key: cord-1042584-zfp04hgx
authors: Flores-León, Diana; Quino, Willi; Cúneo, Alejandra E.; Caro-Castro, Junior; Cucho-Meza, Evans; Gavilan, Ronnie G.
title: Evaluation of the SMARTCHEK Genesystem RT-qPCR assay for the detection of SARS-CoV-2 in clinical samples
date: 2022-04-04
journal: BMC Infect Dis
DOI: 10.1186/s12879-022-07319-0
sha: d70d591bc899d8fda602003d06928ab1ffaed06e
doc_id: 1042584
cord_uid: zfp04hgx

BACKGROUND: The COVID-19 pandemic remains the main public health problem, due to the quick and easy dissemination of the causal agent, SARS-CoV-2 virus, around the world. Since the beginning of the pandemic, an opportune laboratory diagnosis has been critical to respond this emergency, and RT-qPCR has been used as reference molecular tests for detection of SARS-CoV-2. METHODS: In this study, we performed the evaluation of a RT-qPCR SMARTCHEK platform (SMARTCHEK, Genesystem) for SARS-CoV-2 detection based on the amplification of RdRp and N gene markers. The platform was evaluated with nasopharyngeal swab samples corresponding to 360 suspected cases of COVID-19 which were remitted to Instituto Nacional de Salud in Peru. This quick method was compared with conventional RT-qPCR as gold standard. RESULTS: The RT-qPCR SMARTCHEK showed a 98.1% sensitivity (CI: 93.3–99.8%), a 98.8% specificity (CI: 96.6–99.8%), a 97.2% positive predictive value (CI: 92–99.4%) and a 99.2% negative predictive value (CI: 97.2—99.9%). The assay demonstrated a strong agreement between the RT-qPCR SMARTCHEK and conventional RT-qPCR (kappa value ≥ 0.966). CONCLUSION: The RT-qPCR SMARTCHEK is a platform that gives reliable and fast results, with high sensitivity and specificity for the detection of SARS-CoV-2, and it will be considered a suitable alternative to COVID-19 diagnosis in low-resource settings. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-022-07319-0.

The Coronavirus disease 2019 is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which emerged in Wuhan, province of Hubei, China in December, 2019, affecting more than 190 countries all over the world [1, 2] . Until February 15th, 2022, more than 413.7 million cases of COVID-19 have been reported in 191 countries and approximately more than 5.8 million deaths from this disease [3] . Peru is experiencing one of the largest COVID-19 epidemics in Latin America, reporting 3.36 million of confirmed cases and 206 thousand deaths until February 15th, 2022 [4] . Due to the increasing cases of COVID-19, Peruvian government has launched an epidemiological alert to intensify early detection of cases [5] .

In most countries, one of the main preventive measures include the rapid diagnosis of COVID-19 cases, thus World Health Organization recommend reverse transcription-PCR real-time (RT-qPCR) as standard test [6] [7] [8] . Recently, U.S. Food and Drug Administration (FDA) authorized over 30 in vitro diagnostic methods

*Correspondence: rgavilan@ins.gob.pe 1 Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Peru Full list of author information is available at the end of the article for SARS-CoV-2 detection under an Emergency Use Authorization (EUA), providing various qPCR alternatives including quantitative reverse transcription-PCR (RT-qPCR), reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and direct rapid RNA extraction-free RT-qPCR [9] . However, most molecular methods present some limitations, including the need of expensive equipment, long periods of processing time, trained laboratory-personal and specialized laboratory infrastructure. Likewise, the access to molecular testing in low-resource communities in Peru is very scarce. For this reason, the developing of faster and precise diagnosis molecular methods applied on in low-resource settings are necessary to improve early detection and control of COVID-19 [10] .

To address these limitations, rapid molecular methods as viral RNA detection kit for SARS-CoV-2 (SMARTCHEK, Genesystem) are available, based on a biochip sample format on the amplification of RdRp and N gene markers which gives around 45-min response time with a simple workflow, offering all the benefits of a RT-qPCR at the same time [11] . Thus, the aim of this study was to evaluate the platform for quick detection of SARS-CoV-2 based on RT-qPCR from clinical samples.

