key: cord-0430366-3x3w6aqw
authors: Salinas, M.; Aguirre, D.; De la Torre, D.; Perez-Galarza, J.; Pibaque, R.; Beltran, P.; Veloz, T.; Baldeon, L.
title: Development and performance evaluation of a low-cost in-house rRT-PCR assay in Ecuador for the detection of SARS-CoV-2
date: 2021-08-06
journal: nan
DOI: 10.1101/2021.08.03.21260966
sha: d39246bef0a97b4c460b0d1b2380c184cc43652b
doc_id: 430366
cord_uid: 3x3w6aqw

Antecedents: Ecuador has had the greatest fatality rate from Coronavirus (COVID-19) in South America during the SARS-CoV-2 pandemic. To control the pandemic, it is necessary to test as much population as possible to prevent the spread of the SARS-CoV-2 infection. For the Ecuadorian population, accessing a PCR test is challenging, since commercial screening kits tend to be expensive. Objective: the objective of this study was to develop an in-house duplex rRT-PCR protocol for the detection of SARS-CoV-2 that contributes to the screening while keeping quality and low testing costs. Results: An in-house duplex rRT-PCR protocol based on the viral envelope (E) gene target of SARS-CoV-2 and a human ribonuclease P gene (RP) as an internal control is reported. The protocol was optimized to obtain primers E with an efficiency of up to 94.45% and detection of 100% of SARS-CoV-2 up to 15 copies per uL. The clinical performance was determined by a sensibility of 93.8% and specificity of 98.3%. Conclusion: we developed, standardized, and validated a low-cost, sensitive in-house duplex rRT-PCR assay that may be utilized in low-income countries.

disease is caused by a novel coronavirus, which is highly infectious and can induce severe acute 27 respiratory syndrome (SARS-CoV2). Until June 2021, the number of cases reported is more than 180 28 million infections and more than 3 million deaths around the world [2] . The World Health 29

Organization (WHO) recorded 360 thousand confirmed illnesses and over 18 thousand fatalities in 30

Ecuador. 31

Ecuador has the highest case fatality rate in the area (4.9 percent) [3] . Furthermore, the country's 32 death rate increased by 24% in the first trimester of 2021 (mortality comparison between December 33 2019 and March 2020 vs. December 2020 and March 2021) [4] . 34

The existence of asymptomatic persons confuses the precise number of infected patients, which is a 35 possible danger that stays latent for the disease's spread. As a result, the rate of SARS-CoV-2 36 infection may be greater than previously thought. [5] . This scenario emphasizes population-wide 37 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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testing and, as a result, the availability of specialized tests for early viral identification and the 38 implementation of pandemic control epidemiological methods. [6] . Ecuador is one of the countries in 39

South America with the lowest number of tests per capita (0.2 per 1000 persons each day) [7] . 40

The gold standard for SARS-CoV-2 detection is a viral nucleic acid detection technique based on 41 real-time reverse transcriptase-PCR (rRT-PCR). Commercial kits based on rRT-PCR have been in 42 great demand across the world due to the technique's excellent sensitivity and specificity in 43 accurately detecting the virus [8] . The US Food and Drug Administration (FDA) has approved 44 various rRT-PCR assays targeting viral genes such as nucleocapsid (N), envelope (E), and RNA-45 dependent RNA polymerase (RNA-dependent RNA polymerase) (RdRp) [9] . However, numerous 46

tests have shown that the E gene is somewhat more sensitive than other genetic targets, and the Pan 47

American Health Organization (PAHO) has advised that the E gene be utilized exclusively for viral 48 population screening in the Americas during this emergency [10] . 49

Because of the COVID-19 pandemic, there has been an increase in the global demand for rRT-PCR 50 commercial kits for detecting SARS-CoV-2. The scarcity of kits is worse in low-income countries 51

like Ecuador, where the cost of these kits makes them inaccessible to the government and the general 52

population. Many laboratories have created in-house assays with excellent sensitivity and specificity 53 to tackle this challenge by emphasizing test cost reduction. In addition, to minimize the transmission 54 of the virus by asymptomatic persons, the majority of the population must be screened. Our goal was 55

to develop an in-house duplex rRT-PCR test that would identify SARS-CoV-2 in human respiratory 56 samples using the E gene. 57

