Optimizacija reakcije RT-LAMP za detekcijo SARS-CoV-2 v slini.

Rajh, Eva (author), Benčina, Mojca (mentor), Gunčar, Gregor (comentor)

Konec leta 2019 so se v provinci Wuhan na Kitajskem pojavila prva poročila o izbruhu novega koronavirusa, poimenovanem SARS-CoV-2. Virus se je hitro razširil po celem svetu in predstavlja eno največjih globalnih zdravstvenih kriz 21. stoletja. Množično testiranje populacije je ključno za ustavitev širjenja pandemije covid-19. RT-qPCR, ki je zlati standard molekularnih diagnostičnih orodij, ima omejitve, ki so predvsem zaradi povečanih potreb po testiranju postale očitne. RT-qPCR je dolgotrajen, zahteva dobro opremljen diagnostični laboratorij in v primeru množičnega testiranja, pri katerem se vsi zanašamo na isto metodo, lahko pride do primanjkljaja komercialnih reagentov in opreme. Zaradi preprostosti uporabe, hitrosti in ker za reakcijo ne potrebujemo hitrega spreminjanja temperature, je metoda obratne transkripcije in z zankami posredovanega pomnoževanja (RT-LAMP) zanimiva alternativa klasičnemu testiranju RT-qPCR. Slina je v primerjavi z nosno žrelnimi vzorci preprostejša za odvzem in neinvazivna, vendar vsebuje motilce in inhibitorje encimov RT-LAMP in RT-qPCR. Cilj raziskave je bil oblikovati diagnostično metodo na osnovi kolorimetričnega RT-LAMP za hitro detekcijo SARS-CoV-2 s prostim očesom v vzorcu sline brez izolacije virusne RNA. Optimirali smo sestavo vzorčnega pufra, reakcijske pogoje enostopenjske reakcije RT-LAMP ter določili analitično občutljivost in specifičnost metode. Vzporedno smo optimirali še metodo RT-qPCR za detekcijo virusne RNA na vzorcih neobdelane sline. Diagnostična metoda je hitra in od začetka procesiranja vzorcev do rezultata traja le 50 minut. Ugotovili smo, da je ključna komponenta vzorčnega pufra RNA stabilizacijski pufer. Z inhibitorji RNaz (RNasecure) in odstranjevalci nečistoč (Chelex100) smo dodatno izboljšali kvaliteto vzorca. Z metodo RT-LAMP smo na tarčni regiji N2 dosegli analitično občutljivost 615 molekul/µL vzorca in analitično specifičnost 97,5 %. Ko smo metodo RT-LAMP preizkusili v realnem okolju s testiranjem vzorcev sline zbranih na COVID vstopni točki, smo dosegli 61 % občutljivost in 89 % specifičnost. Z metodo RT-qPCR smo na istih vzorcih dosegli 95 % občutljivost in 100 % specifičnost. Ugotovili smo, da je slina tudi brez izolacije virusne RNA primerna za detekcijo virusne RNA, vendar je metoda RT-LAMP za ta namen manj uporabna.

• Language: Slovenian
• Keywords: Ključne besede: RT-LAMP, SARS-CoV-2, diagnostična metoda
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-129646
• COBISS.SI-ID: 79054851
• Publication date in RUL: 06.09.2021

Secondary language

• Language: English
• Title: Optimisation of RT-LAMP reaction for detection of SARS-CoV-2 in saliva.

Abstract

At the end of 2019, the first reports of an outbreak of a new coronavirus called SARS-CoV-2 appeared in Wuhan province, China. The virus has spread rapidly around the world, and it became one of the greatest global health crises of the 21st century. Mass population testing is key to preventing the spread of the covid-19 pandemic. RT-qPCR, the golden standard of molecular testing, has limitations that have become apparent mainly due to the significant increase in need for testing. RT-qPCR is time consuming, requires a well equipped diagnostic laboratory, and in the case of mass testing, in which we all rely on the same method, can lead to a shortage in commercial reagents and equipment. Due to ease of use, speed, cost-effectiveness, and because the reaction does not require a rapid change in temperature for amplification, reverse transcription and loop-mediated isothermal amplification (RT-LAMP) is an interesting alternative to classical RT-qPCR testing. Collection of saliva is easy and non-invasive to collect, but it does contain disruptors and inhibitors of the enzymes in RT-qPCR and RT-LAMP. The aim of this study was to design a diagnostic method, based on colorimetric RT-LAMP for rapid detection of SARS-CoV-2 in saliva samples without prior isolation of viral RNA. We optimised the composition of the sample buffer, conditions for one-step colorimetric RT-LAMP reaction, and determined analytical sensitivity and specificity. In parallel, we also optimised the RT-qPCR method for the detection of viral RNA in saliva samples without the extraction of RNA. RT-LAMP diagnostic method was rapid, and it took only 50 minutes from sample collection to a readout of results. We found that the key component of the sample buffer was the RNA stabilisation buffer, and with the addition of RNase inhibitor (RNasecure) and chelating agent for removal of impurities (Chelex100), we further improved the quality of samples. RT-LAMP method targeting region N2 of SARS-CoV-2 genome was used to achieve analytical sensitivity of 615 molecules/ µl of the sample and an analytical specificity of 97.5 %. When the RT-LAMP method was tested in a real environment by testing saliva samples collected at the COVID entry point, we achieved 61 % sensitivity and 89 % specificity. With the RT-qPCR method, we achieved 95 % sensitivity and 100 % specificity on the same set of samples. We found that saliva is suitable for viral RNA detection even without viral RNA isolation, but the RT-LAMP method is less useful for this purpose than conventional RT-qPCR.

• Keywords: LAMP, SARS-CoV-2, diagnostic method