A total of 360 nasopharyngeal swab samples were received by the Instituto Nacional de Salud (INS) for molecular diagnosis by RT-qPCR under the SARS-CoV-2 surveillance system in Peru.

The viral RNA of clinical samples was extracted and purified using QIAamp Viral RNA Mini kit (Qiagen, Germany), following the manufacturer recommendations. RNA quantification and quality assessment were evaluated by spectrophotometry using DS-11 FX (DeNovix, USA) and fluorometry using Qubit 3.0 (Invitrogen, USA).

The conventional reverse transcription-qPCR (RT-qPCR) was performed using the primers and probes described by Corman et al. [7] which are recommended for SARS-CoV-2 detection by the Centers for Disease Control and Prevention (CDC). RT-qPCR amplification of the SARS-CoV-2 which includes E (0.4 μM) and RdRp (0.8 μM) as protein gene targets were performed using one step RT-qPCR kit Superscript III with Platinum Taq Polymerase (Invitrogen, Germany). The assay uses a human reference gene as amplification control. Cycling conditions were as follows: 15 min at 50 °C, 2 min at 95 °C, and 45 cycles, with 1 cycle consisting of 15 s at 95 °C and 30 s at 58 °C. Rotor Gene Q Qiagen thermocycler was employed for the amplification. To interpret the results, SARS-CoV-2 positive samples was considered when there was an amplification in at least one of two markers with Ct value < 40, and considered negative with Ct values ≥ 40. In case of internal control (GAPDH), positive reaction was considered with Ct value < 40 and negative reaction with Ct values ≥ 40. In all reactions, the internal control must be positive before the final interpretation.

The samples were analyzed with RT-qPCR SMARTCHEK (Genesystem, South Korea) detection kit for the novel coronavirus (SARS-CoV-2) following the manufacturer recommendations. The kit is based on a biochip sample format, where primers and probes employed for the detection of RdRp and N genes specific to SARS-CoV-2 are dehydrated on the chip wells. For each Biochip, 04 patient samples can be detected simultaneously (well 2-9) and an internal control is evaluated in each well. Well number 1 corresponds to the not template control (contains pre-labeled primers and probes) and well number 10 is assigned to the positive control (contains pre-labeled primers and probes along with positive templates) (Additional file 1: Fig S1) .

For the preparation of the sample reaction for RT-qPCR, a mix of 10 μL of Premix and 10 μL of viral RNA was used. The mix was inoculated in duplicate on the chip; each single assay allows detecting N and RdRp genes, respectively. Each Biochip can detect 4 patient samples simultaneously for two markers, including internal and negative control.

The RT-qPCR assay was performed in the Genechecker UF-300 (Genesytem, South Korea) platform, using temperature condition including 10 min at 50 °C, 30 s at 95 °C, and 40 cycles, with 1 cycle consisting of 5 s at 95 °C, 20 s at 58 °C and 5 s at 72 °C. The presence of SARS-CoV-2 virus was detected by the identification of an amplification curve with Ct ≤ 37 value for RdRp gene, N gene and the amplification of internal control. In all reactions, the internal control must be positive to allow for results to be interpreted.

The limit of detection (LOD) of RT-qPCR and RT-qPCR SMARTCHEK were compared. LoD was calculated using the positive control 2019-nCoV_RdRp (ORF1ab) (Integrated DNA Technologies), which contains the envelope gene and a portion of the RNA-dependent RNA polymerase (RdRp) and the positive control 2019-nCoV_E (Integrated DNA Technologies), both controls have been synthesized at a concentration of 200,000 copies/μL. Predetermined copy numbers of biochemically synthesized RNA were serially diluted ten-fold from 10 6 copies to 10 -1 copies of the target gene per reaction. The linearity test of both methodologies was evaluated by means of linear regression analysis (average of the Ct values versus logarithmic concentration values).

Cross-reactivity of SMARTCHEK RT-qPCR was evaluated from fourteen viral RNAs: influenza viruses (A and B), respiratory syncytial virus, human metapneumovirus, rhinovirus, Zika virus, and Human Immunodeficiency Virus (HIV).

