key: cord-0688826-75n33q5d authors: da Silva Queiroz, Jackson Alves; de Cássia Pontello Rampazzo, Rita; da Silva Filho, Edivá Basílio; Oliveira, Gabriella Sgorlon; da Costa Oliveira, Suyane; Botelho Souza, Luan Felipo; dos Santos Pereira, Soraya; de Souza Rodrigues, Moreno Magalhães; Salvador Maia, Adriana Cristina; da Silva, Cicileia Correia; de Melo Mendonça, Aline Linhares Ferreira; Lugtenburg, Celina Aparecida Bertoni; de Assis Araújo Aguiar, Francisco; de Souza Soares Rodrigues, Rosiane; Nemeth Santos, Caio Henrique; Di Sabatino Guimarães, Alice Paula; Máximo, Fernando Rodrigues; de Oliveira dos Santos, Alcione; Krieger, Marco Aurélio; Salcedo, Juan Miguel Villalobos; Dall’Acqua, Deusilene Souza Vieira title: Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix date: 2021-01-09 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2021.01.001 sha: 0b22ef7af107f786a02004c1fc268e62788600a1 doc_id: 688826 cord_uid: 75n33q5d INTRODUCTION: COVID-19 is a disease caused by Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) which emerged in China in late 2019. The rapid viral spread has made the disease a public health emergency of worldwide concern. The gold standard for diagnosing SARS-CoV-2 is reverse transcription followed by qualitative real-time polymerase chain reaction (RT-qPCR); however, the role of viral load quantification has not been thoroughly investigated yet. OBJECTIVE: The aim of this study was to develop a high-precision quantitative one-step RT-qPCR reaction using the association of the viral target and the human target in the same reaction. METHODS: The assay standardization involved the absolute quantification method with serial dilutions of a plasmid with the N gene in a biological matrix to build a standard curve. RESULTS AND DISCUSSION: The results demonstrated the possibility of quantifying as few as 2.5 copies/reaction and analysis of 244 patients with known results selected by cross-section revealed 100% agreement with a qualitative RT-qPCR assay registered by Anvisa. In this population, it was possible to quantify patients with between 2.59 and 3.5 × 10(7) copies per reaction and negative patients continued to indicate the same result. CONCLUSION: This assay can be a useful tool for the proper patients management, since the level and duration of viral replication are important factors to assess the risk of transmission and to guide decisions regarding the isolation and release of patients also, an accurate diagnosis is critical information whereas the current COVID-19 pandemic represents the biggest current global health problem. The Coronavirus genus comprises a variety of viral species that infect vertebrates, including human beings (1) . As of May 2019, approximately 2,505 species belonging to this genus have been cataloged; however, only seven of these have been isolated in humans, all of which were linked to respiratory infections. Four of these species are considered common worldwide and trigger mild conditions (HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1) ( This disease causes mild respiratory symptoms in about 80% of those infected, where the basic recommendation is to stay home and observe social isolation. Severe cases with complications characterized by acute respiratory distress syndrome, arrhythmia, septic shock, acute kidney, heart damage, liver dysfunction, and secondary infections are reported in about 20% of cases; 14% being considered serious and 5% considered critical (6, 7) . The viral dynamic of the new coronavirus is not fully understood; however, the severity of the disease may be related to numerous factors such as pre-existing comorbidities and advanced age (8) . The vast majority of patients who develop severe conditions and require hospitalization have an association of at least one comorbidity, of which the main ones are hypertension, diabetes, cardiovascular diseases, and respiratory system diseases (9), or even smoking, malignancies and chronic kidney disease (10) . In the absence of vaccines and pharmaceutical products proven to be effective, the implementation of control measures is an alternative to battle SARS-CoV-2 infection (11). For this purpose, in view of the pandemic scenario, a sensitive and specific diagnosis performed in the right period to avoid false negative results, prove to be efficient in coping with COVID-19. The gold standard for the diagnosis of COVID-19 is reverse transcription followed by qualitative real-time polymerase chain reaction (RT-qPCR) J o u r n a l P r e -p r o o f (12). This method is able to indicate whether or not the patient is infected; however, it is not able to measure viral load, whereas a quantitative RT-qPCR method is able to determine the viral particles number in an infected individual. In viral diagnostics, depending on the target or individual characteristic of each disease, quantitative RT-qPCR assay can be used in a number of ways, such as for screening, diagnosis or confirmatory testing, treatment decisions, monitoring or even evaluation of cure rates after therapeutic intervention (13) . In this context, the objective of the study was to develop a quantitative one-step RT-qPCR reaction that is sensitive and specific to SARS-CoV-2. The study was carried out in accordance with the ethical principles stipulated by The multiplex reaction was standardized using TaqMan Reproducibility and repeatability were measured through 3 assays with 5x10 6 ; 5x10 5 ; 5x10 4 ; 5x10 3 ; 5x10 2 ; 50; 5; 2.5; 1.25 copies/reaction executed on consecutive days, where each point was performed in technical octuplicates. This was the basis for determining analytical sensitivity. Diagnostic sensitivity and specificity were assessed by testing 244 samples, 192 from patients known to be positive and 52 from patients known to be negative for SARS-CoV-2, with a result previously confirmed by LACEN/RO, using the SARS-CoV-2 Molecular Kit (BioManguinhos ® , Brazil). This qualitative kit is based in three singleplex reactions of one step real-time PCR (RT-qPCR), for detection