ABSTRACT
For field-identification of taeniid cestodes in canine animals in Tibetan area, loop-mediated isothermal amplification (LAMP) assays for Echinococcus multilocularis, E. shiquicus, Taenia hydatigena, T. multiceps, T. pisiformis and T. crassiceps were developed and evaluated along with the reported assay for E. granulosus. The LAMP assays showed specific reaction with their corresponding target species DNA with the detection limit of 1 to 10 pg. Moreover, the assays for E. granulosus, E. multilocularis, T. hydatigena and T. multiceps could detect DNA extracted from 3 or more eggs of their corresponding target species. Then, the LAMP assays were applied on samples containing 3 to 35 taeniid eggs obtained from 61 field-collected canine feces in Qinghai, and the result was compared with a reported multiplex PCR and sequence analysis. The LAMP assays and the PCR detected single species DNA of E. granulosus, E. shiquicus, T. hydatigena and T. multiceps in 5, 2, 44 and 2 samples, respectively. In the rest 8 samples, DNA of both E. granulosus and T. hydatigena were detected by the PCR but the LAMP assays detected those DNAs in 2 samples and only T. hydatigena DNA in 6 samples. It was assumed that less than 3 E. granulosus eggs were mixed in the samples although the samples contained 21 to 27 eggs in total. In conclusion, the LAMP assays were less sensitive than the multiplex PCR, but would have adequate sensitivity for field use in Tibetan area.
Subject(s)
Dog Diseases/parasitology , Nucleic Acid Amplification Techniques/veterinary , Taeniasis/veterinary , Animals , DNA, Helminth/genetics , Dog Diseases/diagnosis , Dog Diseases/epidemiology , Dogs , Echinococcosis/diagnosis , Echinococcosis/epidemiology , Echinococcosis/veterinary , Echinococcus/genetics , Echinococcus multilocularis/genetics , Multiplex Polymerase Chain Reaction/veterinary , Nucleic Acid Amplification Techniques/methods , Sensitivity and Specificity , Taenia/genetics , Taeniasis/diagnosis , Taeniasis/epidemiology , Tibet/epidemiologyABSTRACT
A novel method which involved dispersive liquid-liquid microextraction (DLLME)-back extraction based on ionic liquid (IL) was developed for the determination of three phosphodiesterase-5 (PDE-5) inhibitors, sildenafil (SD), vardenafil (VD) and aildenafil (AD), in human plasma. DLLME based on IL as the extractant solvent and methanol as the dispersive solvent was the first step to extract PDE-5 inhibitors from sample solution; the other step of back extraction was followed by transferring target analytes from the IL to acidified aqueous solution. This two-step extraction ensured the compatibility of the final extractant phase, acidified aqueous solution herein, with the reversed phase high performance liquid chromatography-UV detection, and afforded clean extractant phase. The optimal extraction condition was obtained after systematical optimization. The sample solution (960µL) was extracted by 20µL of 1-octyl-3-methylimidazolium hexafluorophosphate in the presence of 20µL methanol and 300mgmL(-1) NaCl with the assistance of vortex; IL phase enriched with the target analytes was then extracted by 10% acetic acid aqueous solution. Good linearity ranges (SD 1-500ngmL(-1), VD 2-2000ngmL(-1) and AD 2-2000ngmL(-1)) with suitable r(2) (=0.9999) were achieved. Limits of detection (LODs) in pure water were 0.15ngmL(-1), 0.30ngmL(-1) and 0.43ngmL(-1) for VD, SD and AD, respectively. Intra-day and inter-day relative standard deviations were below 6.38%. Finally, this method was applied for the determination of PDE-5 inhibitors in human plasma with satisfactory LODs of 0.92ngmL(-1), 1.19ngmL(-1) and 2.69ngmL(-1) for VD, SD and AD, respectively. Acceptable absolute recoveries were obtained from 100.4% to 103.9%. The developed method afforded a convenient, fast and cost-saving operation with high extraction efficiency for the test analytes. It has potential to be applicable to biological samples.