In the evaluation of the diagnostic sensitivity and specificity of RT-qPCR SMARTCHEK, the primers and probes described by Corman et al. [7] were used, taking the conventional RT-qPCR as reference method. A total of 360 nasopharyngeal swab samples were used for the study. An estimation of diagnostic sensitivity, specificity, positive predictive value and negative predictive value of RT-qPCR SMARTCHEK was performed.

The analysis of the diagnostic accuracy data was performed with the statistical program Stata v16.0 (Stata Corporation, College Station, TX, USA). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of SMARTCHEK RT-qPCR were estimated. Likewise, the results obtained between both methodologies were compared. All point estimators obtained were accompanied by their 95% confidence interval (95% CI).

To compare the linearity of the RT-qPCR SMARTCHEK assay and the RT-qPCR, the concentrations of the serial dilutions were plotted on the X axis and the average Ct of the three replicates performed on the Y axis. Correlation coefficient R 2 obtained in the linear regression was used to determine the linearity of the RT-qPCR assay. LoD was calculated by Probit regression analysis, at 95% probability to detect the analytic limit that can be reliably detected by molecular assays, using MedCalc v19.21 software. The degree of agreement was quantified by Cohen's kappa statistic and correlation between methodologies was evaluated using the Spearman's correlation coefficient. Also, Ct values were evaluated using the intraclass correlation coefficient and Bland-Altman analysis. On the Y axis, the differences in Ct between both methods (AB) were represented, while on the X axis, the average of the two measurements was represented (A + B/two).

Detection limits were compared by RT-qPCR and RT-qPCR SMARTCHEK for theRdRp gene, obtaining a LoD of 20 copies/reaction and 200 copies/reaction with R 2 = 0.9925 and 0.9973, respectively (Fig. 1A, B) . In the same way, the N gene and the E gene were evaluated (RT-qPCR SMARTCHEK), both obtained a LoD of 20 copies/reaction with an R 2 = 0.9952 and 0.9959 (Fig. 1C, D) , respectively. The results showed a lower detection range of RT-qPCR SMARTCHEK compared to RT-QPCR for the RdRp gene. A minimum range of difference between N and E gene detection was also evidenced by RT-qPCR SMARTCHEK and RT-qPCR.

In Fig. 2 , the LoD (95% probability) of the RdRp gene was identified, being 373.74 (95% CI: 299.74-447.75) copies/reaction using RT-qPCR SMARTCHEK, and 40.68 (95% CI: 32.62-48.74) copies/reaction using RT-qPCR ( Fig. 2A, B) . In the case of E gene by RT-qPCR and N gene by RT-qPCR SMARTCHEK, they also presented a LoD of 40.68 (95% CI: 32.62-48.74) copies/ reaction. The detection of SARS-CoV-2 at a probability of 95% presents a very similar detection limit for both methodologies.

From the evaluation of the analytical specificity of the RT-qPCR SMARTCHEK, it has been shown that the fourteen viral RNAs: influenza viruses (A and B), respiratory syncytial virus, human metapneumovirus, rhinovirus, Zika virus and HIV were not detected by the methodology evaluated (Table 1) .

The conventional RT-qPCR based on the RdRp and E genes detected a total of 100 positives and 260 negatives. This method was used as gold standard on the evaluation of the RT-qPCR SMARTCHEK based on the RdRp and N genes amplification, which detected 105 positives and 255 negatives; the quick RT-qPCR showed 98.1% sensitivity (CI: 93.3-99.8%), 98.8% specificity (CI: 96.6-99.8%), 97.2% positive predictive value (CI: 92-99.4%) and 99.2% negative predictive value (CI: 97.2-99.9%) ( Table 2 ). The RT-qPCR SMARTCHEK is a platform that gives reliable and fast results, with high sensitivity and specificity for the detection of SARS-CoV-2.

In the comparative analysis of the RdRp gene by both methodologies, 7 false negatives and 2 false positives samples were observed (Fig. 3A) . The N gene of the RT-qPCR SMARTCHEK was also compared with the RdRp gene of the RT-qPCR, obtaining a lower number of false positives (n = 8) and a higher number of false negatives (n = 5) (Fig. 3B) . The RdRp gene of the RT-qPCR SMARTCHEK was also compared with the E gene of the RT-qPCR, identifying 10 false negatives and 4 false positives (Fig. 3C) , likewise the N and E genes were compared, showing a lower number of false positives (n = 3) (Fig. 3D) .

