Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

Soon Keong Wee; Suppiah Paramalingam Sivalingam; Eric Peng Huat Yap

PCR directly from crude samples without nucleic acid purification has been attempted before using inhibitor-resistant enzymes, modified buffers and additives in the mastermix. Our group (Sivalingam et al, unpublished) and others (18) have previously detected whole dengue virus in a single tube reaction containing serum and plasma in up to 8% (v/v). Direct amplification from samples has also been reported for the detection of other RNA viruses, including African Chikungunya virus (19), noroviruses (20) , and bovine viral diarrhea virus (21) and from a variety of matrices, including serum, throat swab and faeces (22) . However the presence of PCR inhibitors, such as mucin and proteins, poses a challenge for direct amplification from respiratory samples (23) , and there are limited studies amplifying coronaviruses directly from such specimens. We have developed a DIRECT-PCR protocol using widely used and validated PCR primers, established its analytical performance with both DNA and RNA templates in respiratory samples, and transferred the protocols from benchtop to a portable thermocycler.