Open AccessDevelopment of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2
Author(s) -
Hüseyin Tombuloğlu,
Hussein Sabit,
Ebtesam A. Al-Suhaimi,
Reem Al Jindan,
Khaled R. Alkharsah
Publication year - 2021
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.0250942
Subject(s) - multiplex , virology , covid-19 , molecular diagnostics , real time polymerase chain reaction , biology , reverse transcription polymerase chain reaction , polymerase chain reaction , multiplex polymerase chain reaction , computational biology , coronavirus , gene , medicine , genetics , messenger rna , infectious disease (medical specialty) , disease , pathology
The outbreak of the new human coronavirus SARS-CoV-2 (also known as 2019-nCoV) continues to increase globally. The real-time reverse transcription polymerase chain reaction (rRT-PCR) is the most used technique in virus detection. However, possible false-negative and false-positive results produce misleading consequences, making it necessary to improve existing methods. Here, we developed a multiplex rRT-PCR diagnostic method, which targets two viral genes ( RdRP and E ) and one human gene ( RP ) simultaneously. The reaction was tested by using pseudoviral RNA and human target mRNA sequences as a template. Also, the protocol was validated by using 14 clinical SARS-CoV-2 positive samples. The results are in good agreement with the CDC authorized Cepheid`s Xpert ® Xpress SARS-CoV-2 diagnostic system (100%). Unlike single gene targeting strategies, the current method provides the amplification of two viral regions in the same PCR reaction. Therefore, an accurate SARS-CoV-2 diagnostic assay was provided, which allows testing of 91 samples in 96-well plates in per run. Thanks to this strategy, fast, reliable, and easy-to-use rRT-PCR method is obtained to diagnose SARS-CoV-2.