The StepOne real-time polymerase chain reaction detection of Salmonella sp., Salmonella enterica ser. typhimurium and enteritidis in milk and meat

The aim of this study was to follow contamination of ready to eat milk and meat products with Salmonella spp. by using the StepOne real-time polymerase chain reaction (PCR). Classical microbiological methods for detection of foodborne bacteria involve the use of pre-enrichment and/or specific enrichment, following isolation of bacteria in solid media and the final confirmation by biochemical and/or serological tests. We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and MicroSEQ® Salmonella spp. Detection Kit for pursuance of the real-time PCR (Applied Biosystems). In samples without incubation we detected strain of Salmonella sp. in 5 out of 25 samples (swabs), as well as in the internal positive control (IPC), which was positive in all samples. This StepOne real-time PCR assay is extremely useful for any laboratory equipped by real-time PCR. It is a fast, reproducible, simple, specific and sensitive way to detect nucleic acids, which could be used in clinical diagnostic tests in the future. Our results indicated that real-time PCR assay developed in this study could sensitively detect Salmonella spp. in ready-to-eat food. This could prevent infection caused by Salmonella, and also could benefit food manufacturing companies by extending their product's shelf-life as well as saving the cost of warehousing their food products while awaiting pathogen testing results.     

Combining Flow Cytometry and Real-Time PCR Methodology to Demonstrate Consumption by Prymnesium parvum1

Bowers, Holly A., Andreas Brutemark, Wanderson F. Carvalho, and Edna Granéli, 2010. Combining Flow Cytometry and Real-Time PCR Methodology to Demonstrate Consumption by Prymnesium parvum. Journal of the American Water Resources Association (JAWRA) 46(1):133-143. DOI: 10.1111/j.1752-1688.2009.00397.x Abstract: Harmful algal bloom species can persist in the environment, impacting aquatic life and human health. One of the mechanisms by which some harmful algal bloom species are able to persist is by consumption of organic particles. Methods to demonstrate and measure consumption can yield insight into how populations thrive. Here, we combine flow cytometry and real-time PCR to demonstrate consumption of a cryptophyte species (Rhodomonas sp.) by a toxic mixotrophic haptophyte (Prymnesium parvum). Using flow cytometry, the feeding frequency of a population of P. parvum cells was calculated using the phycoerythrin (PE) fluorescence signal from Rhodomonas sp. and the fluorescence of an acidotropic probe labeling the food vacuoles. Feeding frequency increased in the beginning of the experiment and then began to decline, reaching a maximum of 47.5% of the whole P. parvum population after 212 min. The maximum number of consumed Rhodomonas sp. cells was 0.8 per P. parvum cell, and occurred after 114 min corresponding to an ingestion rate of 0.4 Rhodomonas sp. cells/P. parvum/h. Cells from the feeding P. parvum population were sorted, washed, and subjected to a real-time PCR assay targeting the cryptophyte 18S locus. There was a correlation between cycle threshold (Ct) values and number of consumed prey cells calculated by fluorescence. Overall, this study shows that flow cytometric analysis, of the acidotropic probe and prey pigments, is an efficient and rapid tool in enumerating food vacuoles and the number of prey cells consumed. Furthermore, we suggest that real-time PCR can be applied to cells sorted by flow cytometry, thus allowing for the detection and potential quantification of the targeted prey cells.     

Wildlife identified as major source of Escherichia coli in agriculturally dominated watersheds by BOX A1R-derived genetic fingerprints

The presence of Escherichia coli in recreational and potable waters is a major concern to the general public as elevated levels of E. coli suggest the presence of pathogenic bacteria and viruses. Unfortunately, traditional microbial techniques do not allow specific identification of the source of E. coli. This reduces the ability to target management practices that reduce bacterial contamination. In the Finger Lakes region of western New York, USA, wildlife resides in relatively high densities on watersheds dominated by people and dairy farms, and as a result, the sources of fecal degradation of potable and recreational waters are often unknown. In the Conesus Lake watershed, the sources of microbial contamination were assessed using Rep-PCR molecular tools, a method of amplifying repetitive DNA sequences found throughout the E. coli genome to produce distinct fingerprints for a given ecotype. Molecular fingerprints of E. coli isolated from regional populations of cattle, humans, geese and deer were compared to E. coli isolated from stream water samples. Canonical discriminant function analysis indicated that the DNA fingerprints of the original source group isolates were correctly predicted 90.2% of the time. Since land use in the sub-watersheds was dominated by dairy and cash crop farms, it was expected that the majority of E. coli isolated would be identified as cows; however, an unexpectedly high percentage of isolates were identified as wildlife (geese and deer). Geese were the dominant source of E. coli (44.7-73.7% of the total sources) in four sub-watersheds followed by cows (10.5-21.1%), deer (10.5-18.4%), humans (5.3-12.9%) and unidentifiable sources (0.0-11.8%). Management practices intended to decrease the number of cattle or the amount of manure spread in a sub-watershed were reflected in a decrease of E. coli ecotypes associated with dairy cows.     

Case report: birth after preimplantation genetic diagnosis of a subtle mutation in SMN1 gene

Spinal muscular atrophy (SMA) preimplantation genetic diagnosis (PGD) has been available since 1998. Protocols are based on the detection of the homozygous deletion of exon 7, which are present in 90–98% of SMA patients. A couple where the woman was a heterozygous carrier of the usual SMN1 Del7 mutation and the man was a heterozygous carrier of pMet263Arg substitution in exon 6 of SMN1 gene was referred for PGD. The usual PGD test being unsuitable for this couple, we developed a novel duplex polymerase chain reaction (PCR)-based PGD test for the detection of the mutation pMet263Arg by allele specific amplification, combined with the amplification of D5S641 extragenic polymorphic marker. PCR conditions were established using single control lymphoblasts and lymphocytes from the pMet263Arg substitution carrier. Amplification was obtained in 100% of the 86 single cells tested, amplification refractory mutation system (ARMS) PCR was specific in 100% of single cells tested and a complete genotype (mutation plus D5S641) was achieved in 88% of them. A PGD cycle was performed successfully and a pregnancy was obtained. An unaffected girl was born and postnatal diagnosis confirmed PGD results. This is the first PGD described for SMA because of another mutation than the major homozygous exon 7 deletion of SMN1. In the future, a similar strategy could be adopted for other subtle mutations of this gene. Copyright © 2006 John Wiley & Sons, Ltd.