Analogue transformations in physics and their application to acoustics.

Transformation optics has shaped up a revolutionary electromagnetic design paradigm, enabling scientists to build astonishing devices such as invisibility cloaks. Unfortunately, the application of transformation techniques to other branches of physics is often constrained by the structure of the field equations. We develop here a complete transformation method using the idea of analogue spacetimes. The method is general and could be considered as a new paradigm for controlling waves in different branches of physics, from acoustics in quantum fluids to graphene electronics. As an application, we derive an "analogue transformation acoustics" formalism that naturally allows the use of transformations mixing space and time or involving moving fluids, both of which were impossible with the standard approach. To demonstrate the power of our method, we give explicit designs of a dynamic compressor, a spacetime cloak for acoustic waves and a carpet cloak for a moving aircraft.

A real-time PCR approach based on SPF10 primers and the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus.

The SPF10 PCR targets a conserved 65bp region of the HPV L1 gene for broad-spectrum amplification. The LiPA assay allows subsequent genotyping of the HPV amplicons. This study aims to develop a SPF10 real-time PCR to achieve simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. The real-time PCR shows an analytical sensitivity of 29.7 copies for HPV 6, 16, 18 and 31 and an HPV-specific melting peak. Thirty-one HPV DNA plasmids were genotyped correctly using the SPF10 real-time PCR in combination with the LiPA. Here, the LiPA assay was performed at an increased hybridisation temperature (49.5°C) in combination with a reduced amplicon volume (1µl) to avoid cross-reactivity. In conclusion, the SPF10 real-time PCR proves to be very sensitive and generates amplicons, which are compatible with the LiPA.

WITHDRAWN: A real-time PCR approach based on SPF10 primers and the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus.

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Experimental demonstration of the supersonic-subsonic bifurcation in the circular jump: a hydrodynamic white hole.

We provide an experimental demonstration that the circular hydraulic jump represents a hydrodynamic white hole or gravitational fountain (the time reverse of a black hole) by measuring the angle of the Mach cone created by an object in the "supersonic" inner flow region. We emphasize the general character of this gravitational analogy by showing theoretically that the white hole horizon constitutes a stationary and spatial saddle-node bifurcation within dynamical-systems theory. We also demonstrate that the inner region has a "superluminal" dispersion relation, that is, that the group velocity of the surface waves increases with frequency, and discuss some possible consequences with respect to the robustness of Hawking radiation. Finally, we point out that our experiment shows a concrete example of a possible "trans-Planckian distortion" of black or white holes.

Design of a 5' exonuclease-based real-time PCR assay for simultaneous detection of Bacillus licheniformis, members of the 'B. cereus group' and B. fumarioli in gelatine.

AIMS: The design of a fast, sensitive and specific detection method for Bacillus licheniformis, members of the 'B. cereus group' and B. fumarioli in gelatine. METHODS AND RESULTS: Specific Taqman probes were designed and tested in a real-time PCR setting. A specific fluorescent signal could be obtained for all gelatine isolates attributed to these species in one single real-time PCR reaction. After sample preparation, a gelatine sample spiked with 1 CFU provided enough template DNA for a significant signal. CONCLUSION: The potential of a real-time PCR assay for simultaneous detection of B. licheniformis, members of the 'B. cereus group' and B. fumarioli in gelatine is demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of the assay in gelatine producing plants may shorten delivery terms and inform on hazards to public health and suitable remediation procedures.

Evaluation of a polymerase chain reaction reverse hybridization line probe assay for the detection and identification of medically important fungi in bronchoalveolar lavage fluids.

