Erroneous or Arrhenius: A Degradation Rate-Based Model for EPDM during Homogeneous Ageing.

To improve the predictive capability of long-term stress relaxation of elastomers during thermo-oxidative ageing, a method to separate reversible and irreversible processes was adopted. The separation is performed through the analysis of compression set after tempering. On the basis of this separation, a numerical model for long-term stress relaxation during homogeneous ageing is proposed. The model consists of an additive contribution of physical and chemical relaxation. Computer simulations of compression stress relaxation were performed for long ageing times and the results were validated with the Arrhenius treatment, the kinetic study and the time-temperature superposition technique based on experimental data. For chemical relaxation, two decay functions are introduced each with an activation energy and a degradative process. The first process with the lower activation energy dominates at lower ageing times, while the second one with the higher activation energy at longer ageing times. A degradation-rate based model for the evolution of each process and its contribution to the total system during homogeneous ageing is proposed. The main advantage of the model is the possibility to quickly validate the interpolation at lower temperatures within the range of slower chemical processes without forcing a straight-line extrapolation.

Analysis of O-Ring Seal Failure under Static Conditions and Determination of End-of-Lifetime Criterion.

Determining a suitable and reliable end-of-lifetime criterion for O-ring seals is an important issue for long-term seal applications. Therefore, seal failure of ethylene propylene diene rubber (EPDM) and hydrogenated nitrile butadiene rubber (HNBR) O-rings aged in the compressed state at 125 °C and at 150 °C for up to 1.5 years was analyzed and investigated under static conditions, using both non-lubricated and lubricated seals. Changes of the material properties were analyzed with dynamic-mechanical analysis and permeability experiments. Indenter modulus measurements were used to investigate DLO effects. It became clear that O-rings can remain leak-tight under static conditions even when material properties have already degraded considerably, especially when adhesion effects are encountered. As a feasible and reliable end-of-lifetime criterion for O-ring seals under static conditions should include a safety margin for slight dimensional changes, a modified leakage test involving a small and rapid partial decompression of the seal was introduced that enabled determining a more realistic but still conservative end-of-lifetime criterion for an EPDM seal.

Scission, Cross-Linking, and Physical Relaxation during Thermal Degradation of Elastomers.

Elastomers are susceptible to chemical ageing, i.e., scission and cross-linking, at high temperatures. This thermally driven ageing process affects their mechanical properties and leads to limited operating time. Continuous and intermittent stress relaxation measurements were conducted on ethylene propylene diene rubber (EPDM) and hydrogenated nitrile butadiene rubber (HNBR) samples for different ageing times and an ageing temperature of 125 °C. The contributions of chain scission and cross-linking were analysed for both materials at different ageing states, elucidating the respective ageing mechanisms. Furthermore, compression set experiments were performed under various test conditions. Adopting the two-network model, compression set values were calculated and compared to the measured data. The additional effect of physical processes to scission and cross-linking during a long-term thermal exposure is quantified through the compression set analysis. The characteristic times relative to the degradation processes are also determined.

Initial Reverse Transcription is Essential for Reliable Helicobacter pylori 23S rDNA Real-Time PCR Diagnostics from Fecal Samples.

BACKGROUND: A sensitive, non-invasive molecular test for identifying Helicobacter pylori (H. pylori) in stool samples is important in the context of a "test-and-treat strategy". The goal of this study was to elucidate the benefit of an initial reverse transcription (RT) step for the sensitivity of H. pylori 23S rDNA Real-time PCR in stool samples. METHODS: We compared a LightMix® Modular Helicobacter 23S rDNA PCR assay without (LightMix® PCR) and with an initial reverse transcription step (LightMix® RT-PCR) assay for rapid detection of Helicobacter pylori in fecal samples. For maximum sensitivity, all Lightmix® RT-PCR assays were performed in triplicate for each sample. Additionally, the LightMix® RT-PCR was compared to H. pylori AG ELISA. RESULTS: Direct detection of H. pylori in feces by Lightmix® PCR was less sensitive than LightMix® RT-PCR. Only 2 out of 44 stool samples (4.5%) from patients with diarrhea were positive for H. pylori in the LightMix® PCR assay. In contrast, a reverse transcription step prior Lightmix® PCR increased the assay sensitivity markedly (19 out of 44 positives, 43.2%). When re-testing 65 samples initially analyzed with H. pylori AG ELISA with LightMix® RT-PCR, the detection rate for H. pylori was similarly increased from 23.1% (15/65) with H. pylori AG ELISA to 43.1% (28/65) with H. pylori LightMix® RT-PCR. 21.5% of the 28 H. pylori positive samples were positive in 1/3, 32% in 2/3, and 46.5% in 3/3 triple RT-PCR approaches. When re-testing the 28 LightMix® RT-PCR positive samples by LightMix® PCR, only 17.9% (5/28) RT-PCR positive samples were positive in PCR. CONCLUSIONS: LightMix® RT-PCR is much more sensitive than Lightmix® PCR. In comparison to H. pylori AG ELISA, the LightMix® RT-PCR shows a markedly higher sensitivity. An initial reverse transcription step is crucial for reliable Helicobacter pylori 23S rDNA Real-time PCR diagnostics. The triple RT-PCR approach can additionally improve the detection of H. pylori in fecal samples. Thus, the method presented provides a highly sensitive, noninvasive assay to detect H. pylori in fecal samples with potential advantages compared to other H. pylori detection methods currently used in routine diagnostics.

