Infectious diseases caused by viruses such as SARS-CoV-2 and HPV have greatly endangered human health . The nucleic acid detection is essential for the early diagnosis of diseases . Here, we propose a method called PLCR (PfAgo coupled with modified Ligase Chain Reaction for nucleic acid detection) which utilizes PfAgo to only use DNA guides longer than 14-mer to specifically cleave DNA and LCR to precisely distinguish single-base mismatch . PLCR can detect DNA or RNA without PCR at attomolar sensitivities, distinguish single base mutation between the genome of wild type SARS-CoV-2 and its mutant spike D614G, effectively distinguish the novel coronavirus from other coronaviruses and finally achieve multiplexed detection in 70 min . Additionally, LCR products can be directly used as DNA guides without additional input guides to simplify primer design . With desirable sensitivity, specificity and simplicity, the method can be extended for detecting other pathogenic microorganisms.