Detecting and Sequencing crAssphages

To detect crAssphages, we employed two previously reported real-time qPCR assays, CPQ056 (Stachler et al., 2017 (link)) and TN201-203 (Park et al., 2020 (link)). Both assays were used to detect crAssphages in leafy greens, environmental water (stream and irrigation water), and fecal samples. The oligonucleotide primers and probes used in the assays are summarized in Table 1. The qPCR assays were performed as previously described (Stachler et al., 2017 (link); Park et al., 2020 (link)) on a 7500 Fast Real-Time PCR system (Applied Biosystems, USA). To quantify crAssphages, a 10-fold serially diluted quantified amplicon was used to generate a standard curve. Based on the standard curve and the cut-off of Ct > 40, the Limit of Quantification (LOQ) was estimated as 1.7 × 10³ and 2.0 × 10³ copies for CPQ056 and TN201-203, respectively.
For sequencing, PCR-positive crAssphage samples were amplified using oligonucleotide primers (JP1crasF/TN203) to generate a 1089-bp PCR amplicon (Table 1). The purified PCR products were sequenced using an ABI Prism 3500 × L genetic analyzer and a BigDye Terminator cycle sequencing mix (Applied Biosystems, Foster City, CA, USA).