The concordance between the RdRp and N genebased RT-qPCR SMARTCHEK and conventional RT-qPCR which targeted the RdRp and E gene were further evaluated through the Cohen's kappa index, obtaining a value of 0.966 (CI: 95%: 0.937-0.996) (p < 0.001), indicating a good statistic concordance between the evaluated diagnostic tests. Also, the correlation of the individual tests using the Spearman's correlation between RdRp gene-based RT-qPCR SMARTCHEK, N gene-based RT-qPCR SMARTCHEK, RdRp gen-based conventional RT-qPCR and E gene-based conventional The expected values (converted to log 10 ) were plotted on the X axis, and the average of the RT-qPCR Ct obtained from the three replicates was plotted on the Y axis (A is for RdRp gene evaluated by RT-qPCR SMARTCHEK and B is for RdRp gene evaluated by RT-qPCR). C, D ( Control 2019-nCoV_E): Expected values (converted to log 10 ) were plotted on the X axis, and the average of the RT-qPCR Ct obtained from the three replicates were plotted on the Y axis (C is for N gene and D is for E gene). Data are representative of three independent experiments with 3 replicates for each concentration. The software used was MedCalc v19.2.1

RT-qPCR showed a high correlation between them (CI: 0.93 and 0.97 (Fig. 4A) .

Using the Bland-Altman representation, the Ct values were evaluated by comparing the results obtained between the N marker of the RT-qPCR SMARTCHEK and the E marker of the RT-qPCR; in the same way, the RdRp marker was compared between both methodologies (Fig. 4B, C) . Despite the presence of outliers, a similar sensitivity was obtained between the RT-qPCR SMARTCHEK and the RT-qPCR.

COVID-19 pandemic has demonstrated being an important public health problem for its quick and easy dissemination in the world, showing the importance to count with reliable diagnostic methods for the detection of the SARS-CoV-2 virus. Currently, a lot of investigation institutions around the world are focusing on implement new detection technologies based on RT-qPCR, in order to contribute with the opportune and fast diagnose of patient with suspected of COVID-19 [12] .

Since the pandemic started, an important number of prevention measures have been proposed, which include providing an opportune diagnosis [6, 8, 13] . The RT-qPCR test is recommended by the World Health Organization (WHO) as a standard test [6] and is used as a reference for the validation of alternative molecular test for diagnosis such as the validation of commercial kits with clinical samples. In this context, it is necessary to implement new alternatives of molecular tests to RT-qPCR that are low cost, easy to use, and that can bring quick results with a high sensitivity and specificity, in order to strengthen prevention in areas where the access to molecular test is limited. During the pandemic, different specific primers and probes for the SARS-CoV-2 detection had been accomplished, showing that the ORF1ab gene, in spite of being conserved, is less sensitive than other target genes [14] . The RT-qPCR SMARTCHEK has been raised for the detection of the RNA-dependent RNA polymerase (RdRp) which encodes a fragment of the ORF1b region and shows a high level of intragroup conservation; therefore, it is an ideal target for its application on the diagnosis [15, 16] . Also, the RT-qPCR detects the N gene, which is less conserved but more sensitive than other target genes [7] . In the current validation, it has been shown that the RdRp gene is more sensitive and specific than the N gene using as gold standard the RT-qPCR recommended by the WHO [6, 8] . The RT-qPCR SMARTCHEK provides a mixed result of both genes achieving a sensitivity of 98.1% and a specificity of 98.8%, highly comparable by the methodology proposed by Corman et al. who obtained a sensitivity of 95% and a specificity of 100% [7] . In other investigations, it had also been proved that diagnostic tests enhance the efficacy of the detection [17, 18] .Despite having sensitivity and specificity values above 98%, false positives and negatives have been detected oriented to samples with a low viral load of SARS-CoV-2 with very low Ct values; these results also tend to appear in other methodologies for detecting the SARS-CoV-2 virus [23, 24] , being the main inconvenience transporting the sample to the processing laboratories.