Overnight Enrichment is Essential for Reliable Salmonella PCR Testing from Fecal Samples.

BACKGROUND: A rapid molecular test for identifying cases of infectious diarrhea might be beneficial for patients. METHODS: We established a LightMix modular multiplex PCR assay for rapid detection of Campylobacter, Salmonella, Yersinia, and Shigella species from fecal samples. RESULTS: Unlike Campylobacter sp., direct detection of Salmonella from feces by multiplex PCR was significantly less sensitive than culture. Only 11 out of 21 (52%) Salmonella culture positive specimens were positive for Salmonella in the direct multiplex PCR assay (Campylobacter: 52/53 samples [98%]). In contrast, an overnight selenite enrichment step prior to multiplex PCR increased the Salmonella assay sensitivity significantly: 18 out of 18 Salmonella culture positive samples were then also detected by PCR. CONCLUSIONS: An overnight enrichment step is necessary for reliable PCR detection of Salmonella from fecal samples.

Presence of murine leukemia virus (MLV)-related virus gene sequences in a commercial RT-PCR reagent.

BACKGROUND: The recent identification of murine leukemia virus (MLV)-related viruses in patients with chronic fatigue syndrome (CFS) has aroused much interest, not least among sufferers. However, other studies failed to detect these viruses in CFS patients. METHODS: We wanted to establish a MLV-related virus real-time PCR for routine diagnostics. RESULTS: Our study identified false positive MLV-related virus results due to a contamination of Superscript III Platinum One-Step Quantitative RT-PCR System (Invitrogen). CONCLUSIONS: This observation may be helpful to elucidate discrepant results for the detection of MLV-related virus like xenotropic MLV-related virus (XMRV) in recently published studies.

Evaluation of a real-time PCR assay for the detection, genotyping, and quantification of Borrelia burgdorferi sensu lato in Ixodes ticks in a routine laboratory setting.

BACKGROUND: Testing of saved Ixodes ticks for the presence of Borrelia burgdorferi after biting a patient may be helpful to assess the risk of bacterial transmission and to decide upon preventive antibiotic treatment. A commercially available real-time PCR assay for the identification and quantification of Borrelia burdorferi sensu lato species (LightMix Kit for the detection of Borrelia spp., TIB MOLBIOL) was evaluated. METHODS: Clinical and laboratory specimens were analysed for Borrelia DNA, including seven official proficiency panels (INSTAND, Germany, 2004 to 2009)), kit standards and 1121 ticks saved from bitten patients from all over Germany. RESULTS: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia spielmanii, and Borrelia valaisiana were reliably detected by the real-time PCR. The recently described Borrelia lusitaniae (not pathogenic for humans) was not detected. Melt curve analysis of the PCR products allowed for a reliable Borrelia genospecies differentiation. Dilution series of proficiency panel samples revealed a detection limit of approximately 10(1) genomes/mL. Based on CP values the mean interassay coefficient of variation of the assay was 3.6%, while the intraassay coefficients of variation were 1.6% and 0.8% for a strong positive and a weak positive kit standard, respectively. 20.2% of the clinical ticks revealed detectable Borrelia burgdorferi DNA, with Borrelia afzelii being the most prominent subspecies. The quantification of Borrelia genomes in individual ticks showed a broad distribution ranging from 0.5 x 10(1) to 2.6 x 10(7) Borrelia equivalents per tick (median 1.6 x 10(4)). CONCLUSIONS: The LightMix Kit for the detection of Borrelia spp. represents an excellent tool for the identification and quantification of Borrelia species in ticks. It can serve to further elucidate the relevance of preventive antibiotic treatment following tick bites in upcoming clinical studies.

