Abi prism 3500 l genetic analyzer

Viral RNA was reverse transcribed to cDNA using the SuperScript III First-Stand Synthesis kit (Invitrogen, Carlsbad, CA, USA). Generated cDNA was subjected to amplification of the viral gene for both the PR and RT encoding regions in a separate reaction by nested PCR using Ex Taq (Takara Bio, Shiga, Japan). Primer information is shown in Additional file 3. If a viral gene fragment failed to be amplified from the cDNA even after multiple attempts, it was amplified instead from DNA extracted from PBMC. In order to examine the genomic fragment of the major viral population in a sample, PCR products amplified at the end-point dilution of DNA templates were subjected to sequencing analysis.
Sequencing analysis of the amplified fragment was performed using the BigDye Terminator v3.1 Cycle Sequencing kit with an ABI PRISM 3500 × l genetic analyzer (Applied Biosystems, Foster City, CA, USA). Data were assembled and aligned using Genetyx ver. 10 software (Genetyx, Tokyo, Japan). Nearly the full length of the PR gene [(280 bp; corresponding to nucleotides 2262 to 2541 of a HIV-1 reference strain, HXB2 (GenBank accession no. K03455)] and part of the RT gene (762 bp; nucleotides 2550 to 3311) were sequenced and subjected to subsequent analysis.

Genomic DNA was extracted from the whole blood samples with EDTA and DBS using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The exons 1–20 of the HADHA gene and the adjacent intronic sequences as well as the exons 1–20 of the VLCAD gene and the adjacent intronic sequences were analyzed using Sanger sequencing (the primer sequences are available upon request) on ABI PRISM 3500×L Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

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).