| 1. |
- Johansson, Anna-Lena, et al.
(författare)
-
Francisella
- 2015. - 2
-
Ingår i: Molecular medical microbiology. - : Academic Press. - 9780123971692 - 9780123977632 ; , s. 1991-2009
-
Bokkapitel (refereegranskat)abstract
- Francisella tularensis, the causative agent of tularaemia, is a zoonotic intracellular pathogen that can be found in a very large number of species ranging from large mammals and vertebrates to invertebrates, arthropods and amoebas. Disease in humans often occurs in parallel with tularaemia in wild animals. Human infection can occur through aerosolization, direct contact with infected animals via arthropod vectors like ticks, mosquitos and biting flies. F. tularensis subspecies tularensis (type A) is one of the most infectious bacteria known and inhalation of as few as ten organisms can be sufficient to establish an infection in humans that if left untreated could be fatal. The success of F. tularensis as a pathogen lies in its unique ability to adapt a lifestyle as an intracellular pathogen and to very timely subvert host defences both at extracellular and intracellular levels. Key events in the infection process are the ability to rapidly escape the phagosome after uptake by phagocytic cells and the ability to replicate to high levels inside the host cells without evoking a host inflammatory response.
|
|
| 2. |
- Sidstedt, Maja, et al.
(författare)
-
Humic substances cause fluorescence inhibition in real-time PCR.
- 2015
-
Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 487, s. 30-37
-
Tidskriftsartikel (refereegranskat)abstract
- Real-time PCR (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, i.e. amplification inhibition. Humic substances (HS) are well known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, i.e. quench the fluorescence signal of dsDNA binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I and SYTO 82, generating lowered amplification plots although amplicon production was unaffected. For EvaGreen, 500 ng HA quenched almost all fluorescence, whereas 1000 ng HA completely inhibited amplification when applying Immolase DNA polymerase with BSA. Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional, and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor, but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays.
|
|
