Optimisation of RT-LAMP reaction for detection of SARS-CoV-2 in saliva.

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.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

Social inclusion of people with intellectual disabilities employed under the employment support scheme

The right to employment is the right of every single person. Employment is also an indicator of a person's quality of life. Nowadays, we try to provide the best possible quality of life for people with intellectual disabilities. An important factor in this is also social inclusion in regular environments. Until recently, regular employment of people with MDRs was not possible because the legislation did not allow it. With the adoption of the Social Inclusion Act (2018), the possibility of finding employment for people with MDR in a regular work environment is explained for the first time in Slovenia. They were involved in forms of employment in regular environments run by protective work centres. Persons employed in such forms of employment have more opportunities to interact with persons without MDR. Previous research, therefore, has established a link between the employment of people with MDR in regular settings and mostly socially included personality with MDR in all areas. The selected areas are important parts of the quality of life of people with MDR and the essence of the newly adopted law on social inclusion of the disabled (2018). In this research with the help of four user employees in supportive forms of employment in a regular work environment, I investigate user satisfaction with employees and factors of social inclusion of persons with a man in mental development. I collected data through a partially structured interview and analysed them using a qualitative research method. In doing so, I conducted 12 interviews, 4 with users and their employment mentor and group user habilitator. In the research, I find that users are satisfied with their employee in supportive forms of employment in a regular environment. Above all, so pleased that the work takes place outside the institution. Mentors perceive them as good workers and are happy with them. Users can be heard both in the collective and in the wider social environment. The amount of time spent in a regular employment environment does not affect the experience of user acceptance. Accepted are heard by both users who perform work over a longer period of time and users who perform work several times a week for several hours

Robust saliva-based RNA extraction-free one-step nucleic acid amplification test for mass SARS-CoV-2 monitoring

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections