Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to 3 μM or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, 2 μM of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.

Antibiotic Detection Kit (Combination I), a lateral flow immunoassay (LFIA) developed for detecting antibiotic residues in milk, was utilized for the analysis of antibiotic residues in the muscle tissue of olive flounder. After 5-h treatment of samples by placing them in water dosed with sulfadimethoxine (SDM; 200 g/ton water), the residue depletion of SDM was investigated in 25 cultured olive flounders (Paralichthys olivaceus). Muscles from fish were sampled before treatment and on the 1st, 2nd, 3rd, 4th and 5th days after treatment. The concentration of SDM in the muscle was then determined by LFIA. The absorbance ratio of the sample to the control blank (Bs/Bo) was employed as an index to determine the residue in olive flounder muscle. To investigate the recovery rate, standard solutions were added to muscle samples to obtain final concentrations of 25 and 50 ng/mL in the muscle. The recovery rates of all spiked samples were >96.6% of the spiked value. SDM was detectable in the muscle of fish treated with the drug until the 1st day of the withdrawal period. The present study shows that the LFIA can be easily adopted to detect SDM residues in the tissue of farmed fish.

Antibiotic Detection Kit (Combination I), a lateral flow immunoassay (LFIA) developed for the detection of antibiotic residues in milk, was utilized for the analysis of antibiotic residues in the muscle tissue of olive flounder. After 60-min treatment by dipping in water dosed with ampicillin (200-g/ton water), the residue depletion of ampicillin was investigated in 25 cultured olive flounder (Paralichthys olivaceus). Muscles of fish were sampled on the 1st, 2nd, 3rd, 4th and 5th day after drug treatment. The concentration of ampicillin in the muscle was determined by LFIA. The absorbance ratio of the sample to the control blank (Bs/Bo) was employed as an index to determine the muscle residues in olive flounder. To investigate the recovery rate, standard solutions were added to muscle samples to give final concentrations in the muscle of 4 and 8 ng/ml. The recovery rates of all spiked samples were > 96% of the spiked value. Ampicillin was detected in the muscle of fish treated with the drug until the 2nd day of the withdrawal period. The present study showed that the LFIA can be easily adopted to predict ampicillin residues in tissue of farmed fishes.