For the duplex standardization experiment, two sets of primer pairs and probes for the viral E gene 60 and human ribonuclease P (RP) (internal control) were employed. A multiplex assay was also 61 attempted using primers targeting RdRp gene. E and RdRp primers/probes were obtained from the 62 study published by Charité-Universitatsmedizin Berlin Institute of Virology [11] . RP primer/probe 63 was obtained from the United States of America Centre for Disease Control and Prevention (CDC) 64 [12] . The probes were labelled with FAM, ROX, and VIC for E, RdRp, and RP, respectively (see 65 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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To develop the assay, a SARS-CoV-2 RNA source (RNA standardization solution) with a known 84 concentration (3.81 x 105 viral copies per uL) was used. To optimize the final reaction setup, 85 simplex assays were done. Three sets of primers/probes were used in different concentrations. The 86

thermocycler program was set up using temperature gradient at 56, 58, and 60 °C. Amplification 87 products were observed by electrophoresis (2% agarose gel) to verify the specificity of the primers. 88

With 5 and 3.3 uL of RNA standardization solution, respectively, a final volume of 20 and 10 uL 89 was tested. The master mix was prepared with the GoTaq Probe 1-step RT-qPCR System reagent 90

(Promega, USA were added to the duplex setup and RNA standardization solution was decreased to 2.5 uL. 99

To assess the efficiency of E primers, successive dilutions of the RNA standardization solution were 101 produced in the range of 512 to 16 copies per uL. Serial dilutions were reduced from 64 to 1 copy 102 per uL for the LoD of the duplex test. Three distinct operators carried out each test independently, 103 and the reactions were produced in triplicate. 104

The in-house assay was created and tested at the Research Institute in Biomedicine of Central is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The sample was calculated to achieve a 95 percent level of significance and an 8 

We calculate the positive percent agreement (sensitivity), negative percent agreement (specificity), 126

positive and negative predictive values, likelihood ratio, and Cohen's Kappa to assess the duplex 127

rRT-PCR test in clinical samples. SPSS software version 23 was used for the analysis (IBM). 128

Simplex rRT-PCR experiments were prepared to find the optimal primers and probes concentration.

The amplicons of these assays were located at the expected positions for the fragment lengths which 132

were 113 bp, 100 bp, and 50 bp for the E, RdRp, and RP respectively. For the RP set, a low variation 133

in the Ct was observed when changing RP primer concentrations from 600 nM to 200 nM (with half 134 of the concentration of its probe). For this reason, 200/100 nM primers/probe concentration was 135 chosen for all following assays. In the case of E and RdRp sets, 400/200 nM and 300/150 nM, 136

respectively, was chosen for the remaining experiments because this primers/probe concentration 137 had one of the lowest Ct (E=18.40, RdRp=25.65). As for the annealing temperature, the lowest Ct 138

for the E pair of primers was prioritized. The temperature was 60 °C (Data not shown). 139

Upon optimization of reaction conditions on the simplex set-up we performed a duplex protocol, 141

which had more consistent results than the multiplex protocol. When the Ct value of a target changed 142

in more than one of the E and RP sets of primers/probes (Duplex) targets, although their Ct values were within a respectable margin of error between replicates (no 148 more than 0.5) (see Figure 1 ). At low quantities of viral RNA (15 viral copies/reaction), the RdRp 149 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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Serial dilutions of the RNA standardization solution (64, 32, 15, 10, 5, 2, 1 copies/uL) were prepared 160

for LoD evaluation. 15 copies/uL (49.5 copies/reaction) with a mean Ct value of 35.99 (CI95 percent 161 35.33 -36.66) was the lowest concentration at which all nine replicates (100%) showed 162 amplification curves. (see Table 2 ). Moreover, the primer efficiency was 94.45%, with an R2 of 98.1 163 percent. (see Figure 2 ). 164 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted August 6, 2021. 

