[Hepatitis A virus detection in shellfish from Tunisia by reverse transcription-nested PCR--investigation of a correlation between viral and bacterial contamination]. / Détection du virus de l'hépatite A dans les coquillages en Tunisie par reverse transcription-nested PCR - recherche de corrélation entre la contamination virale et bactérienne.

OBJECTIVES: We aimed at evaluating the contamination by hepatitis A virus (HAV) of 54 shellfish samples collected from five Tunisian shellfish harvesting areas and finding a correlation between bacterial and viral contamination. MATERIAL AND METHODS: Fifty-four shellfish samples were analysed in our study. Two methods of viral extraction were evaluated by reverse transcription-nested PCR. The first one was based on elution by glycine solution and the second one used a beef extract solution. Bacteriological determination (Samonella and E. coli) was carried out for all shellfish samples. RESULTS: Glycine extraction showed a higher detection rate of HAV compared to the saline beef extraction method. The hepatitis A virus was detected in 32 % of shellfish samples analysed. None of the samples revealed the presence of Samonella. From 17 samples positive for HAV, we found six samples showing a number of E. coli below the European legislation. CONCLUSION: An important HAV contamination was observed in our study. No correlation between bacterial and viral contamination was found.

Real-time RT-PCR for norovirus screening in shellfish.

Real-time RT-PCR, combining amplification and detection of virus-specific amplicons, is a promising tool for norovirus detection in environmental or food samples such as shellfish. We developed a real-time RT-PCR assay based on one-step detection using single primer sets and probes for norovirus genogroups I and II. Seventy and seven RT-PCR units of genogroup I and II reference norovirus strains, respectively, were detected in artificially contaminated oysters. Validation of the new method on 150 archived naturally contaminated shellfish confirmed the utility of the genogroup II primer set to detect a large range of different strains circulating in France since 1995, but genogroup I strains were detected infrequently.

An improved method for the detection of Norwalk-like caliciviruses in environmental samples.

An original magnetic beads RNA capture was developed for the detection of Norwalk-like virus by RT-PCR. The same oligonucleotide was used both for capture and reverse transcription of the viral RNA. The optimization studies showed that the most important parameter for sensitivity is the biotin-binding capacity of the beads. This method was found to be efficient for eliminating inhibitors in sewage samples compared with the classic RT-PCR. Moreover, the sensitivity was greatly enhanced, allowing the detection of 42% positive sample after gel electrophoresis, which is fourfold greater than classic RT-PCR (11%). Beads-RT-PCR sensitivity is the same as classic RT-PCR and hybridization. Thus, this method, which is easy to perform, should be of particular interest for developing quantitative RT-PCR and sequencing.

Bulk analysis by IR laser ablation inductively coupled plasma atomic emission spectrometry.

Two laser ablation systems dedicated to bulk analysis were evaluated for steel and PVC samples, using inductively coupled plasma atomic emission spectrometry detection. These systems were characterized by the use of a Nd:YAG laser operating at 1064 nm, the absence of observation device and a large laser spot size. The 1064 nm wavelength was selected to avoid the use of frequency-multiplying optics, and to be less critical to the sampling position. Calibration graphs and limits of detection are given for both types of materials. LODs were in the range 3-120 microg/g for steel, and in the range 0.07-15 microg/g for PVC. In the case of steel samples, similar calibration graph slopes were obtained between polished and unpolished samples.