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SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCRbased protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of speciﬁc primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for speciﬁc hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-ﬁdelity screening of asymptomatic people, even without any high-speciﬁcation equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.
Purpose: We aimed to identify the relationship between the serum testosterone (TS) axis and the clinically localized prostate cancer and to evaluate the changes in hormone concentrations after radical prostatectomy (RP). Materials and Methods: Blood samples were drawn from 699 patients with prostate cancer before and after RP, without hormone or radiation therapy, and from 700 age-matched healthy men between 7:00 and 9:00 A.M., and their serum concentrations of total TS and sex hormone-binding globulin (SHBG) were measured. Results: Patients with prostate cancer had lower mean SHBG (55.8±22.0 vs. 61.4±24.2 nmol/L) and higher mean free TS (FT) (7.1±2.5 vs. 6.3±2.6 ng/dl), bioavailable TS (BAT) (158.2±55.2 vs. 138.1±56.5 ng/dl), and free TS index (FTI) (31.3±12.0 vs. 27.1±11.6) than healthy controls (all, p<0.001), while total TS did not differ. Following RP, patients with prostate cancer showed significant increases in SHBG (65.6±26.3 vs. 56.3±21.6 nmol/L) and subsequent decreases in FT (6.4±4.4 vs. 7.2±2.5 ng/dl), BAT (138.2±50.3 vs. 158.7±55.3 ng/dl), and FTI (26.5±11.1 vs. 31.2±11.7) (all, p<0.001), whereas total TS remained unchanged. Preoperative SHBG concentration was significantly lower in patients with pathologic Gleason score (GS) <7 than in those with GS ≥7 (p<0.001). SHBG was an independent predictor of pathologic GS <7 (p=0.003), along with preoperative prostatic specific antigen (PSA) concentration and biopsy GS. Conclusions: Prostate cancer influences the sex hormonal axis, modulating SHBG concentration and increasing the utilization of bioactive TS.