For clinical comparison, we used the commercial LightMix SarbecoV E-gene + EAV control kit, 171

which identified 64 positives and 60 negatives in nasopharyngeal samples. Sixty-four out of sixty-172 four samples were found to be positive using an in-house duplex method. The in-house duplex 173 procedure found 60 out of 64 samples to be positive (see Table S1 in the Supplementary Material).

Four samples were missed by the duplex test, resulting in 93.8 % sensitivity, 98.3 % specificity, a 175

Likelihood ratio of 56.25, and a Cohen's K of 0.92 (see Table 3 ). 176 

Ecuador is one of the South American countries worst hit by the COVID-19 pandemic. Molecular 182 diagnostics using rRT-PCR is the best approach to detect SARS-CoV-2 virus to restrict its spread 183 until we achieve herd immunity in the nation. An improved and reproducible rRT-PCR methodology 184

for the diagnosis of COVID-19 using a viral gene (E) and a human gene (Rp) in a simple reaction 185

was designed in this work, using primers built according to recognized international guidelines 186

[11 ,12] . As a result, 113-bp bands for the E gene and 55-bp bands for the Rp gene were produced 187

without nonspecific products such as dimers or overlapping sequences that might cause false 188 positives during the amplification process [13] . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The sets of primers/probes for the detection of the E and RdRp genes employed to standardize the 190 technique have a reasonably low nonselective mutation rate and have been reported in similar 191 researches [14, 15] . Following the guidelines of the US Centers for Disease Control and Prevention 192

(CDC), the Rp gene was utilized as an internal control of RNA extraction [16] . The E gene showed 193 little variability in duplex and multiplex reactions, according to the amplification graph ( Figure 1) The limitation of the in-house duplex test is that it can only identify one viral gene (E) and one 210

internal control gene (Rp). However, the kappa index (0.92) shows that our protocol is comparable to 211 commercial kits (LightMix E / RdRp) that screen two or more viral genes. Furthermore, because the 212 E gene is unique to all Sarbecoviruses and because SARS-CoV-2 is the only member of the family 213 now circulating in humans, the WHO has recommended that the E gene be prioritized as a target. In 214 this approach, a single viral genetic target suffices for case confirmation in the lab. 215

In conclusion, we designed, standardized, and validated an in-house duplex rRT-PCR assay that can 217 detect SARS-CoV-2 virus presence up to 15 copies/uL. This approach can assist our country enhance 218 its capacity to screen both symptomatic and asymptomatic carriers by making SARS-CoV-2 rRT-219 PCR more accessible. 220 

[1] World Health Organization (WHO), "WHO Director-General's opening remarks at the media 241 briefing on COVID-19 -11 March 2020," 2020. https://www.who.int/director-242 general/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-243

covid-19---11-march-2020. 244 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Recent advances and perspectives of nucleic acid detection for coronavirus

Understanding, verifying, and implementing emergency use 263 authorization molecular diagnostics for the detection of sars-cov-2 RNA

Laboratory Guidelines for the Detection and Diagnosis 266 of COVID-19 Virus Infection

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-270

US CDC real-time reverse transcription PCR panel for detection of severe acute 272 respiratory syndrome Coronavirus 2

Optimization of primer sets and detection 275 protocols for SARS-CoV-2 of coronavirus disease 2019 (COVID-19) using PCR and real

RdRp mutations are 279 associated with SARS-CoV-2 genome evolution

Comparison of commercial realtime reverse transcription PCR assays for the 282 detection of SARS-CoV-2

CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel For

Emergency Use Only Instructions for Use

Comparative performance of SARS-CoV-2 detection assays using seven 288 different primer-probe sets and one assay kit

Analytical sensitivity and efficiency comparisons of SARS-CoV-2

RT-qPCR primer-probe sets

Evaluation of a quantitative RT-294