An assay system in which polymerase chain reaction (PCR) amplification of the ITS-1 region of ribosomal DNA (rDNA) is combined with a reverse-hybridization line probe assay (LiPA) was used for the identification of six Candida species and four Aspergillus species in pure cultures of clinical isolates, as well as in bronchoalveolar lavage (BAL) fluid samples from 42 patients with various underlying diseases. The results were compared with the results obtained with conventional routine identification methods as well as with a commercial enzyme-linked immunosorbent assay (ELISA) galactomannan detection assay and an Aspergillus-specific PCR. No discrepancies between the PCR-LiPA system and routine methods were found for pure cultures of Candida and Aspergillus species except in the case of Aspergillus versicolor. In BAL fluid samples in which Candida species were cultured, the PCR-LiPA system identified more species than did the routine methods. When routine analyses of patient samples were supplemented by adding data obtained by repurifying and re-identifying cultures and by taking isolates obtained from other body sites into account, the results agreed with PCR-LiPA system results in 81% of the cases (34/42). Most of the remaining discrepancies (6/8) involved cases in which such supplementary data were not available. In BAL fluid samples from which A. fumigatus was cultured, the agreement between the PCR-LiPA system and the routine methods was low. Only 2 of 11 BAL samples shown to contain A. fumigatus in ELISA and genus-specific PCR assays were positive in PCR-LiPA system. The PCR-LiPA system enables the simultaneous detection and identification of different fungal species present in pure or mixed populations within 6 h in a single assay. Optimization is required, however, before it is useful as a diagnostic tool in the clinical microbiology laboratory.

Rapid identification of Mycobacteria to the species level using INNO-LiPA Mycobacteria, a reverse hybridization assay.

INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium) is a kit for the simultaneous detection and identification of Mycobacterium species in culture and identifies the Mycobacterium tuberculosis complex, the M. avium complex (MAC), and the following Mycobacterium species: M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, and the M. chelonae-M. abscessus complex. The assay, which targets the 16S-23S rRNA spacer region, was evaluated on 157 mycobacterial strains that had been identified by conventional techniques and PCR-restriction enzyme analysis of the hsp65 gene (PRA). Forty-seven reference strains consisting of 37 different species and 110 human clinical isolates were submitted to the test, and all were hybridized with the Mycobacterium genus probe (MYC) on the LiPA strip (100% sensitivity). Ninety-four isolates hybridized to their corresponding species- or complex-specific probes; only one isolate phenotypically identified as M. gordonae did not react with its specific probe (99.4% accuracy). Thirty-seven MAC strains were phenotypically identified to the complex level and to the species level by LiPA as M. avium (n = 18) or M. intracellulare (n = 7) or as belonging to the M. avium-M. intracellulare-M. scrofulaceum complex (n = 12). Of the last 12 strains, 10 had M. avium PRA patterns and 2 had M. intracellulare PRA patterns. Three isolates that had been identified as a single species by conventional identification were proven to be mixed cultures by the LiPA assay. The whole procedure can be performed in 1 working day, starting with the supernatant of a small amount of bacterial mass that had been treated by freezing and then boiling.

Development of a PCR-based line probe assay for identification of fungal pathogens.

We report on a reverse-hybridization line probe assay (LiPA) which when combined with PCR amplification detects and identifies clinically significant fungal pathogens including Candida, Aspergillus, and Cryptococcus species. DNA probes have been designed from the internal transcribed-spacer (ITS) regions of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Candida dubliniensis, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus versicolor, Aspergillus nidulans and Aspergillus flavus. The probes were incorporated into a LiPA for detection of biotinylated ITS PCR products, and the specificity of the probes was evaluated. We established LiPA detection limits for ITS 1 and for full ITS amplicons for genomic DNA from C. albicans, A. fumigatus, and C. neoformans. Further evaluation of the LiPA was carried out on clinical fungal isolates. One hundred twenty-seven isolates consisting of dimorphic yeasts and dermatophytic and filamentous fungi were tested by the LiPA, which correctly identified 77 dimorphic yeasts and 23 of the filamentous isolates; the remaining 27 isolates represented species of fungi for which probes were not included in the LiPA. The fungal-PCR-LiPA technology was applied to blood samples inoculated with Candida cells which were pretreated by minibead beating to mechanically disrupt the cells, with the DNA extracted by either a previously described guanidium thiocyanate-silica method or the commercially available QIAmp tissue kit. PCR amplification of the extracted DNA and subsequent DNA probe hybridization in the LiPA assay yielded detection limits of 2 to 10 cells/ml. An internal standard control was included in the PCR amplification to monitor for PCR inhibition. This fungal PCR-LiPA assay is robust and sensitive and can easily be integrated into a clinical-testing laboratory with the potential for same-day diagnosis of fungal infection.

