Singleplex real-time RT-PCR for detection of influenza A virus and simultaneous differentiation of A/H1N1v and evaluation of the RealStar influenza kit.

BACKGROUND: A novel influenza A virus, subtype A/H1N1v emerged in April 2009 and caused the first influenza pandemic of the 21st century. Reliable detection and differentiation from seasonal influenza viruses is mandatory for appropriate case management as well as public health. OBJECTIVES: To develop and technically validate a novel one-step real-time RT-PCR assay which can be used for influenza A virus screening and subtyping of A/H1N1v in a singleplex fashion. To assess the clinical performance of a novel commercial influenza RT-PCR kit based on the in-house version. STUDY DESIGN: A real-time RT-PCR assay targeting the matrix gene of influenza A viruses was developed and validated using in vitro transcribed RNA derived from influenza A/H1N1v, A/H1N1 and A/H3N2 virus as well as plaque-quantified influenza A/H1N1v, A/H1N1 and A/H3N2 virus samples. After validation of the in-house version the commercial RealStar kit was used to assess the clinical performance and specificity on a panel of influenza viruses including A/H1N1v, A/H1N1, swine A/H1N1, A/H3N2, avian A/H5N1 as well as patient specimens. RESULTS: The lower limit of detection of the in-house version was 2149, 1376 and 2994 RNA copies/ml for A/H1N1v, A/H1N1 and A/H3N2, respectively. The RealStar kit displayed 100% sensitivity and specificity and could reliably discriminate influenza A viruses from A/H1N1v. No cross reaction with swine A/H1N1 and A/H1N2 was observed with the RealStar A/H1N1v specific probe. CONCLUSION: Both assays demonstrated high sensitivity and specificity and might assist in the diagnosis of suspected influenza cases.

Catanionic micelles as a model to mimic biological membranes in the presence of anesthetic alcohols.

We show here the influence of n-alcohols (C(2)OH-C(8)OH) on the solubility behavior of cationic-anionic surfactant mixtures, so-called "catanionics". We studied catanionics of different compositions composed of sodium dodecyl sulfate (SDS)/cetyltrimethylammonium bromide (CTAB) and sodium dodecanoate (SDod)/CTAB mixtures. Interestingly, with a molar excess of SDS, long chain n-alcohols (C(4)OH-C(8)OH) significantly depress the solubility temperature of the SDS+CTAB catanionic and increase the kinetic stability of the solution. The visual observations of solubility temperatures of catanionics were further confirmed by differential scanning calorimetry (DSC) measurements. For the catanionics a multistep solubilization was observed by DSC, for which the sulfate headgroup is responsible. This was probed by replacing SDS by SDod. A remarkable analogy was found between the influence of the alcohols on the solubility patterns of the catanionic mixtures and on the anesthesia of tadpoles. Possible reasons for this analogy are discussed also in this paper.

Diagnostic reverse-transcription polymerase chain reaction kit for filoviruses based on the strain collections of all European biosafety level 4 laboratories.

A network of European biosafety level 4 laboratories has designed the first industry-standard molecular assay for all filoviruses species, based on the strain collections of all participants. It uses 5 optimized L gene primers and 3 probes, as well as an internal control with a separate detection probe. Detection limits (probit analysis, 95% detection chance) were as follows: Zaire ebolavirus, 487 copies/mL of plasma; Sudan ebolavirus Maleo, 586 copies/mL; Sudan ebolavirus Gulu, 1128 copies/mL; Cote d'Ivoire ebolavirus, 537 copies/mL; Reston ebolavirus, 4546 copies/mL; Lake Victoria marburgvirus Musoke, 860 copies/mL; and Lake Victoria marburgvirus Ravn, 1551 copies/mL. The assay facilitates reliable detection or exclusion screening of filovirus infections.