Quickdrop micro volume spectrophotometer

The limit of detection (LOD) of the multiplex reaction was determined based on a standard curve. The standard template was prepared as follows. Two large (874 bp) fragments of the TK gene and 1315 bp of the Or01 gene were amplified using the primer sets shown in Appendix A Table A1. The large fragments contained binding sites for 5ILTV.233F/R and 6ORT.467F/R primers. The 874 bp and 1315 bp PCR fragments were excised from the agarose gel and purified using GeneJET Gel Extraction (K0691, Thermo Fisher Scientific, Waltham, MA, USA). The DNA concentration of the purified fragments (37 ng/µL for 874 bp fragments; 31 ng/µL for 1315 bp fragments) was determined using a QuickDrop Micro-Volume Spectrophotometer (Molecular Devices). The template copy number was calculated using the following equation: template copies/µL = [amount of DNA (ng/µL) × (6.022 × 10²³)]/[fragment length (bp) × 10⁹ × 650], which is available from http://cels.uri.edu/gsc/cndna.html (accessed on 1 October 2022). Purified PCR fragments with known copy number (3.92 × 10¹⁰ for 874 bp fragment; 2.18 × 10¹⁰ for 1315 bp fragment) were serially diluted ten-fold from 10⁻¹ to 10⁻¹⁰ to create standard curves. The LOD of the multiplex PCR assay was determined under optimized conditions of the annealing temperature and primer concentration.