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Primerjava standardne in molekularne metode za določitev koncentracije arbovirusov
ID Primožič, Ana (Author), ID Avšič Županc, Tatjana (Mentor) More about this mentor... This link opens in a new window, ID Korva, Miša (Co-mentor)

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PID: 20.500.12556/rul/7498e5bd-dbc2-4526-982c-935db47f4807

Abstract
Arbovirusi se v naravi ohranjajo v kronično okuženih členonožcih. Pri ljudeh in živalih lahko povzročijo širok spekter bolezni: od blagih, vročinskih oblik, do zelo hudih oblik bolezni, kot sta meningoencefalitis ali hemoragična mrzlica. Za spremljanje rasti virusov v celičnih kulturah se uporabljata predvsem klasični virološki tehniki: 50-odstotna infektivna doza (TCID50) in metoda štetja plakov, vendar za obe tehniki potrebujemo visoko usposobljeno tehnično osebje ter dostop do laboratorija 3. stopnje biološke varnosti. Poleg zahtevnosti, sta metodi tudi časovno zamudni. Zato, se vse pogosteje za merjenje infektivnosti virusov uporabljajo sodobni molekularni pristopi, vendar natančnih primerjav med posameznimi tehnikami in klasičnimi metodami še ni na voljo. Za določitev koncentracije arbovirusov v celičnih kulturah smo primerjali rezultate metod TCID50 in digitalne verižne reakcije s polimerazo (dPCR) ter skušali ugotoviti ali bi lahko dPCR za oceno koncentracije v celoti nadomestil klasične virološke tehnike v laboratoriju. V analizo smo vključili 4 arboviruse: virus klopnega meningoencefalitisa (KMEV), virus Zahodnega Nila (WNV), virus Usutu (USUV) in virus rumene mrzlice (YFV). Ugotovili smo, da bi v nekaterih primerih lahko zamenjali dPCR za TCID50, saj se koeficient primerjave ohranja skozi pasaže, pri čemer smo morali uspešno vpeljati molekularne metode in izračunati razmerja med vrednostmi TCID50/ml in številom kopij RNA/ml. V našem primeru smo uspešno vpeljali molekularno metodo in koeficient za KMEV, WNV (linija 2), YFV in USUV. Vsekakor pa molekularne metode ne morejo v celoti nadomestiti klasičnih tehnik, lahko so le v pomoč pri rutinski diagnostiki, saj so hitrejše, bolj ponovljive in manj nevarne za tehnično osebje.

Language:Slovenian
Keywords:virusi, arbovirusi, flavivirusi, klasične virološke metode, 50-odstotna infektivna doza, molekularne metode, digitalni PCR, primerjava metod
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Publisher:[A. Primožič]
Year:2017
PID:20.500.12556/RUL-96712 This link opens in a new window
UDC:578.833.2.083.2
COBISS.SI-ID:4830328 This link opens in a new window
Publication date in RUL:12.10.2017
Views:1928
Downloads:674
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Secondary language

Language:English
Title:Comparison of standard and molecular method for determination of arboviral concentration
Abstract:
Arboviruses are maintained in nature in chronically infected arthropods. In humans and animals, they can cause a wide range of diseases, from mild, febrile forms, to very serious diseases such as encephalitis or hemorrhagic fever. Viral growth in cell cultures is traditionally monitored by classical virological techniques such as 50 % tissue culture infective dose (TCID50) and plaque forming units, but both techniques require highly trained staff and access to biosafety level 3 laboratory. In addition to the exceptional complexity, the methods are also time-consuming. Recently, several laboratories decided to use modern molecular approaches to determine viral titters, but the comparison or standardization between molecular techniques and classical virological techniques is still not-existent. We compared results obtained with TCID50 and digital polymerase chain reaction (dPCR) for determination of arboviral concentration in cell culture. We have included 4 different arboviruses: tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Usutu virus (USUV) and yellow fever virus (YFV). In conclusion, we have found that in some cases dPCR could be substituted for TCID50, since the coefficient of comparison was maintained through passages, whereby molecular methods were successfully applied, and the ratios between the values of TCID50/ml and the number of RNA copies/ml were calculated. In our case, we successfully implemented the molecular method and the coefficient for TBEV, WNV (lineage 2), YFV and USUV. Definitely, molecular methods can not completely replace classical techniques, they can only be helpful in routine diagnostics, as they are faster, more repeatable and less dangerous for technical staff.

Keywords:viruses, arboviruses, flaviviruses, classical virological methods, 50 % tissue culture infective dose, molecular methods, digital PCR, comparison of methods

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