Optimiranje komercijalno dostupnih metoda izolacije RNK i detekcija koronavirusa (SARS-COV-2) u lancu hrane

doctoral thesis

• Title (english): Optimization of commercially available methods for RNA isolation and detection of coronaviruses (SARS-CoV-2) in the food chain
• Author: Zdenko Mlinar
• Mentor: Jadranka Frece (mentor)
• Committee member: Ksenija Markov (predsjednik povjerenstva)
• Committee member: Jasna Novak (član povjerenstva)
• Committee member: Jasna Bošnir (član povjerenstva)
• Committee member: Jasna Mrvčić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for General Microbiology and Food Microbiology); Zagreb
• Defense date and country: 2023, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology
• Universal decimal classification (UDC): 57 - Biological sciences. Physical anthropology. Biodiversity

Abstract

Novi koronavirus SARS-CoV-2 primarno je respiratorni virus, no putevi prijenosa nisu u potpunosti razjašnjeni pa tako niti onaj kroz lanac hrane. Tehnika izolacije ribonukleinskih kiselina (RNK) jedan je od najvažnijih elemenata u otkrivanju SARS-CoV-2. U ovom radu napravljeno je eksperimentalno ispitivanje i procjena četiri najprikladnija komercijalno dostupna izolacijska kita s izvornim protokolom proizvođača i modificiranim protokolom u koraku precipitacije u kojem se koristio etanol, odnosno izopropanol. Na osnovu tih rezultata izdvojen je najprikladniji kit kojim je napravljena izolacija RNK iz uzoraka briseva površina pakiranja hrane, površine hrane i gotovih jela, a zatim i detekcija virusa putem RT-PCR-a u stvarnom vremenu. Dobiveni rezultati dali su uvid u stvarno stanje prisutnosti virusa SARS-CoV-2 u lancu hrane te uz pregled dostupne literature omogućili pojašnjavanje uloge i značaja hrane kao mogućeg vektora za prijenos virusa SARS-CoV-2. Uz ispitivanje prisutnosti virusa, u svim uzorcima ispitana je bakterijska flora, u brisevima indikatorski mikroorganizmi, a u hrani mikroorganizmi prema kriterijima nacionalnog Vodiča za mikrobiološke kriterije. Posebno su znakoviti rezultati potvrde primjenjivosti odabranog kita koji ukazuju na značajan udio uzoraka površina pozitivnih na virus SARS-CoV-2 s naglaskom da su ti uzorci iz prostora u kojima su boravile oboljele osobe s manje izraženim simptomima ili bez simptoma. Prema rezultatima zaključnog dijela studije u kojoj su ispitani uzorci koji se odnose na lanac hrane, može se zaključiti da je mogućnost infekcije neznatna s obzirom na to da virus i virusne čestice u tim uzorcima nisu otkriveni.

Abstract (english)

The new coronavirus SARS-CoV-2 is primarily a respiratory virus, but the routes of transmission have not been fully clarified, including the one through the food chain. The ribonucleic acid (RNA) isolation technique is one of the most important elements in the detection of the SARS-CoV-2. In this paper, experimental testing and evaluation of the four commercially available extraction kits with the original protocol of the manufacturer and a modified protocol in the precipitation step in which ethanol and isopropanol were used were made. The selected kit was used to isolate RNA from swab samples of food packaging surfaces, food surfaces and ready-to-eat meals, followed by virus detection via real-time RT-PCR. The obtained results gave an insight into the actual situation of the presence of the SARS-CoV-2 virus in the food chain, and with a review of the available literature, made it possible to clarify the role and significance of food as a possible vector for the transmission of the SARS-CoV-2 virus. In addition to testing the presence of viruses, all samples were tested for bacterial flora, indicator microorganisms in swabs, and in food for microorganisms according to the criteria of the national Guide for microbiological criteria. The results of the confirmation of the applicability of the selected kit are particularly significant, indicating a significant proportion of surface samples positive for the SARS-CoV-2 virus, with the emphasis that these samples are from areas where sick persons with less pronounced symptoms or no symptoms have stayed. According to the results of the final part of the study in which the samples related to the food chain were examined, it can be concluded that the possibility of infection is trivial since the virus and virus particles were not detected in these samples.

• Keywords: SARS-CoV-2; hrana; RT-PCR; koronavirus; prijenos hranom; izolacija RNK
• Keywords (english): SARS-CoV-2; food; RT-PCR; coronavirus; foodborne transmission; RNA extraction
• Language: croatian
• URN:NBN: urn:nbn:hr:159:128710
• Promotion: 2023
• Study programme: Title: Postgraduate PhD Study in Nutrition; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje biotehničkih znanosti, polje nutricionizam (doktor/doktorica znanosti, područje biotehničkih znanosti, polje nutricionizam)
• Type of resource: Text
• Extent: 104 str. ; 30 cm
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-10-02 10:41:36