Research Article Impact of Thermal Pretreatment of Saliva on the RT-PCR Detection of SARS-CoV-2 Orlando Miguel Morais , 1 Manuel Rui Azevedo Alves , 1,2 and Paulo Alexandre da Costa Fernandes 1,2 1 Escola Superior de Tecnologia e Gestão, Instituto Polit´ ecnico de Viana do Castelo, Rua Escola Industrial e Comercial de Nun’ ´ Alvares, Viana do Castelo 4900-347, Portugal 2 CISAS, Escola Superior de Tecnologia e Gestão, Instituto Polit´ ecnico de Viana do Castelo, Rua Escola Industrial e Comercial de Nun’ ´ Alvares, Viana do Castelo 4900-347, Portugal Correspondence should be addressed to Paulo Alexandre da Costa Fernandes; paulof@estg.ipvc.pt Received 10 December 2021; Revised 25 April 2022; Accepted 6 May 2022; Published 1 June 2022 Academic Editor: Leny Jose Copyright © 2022 Orlando Miguel Morais et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e use of saliva directly as a specimen to detect viral RNA by RT-PCR has been tested for a long time as its advantages are relevant in terms of convenience and costs. However, as other body ﬂuids, its proven inhibition eﬀect on the ampliﬁcation reaction can be troublesome and compromise its use in the detection of viral particles. e aim of the present work is to demonstrate that saliva pretreatment may inﬂuence the RT-PCR ampliﬁcation of three gene targets of SARS-CoV-2 signiﬁcantly. A pool of RNA from conﬁrmed COVID-19 patients was used to test the inﬂuence of heat pretreatment of saliva samples at 95 ° C for 5, 10, 15 and 20 min on the ampliﬁcation performance of ORF1ab, E, and N SARS-CoV-2 genes. Prolonged heating at 95 ° C signiﬁcantly improves the Ct value shift, usually observed in the presence of saliva, increasing the limit of detection of viral genes ORF1ab, E, and N. When tested using a cohort of COVID-19 patients’ saliva, the increased time of heat pretreatment resulted in a signiﬁcant increase in the detection sensitivity. 1. Introduction Saliva is a complex ﬂuid composed of a mixture of com- ponents produced by major and minor salivary glands [1] and other constituents from the oral mucosa and micro- biome [2, 3]. Its suitability as a specimen for biomonitoring [2] and to detect respiratory viruses has been tested over several years [4–6], and its use in routine testing and screening campaigns to detect SARS-CoV-2 has also been subjected to assessment [4, 7–16]. A recent meta-analysis using 25 published studies involving RT-PCR detection of SARS-CoV-2 in saliva samples highlighted considerable discrepancies in reported ﬁndings, pointing out that many of these discrepancies could probably be attributed to exper- imental procedures, such as target populations, sample collection, and saliva processing protocols [17]. e US Food and Drug Administration has recently issued an emergency use authorization (EUA) for the so- called SalivaDirect assay, a method using saliva directly without extraction in the RT-PCRs [18]. However, the inhibition potential of saliva has been described in the literature [19] and has been a relevant obstacle to more widespread use of this specimen in diagnostic tests. Saliva, as other body ﬂuids, is known to have constituents that may inhibit the RT-PCR [19]. In fact, RT-PCR is a very sensitive and powerful technique based on the ampliﬁca- tion of small amounts of nucleic acids present in a sample. However, due to its enzymatic nature and complex mixture of components including ﬂuorophores and oligonucleo- tides, several substances may aﬀect the reaction mainly by interfering with the DNA polymerase activity/stability, nucleotide/nucleic acid stability, and ﬂuorescence intensity [20, 21]. In the present work, saliva is used directly without ex- traction in RT-PCRs to detect 3 viral targets of SARS-CoV-2, and it is demonstrated that the inhibition potential of this Hindawi Advances in Virology Volume 2022, Article ID 7442907, 8 pages https://doi.org/10.1155/2022/7442907