key: cord-0733910-ytntkgvb
authors: Pessoa-e-Silva, Rômulo; de Oliveira, Priscilla Stela Santana; Gonçalves, Sayonara Maria Calado; Guarines, Klarissa Miranda; Carvalho, Lidiane Vasconcelos do Nascimento; Correia, Maria Andreza Bezerra; da Rosa, Michelle Melgarejo; Rêgo, Moacyr Jesus Barreto de Melo; Pitta, Maira Galdino da Rocha; Pereira, Michelly Cristiny
title: Enhanced rapid commercial DNA extraction kit for the molecular detection of severe acute respiratory syndrome coronavirus 2: Easy adaptation to current protocols
date: 2021-11-12
journal: Revista Da Sociedade Brasileira de Medicina Tropical
DOI: 10.1590/0037-8682-0270-2021
sha: 4a36c10738a60184e24070beaeaea0db35a845ec
doc_id: 733910
cord_uid: ytntkgvb

INTRODUCTION: Herein, the authors describe a simple enhancement to a commercial rapid DNA extraction kit based on simple viral lysis for detecting COVID-19 via RT-qPCR. METHODS: After testing several different modifications, the adapted protocol with the best results in preliminary experiments was statistically evaluated in comparison with an automated robotic protocol. RESULTS: Processing and testing of 119 nasopharyngeal samples ultimately yielded near-perfect agreement with the automated protocol (κ = 0.981 [95% confidence interval 0.943-1.000]). CONCLUSIONS: The low cost and rapidity of the enhanced protocol makes it suitable for adoption in laboratories diagnosing COVID-19, especially those with high demand for examinations.

In late 2019, the emergence of a novel virus precipitated a global public health crisis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), whose transmission began in Wuhan, China, was identified as the causative agent of coronavirus disease 2019 (COVID-19). By June 12, 2021, the virus had been registered in 222 countries and territories, and caused 3.14 million deaths worldwide 1,2 .

Robust testing using reverse transcription followed by realtime quantitative polymerase chain reaction (RT-qPCR) ranks among the most used strategies adopted by health care entities and governments to monitor cases of COVID-19 and prevent the spread of the virus, primarily via the identification of asymptomatic individuals [3] [4] [5] [6] . Extensive testing of the population must be performed using a reliable and accurate diagnostic protocol. Rapid DNA and RNA extraction protocols based on simple viral lysis are highly useful for increasing the testing capacity of clinical laboratories. However, these extraction procedures generally do not guarantee purity or sufficient removal of PCR inhibitors, which decreases their sensitivity and can lead to false-negative results [7] [8] [9] . In this article, we describe an adaptation to a commercial rapid extraction kit for COVID-19 detection via RT-qPCR, ensuring a sensitivity comparable to that of an automated commercial protocol.

In total, five assays were performed sequentially (Figure 1) . First, a preliminary test was performed with 20 nasopharyngeal swab samples collected for COVID-19 investigation and chosen at random. The aim of this preliminary test was to determine whether the rapid extraction protocol would be able to extract viral RNA of sufficient quality and quantity to detect SARS-CoV-2 in nasopharyngeal samples. Swabs were placed in phosphate-buffered saline (PBS, 4 mL). For processing, the tubes were vortexed, and RNA extraction was performed using two methods simultaneously (different aliquots): an automated procedure; and a rapid manual extraction procedure. The kits used were the Maxwell® RSC Viral Total Nucleic Acid kit (AS1330), for use in robotic extraction with The results yielded 18 positive samples (90%) in the automated procedure, seven (38.89%) of which were positive using the rapid protocol. All samples extracted using both protocols yielded amplification of RP, with Ct values < 36.0. The rapid extraction protocol yielded viral RNA of sufficient quality and quantity to be detected in some samples. However, given the loss in sensitivity compared to the automated procedure, and the need for a quick and reliable extraction method in routine laboratory protocols, further tests were performed.

