Development of a novel crAss-like phage detection method with a broad spectrum for microbial source tracking

CrAssphage has been recognized as the most abundant and human-specific bacteriophage in the human gut. Consequently, crAssphage has been used as a microbial source tracking (MST) marker to monitor human fecal contamination. Many crAss-like phages (CLPs) have been recently discovered, expanding the classification into the new order Crassvirales. This study aims to assess CLP prevalence in South Korea and develop a detection system for MST applications. Thirteen CLPs were identified in six human fecal samples and categorized into seven genera via metagenomic analysis. The major head protein (MHP) displayed increased sequence similarity within each genus. Eight PCR primer candidates, designed from MHP sequences, were evaluated in animal and human feces. CLPs were absent in animal feces except for those from raccoons, which hosted genera VI, VIIa, and VIIb. CLPs were detected in 91.52% (54/59) of humans, with genus VI (38 out of 59) showing the highest prevalence, nearly double that of p-crAssphage in genus I (22 out of 59). This study highlights genus VI as a potent MST marker, broadening the detection range for CLPs. Human-specific and selectively targeted MST markers can significantly impact hygiene regulations, lowering public health costs through their application in screening liver, sewage, wastewater, and various environmental samples.