ABSTRACT
Background@#African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. @*Objectives@#The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. @*Methods@#Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5′untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. @*Results@#The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10 0 copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. @*Conclusions@#The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.
ABSTRACT
The chemical structures of mequindox related metabolites in chicken plasma had been investigated using high performance liquid chromatography combined with linear ion trap quadrupole(LC-ESI/LTQ) and high performance liquid chromatography combined with ion trap-time of flight-mass spectrometry (LC-ESI/IT-TOF).Samples were separated by Hypersil BDS C_(18) and symmetry Shield columns, respectively, and 0.01% formic acid aqueous(A) and methanol(B) were used as mobile phase with gradient elution.Electros pray ionization mass spectrometric(ESI) source was used and operated in positive ion mode.When chickens were orally administered with mequindox at dosage of 20 mg/kg, blood samples were collected from the brachi al vein.Mequindox and its metabolites were extracted by the mixture of acetonitrile and acetoacetate (3:2, V/V).After solvent evaporated, the residue was dissolved in 30% methanol aqueous and the solution was detected by LC/IT-TOF MS and LC-ESI/LTQ.The molecule weight from LC-ESI/IT-TOF was analyzed by software Shimadzu's Composition and the mass chromatogram from LC-ESI/LTQ was analyzed by software Xcalibur 2.0.7.According to the molecular weight and MS~n data, referring the metabolic reaction rules, five chemical structures of mequindox related metabolites in chicken plasma were identified.Metabolites (M1-M4) were synthesized to verify the structure of metabolites.The metabolites are 3-methyl-2-(1-hydroxy) ethyl-qui-noxaline-N~1,N~4-dioxide(Ml), 3-methyl-2-(1-hydroxy) ethyl-quinoxaline-N~4-oxide(M2), 3-methyl-2-acetyl-quinoxaline-N~4-oxide, 3-methyl-2-acetyl-quinoxaline (M4), 3-hydroxymethyl-2-(1-hydroxy) ethyl-quinoxa-line-N~1,N~4-dioxide (M5).