Triplex real-time PCR ZKIR-T assay for simultaneous detection of the species complex and identification of sensu stricto.

Unlabelled: species complex (KpSC) members, including the most important species (phylogroup Kp1 of the KpSC), are important opportunistic pathogens that display increasing rates of antimicrobial resistance worldwide. As they are widespread in food and the environment, there is a need for fast, sensitive, and reliable methods to detect KpSC members in complex matrices. Previously, the ZKIR real-time PCR assay was developed to detect all KpSC members without distinction. Given that Kp1 is the clinically most significant phylogroup of the KpSC, here, we aimed to simultaneously identify Kp1 while detecting all KpSC members. Three TaqMan probes were developed and used: the Kp1 probe to specifically identify this phylogroup; the P1 probe to detect phylogroups Kp1-Kp5 and Kp7; and the P2 probe to detect phylogroup Kp6. The new triplex assay was tested on a total of 95 KpSC and 19 non-KpSC strains from various sources, representing the different phylogroups as defined by whole genome sequencing. The results showed almost complete specificity, as the expected PCR results were obtained for 112 (98%) strains. The new triplex real-time PCR assay, called ZKIR-T, enables the detection of all KpSC taxa while discriminating Kp1, which will be useful for rapid screening and to focus downstream analyses on chosen phylogroups of the KpSC.

Importance: The pathogens of the species complex are widespread in food and animals and are among the main pathogens responsible for multidrug resistant infections in humans. In this study, we developed a highly sensitive detection assay that enables detection of this group of bacteria, with the simultaneous identification of the most common and clinically important species. This triplex one-reaction assay was shown to be highly sensitive and precise, enabling fast screening of varied samples for the presence of KpSC and sensu stricto.