Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain

Background  

5-hydroxytryptamine (5 HT, serotonin) is one of the key neuromodulators in mammalian brain, but many fundamental properties of serotonergic neurones and 5 HT release remain unknown. The objective of this study was to generate an adenoviral vector system for selective targeting of serotonergic neurones and apply it to study quantal characteristics of 5 HT release in the rat brain.     

Development of an in vitro method for the prediction of mycotoxin binding on yeast-based products: case of aflatoxin B1, zearalenone and ochratoxin A

To date, no official method is available to accurately define the binding capacity of binders. The goal is to define general in vitro parameters (equilibrium time, pH, mycotoxin/binder ratio) for the determination of binding efficacy, which can be used to calculate the relevant equilibrium adsorption constants. For this purpose, aflatoxin B₁ (AFB₁), zearalenone (ZEA) or ochratoxin A (OTA) were incubated with one yeast cell wall in pH 3, pH 5 or pH 7 buffers. The percentage of adsorption was recorded by quantitation of remaining mycotoxins in the supernatant and amount of mycotoxin adsorbed on the residue. The incubation of yeast cell wall in the presence of mycotoxins solved in buffer, lead to unexpected high adsorption percentage when the analysis was based only on remaining mycotoxins in the supernatant. The decrease of mycotoxins in the supernatant was not correlated to the amount of mycotoxins found in the residue. For this reason we modified the conditions of incubation. Yeast cell wall (5 mg) was pre-incubated in buffer (990 μl) at 37 °C during 5 min and then 10 μl of an alcoholic solution of mycotoxin (concentration 100 times higher than the final concentration required in the test tube) were added. After incubation, the solution was centrifuged, and the amount of mycotoxins were analysed both in the supernatant and in the residue. A plateau of binding was reached after 15 min of incubation whatever the mycotoxins and the concentrations tested. The adsorption of ZEA was better at pH 5 (75 %), versus 60 % at pH 3 and 7. OTA was only significantly adsorbed at pH 3 (50 %). Depending on the pH, the adsorptions of OTA or ZEA were increased or decreased when they were together, indicative of a cooperative effect.     

Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.