Open AccessDetection of SARS-CoV-2 from raw patient samples by coupled high temperature reverse transcription and amplification
Author(s) -
Johannes W. P. Kuiper,
Timo Baade,
Marcel Kremer,
Ramon Kranaster,
Linda Irmisch,
Marcus Schuchmann,
Johannes Zander,
Andreas Marx,
Christof R. Hauck
Publication year - 2020
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0241740
Subject(s) - rna , reverse transcriptase , computational biology , rna extraction , biology , reverse transcription polymerase chain reaction , nucleic acid , virus , dna , virology , genome , transcription (linguistics) , polymerase chain reaction , covid-19 , rna virus , swap (finance) , gene , genetics , messenger rna , medicine , infectious disease (medical specialty) , business , disease , pathology , finance , linguistics , philosophy
SARS-CoV-2 is spreading globally with unprecedented consequences for modern societies. The early detection of infected individuals is a pre-requisite to contain the virus. Currently, purification of RNA from patient samples followed by RT-PCR is the gold standard to assess the presence of this single-strand RNA virus. However, these procedures are time consuming, require continuous supply of specialized reagents, and are prohibitively expensive in resource-poor settings. Here, we report an improved nucleic-acid-based approach to detect SARS-CoV-2 with the ability to detect as little as five viral genome equivalents. The approach delivers results without the need to purify RNA, reduces handling steps, minimizes costs, and allows evaluation by non-specialized equipment. The use of unprocessed swap samples is enabled by employing a heat-stable RNA- and DNA-dependent DNA polymerase, which performs the double task of stringent reverse transcription of RNA at elevated temperatures as well as PCR amplification of a SARS-CoV-2 specific target gene. As results are obtained within 2 hours and can be read-out by a hand-held LED-screen, this novel protocol will be of particular importance for large-scale virus surveillance in economically constrained settings.