In the next test, eight positive samples not included in the first test and with different Ct values in the automated procedure were extracted from a new aliquot (stored at -80°C) in duplicate using the rapid procedure. In one of the duplicates, 160 µL of RNase-free water was added to 40 µL of extracted sample (20 µL of sample + 20 µL of extraction solution) to evaluate possible interference from PCR inhibitors. Three samples with late Ct values (N1 and N2 > 32.0) were negative in both duplicates (with and without dilution). The remaining five samples were positive ( Table 1) Thereafter, to verify the performance of the adapted rapid extraction protocol in the routine laboratory protocol, 156 samples were randomly selected, with all swabs placed in PBS, and extracted using the optimal proportion (i.e., 80:20 µL). As a result, 100 samples were negative and, of the remaining 56, five were inconclusive. Samples that were negative or inconclusive were re-extracted from a new aliquot (stored at -80°C) using an automated procedure. After re-extraction, the five inconclusive samples tested positive, whereas the other 100 samples remained negative. These results indicated that no false-negative results were obtained after using the modified rapid extraction protocol.

Finally, to confirm the applicability of the adapted protocol according to analysis of agreement, 119 nasopharyngeal swab samples, all stored in PBS, were randomly selected and extracted using the automated procedure and the rapid protocol. After RT-qPCR and data analysis, the automated extraction protocol identified 52 positive and 67 negative samples, as with the adapted rapid protocol, 41 positive, 68 negative, and 10 inconclusive samples were obtained. Considering only positive and negative samples, 99.08% agreement between the extraction protocols was observed. Calculation of the kappa (κ) coefficient revealed very good agreement (κ = 0.981 [95% confidence interval 0.943-1.000]), as shown in Table 2 The results highlight that the adapted rapid extraction protocol was as sensitive as the automated protocol, ultimately reflected by near-perfect agreement. This finding reinforces the applicability of the rapid protocol in routine laboratory procedures for detecting COVID-19 using RT-qPCR. The rapid protocol requires only heat treatment of the sample mixed with an extraction solution to lyse the virus and release the genomic content. Previous studies have demonstrated the possibility of directly detecting SARS-CoV-2 via RT-qPCR by using only heated and/or lysed samples and without any significant loss in detection capability [12] [13] [14] . Furthermore, in addition to its speed and practicality, the cost per patient of the rapid extraction kit can be 20 times less than that of the automated extraction kit (prices based on quotes from December 2020).

These characteristics make the quick protocol suitable for adoption in laboratories that diagnose COVID-19, especially those with a high demand for examinations such as central reference and public health laboratories. Robotic extraction can be used specifically to retest samples with inconclusive results.

In this study, all tests were performed using nasopharyngeal swabs soaked in PBS. For samples in viral transport medium or other types of media, however, it is critical to execute an appropriate test before using the adapted rapid protocol due to the possible presence of PCR inhibitors.

The adaptation proposed in the tested protocol-given its simplicity and efficiency-can be used to improve the sensitivity of rapid extraction kits from different manufacturers. If an automated/ robotic extraction procedure is not accessible, the rapid protocol can be compared using column-based RNA extraction kits available in the laboratory. Moving forward, it is mandatory to critically review all stages of any extraction protocol in use and to perform statistical comparisons with different extraction kits to confirm its applicability before use.

Timeline: WHO's COVID-19 response

World Health Organization. WHO Coronavirus Disease (COVID-19) Dashboard; 2021

Modelling the impact of testing, contact tracing and household quarantine on second waves of COVID-19

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

Effectiveness of mass testing for control of COVID-19: a systematic review protocol

Lessons learnt from easing COVID-19 restrictions: an analysis of countries and regions in Asia Pacific and Europe

Detection of Herpes Simplex Virus and Varicella-zoster Virus in Clinical Swabs: Frequent Inhibition of PCR as Determined by Internal Controls

Falsenegative result in molecular diagnosis of SARS-CoV-2 in samples with amplification inhibitors

PCR inhibitorsoccurrence, properties and removal

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

Coefficient of agreement for nominal scales

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step

A Direct Method for RT-PCR Detection of SARS-CoV-2 in Clinical Samples

Massive and rapid COVID-19 testing is feasible by extractionfree SARS-CoV-2 RT-PCR

We appreciate the COVID-19 diagnostic team at the Research Center for Therapeutic Innovation Suely-Galdino (NUPIT-SG) for carrying out the processing of samples, extractions and RT-qPCR.

We appreciate the UFPE Development Support Foundation (Fundação de Apoio ao Desenvolvimento da UFPE -FADE), the Recife City Hall Agency (Agência da Prefeitura do Recife), and the Associação Municipalista de Pernambuco (AMUPE) for the purchase of general supplies, reagents and equipment that allowed the tests to be carried out.