Detecting and resolving position-dependent temperature effects in real-time quantitative polymerase chain reaction

Real-time quantitative polymerase chain reaction (qPCR) depends on precise temperature control of the sample during cycling. In the current study, we investigated how temperature variation in plate-based qPCR instruments influences qPCR results. Temperature variation was measured by amplicon melting analysis as a convenient means to assess well-to-well differences. Multiple technical replicates of several SYBR Green I-based qPCR assays allowed correlation of relative well temperature to quantification cycle. We found that inadequate template denaturation results in an inverse correlation and requires increasing the denaturation temperature, adding a DNA destabilizing agent, or pretreating with a restriction enzyme. In contrast, inadequate primer annealing results in a direct correlation and requires lowering the annealing temperature. Significant correlations were found in 18 of 25 assays. The critical nature of temperature-dependent effects was shown in a blinded study of 29 patients for the diagnosis of Prader-Willy and Angelman syndromes, where eight diagnoses were incorrect unless temperature-dependent effects were controlled. A method to detect temperature-dependent effects by pairwise comparisons of replicates in routine experiments is presented and applied. Systematic temperature errors in qPCR instruments can be recognized and their effects eliminated when high precision is required in quantitative genetic diagnostics and critical complementary DNA analyses.

Real-time adaptive models for the personalized prediction of glycemic profile in type 1 diabetes patients

Prediction of glycemic profile is an important task for both early recognition of hypoglycemia and enhancement of the control algorithms for optimization of insulin infusion rate. Adaptive models for glucose prediction and recognition of hypoglycemia based on statistical and artificial intelligence techniques are presented.

Occurrence of Chlamydiaceae, Mycoplasma conjunctivae, and pestiviruses in Alpine chamois (Rupicapra r. rupicapra) of Grisons, Switzerland

Because interactions between livestock and chamois occur on Alpine pastures, transmission of infectious diseases is considered possible. Thus, the occurrence of Chlamydiaceae, Mycoplasma conjunctivae, and pestiviruses in Alpine chamois (Rupicapra r. rupicapra) of the Surselva region (eastern Swiss Alps) was investigated. In total, 71 sera, 158 eye swabs, 135 tissue samples, and 23 fecal samples from 85 chamois were analyzed. The sera were tested by 2 enzyme-linked immunosorbent assay (ELISA) kits specific for Chlamydophila abortus. Eye swabs, tissue, and fecal samples were examined by a Chlamydiaceae-specific real-time polymerase chain reaction (PCR). Positive cases were further investigated by microarray method. One serum sample (1.4%) was positive in 1 of the ELISAs. Eye swabs of 3 chamois (3.8%) were positive for Chlamydiaceae. The microarray method revealed the presence of Chlamydophila abortus, C pecorum, and C pneumoniae. All tissue and fecal samples were negative. With real-time PCR, 3.9% of the chamois tested positive for Mycoplasma conjunctivae. One chamois had a simultaneous infection with Al. conjunctivae and 2 chlamydial species (C abortus, C. pecorum). Skin and tongue tissue samples of 35 chamois were negative for pestivirus antigen by immunohistochemistry. It was concluded that in contrast to the findings in Pyrenean chamois (Capra p. pyrenaica) of Spain, the occurrence of Chlamydiaceae in Alpine chamois of the Surselva region is low, and the transmission between domestic and wild Caprinae seems not to be frequent. Comparably, persistent pestiviral infections do not seem to be common in chamois of the Surselva region.

Real-time ultrasound-guided spinal anaesthesia: a prospective observational study of a new approach

Identification of the subarachnoid space has traditionally been achieved by either a blind landmark-guided approach or using prepuncture ultrasound assistance. To assess the feasibility of performing spinal anaesthesia under real-time ultrasound guidance in routine clinical practice we conducted a single center prospective observational study among patients undergoing lower limb orthopaedic surgery. A spinal needle was inserted unassisted within the ultrasound transducer imaging plane using a paramedian approach (i.e., the operator held the transducer in one hand and the spinal needle in the other). The primary outcome measure was the success rate of CSF acquisition under real-time ultrasound guidance with CSF being located in 97 out of 100 consecutive patients within median three needle passes (IQR 1-6). CSF was not acquired in three patients. Subsequent attempts combining landmark palpation and pre-puncture ultrasound scanning resulted in successful spinal anaesthesia in two of these patients with the third patient requiring general anaesthesia. Median time from spinal needle insertion until intrathecal injection completion was 1.2 minutes (IQR 0.83-4.1) demonstrating the feasibility of this technique in routine clinical practice.

Real-time evaluation of diffusion of the local anesthetic solution during peribulbar block using ultrasound imaging and clinical correlates of diffusion

The aims of this prospective observational study were to assess the incidence of intraconal spread during peribulbar (extraconal) anesthesia by real-time ultrasound imaging of the retro-orbital compartment and to determine whether a complete sensory and motor block (with akinesia) of the eye is directly related to the intraconal spread.

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.

PCR-based diagnosis of Naegleria sp. infection in formalin-fixed and paraffin-embedded brain sections

We developed a real-time PCR which allowed the highly sensitive detection of Naegleria fowleri in histological brain tissue sections from experimentally infected mice. This genus-specific small-subunit (18S) rRNA gene-based PCR can complement conventional (immuno-) histology for the diagnosis of primary amoebic meningoencephalitis in paraffin-embedded brain necropsy specimens that had been fixed in formalin buffered with phosphate-buffered saline.

