A brand new examine has discovered that the Novaplex SARS-CoV-2 Variant I, II, and IV real-time PCR assays (from Seegene Know-how) can reliably detect SARS-CoV-2 in affected person samples and establish recognized variants of curiosity and concern. Outcomes from the PCR assays have been corresponding to these from the “gold customary” spike gene Sanger sequencing technique. Researchers have been additionally in a position to efficiently streamline testing and cut back value and turnaround time by processing samples with out extracting RNA for testing. Their findings seem in The Journal of Molecular Diagnostics, printed by Elsevier.
“Actual-time PCR (RT-PCR) methodology for variant detection is accessible, fast, less complicated, and correct in comparison with conventional sequencing,” stated lead investigator Ping Ren, PhD, Division of Pathology, College of Texas Medical Department, Galveston, TX, USA. “Combining an extraction-free processing technique with RT-PCR expertise may help laboratories with out sequencing capabilities monitor circulating variants and examine variant-dependent results on remedy efficacy and illness severity.”
The I, II, and IV assays are designed to detect genetic mutations related to the alpha, beta, delta, and epsilon variants of SARS-CoV-2. On the time of the examine, the omicron variant had not but emerged. RNA was extracted from every pattern for testing by the Novaplex RT-PCR assays and Sanger sequencing. The samples have been additionally immediately examined with out extraction of RNA by the Novaplex assays.
Of the 156 samples processed with RNA extraction, the RT-PCR assays recognized 109 variants. The outcomes have been 100% in settlement with the Sanger sequencing check. The RNA extraction-free technique was 91.7% as delicate as the normal RNA extraction technique. In samples with a decrease viral load, the extraction-free RT-PCR assays didn’t detect some mutations, presumably due to decrease nucleic acid concentrations within the authentic samples.
“A significant limiting issue for molecular SARS-CoV-2 assays is the scarcity of RNA extraction reagents,” defined co-lead creator Marisa C. Nielsen, PhD, Division of Pathology, The College of Texas Medical Department, Galveston, TX, USA. “Typical extraction stays a time-consuming facet of molecular prognosis of SARS-CoV-2. Current CDC steering recommends sequencing just for instances with a cycle threshold (Ct) worth decrease than 28, which signifies a better viral load, as a result of sequencing is much less dependable in samples with decrease viral masses.”
“Though decrease sensitivity was noticed with the extraction-free technique, it nonetheless represents a viable different,” Dr. Nielsen added. “Spike sequencing continues to be essential for detecting new variants.”
RT-PCR assays might be tailor-made to incorporate extra consultant genes as totally different variants emerge and permit for extra accessible variant detection and monitoring to tell public well being and remedy choices. Whereas not included on this examine, assays at the moment are obtainable to establish omicron-specific mutations.
“Figuring out the SARS-CoV-2 variant in particular person affected person samples may help information remedy since some variants are extra proof against present remedy regimens,” Dr. Ren noticed. “Nevertheless, the potential influence extends past particular person sufferers and into the general public well being realm. You will need to monitor variant unfold as a part of public well being surveillance due to variant-dependent transmission, illness severity, and remedy choices.
Supplies offered by Elsevier. Notice: Content material could also be edited for type and size.