Column purification method

The plasmid constructs containing full-length HEV genome (pSHEV-3 cDNA clone of HEV genotype 3 accession number-AY575859.1, a kind gift from SU Emerson, NIH, USA) and sub-genomic HEV carrying luciferase gene (GenBank accession number JQ679013) were used to prepare in vitro transcripts. The full-length cDNA and HEV replicon carrying the Renilla luciferase gene was linearized using XbaI and MluI, respectively. The linearized DNA was purified using the column purification method (Qiagen, Germany). Briefly, 5 μg of linearized DNA was used for in vitro transcription [41 (link)], and the reaction was performed in 50 μL of reaction mixture containing 1× transcription buffer (Promega, Madison, WI, USA). The reaction mix consisted of 0.5 mM concentration of nucleoside triphosphates (ATP, CTP and UTP and 0.05 mM concentration of GTP (Promega, WI, USA), 5 mM DTT, 2 μL of RNasin (10 U/mL) and T7 RNA polymerase (10U/mL) (Promega, WI, USA). The reaction was performed at 37 °C for 2 h, and 1 μL of T7 RNA polymerase was added after an hour. The RNA was capped using a 0.5 mM Ribom⁷G cap analogue (Promega, Madison, WI, USA), and the transcripts were purified using an RNA cleanup kit (Qiagen, Germany).

CF-LAMP assay was carried out as per the manufacturer’s instructions (EIKEN chemicals, Japan). The master mix was prepared with the final reaction volume of 12.5 µl containing 10 µM of FIP, 3 µM of BIP and 0.5 µM of F3, B3 primers. For enrichment of the copies of target cfDNA, an initial PCR amplification was performed using 100 ng cfDNA as template in a 50 µl reaction cocktail. The PCR products were purified using column purification method (Qiagen, Germany) and the concentration was measured using a nanophotometer (Implen, Germany). For each sample, 30–50 ng of enriched cfDNA was used as template for LAMP experiments. The final detection was done by gel electrophoresis. All the experimental procedures were performed on ice.