded DNA (dsDNA) binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I, and SYTO 82, generating lowered amplification plots, although amplicon production was unaffected. For EvaGreen, 500ng of HA quenched nearly all fluorescence, whereas 1000ng of HA completely inhibited amplification when applying Immolase DNA polymerase with bovine serum albumin (BSA). Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays.

Objectives To determine the genetic and environmental influence on the liability for stress urinary incontinence and pelvic organ prolapse surgery. Methods We used the Swedish Twin Registry to identify all same-sex female twin pairs with known zygosity born from 1926 to 1958. Occurrence of stress urinary incontinence and pelvic organ prolapse surgery was determined by cross-linkage to the Swedish Inpatient Registry. By comparing mono- and dizygotic female twins, we determined twin similarity and the relative proportions of phenotypic variance resulting from genetic and environmental factors. Using statistical modelling, the liability for pelvic floor surgery explained by heritability versus shared and non-shared environmental effects was estimated. Results We identified 3376 complete monozygotic and 5067 dizygotic same-sex female twin pairs. There was greater twin similarity among monozygotic compared with dizygotic twins, which indicates the influence of a genetic component to the aetiology of both stress urinary incontinence and pelvic organ prolapse surgery. For both disorders, genetic and non-shared environmental factors equally contributed about 40% of the variation in liability. Shared environment accounted for approximately one fifth of the total variance for the two disorders. Conclusions Genetic effects contribute to the occurrence of both stress urinary incontinence and pelvic organ prolapse, but the influence of environmental factors is substantial. Significant environmental effects suggest that the liability for benign pelvic floor surgery is amenable to intervention.

Tularemia is a plague-like infection caused by Francisella (F.) tularensis classified as a biological warfare agent. F. tularensis subsp. tularensis is the most virulent subspecies demanding rapid diagnosis. Typing systems for this fastidious bacterium to the subspecies level are laborious and time consuming. Therefore, the aim of this study was to develop a real-time PCR for the rapid and specific identification of F. tularensis subsp. tularensis. The specificity of the assay was determined using a comprehensive panel of Francisella strains, clinically relevant bacteria, and DNA preparations of potential hosts. F. tularensis subsp. tularensis was specifically detected but no other organisms. The range of linearity was determined to be 100 fg to 10 ng, the lower limit of detection was 25 fg of DNA (13 genome equivalents). An internal amplification control PCR system targeting lambda phage DNA was included. Neither the internal amplification control nor host DNA influenced the cycle threshold values obtained for F. tularensis subsp. tularensis. In conclusion, we have developed a highly sensitive and specific assay that can be integrated into real-time PCR-based identification procedures for biological agents. This is a major diagnostic improvement, as all other methods for the specific identification of F. tularensis subsp. tularensis are more time consuming.

Polycyclic aromatic hydrocarbons (PAHs) are of environmental concern, for instance when found in contaminated soils at sites where industrial activities have occurred. For efficient screening of su ...

With the objective of monitoring xenobiotic degrading bacteria in soil, a method for rapid extraction of DNA from soil, amenable to amplification by PCR, was developed. The method was based on lysis by freeze-thawing and subsequent addition of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide and proteinase K. The extraction method required 2 h and was tested on six different soils differing in organic content, water holding capacity and pH, including ones from which DNA extraction is difficult. DNA yields from the soils ranged from 6.1 to 54.0 micrograms of DNA per g soil. The efficiency and reproducibility of the DNA extraction method were evaluated by competitive PCR. The organic content in the soils was a major factor affecting the amount of obtained DNA amenable for amplification by PCR. A PCR primer-pair was designed on the basis of the known nucleotide sequences of several catechol 2,3-dioxygenase genes. The specificity of the primer-pair was demonstrated on different sequenced catechol 2,3-dioxygenase genes and on site-specific bacterial isolates from polycyclic aromatic hydrocarbon (PAH)-contaminated soil. The concentration of catechol 2,3-dioxygenase DNA in PAH-contaminated sediment undergoing an ex situ compost process was quantified by competitive PCR over a period of 16 weeks. The concentration of PAHs and catechol 2,3-dioxygenase DNA in the soil samples, was found to correlate.

The Streptomyces fradiae β lactamase promoter (PblaF) was sequenced and characterized by promoter probing, primer extension, and exonuclease III-mediated deletions. The transcription start point (tsp) was the same in both S. lividans and S. fradiae. Oligodeoxyribonucleotide-directed random mutations and site-specific mutations were introduced in the promoter region. The effects of these mutations on transcription were assayed by an RNA colony hybridization method. This analysis identified cis-acting sequence determinants located similarly to the −10 and −35 regions of a typical Escherichia coli promoter. Also, a change in the distance between these regions from 19 to 17 bp drastically reduced promoter activity. PblaF was shown not to be recognized by sigma-whiG or by sigma-hrdA, hrdC., or hrdD. Sequence alignment of PblaF to sigma factor-classified Streptomyces promoters revealed little homology. Thus, PblaF is probably recognized by an as yet unidentified sigma factor.