Thereby, the aim of this study was the standardization of alternative molecular methods, with lower cost and easier availability of reagents, for the diagnosis of COVID-19. The SARS-CoV-2 testing capacity is a major issue worldwide and the development of alternative tests is urgently needed, particularly in developing countries where availability of supplies or infrastructure to carry out the real-time PCR test is limited. However, only severe cases were tested due to a lack of testing reagents in the market and the number of cases can be far higher.įor this reason, the WHO advises that, in addition to isolation, massive diagnostic testing must be implemented in affected areas. Until May 2, 2020, the total number of cases was 96.559 with 6.750 deaths (lethality rate of ~ 6.8%). ![]() Since then, the number of confirmed cases has been growing, steadily reaching the highest number of cases by day at the beginning of April, with around 1000 cases per day (Brazil, Ministry of Health, 2020 - ). In Brazil, the first case of COVID-19 was confirmed on February 26 in the State of São Paulo, considered the epicenter of the disease in the country. According to WHO (last update: ), 215 countries or territories were affected with 238.628 deaths and more than 3.349.786 confirmed cases. In March, WHO declared COVID-19 a pandemic infection. The progressive number of cases and the fast dissemination to other countries led the World Health Organization (WHO) to officially declare the COVID-19 epidemic as a public health emergency of international concern. In January, the causative agent was identified as a new β-coronavirus, the SARS-CoV-2. In December 2019, an increase in the number of severe pneumonia cases was reported in China, more specifically in the Hubei Province. Both of the proposed assays are less sensitive than the current gold standard however, our data shows a low sensibility variation, suggesting that these methods could be used by laboratories as a lower cost molecular method for SARS-CoV-2 diagnosis. The average Ct variation between SYBR Green and TaqMan was 1.92 and the highest Ct detected by conventional PCR was 35.98. The conventional PCR detected until 10 7 dilution, both assays detected the majority of the 63 samples, 98.42% of positivity in SYBR Green, and 93% in conventional PCR. The SYBR Green-based assay was able to detect all 8 dilutions of the isolate. ![]() The SYBR Green assays and conventional RT-PCR have not shown amplification of the 17 respiratory samples positives for other viruses. The cross-reactivity test was performed with 17 positive samples for other respiratory viruses, and the sensibility test was performed with 8 dilutions (10 based) of SARS-CoV-2 isolated and 63 SARS-CoV-2-positive samples. (2020) were adapted in SYBR Green assay and RT-PCR conventional. E gene primers previously determined for TaqMan assays by Colman et al. The objective of this study was to standardize lower cost molecular methods for SARS-CoV-2 identification. However, it is a high-cost assay, inaccessible for the majority of laboratories around the world, making it difficult to diagnose on a large scale. TaqMan-based real-time RT-qPCR is currently the gold standard for COVID-19 diagnosis. In March 2020, WHO declared a pandemic state due to SARS-CoV-2 having spread. Daniella Gregorio Bonfim Prado Silva 3,.Lower cost alternatives for molecular diagnosis of COVID-19: conventional RT-PCR and SYBR Green-based RT-qPCR
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