Preliminary application of recombinase -aided amplification in detection of Clonorchis sinensis metacercariae in freshwater fish / 中国血吸虫病防治杂志

OBJECTIVE@#To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay.@*METHODS@#Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples.@*RESULTS@#Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ2 = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ2 = 11.20, P < 0.05) and fluorescent RAA assay (χ2 = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05).@*CONCLUSIONS@#Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.

Identification and Characterization of Carbon-Centered Free Radicals Formed from Lipoxygenase-Catalyzed Lipid Peroxidation of Dihomo-γ-linolenic Acid / 分析化学

A combination technique of LC/ESR, LC/MS and spin trapping was used to identify and characterize the carbon-centered free radicals formed from lipoxygenase-catalyzed peroxidation of dihomo-γ-linolenic acid(DGLA). The spin trap, α-[4-pyridyl-1-oxide]-N-tert-butyl nitrone(POBN), could react with short-lived carbon-centered radicals to form relatively stable adducts. Based on the same retention time of POBN radical adduct in LC/UV/ESR and LC/MS, molecular weight of adduct could be confirmed and the structure of adduct could be confirmed by their LC/MS2 fragment pattern. The results showed that free radicals formed in lipoxygenase-catalyzed peroxidation of DGLA, including ~·C_7H_(13)O_2, ~·C_(10)H_(17)O_2 and ~·C_5H_(11), all stemmed from β-scission of DGLA alkoxyl radicals(8-, 11-, 15-LO~·). The results were helpful for further study of biological activities of these radicals in vivo.