Browsing by discipline "Microbiology (Food Safety)"
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(2013)Campylobacteriosis, the most common bacterial food-borne disease worldwide, is mainly caused by Campylobacter jejuni and C. coli. Most studies have focused on the genetic diversity of C. jejuni, but little is known about C. coli. The aim of this work was to characterize C. coli from different sources, by evaluating the distribution and/or diversity of certain genetic markers. A total of 145 C. coli isolates from different sources (2 goose, 18 poultry, 35 human and 90 swine) were screened for fucP, ggt, cytC, sialyltransferases genes and CRISPRs. Additionally, the diversity of the LOS loci and of the CRISPRs, were assessed. A frequency of 90.34% was observed for fucP and CRISPRs among C. coli. Conversely, the frequency of GGT phenotype, and cytC, and cst-I genotype was 1.38%, while cst-V genotype was 0.69%. Only one isolate was positive for all markers except fucP; no source association was observed. LOS and CRISPRs exhibited a wide diversity. In conclusion, ggt, fucP, and cytC seem to be lineage related in C. coli, and not host associated. CRISPRs were too discriminatory to be of use in epidemiological investigations. Results suggest a high diversity of the LOS, and there may exist more classes than those previously described.
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(2014)Quantitative reverse transcription PCR (RT-qPCR) assay is widely used for the detection of RNA viruses in environmental water samples. However, a major limitation of using RT-qPCR assay to quantify virus titers is its inability to discriminate between infectious and non-infectious viruses, resulting in overestimation of viral infectivity. Thus, the aim of this study was to develop a reliable molecular method for rotavirus detection with information on viral infectivity, and which may contribute to the development of molecular detection methods for correct estimation of infectivity of non-cultivable viruses. In experimental work, the potential of using propidium monoazide (PMA) or RNase treatment prior to RT-qPCR assay was evaluated to measure the infectivity of human rotavirus. In brief, original human rotavirus (HRV) stock was produced by propagating viruses in MA-104 cells. The virus stocks (including HRV stock A and B) were thermally treated at 80 °C at different time points. The virus titer was measured by (1) cell culture-based infectivity assay, (2) RT-qPCR assay, and (3) RT-qPCR assay with PMA or RNase pretreatment. The result of cell culture-based infectivity assay showed that heat exposure for 5 min at 80 °C was sufficient to inactivate the HRV, while RT-qPCR assay alone overestimated the viral infectivity. The results of RT-qPCR assay with pre-treatments showed that, for thermally-inactivated HRV stock A, similar level of false-positive results was reduced with PMA treatment regardless of inactivation time (ranges from 1.04 to 1.18 log10 PCR-units), while higher reduction level was observed with RNase treatment (ranges from 2.64 to 2.89 log10 PCR-units). On the other hand, the effects of both pre-treatments on thermally-inactivated HRV stock B were negligible. In conclusion, both PMA and RNase pre-treatments eliminated the false-positive results of RT-qPCR assay to some extent in defined conditions, while the discrepancy between the infectivity assay and RT-qPCR assay even with PMA or RNase treatment was observed. In order to confirm the potential of using RT-qPCR assay combined with pre-treatments to measure the infectivity of rotavirus, further studies on optimization of PMA and RNase treatments and production of optimal virus stock would be necessary.
Now showing items 1-2 of 2