| 1. |
- Hedman, Johannes, et al.
(author)
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Validation guidelines for PCR workflows in bioterrorism preparedness, food safety and forensics
- 2018
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In: Accreditation and Quality Assurance. - : Springer Science and Business Media LLC. - 0949-1775 .- 1432-0517. ; 23:3, s. 133-144
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Journal article (peer-reviewed)abstract
- The polymerase chain reaction (PCR) is the backbone of contemporary DNA/RNA analysis, ideally enabling detection of one or just a few target molecules. However, when analysing food or forensic samples the analytical procedure is often challenged by low amounts of poor quality template molecules and complex matrices. Applying optimised and validated methods in all steps of the analysis workflow, i.e. sampling, sample treatment, DNA/RNA extraction and PCR (including reverse transcription for RNA analysis), is thus necessary to ensure the reliability of analysis. In this paper, we describe how in-house validation can be performed for the different modules of the diagnostic PCR process, providing practical examples as tools for laboratories in their planning of validation studies. The focus is analysis of heterogeneous samples with interfering matrices, with relevance in food testing, forensic DNA analysis, bioterrorism preparedness and veterinary medicine. Our objective is to enable rational in-house validation for reliable and swift quality assurance when results are urgent, for example in the event of a crisis such as a foodborne outbreak or a crime requiring the analysis of a large number of diverse samples. To that end, we explain the performance characteristics associated with method validation from a PCR and biological sample matrix perspective and suggest which characteristics to investigate depending on the type of method to be validated. Also, we include a modular approach to validation within the PCR workflow, aiming at efficient validation and a flexible use of methods.
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| 2. |
- Kaden, Rene, 1975-, et al.
(author)
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Brucella abortus : determination of survival times and evaluation of methods for detection in several matrices
- 2018
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In: BMC Infectious Diseases. - : Springer Science and Business Media LLC. - 1471-2334. ; 18
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Journal article (peer-reviewed)abstract
- BACKGROUND: Brucella abortus is a highly pathogenic zoonotic agent, tempting for the development of a rapid diagnostic method to enable adequate treatment and prevent further spread. Enrichment of the bacteria is often used as a first step in diagnostics to increase the bacterial number above the detection limit of the real-time PCR. The enrichment of Brucella spp. takes at least 3 days, which might be avoidable if sensitive PCR methods can be used. Since many matrices contain PCR inhibitors, the limit of detection (LOD) must be determined for each separate matrix. Another aim of this study was the determination of survival of Brucella abortus in the analyzed matrices. METHODS: The LOD for the detection of B. abortus in 14 matrices, relevant for human medicine, veterinary medicine and food and feed safety, was determined to evaluate the need of a pre-enrichment step prior to real-time PCR. The survival of B. abortus in the spiked matrices was tested by plate count in a 7-day interval for 132 days. RESULTS: The limit of detection for B. abortus in most matrices was in the range of 10(3)-10(4) CFU/g for cultivation and 10(4)-10(5) CFU/g for direct real-time PCR. The survival time of B. abortus was less than 21 days in apple puree and stomach content and 28 days in water while B. abortus remained viable at day 132 in milk, blood, spinach and minced meat. CONCLUSIONS: A direct PCR analysis without enrichment of bacteria saves at least 3 days. However, the limit of detection between direct PCR and plate count differs in a 10 fold range. We conclude that this lower sensitivity is acceptable in most cases especially if quick analysis are required.
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| 3. |
- Loftsdottir, Heidur, et al.
(author)
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Dynamics of insertion sequence element IS629 inactivation of verotoxin 2 genes in Escherichia coli O157:H7
- 2017
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In: FEMS Microbiology Letters. - : Oxford University Press. - 0378-1097 .- 1574-6968. ; 364:8
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Journal article (peer-reviewed)abstract
- There are several anecdotal reports of insertion sequence (IS) element inactivation of verotoxin genes among enterohaemorrhagic Escherichia coli of the serotype O157:H7, a pathogen causing severe gastrointestinal disease in infected humans. These insertions can be expected to drastically reduce the virulence of the bacteria. IS element inactivation has been shown to be reversible in model systems, suggesting the possibility of spontaneous restoration of virulence. In this study, traditional and high-throughput sequencing was used to characterise three patterns of IS629 inactivation of verotoxin 2 genes in EHEC O157:H7, caused by insertion or insertion followed by partial deletion. At least one of the patterns of inactivation appears to have persisted several years among cattle O157:H7, indicating it has no major effect on fitness in the animal reservoir. Digital PCR was used to directly quantify the reversal rates of the insertional inactivation of a selected isolate under laboratory conditions. Inserts were found to be absent from in the order of 1/10(5) of individual genomes, with significantly higher loss frequencies observed in cultures under nutrient-poor conditions. We conclude that strains with this type of inactivation found in food or animal samples should be considered a threat to human health, and may pose a challenge for PCR-based detection methods.
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| 4. |
- Ström Hallenberg, Gunilla, et al.
(author)
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Manure management and public health : Sanitary and socio-economic aspects among urban livestock-keepers in Cambodia
- 2018
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In: Science of the Total Environment. - : Elsevier BV. - 0048-9697 .- 1879-1026. ; 621, s. 193-200
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Journal article (peer-reviewed)abstract
- Livestock manure is a valuable source of nutrients for crop production, but can also pose a public health hazard and have negative environmental impacts. This study investigated manure management practices among urban and peri-urban livestock keepers in Cambodia, to identify risk behaviours and socio-economic aspects associated with the handling of manure. A survey including 204 households was conducted, using a structured questionnaire with questions on demographics, socio-economic characteristics and household practices related to manure management. Faecal samples were obtained from pig pens and pig manure storage units for analysis of the potential zoonotic pathogens Salmonella enterica (Polymerase Chain Reaction (PCR)), Ascaris suum and Trichuris suis (McMaster flotation technique). The survey revealed a difference in management between cattle and pig manure. Cattle manure was most commonly used as fertiliser for crop production (66%) (p < 0.001), whereas pig manure was most commonly dumped in the environment (46%) (p < 0.001). Logistic regression models showed that households with a lower socio-economic position were more likely to dump pig manure (p < 0.001), with scarcity of agricultural land (p < 0.001) and lack of carts for transportation of manure (p < 0.01) being identified as contributing factors. Salmonella enterica was detected in 9.7% of manure samples, while Ascaris suum and Trichuris suis were detected in 1.6% and 2.4% of the samples, respectively. The results presented in this study indicate that manure management by urban and peri-urban households may pose a public health threat and an environmental hazard. There is evidently a need for further knowledge support to the livestock keepers to promote good management practices.
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