of two viral targets (N1 and N2 region) and an internal human endogenous control for a specific gene RNase P. To summarize the information about the samples obtained, we used descriptive statistical techniques; in this case, we used proportions followed by their respective The The plasmid was diluted in intervals from 5x10 6 copies to 1.25 copies per reaction (5x10 6 ; 5x10 5 ; 5x10 4 ; 5x10 3 ; 5x10 2 ; 50; 5; 2.5; 1.25), to determine amplification efficiency and linearity (figure 1). All points on the quantification curve up to 2.5 copies per reaction had 100% amplification. The slope of the initial quantitative assay was -3.32 with an efficiency of 99.9%, a correlation coefficient of the line (R 2 ) equal to 0.99, and an amplification factor equal to 2. Table 2 shows the Cts of 3 distinct curves that were the basis for determining the assay reproducibility and repeatability. Linear regression analysis of 9 serial dilutions using a recombinant plasmid with a N1 region diluted in RNA extracted from a human biological matrix, ranging from 1.25* to 5x10 6 copies per reaction, tested by RT-qPCR for SARS-CoV-2; Ct: cycle threshold. The 3 assays were carried out with the standard dilutions evaluated individually and together presenting a Coefficient of Variation (CV%) less than 10%. The data for slope, intersection of the line and correlation between points are described in Table 2 . Repeatability and Reproducibility Assays. The reproducibility and repeatability assays were performed in three runs on alternate days with dilutions in technical octuplicates, with Coefficient of Variation (CV%) less than 10% between runs. The analytical sensitivity was determined using the limit of optical quantification (LOQ) at 2.5 copies per reaction and limit of optical detection (LOD95%) at 1.41 copies per reaction; this is the minimum viral load value for qualitative detection ( figure 2 ). Molecular tests are the gold standard for laboratory diagnosis of COVID 19 due to the short time gap for viral RNA detection (16, 17) . This study developed a quantitative assay based on one-step RT-qPCR for the detection of SARS-CoV-2 in a biological matrix using 244 symptomatic patients with qualitative molecular diagnosis, selected by cross-section. Most published RT-qPCR assays for the diagnosis of SARS-CoV-2 are qualitative (18) (19) (20) (21) (22) (23) . The assays most commonly described in the literature involve the N, E, RdRP, and S genes for viral detection (14, 17, 24) . The construction of this quantitative multiplex assay was based on the choice of the viral target N1 (14, 21) , with the addition of an J o u r n a l P r e -p r o o f endogenous human control, a single copy gene RNase P, as a reference gene to monitor the nucleic acids extraction and the RT-qPCR assay in order to avoid false-negative results, thus preserving the reaction quality (25, 26) . Regarding the sensitivity of the developed method, the current published assays for SARS-CoV-2 detection by RT-qPCR report LOD95% values between 2.9 and 275.72 copies per reaction, which confirms that the developed assay has high levels of analytical sensitivity, since the reactions can be quantified up to 2.5 copies/reaction and still have a LOD95% of 1.41 copies per reaction (20, 24, 27, 28) . As for the cross-section, it was found that all 244 patients evaluated presented 100% agreement with the qualitative results, with the lowest quantified viral number at 2.59 copies/reaction (0.4 log10 copies/mL), and the highest at 3.5x10 7 copies/reaction (7.6 log10 copies/mL). The high value of the detected viral load is similar to the viral levels reported in other studies using samples from the respiratory tract (29, 30) . The negative patients continued to demonstrate the same result, guaranteeing the test diagnostic accuracy, since these patients also had clinical symptoms for respiratory diseases. The quantitative RT-qPCR technique has been applied to other respiratory viruses and has shown advantages for the management of patients with respiratory syncytial virus, since this method permits the association of viral load and the development of obstructive pulmonary disease (31) . In some studies with COVID-19 the subject is still controversial and, in some cases, the infected patients monitoring uses qualitative assays mistakenly considering the quantitative methods for monitoring these patients, based only on the Ct values used as a viral load quantification unit, in addition to the absence of a standard curve as reference material and internal control (32) (33) (34) (35) . The viral replication level and duration are important factors to assess the risk of transmission and to guide decisions regarding the isolation of patients (36) and, consequently, to establish measures to control the virus spread; thus, the quantitative assay could be used as a strategy for assessing the viral load of patients in this situation, considering that IgM and IgG antibodies are often not able to be detected after the 14-day period (37) , with reports of viral transmission from post-isolation individuals to close contacts (38) . In some studies on the diagnosis of COVID-19, it was observed that the first qualitative RT-qPCR assays from patients showing signs and symptoms of the disease, J o u r n a l P r e -p r o o f such as high fever and severe pulmonary alterations, showed negative results, and only became positive after subsequent tests during hospitalization (39) (40) (41) (42) . Therefore, monitoring viral load through sensitive molecular tests is essential since these patients could suffer from inadequate management and even compromise hospitals by favoring the virus transmission. Among the limitations faced during the study development, the choice of an adequate human endogenous control stands out, to maintain the same sensitivity of the singleplex reaction after its addition. For this, adjustments were needed in the concentrations of primers, probes of both targets, and annealing temperature. 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