Detection of exon deletions within an entire gene (CFTR) by relative quantification on the LightCycler

BACKGROUND: Cystic fibrosis (CF) is associated with at least 1 pathogen point sequence variant on each CFTR allele. Some symptomatic patients, however, have only 1 detectable pathogen sequence variant and carry, on the other allele, a large deletion that is not detected by conventional screening methods. METHODS: For relative quantitative real-time PCR detection of large deletions in the CFTR gene, we designed DNA-specific primers for each exon of the gene and primers for a reference gene (beta2-microglobulin). For PCR we used a LightCycler system (Roche) and calculated the gene-dosage ratio of CFTR to beta2-microglobulin. We tested the method by screening all 27 exons in 3 healthy individuals and 2 patients with only 1 pathogen sequence variant. We then performed specific deletion screenings in 10 CF patients with known large deletions and a blinded analysis in which we screened 24 individuals for large deletions by testing 8 of 27 exons. RESULTS: None of the ratios for control samples were false positive (for deletions or duplications); moreover, for all samples from patients with known large deletions, the calculated ratios for deleted exons were close to 0.5. In addition, the results from the blinded analysis demonstrated that our method can also be used for the screening of single individuals. CONCLUSIONS: The LightCycler assay allows reliable and rapid screening for large deletions in the CFTR gene and detects the copy number of all 27 exons.

Extracellular nucleotides induce migration of renal mesangial cells by upregulating sphingosine kinase-1 expression and activity

BACKGROUND AND PURPOSE: Extracellular nucleotides act as potent mitogens for renal mesangial cells (MC). In this study we determined whether extracellular nucleotides trigger additional responses in MCs and the mechanisms involved. EXPERIMENTAL APPROACH: MC migration was measured after nucleotide stimulation in an adapted Boyden-chamber. Sphingosine kinase-1 (SK-1) protein expression was detected by Western blot analysis and mRNA expression quantified by real-time PCR. SK activity was measured by an in vitro kinase assay using sphingosine as substrate. KEY RESULTS: Nucleotide stimulation caused biphasic activation of SK-1, but not SK-2. The first peak occurred after minutes of stimulation and was followed by a second delayed peak after 4-24 h of stimulation. The delayed activation of SK-1 is due to increased SK-1 mRNA steady-state levels and de novo synthesis of SK-1 protein, and depends on PKC and the classical MAPK cascade. To see whether nucleotide-stimulated cell responses require SK-1, we selectively depleted SK-1 from cells by using small-interference RNA (siRNA). MC migration is highly stimulated by ATP and UTP; this is mimicked by exogenously added S1P. Depletion of SK-1 by siRNA drastically reduced the effect of ATP and UTP on cell migration but not on cell proliferation. Furthermore, MCs isolated from SK-1-deficient mice were completely devoid of nucleotide-induced migration. CONCLUSIONS AND IMPLICATIONS: These data show that extracellular nucleotides besides being mitogenic also trigger MC migration and this cell response critically requires SK-1 activity. Thus, pharmacological intervention of SK-1 may have impacts on situations where MC migration is important such as during inflammatory kidney diseases.

Real-time closed-loop electrophysiology: towards new frontiers in in vitro investigations in the neurosciences

Reflected at any level of organization of the central nervous system, most of the processes ranging from ion channels to neuronal networks occur in a closed loop, where the input to the system depends on its output. In contrast, most in vitro preparations and experimental protocols operate autonomously, and do not depend on the output of the studied system. Thanks to the progress in digital signal processing and real-time computing, it is now possible to artificially close the loop and investigate biophysical processes and mechanisms under increased realism. In this contribution, we review some of the most relevant examples of a new trend in in vitro electrophysiology, ranging from the use of dynamic-clamp to multi-electrode distributed feedback stimulation. We are convinced these represents the beginning of new frontiers for the in vitro investigation of the brain, promising to open the still existing borders between theoretical and experimental approaches while taking advantage of cutting edge technologies.

Low prevalence of SV40 in Swiss mesothelioma patients after elimination of false-positive PCR results

The association of simian virus 40 (SV40) with malignant pleural mesothelioma is currently under debate. In some malignancies of viral aetiology, viral DNA can be detected in the patients' serum or plasma. To characterize the prevalence of SV40 in Swiss mesothelioma patients, we optimized a real-time PCR for quantitative detection of SV40 DNA in plasma, and used a monoclonal antibody for immunohistochemical detection of SV40 in mesothelioma tissue microarrays. Real-time PCR was linear over five orders of magnitude, and sensitive to a single gene copy. Repeat PCR determinations showed excellent reproducibility. However, SV40 status varied for independent DNA isolates of single samples. We noted that SV40 detection rates by PCR were drastically reduced by the implementation of strict room compartmentalization and decontamination procedures. Therefore, we systematically addressed common sources of contamination and found no cross-reactivity with DNA of other polyomaviruses. Contamination during PCR was rare and plasmid contamination was infrequent. SV40 DNA was reproducibly detected in only 4 of 78 (5.1%) plasma samples. SV40 DNA levels were low and not consistently observed in paired plasma and tumour samples from the same patient. Immunohistochemical analysis revealed a weak but reproducible SV40 staining in 16 of 341 (4.7%) mesotheliomas. Our data support the occurrence of non-reproducible SV40 PCR amplifications and underscore the importance of proper sample handling and analysis. SV40 DNA and protein were found at low prevalence (5%) in plasma and tumour tissue, respectively. This suggests that SV40 does not appear to play a major role in the development of mesothelioma.