Human infection with the important zoonotic foodborne pathogen Toxoplasma gondii has been associated with unwashed raw fresh produce consumption. The lack of a standardised detection method limits the estimation of fresh produce as an infection source. To support method development and standardisation, an extensive literature review and a multi-attribute assessment were performed to analyse the key aspects of published methods for the detection of T. gondii oocyst contamination in fresh produce. Seventy-seven published studies were included, with 14 focusing on fresh produce. Information gathered from expert laboratories via an online questionnaire were also included. Our findings show that procedures for oocyst recovery from fresh produce mostly involved sample washing and pelleting of the washing eluate by centrifugation, although washing procedures and buffers varied. DNA extraction procedures including mechanical or thermal shocks were identified as necessary steps to break the robust oocyst wall. The most suitable DNA detection protocols rely on qPCR, mostly targeting the B1 gene or the 529 bp repetitive element. When reported, validation data for the different detection methods were not comparable and none of the methods were supported by an interlaboratory comparative study. The results of this review will pave the way for an ongoing development of a widely applicable standard operating procedure.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The possibility that Mycobacterium avium subsp. paratuberculosis (MAP) plays some role in the development of Crohn's disease in humans is attracting attention to milk and milk products originating from infected animals. In this study, we focused on the detection of MAP in 220 bulk tank milk (BTM) samples from all dairy cattle herds in Cyprus. In total, 63 (28.6%) BTM milk samples were found to be positive for MAP using quantitative real-time PCR assays for IS900 and F57. The presence of MAP in BTM was low, and was assessed to be several tens of MAP cells per one ml of BTM. Milk samples examined by cultivation were found to be negative for MAP in all 220 BTM. In two BTM samples cultivation and subsequent sequencing of 16S rRNA revealed two isolates of M. fortuitum.
- MeSH
- bakteriální RNA analýza MeSH
- kontaminace potravin analýza MeSH
- lidé MeSH
- mléko mikrobiologie MeSH
- Mycobacterium avium subsp. paratuberculosis izolace a purifikace MeSH
- nemoci skotu diagnóza přenos MeSH
- paratuberkulóza diagnóza přenos MeSH
- plošný screening veterinární MeSH
- počet mikrobiálních kolonií veterinární MeSH
- polymerázová řetězová reakce veterinární MeSH
- prediktivní hodnota testů MeSH
- RNA ribozomální 16S analýza MeSH
- senzitivita a specificita MeSH
- skot MeSH
- zoonózy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Geografické názvy
- Česká republika MeSH
A rapid, cheap and sensitive detection method of Mycobacterium avium subsp. paratuberculosis (MAP) in raw milk was needed for routine usage. We developed two duplex real time qPCR systems specific for MAP detection. These real time qPCR assays amplify the multicopy element IS900 for qualitative analysis and the single copy element F57 for quantitative analysis. Both assays incorporate an internal amplification control amplified with the same primers as the targets and the same probes are used in both assays. The specificity of the assays was confirmed by the testing of 6 different MAP isolates, 12 isolates of other mycobacteria or bacterial species and 4 different mammalian DNAs. The sensitivity of the developed assays and isolation efficiency were demonstrated through the analysis of raw milk samples artificially contaminated with MAP cells and with plasmids containing cloned fragments of the targets (IS900 and F57). The developed assays for milk analysis were applied to samples from one farm with two faecal shedding cows. Three hundred and forty five individual milk samples were tested by real time qPCR assays and by cultivation. Hundred and eleven (32.5%) individual milk samples were positive by the real time qPCR, no milk sample was culture positive. The spread of MAP in individual, tank and bulk tank milk samples was also monitored.
- MeSH
- DNA bakterií MeSH
- druhová specificita MeSH
- kontaminace potravin analýza MeSH
- lidé MeSH
- mléko mikrobiologie MeSH
- Mycobacterium avium subsp. paratuberculosis genetika izolace a purifikace klasifikace MeSH
- počet mikrobiálních kolonií metody MeSH
- polymerázová řetězová reakce metody MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- skot MeSH
- spotřebitelská bezpečnost produktů MeSH
- transpozibilní elementy DNA genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
