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Optimizacija hkratne detekcije genotipov iz neonatalnega vzorca krvnega madeža za namene presejalnega testiranja
ID Zafran, Mojca (Author), ID Hovnik, Tinka (Mentor) More about this mentor... This link opens in a new window, ID Kovac, Jernej (Comentor)

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Abstract
Presejalno testiranje novorojencev predstavlja pomemben doprinos sodobnemu zdravstvu, saj omogoča zgodnje odkritje nekaterih hudih bolezenskih stanj genetskega izvora in s tem hitrejše zdravljenje. Glede na dokaze podprte z medicinskimi podatki in zmožnosti družbe, da zagotovi sredstva za izvajanje presejalnih testiranj novorojencev, ima vsaka država svoj program presejalnega testiranja. Nekatere razvite države imajo v program presejanja že vključeni tudi spinalno mišično atrofijo (SMA) in hudo kombinirano imunsko pomanjkljivost (SCID). V Sloveniji se testiranje za ti dve bolezni izvede šele ob prvem sumu in kadar se prvi simptomi po navadi že kažejo. Z vključitvijo SMA in SCID v program testiranja bi lahko diagnosticirali ti dve bolezni še pred pojavom resnih simptomov in tako preprečili hujši potek ali smrt obolelih. Za namene presejalnega testiranja se uporabi vzorce posušenih krvnih madežev iz katerih se izolira DNA. Cilj magistrskega dela je bil med seboj primerjati dva testa za genotipizacijo z alelno diskriminacijo v qPCR pomnoženih genetskih označevalcih v genu SMN1, krožnega fragmenta TREC in KREC (TaqManTM SCID/SMA Plus Assay proizvajalca Thermo Fisher scientific in EonisTM SCID-SMA kit proizvajalca Perkin Elmer). Na podlagi rezultatov smo ugotovili, da lahko z obema testoma uspešno potrdimo prisotnost ali odsotnost označevalca za SMA. Zaradi pomanjkanja količine vzorcev SCID pozitivnih preiskovancev, smo te testirali le s testom za genotipizacijo genetskih označevalcev za SMA in SCID proizvajalca Perkin Elmer in vzorcem potrdili odsotnost označevalcev SCID. Oba testa sta imela enako občutljivost, medtem ko je imel višjo specifičnost test proizvajalca Thermo Fisher Scientific. S t-testom smo pokazali, da se povprečne vrednosti Cq za označevalec SMN1 vzorcev preiskovancev brez SMA in SCID (negativne kontrole) obeh primerjanih testov med seboj statistično ne razlikujejo. Ugotovili smo, da je test proizvajalca Perkin Elmer natančnejši pri določanju prisotnosti genetskih označevalcev bolezni SMA in SCID.

Language:Slovenian
Keywords:presejalno testiranje novorojencev, spinalna mišična atrofija, hudo kombinirana imunska pomanjkljivost, posušen krvni madež, test, qPCR reakcija, markerji, detekcija
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2023
PID:20.500.12556/RUL-147469 This link opens in a new window
COBISS.SI-ID:157936131 This link opens in a new window
Publication date in RUL:06.07.2023
Views:1144
Downloads:66
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Secondary language

Language:English
Title:Optimisation of simultaneous detection of genotypes from a neonatal dried blood spots in newborn screening
Abstract:
Newborn screening is an important contribution to modern healthcare, as it allows the early detection of some serious genetic conditions and therefore a faster treatment. Each country has its own newborn screening program, depending on the evidence-based medical data and society's ability to provide the resources to implement newborn screening. Some developed countries have already included spinal muscular atrophy (SMA) and severe combined immunodeficiency (SCID) in their screening programme. In Slovenia, testing for these two diseases is only performed at the first suspicion and when the first symptoms are usually already present. By including SMA and SCID in the testing programme, these two diseases could be diagnosed before the appearance of serious symptoms and therefore prevent a more severe outcome or death of the patients. For screening purposes, we use samples of dried blood spots, from which DNA is isolated. The aim of this master thesis was to compare two genotyping assays for allele discrimination in qPCR amplified genetic markers in the SMN1 gene, the TREC and KREC circular fragment (TaqManTM SCID/SMA Plus Assay from Thermo Fisher scientific and EonisTM SCID-SMA kit from Perkin Elmer). Due to the lack of samples from SCID-positive patients, we only tested them with the Perkin Elmer SMA and SCID genetic marker genotyping assay and confirmed the absence of SCID markers. Both tests had the same sensitivity, while the Thermo Fisher Scientific test had a higher specificity. The t-test showed that the mean of Cq values for the SMN1 marker of the samples of people without SMA and SCID (negative controls) of the two tests compared were not statistically different. We found that the Perkin Elmer test is more accurate in determining the presence of genetic markers of SMA and SCID.

Keywords:newborn screening, spinal muscular atrophy, severe combined immunodeficiency, dried blood spot, test, qPCR reaction, markers, detection

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