Correlation of pncA sequence with pyrazinamide resistance level in BACTEC for 21 mycobacterium tuberculosis clinical isolates.

Mutations in the pncA gene, encoding pyrazinamidase, are considered the major mechanism of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis, but resistant strains containing the wild-type gene have been described. The correlation of pncA sequence with PZA resistance level was examined for 21 M. tuberculosis clinical isolates. Susceptibility patterns were determined for 100, 300, and 900 microg/ml concentrations of the drug in BACTEC. Insertions and deletions and a substitution in the putative promoter region led to high-level resistance, whereas substitutions within the open reading frame seemed to confer variable levels of resistance. Variable resistance levels and PZase activities were also observed among isolates lacking pncA mutations. The high-level resistance (900 microg/ml) in pncA wild-type isolates highlights the clinical significance of these isolates. These data also suggest that there may still be more than one alternative mechanism leading to PZA resistance in M. tuberculosis isolates.

Relationship between pyrazinamide resistance, loss of pyrazinamidase activity, and mutations in the pncA locus in multidrug-resistant clinical isolates of Mycobacterium tuberculosis.

Sixty-two Mycobacterium tuberculosis isolates were tested for pyrazinamidase activity, and their pyrazinamide susceptibility was determined by the radiometric method. Sequencing of pncA genes in the 23 resistant strains revealed mutations in 16 pyrazinamidase-negative strains, 11 of which had not been previously described. Six isolates containing wild-type pncA might possess alternative resistance mechanisms.

Rapid detection of stressed Salmonella spp. in dairy and egg products using immunomagnetic separation and PCR.

The rapid detection of an average of 5.9 stressed Salmonella cells in 25 g of food product using immunomagnetic separation (IMS) and PCR is described. For pasteurised egg yolk, egg yolk powder, ice-cream, whole egg, egg white and cheeses made from pasteurised milk PCR was applied after 16 h of preenrichment in buffered peptone water (BPW) using IMS and alkaline lysis as sample preparation method. For whole egg and egg white the BPW was supplemented with iron. For milk powder, and raw milk cheeses, the 16-h preenrichment in BPW was followed by IMS and a 4-h enrichment in Rappaport-Vassiliadis broth. In the latter case, PCR was applied on the enrichment medium after centrifugation and alkaline lysis. For PCR the primers ST11 and ST15 (Aabo et al., 1993) producing a fragment of 429 bp were used. An internal PCR control, designed to be co-amplified with the target DNA using the same primers but producing a smaller fragment of 240 bp, was used.

Unidentified Listeria-like bacteria isolated from cheese.

Unidentified Listeria-like bacteria, which lack only one of the phenotypic characteristics used to confirm Listeria spp., were isolated from cheese during routine analysis for Listeria monocytogenes. The VIDAS Listeria assay and the Listeria specific PCR or DNA probe assays used did not identify these strains as Listeria species. This group of bacteria was studied for its homogeneity using rep-PCR and PFGE. Sequence analysis of the 16S rRNA gene showed a homology of 94% to established Listeria spp., implicating a closer relationship than that between Listeria spp. and Brochothrix spp.

The rrs (16S)-rrl (23S) ribosomal intergenic spacer region as a target for the detection of Haemophilus ducreyi by a heminested-PCR assay.

The intergenic spacer region between the rrs and rrl ribosomal RNA genes of Haemophilus ducreyi was analysed and the DNA sequence was used for the selection of specific PCR primers. A highly sensitive and specific heminested-PCR assay for the identification of H. ducreyi was developed. The assay showed a sensitivity of 96% on genital ulcer specimens from patients with clinically diagnosed chancroid, compared with a sensitivity of 56% for culture methods. These results indicate that this PCR assay has the potential to become an accurate and easy reference method for the detection of H. ducreyi.