Rapid and sensitive detection of Shiga toxin-producing Escherichia coli directly from stool samples by real-time PCR in comparison to culture, enzyme immunoassay and Vero cell cytotoxicity assay.

BACKGROUND: From May until July 2011 a large outbreak of infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) occurred in Germany. More than 800 patients suffered from hemolytic uremic syndrome (HUS), and 49 fatal cases were reported. Obviously, a mandatory requirement for such a clinical situation is the availability of rapid and reliable STEC tests from the investigating laboratory. The standard methods like enzyme immunoassay (EIA), vero cell cytotoxicity assay (VCA), and microbiological culture are, however, hampered by a lack of sensitivity and specificity unless a prior, time consuming broth enrichment step is employed. In order to acelerate the laboratory diagnosis, we evaluated an in-house real-time PCR assay for the detection of the Stx genes (stx1 and stx2) directly from stool specimens without the need of broth enrichment procedures. METHODS: 754 faecal samples were collected from 481 predominantly hospitalised patients with diarrhea from May 23 to June 10, 2011 at the Medical Laboratory Bremen, Germany. The samples were analysed with a direct stx real-time PCR and compared to EIA, VCA and culturing on enterohemolysin, ESBL, and CPS agar after broth enrichment. In addition, artificial samples (n = 12) from three official EHEC/STEC PCR quality proficiency panels (INSTAND, Germany, September 2006, September 2007, and April 2008) were analysed by real-time PCR only. RESULTS: The real-time PCR produced reliable, distinct melting profiles with characteristic peaks for the stx1 and stx2 PCR products. The quality proficiency panels revealed a detection limit of 10 CFU/PCR per reaction. 112, 86, 99, and 122 of 754 clinical samples were positive for culture, EIA, VCA, and real-time PCR, respectively. 121 of 122 PCR samples were positive only for stx2. Compared to culture as the gold standard, sensitivities of EIA, VCA, and real-time PCR were 76.8 %, 83.9%, and 96.4% and specificities were 99.4%, 99.2%, and 97.8%, respectively. CONCLUSIONS: The direct fecal stx real-time PCR proved superior to enrichment based VCA and EIA and can be recommended as a quick and sensitive tool for the early diagnosis of STEC infection in addition to microbiological culture.

Comparison of the Roche COBAS Amplicor Monitor, Roche COBAS Ampliprep/COBAS Taqman and Abbott RealTime Test assays for quantification of hepatitis C virus and HIV RNA.

BACKGROUND: We have evaluated the performance of two newly developed automated real-time PCR assays, the COBAS Ampliprep/COBAS TaqMan (CAP/CTM) and the Abbott RealTime tests, in the quantification of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA. The widely used semi-automated COBAS Amplicor Monitor (CAM) assay served as the reference test. METHODS: Several specimens were analyzed, including 102 plasma samples from HCV patients and 109 from HIV patients and 10 samples from negative donors, as well as Quality Control in Molecular Diagnostics (QCMD) and National Institute for Biological Standards and Controls (NIBSC) proficiency program panels. RESULTS: Good correlation was observed among the three assays, with correlation coefficients (R2) of 0.8 (CAM-CAP/CTM), 0.89 (CAM-RealTime) and 0.91 (CAP/CTM-RealTime) for HCV and 0.83 (CAM-RealTime), 0.85 (CAM-CAP/CTM) and 0.89 (CAP/CTM-RealTime) for HIV. The overall concordance for negative/positive results was 100% for HCV and 98% for HIV. All assays were equally able to quantify HCV genotypes 1, 3, 5 and HIV group M (subtypes A-H) and N from QCMD and NIBSC panels. In terms of workflow, the RealTime assay requires more hands-on-time than the CAP/CTM assay. CONCLUSIONS: The results indicate that real-time PCR assays can improve the efficiency of end-point PCR tests by better covering viral dynamic ranges and providing higher throughput and automation.