Biology subjects

Heldt, F. H.

Publications and source records attributed to Heldt, F. H..

3 recordsLinked to original sources

bioRxiv2020

Comparison Of Different Kits For SARS-CoV-2 RNA Extraction Marketed In Brazil

December 2019 marked the begining of the greatest pandemic since Spanish Flu, the disease named Covid-19 that cause severe pneumonia. Until May 19, 2020 more than 4 million and 700 thousand cases were oficially notified with about 316 thousand deaths. Etiological agent of the disease was identified as being a new coronavirus, Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2). In this study we compared four different manual methods for RNA isolation and purification for detection of SARS-CoV-2 through qRT-PCR, as well as the extraction quality itself through detection of RNAse P. Magnetic beads-based (MagMax) and silica column-based (Biopur(R)) methods presented the better performances. Concerning to the mean delay in CT values when compared to MagMax, TRIzol, Biopur(R) and EasyExtract presented 0,39, 0,95 and 5,23 respectively. Agreement between positive and negative results of different methods when compared with the one with better performance MagMax was 94,44% for silica column-based method (Biopur(R)), 88,89% for phenol-chroloform-based method (TRIzol) and 77,78% for EasyExtract. We aimed to evaluate how reliable each method is for diagnostic purposes and to propose alternatives when usual methods are not available. In this regard, magnectic beads and silica column-based methods are convenient and reliable choices and phenol-chloroform-based method could also be chosen as an alternative.

molecular biology↗

bioRxiv2020

Pre-treatment of the clinical sample with Proteinase K allows detection of SARS-CoV-2 in the absence of RNA extraction

COVID-19 (Coronavirus Disease 2019) outbreak was declared a pandemic, by World Health Organization, on March 11, 2020. Viral detection using RT-qPCR has been among the most important factors helping to control local spread of SARS-CoV-2 and it is considered the "gold standard" for diagnosis. Nevertheless, the RNA extraction step is both laborious and expensive, thus hampering the diagnosis in many places where there are not laboratory staff of funds enough to contribute for diagnosis efforts. Thus, the need to simplify procedures, reduce costs of the techniques used, and expand the capacity of the number of diagnostics of COVID-19 is imperative. In this study, detection of SARS-CoV-2 in the absence of RNA extraction has been successfully achieved through pre-treatment of the clinical sample with Proteinase K. The results show that only the use of proteinase K, without the need to perform the whole standard protocol for sample extraction and purification, can be an efficient technique for the diagnosis of COVID-19, since 91% of the samples matched the results with the standard procedure, with an average increase of 5.64 CT in the RT-qPCR.

microbiology↗

bioRxiv2020

SARS-CoV2 quantification using RT-dPCR: a faster and safer alternative to assist viral genomic copies assessment using RT-qPCR

In this study, serial dilutions of SARS-CoV 2 RNA extract were tested using RT-dPCR using three different primer-probe assays aiming SARS-CoV 2 nucleocapsid coding region. Narrower confidence intervals, indicating high quantification precision were obtained in 100 and 1000-fold serial dilution and RT-dPCR results were equivalent between different assays in the same dilution. High accuracy of this test allowed conclusions regarding the ability of this technique to evaluate precisely the amount of genomic copies present in a sample. We believe that this fast and safe method can assist other researchers in titration of SARS-CoV2 controls used in RT-qPCR without the need of virus isolation.

molecular biology↗