Typing of Helicobacter pylori vacA gene and detection of cagA gene by PCR and reverse hybridization.

The present report describes an analysis of two virulence genes of Helicobacter pylori. Parts of the cagA gene, as well as parts from the signal (s) and middle (m) regions of the mosaic vacA gene, were amplified with biotin-labelled PCR primers and the products were subsequently analyzed by a single-step reverse hybridization line probe assay (LiPA). This assay comprises a strip containing multiple specific probes for the vacA s region (sla, slb, and s2 alleles), the vacA m region (ml and m2 alleles), and the cagA gene. A total of 103 H. pylori-positive materials, including cultured isolates, gastric biopsy specimens, and surgical specimens from patients living in Portugal (n = 55) and The Netherlands (n = 48) were tested by the PCR-LiPA. cagA was detected in 84 and 73% of the Portuguese and Dutch patients, respectively. vacA typing results, as determined by reverse hybridization, were completely concordant with those of sequence analysis. Most Portuguese patients (72%) contained type slb, whereas most Dutch patients (61%) contained type sla (P < 0.001). The method is also very effective at detecting the presence of multiple genotypes in a single biopsy specimen. The prevalence of multiple strains in Portuguese patient samples was significantly higher (29%) than that in Dutch patient samples (8%) (P = 0.001). There was a significant association between the presence of ulcers or gastric carcinoma and the presence of vacA type sl (sla or slb; P = 0.008) and cagA (P = 0.003) genes.

Evaluation of the INNO-LiPA Rif. TB assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium tuberculosis complex and its resistance to rifampin.

Mycobacterium tuberculosis resistance to rifampin results from nucleotide changes in the gene encoding the beta-subunit of the RNA polymerase (rpoB). We developed a reverse hybridization-based line probe assay (LiPA; the INNO-LiPA Rif. TB) carrying one oligonucleotide probe for the detection of M. tuberculosis complex strains and nine probes designed to detect nucleotide changes in the relevant part of rpoB. This assay was evaluated with 107 M. tuberculosis isolates with known rpoB sequences, 52 non-M. tuberculosis complex strains, and 61 and 203 clinical isolates found to be sensitive and resistant, respectively, by in vitro testing. The results indicated that (i) the M. tuberculosis complex probe was 100% specific, (ii) when compared to the results of nucleotide sequencing, no discrepancies with the results of INNO-LiPA Rif. TB were observed, (iii) all strains sensitive by in vitro susceptibility testing were correctly identified, and (iv) among the strains resistant by in vitro susceptibility testing, only 4 (2%) yielded conflicting results. The INNO-LiPA Rif. TB is therefore a reliable and widely applicable assay and a valuable tool for routine diagnostic use, given its simplicity and rapid performance.

Sequence analysis of a highly divergent HIV-1-related lentivirus isolated from a wild captured chimpanzee.

Two strains of simian immunodeficiency viruses (SIV) isolated from chimpanzees (SIVCPZ-GAB and SIVCPZ-GAB2) originating from Gabon have previously been genetically characterized and shown to belong phylogenetically to the same lineage as the human immunodeficiency virus type 1 (HIV-1). We describe the sequence analysis of a third HIV-1-related virus, SIVCPZ-ANT, isolated from a wild captured chimpanzee originating from Zaire. This virus displayed the same genetic organization as HIV-1 and was found to fall on the same lineage as HIV-1 and SIVCPZ-GAB. Protein sequence identity with SIVCPZ-GAB ranged from 72% (Pol) to 48% (Env) for the structural proteins, while a particularly divergent Vpu was found (only 25% identity to SIVCPZ-GAB). The V3 regions of the SIVCPZ isolates were exceptionally conserved in contrast to the high divergence of V3 among HIV-1 isolates. However, SIVCPZ-ANT did not show a greater degree of sequence similarity with SIVCPZ-GAB than with HIV-1 isolates and represents a quite divergent outgroup of the HIV-1 lineage. Our data suggest multiple introductions of HIV-1 in the human population and shed new light on the origin of the HIV-1 pandemic.

Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes.

The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods.