|Tipo di tesi||Tesi di laurea magistrale|
|Titolo||Applicabilità di un kit di estrazione e quantificazione di DNA in real-time PCR per la ricerca di Legionella spp in campioni ambientali|
|Titolo in inglese||Applicability of a DNA extraction and quantification kit in real-time PCR to detect Legionella spp in water samples|
|Struttura||Dipartimento di Scienze della Vita|
|Corso di studi||BIOLOGIA (D.M. 270/04)|
|Data inizio appello||2013-10-10|
|Disponibilità||Accesso limitato: si può decidere quali file della tesi rendere accessibili. Disponibilità mixed (scegli questa opzione se vuoi rendere inaccessibili tutti i file della tesi o parte di essi)|
|Data di rilascio||2053-10-10|
Legionella spp sono batteri diffusi negli ambienti acquatici naturali e nei sistemi idrici artificiali, dove si moltiplicano all’interno di protozoi e sopravvivono come forme a vita libera o associate a biofilm. La trasmissione delle legionelle all’uomo avviene per inalazione di aerosol contaminati ed in soggetti suscettibili può dare origine ad una forma simil-influenzale detta Febbre di Pontiac o ad una polmonite acuta conosciuta come Malattia dei Legionari. Questa severa forma di polmonite, sostenuta nella quasi totalità dei casi dalla specie L. pneumophila, ha un tasso di mortalità del 15-20% che può raggiungere il 50% nei soggetti immunocompromessi.
Legionella organisms are ubiquitous bacteria found in many types of water sources in the environment and their growth is especially favoured in human-made warm water systems. Legionella bacteria replicate as intracellular parasites of amoebae and persist in the environment as free-living microbes or in biofilms. Infection occurs after inhalation of Legionella-contaminated water droplets, formed from aerosol-generating devices. In aerosol form, they enter the lungs and can cause an acute form of pneumonia known as Legionnaires’ disease or a milder form of infection called Pontiac fever. The species L. pneumophila is responsible for the vast majority of the most severe form of this atypical pneumonia. Legionellosis outbreaks are associated with high mortality rates (15 to 20%), which can reach up to 50% among individuals with immune deficiencies. The development of rapid and sensitive methods for the detection and quantification of Legionella spp is essential for monitoring water quality and for legionellosis prevention. Cell culture is the reference method to detect and quantify legionellae. Culture is essential for identifying and typing Legionella strains during epidemics. However, Legionella culture requires long incubation times before results can be scored. This problem makes culture unsuitable for preventive actions and rapid response in emergency situations. Moreover, the Viable But NonCulturable (VBNC) cells are not detectable by culture. The VBNC state is developed by some opportunistic pathogens such as Legionella to survive in unfavourable environmental conditions. The VBNC cells are still alive and retain their virulence. In an attempt to overcome these disadvantages, molecular investigation tools such as Polymerase Chain Reaction (PCR) and especially quantitative PCR (qPCR) were developed. PCR techniques show several advantages including high sensitivity, accuracy and rapid evaluation of germ contamination; nevertheless, the main disadvantage of these molecular methods is that they don’t distinguish between dead and viable cells. A new approach to detect viable cells by DNA-intercalating dyes in combination with qPCR has been proposed over the last ten years. This molecular technique prevents amplification of DNA from dead cells. The aim of our present study was to verify the applicability of a Legionella spp DNA extraction and quantification kit in real-time PCR to analyse environmental samples. This innovative kit is based on the use of membrane filters that allow to not retain any DNA deriving from dead cells present in the water sample. In this way only DNA from viable cells can be amplified and quantified. To evaluate the reliability of the kit, the results were compared with those obtained by conventional culture. Water samples were also analysed by two laboratories to verify the reproducibility of this method. The effectiveness was proven both in liquid pure cultures of L. pneumophila and in natural water samples treated with different disinfection systems. This method may significantly improve the knowledge about the exposure risk to these bacteria, allowing us to optimize the selection of the more appropriate disinfection strategies.