The LoD of the RT-qPCR SMARTCHEK using the synthetic control 2019-nCoV_RdRP (ORF1ab) presented a lower logarithmic unit than the RT-qPCR RdRp gene proposed by Corman et al. [24] ; and the synthetic control 2019-nCoV_E presented the same LoD as the RT-qPCR; these data are very similar to those reported by Chang et al. [15] using an in vitro viral RNA, obtaining a LoD of 11.2 copies/reaction of RNA and 21.3 copies/reaction of RNA on the COVID-19-RdRp and COVID-19-N, respectively. Also, RT-qPCR SMARTCHEK was sensitive enough by detecting the SARS-CoV-2 in a clinical sample, obtaining a LoD for the RdRp and N genes until 10 4 dilution of RNA in the sample, similar to the description made by Zou et al. [20] ; Pan et al. [21] and Wölfel et al. [22] , who have shown that infected people had in their majority a high viral charge (between 10 4 and 10 8 copies of genome/mL for nasopharyngeal or saliva sample) during the first day of initial symptoms and probably during the presyndromic phase.

Finally, this study has some limitations including the necessity to perform RNA extraction before PCR reaction and the limited volume of clinical samples to perform the parallel control. Despite these limitations, RT-qPCR SMARTCHECK is a cost-effective method based on portability that provides a rapid RT-qPCR reaction and a comparable specificity/sensitivity with standard RT-qPCR.

In conclusion, the RT-qPCR SMARTCHEK could be an alternative to give an opportune diagnosis of SARS-CoV-2, being a platform that gives fast results, with high sensitivity and specificity, having the possibility to improve it by using a quick extraction method that can be used in laboratories of the primary health-care attention from countries which do not have an appropriate structure or adequate equipment.

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

First Case of 2019 Novel Coronavirus in the United States

COVID-19 Map In: Coronavirus Resource Center

COVID-19 Perú

Incremento de positividad a pruebas diagnósticas, casos, defunciones y hospitalizados por COVID-19

Directrices de laboratorio para la detección y el diagnóstico de la infección por el virus responsable de la COVID-19

Detection of 2019 novel coronavirus (2019-ncov) by real-time RT-qPCR

Laboratory testing for coronavirus disease (COVID-19) in suspected human cases: interim guidance

Emergency Use Authorization

An emerging coronavirus causing pneumonia outbreak in Wuhan, China: calling for developing therapeutic and prophylactic strategies

Smartchek Novel Coronavirus (SARS-CoV-2) Detection Kit

convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year submit your research ?

Diagnosis and clinical management of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection: an operational recommendation of Peking Union Medical College Hospital (V2.0)

Pruebas diagnósticas para la COVID-19: la importancia del antes y el después

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

The architecture of SARS-CoV-2 transcriptome

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

Sample pooling as a strategy to detect community transmission of SARS-CoV-2

Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-PCR assay validated in vitro and with clinical specimens

SARS-CoV-2 viral load in upper respiratory specimens of infected patients

Viral load of SARS-CoV-2 in clinical samples

Virological assessment of hospitalized patients with COVID-2019

Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification

Stability issues of RT-qPCR testing of SARS-CoV-2 for hospitalized patients clinically diagnosed with COVID-19

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations

We thank the members of the National Reference Laboratory of Entheropathogens/INS for their technical assistance.

The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12879-022-07319-0.Additional file 1: Fig. S1 . Workflow of RT-qPCR SMARTCHECK.Authors' contributions DFL, WQ and JC led on the data analysis and drafted the manuscript. AEC conducted molecular analyses. DFL, JC and ECM were involved in the interpretation of the data. RGG and WQ critically revised the manuscript. All authors read and approved the final manuscript. 

All data generated or analysed during this study are included in this published article.

This study was conducted within the framework of the research project "Platform for the genotyping of SARS-CoV-2 virus" which was approved by the Instituto Nacional de Salud of Peru (D.R. No. 191-2020-OGITT/INS). All procedures and methods were performed in accordance with ethical standards of the Declaration of Helsinki or comparable relevant guidelines and regulation. The approval of an informed consent was waived due to the retrospective nature of this study by the Institutional Committee of Research and Ethics (IRB) of the Instituto Nacional de Salud of Peru, in accordance with the national legislation and the institutional requirements for Public Health Surveillance.

Not applicable.

The authors declare that they have no competing interests.