BACKGROUND: We identified two cases of Omicron BA.5.2.1 that exhibited characteristics of open-reading frame 1a (ORF1a) gene target failure (OGTF) in RNA detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The purpose of this study was to determine the cause of the OGTF in the two samples. METHOD: Samples were specimens obtained from a 37-year-old male and a 43-year-old female who were suspected to have Coronavirus disease 2019. SARS-CoV-2 RNA detection using μTAS Wako g1 system (FUJIFILM Wako Pure Chemical, Osaka, Japan) was performed and it detected S gene but did not detect ORF1a gene. RESULTS: The results of whole genome sequencing of these samples showed that SARS-CoV-2 genome in these two samples matched to Omicron BA.5.2.1. SARS-CoV-2 genomes in the two samples had a deletion of 15 bases and a deletion of 9 bases, so that they overlapped with the 3'-terminal side of the primer binding region in ORF1a gene, and these deletions were considered to be the cause of OGTF. CONCLUSIONS: Since such a gene target failure due to base deletion is not considered rare, it was considered desirable to implement a multi-target detection in SARS-CoV-2 RNA molecular diagnostic system to address the mutation or deletion of the viral gene.