Development and application of a real-time polymerase chain reaction assay for detection of a novel gut bacteriophage (crAssphage)

crAssphage is a novel and by far the most abundant bacteriophage in human gut. This bacteriophage might modulate gut microbiota balance so as to be involved in some diseases like obesity, diabetes, metabolic disorders, hypertension, and cancer. Therefore, a rapid and reliable detection and quantification method for crAssphage is essential for studying its molecular epidemiology and pathogenicity in human diseases. The primers-probes set for the quantitative real-time PCR assay was designed based on the DNA polymerase gene (ORF00018) of crAssphage. The sensitivity and specificity, as well as comparison testing with the conventional PCR and sequencing were evaluated. The assay could specifically detect crAssphage, and no cross-reactions with other gut microbes were observed. The detection limit was 15.6copies/L of clinical samples (46.8copies/reaction). When using clinical samples, the assay showed higher ability to detect samples with low viral DNA copies and had an agreement of 93.33% when compared with the conventional PCR amplification and sequencing. The established real-time PCR assay is a sensitive, specific, and repeatable method for quantitatively detecting crAssphage, and thus is a very useful tool for investigating the molecular epidemiology, dynamics, and pathogenicity of crAssphage in human diseases.