Vzpostavitev metode za ugotavljanje prisotnosti genov sitA, traT in ompT pri sevih Escherichia coli iz perutnine

Lotrič, Marjetka (author), Avguštin, Gorazd (mentor), Kogovšek, Polona (comentor)

Kolibaciloza je najpogostejša bakterijska bolezen pri perutnini in se pogosto konča s poginom živali, zaradi česar nastanejo velike ekonomske izgube. Kolibacilozo povzroča za perutnino patogena bakterija Escherichia coli (APEC). Bolezenski znaki so zelo različni. Sevi APEC povzročajo primarne kot tudi sekundarne okužbe ptic. Vse determinante patogenosti sevov APEC še niso popolnoma pojasnjene, zato sta nadzor nad tovrstnimi okužbami in identifikacija povzročitelja bolezni zahtevna. Trenutno so v uporabi tradicionalne diagnostične metode, ki so časovno zamudne in pogosto cenovno neugodne. Zaradi vse večjih potreb po hitri diagnostiki v ospredje prihajajo molekularne metode, med katerimi sta tudi verižna reakcija s polimerazo - PCR (ang. polymerase chain reaction) in z zanko posredovano izotermalno pomnoževanje - LAMP (ang. loop mediated isothermal amplification). Glavni namen naloge je bil razvoj metode, na osnovi LAMP, ki bi omogočala učinkovito ugotavljanje prisotnosti virulentnih genov pri sevih APEC. Razvili smo teste LAMP-APEC za ugotavljanje prisotnosti genov sitA, traT in ompT, ki se pogosto pojavljajo med virulentnimi sevi. V ta namen smo oblikovali šest kompletov začetnih oligonukleotidov za pomnoževanje teh treh tarčnih genov z LAMP in med njimi izbrali najboljše. Z LAMP dobljene rezultate smo primerjali z rezultati, ki smo jih dobili s PCR in ugotovili smo, da so rezultati obeh metod primerljivi. Pripravili smo protokol priprave različnih vzorcev (tkiva notranjih organov, stelja, krma, iztrebki in zrak), v katerih lahko neposredno potrdimo prisotnost virulentnih genov, brez predhodne osamitve DNA. Z metodo LAMP smo v času krajšem od 30 minut potrdili prisotnost vseh treh genov v različnih vzorcih. Dokazali smo tudi, da je metoda LAMP neobčutljiva za snovi, ki zavirajo potek reakcije PCR in so sicer prisotne v bioloških vzorcih. LAMP se je izkazala za specifično, robustno, selektivno, občutljivo, hitro in enostavno diagnostično metodo za odkrivanje izbranih genov virulentnih dejavnikov sevov APEC. Njena velika odlika je možnost uporabe na terenu.

• Language: Slovenian
• Keywords: Escherichia coli, sevi APEC, bakterijske bolezni, perutnina, kolibaciloza, molekularne metode, z zanko posredovano izotermalno pomnoževanje DNA, LAMP, gen sitA, gen traT, gen ompT
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Lotrič]
• Year: 2018
• PID: 20.500.12556/RUL-102774
• UDC: 579.62:579.842:577.213.3
• COBISS.SI-ID: 4942200
• Publication date in RUL: 08.09.2018

Secondary language

• Language: English
• Title: Development of a method for detection of sitA, traT and ompT genes in Escherichia coli from poultry

Abstract

Colibacillosis is the most common disease in poultry and it is the major cause of death in flocks. Diseases caused by avian pathogenic Escherichia coli strains (APEC) result in significant financial losses to the poultry industry worldwide. Typical clinical signs related to colibacillosis are very diverse. APEC strains cause primary or secondary infections of birds. Several virulence genes were shown to be implicated in avian colibacillosis, but all determinants of pathogenicity are not explained yet, thus the control of infections and identification of the causative E. coli strains are still difficult. Nowaday we use traditional diagnostic methods for detection of APEC. These methods are time-consuming and often priceless. Due to the increasing need for rapid diagnostics, molecular methods come to the forefront, including polymerase chain reaction – PCR and loop mediated isothermal amplification – LAMP. The aim of this master thesis was to developed a method, based on LAMP, for detection of virulence-associated genes in APEC. We developed three LAMP-APEC tests for detection of three virulence-associated genes (sitA, traT and ompT). We designed six sets of primers, of which we end up choosing the most efficient one. We prepared PCR and LAMP protocols to detect the presence of target genes. The analysis revealed that both methods are comparable. We also prepared a protocol for direct detection of virulence-associated genes without prior extraction of DNA. This protocol is suitable for different types of samples (tissues of dead animal’s internal organs, feed, litter, feces and air). With LAMP we were able to detect all three virulence genes in different samples in less than 30 minutes. We also proved that LAMP is not sensitive to PCR inhibitors from animal tissue and other matrix. LAMP has proven to be specific, robust, selective, sensitive, fast and easy to use as a diagnostic tool for detection of the selected virulence genes in APEC. The big advantage is also the fact that the method can be easily performed on the sampling site.

• Keywords: Escherichia coli, strain APEC, bacterial diseases, poultry, colibacillosis, molecular methods, loop-mediated isothermal amplification of DNA, LAMP, gene sitA, gene traT, gene ompT