Furong Tan

Specific, simple and rapid detection of porcine circovirus type 2 using the loop-mediated isothermal amplification method

BackgroundPorcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS), and porcine dermatitis and nephropathy syndrome (PDNS). It has caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS.ResultsA loop-mediated isothermal amplification (LAMP) assay was used to detect PCV2 in this study. Three pairs of primers were specially designed for recognizing eight distinct sequences of the ORF2 gene. This gene lies in the PCV2 virus genome sequence, and encodes the Rep protein that is involved in virus replication. Time and temperature conditions for amplification of PCV2 genes were optimized to be 55 min at 59°C. The analysis of clinical samples indicated that the LAMP method was highly sensitive. The detection limit for PCV2 by the LAMP assay was 10 copies, whereas the limit by conventional PCR was 1000 copies. The assay did not cross-react with PCV1, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, transmissible gastroenteritis of pigs virus or rotavirus. When 110 samples were tested using the established LAMP system, 95 were detected as positive.ConclusionThe newly developed LAMP detection method for PCV2 was more specific, sensitive, rapid and simple than before. It complements and extends previous methods for PCV2 detection and provides an alternative approach for detection of PCV2.

Rapid detection of porcine circovirus type 2 using a TaqMan-based real-time PCR.

Porcine circovirus type 2 (PCV2) and the associated disease postweaning multisystemic wasting syndrome (PMWS) have caused heavy losses in global agriculture in recent decades. Rapid detection of PCV2 is very important for the effective prophylaxis and treatment of PMWS. To establish a sensitive, specific assay for the detection and quantitation of PCV2, we designed and synthesized specific primers and a probe in the open reading frame 2. The assay had a wide dynamic range with excellent linearity and reliable reproducibility, and detected between 102 and 1010 copies of the genomic DNA per reaction. The coefficient of variation for Ct values varied from 0.59% to 1.05% in the same assay and from 1.9% to 4.2% in 10 different assays. The assay did not cross-react with porcine circovirus type 1, porcine reproductive and respiratory, porcine epidemic diarrhea, transmissible gastroenteritis of pigs and rotavirus. The limits of detection and quantitation were 10 and 100 copies, respectively. Using the established real-time PCR system, 39 of the 40 samples we tested were detected as positive.

Screening of specific diagnostic peptides of swine hepatitis E virus

BackgroundSwine hepatitis E virus (swHEV) is a zoonotic disease that is considered a major problem in pig production and presents a threat to human health. Elucidation of the major antigenic epitopes of swHEV is essential for the effective control of swHEV epidemics.ResultsBy bioinformatic analysis, we identified and then synthesized 12 peptides from open reading frames (ORFs) ORF1, ORF2 and ORF3, including swHEV-1 - swHEV-12. Using the results from ELISA, we selected swHEV-11 as the best candidate antigen and used it as a coating antigen for the development of peptide-based swine anti-HEV ELISA kits. The coefficient of variation (CV) the coefficient of variation (CV) varied between 4.3-7.2% in the same batch, and between 8.2-17.7% in six different batches. When comparing our swine peptide-based kit with the commercial recombinant-based kit, the humane anti-HEV IgG test had a 73.4% correspondence rate for them.ConclusionThis is the first systemic study to screen the diagnostic peptides of swHEV and our findings strongly suggest that peptide swHEV-11 is a potent diagnostic reagent of swHEV that could be used in the development of highly efficient diagnostic assays for the specific and highly sensitive detection of anti-HEV activity in swine serum samples.

Enhanced pesticide sensitivity of novel housefly actylcholinesterases: a new tool for the detection of residual pesticide contamination

The full-length cDNA encoding an acetylcholinesterase (AChE) was cloned and sequenced from the housefly, Musca domestica, by reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis revealed that this 2,076xa0bp sequence encodes a mature protein of 612 amino acids (67xa0kDa) and a 79 residue signal peptide. The amino acid sequence shared 52.8–81.4% identity with the AChE proteins of other insects. The cDNA sequence, which lacked the signal peptide was inserted into the vector pPIC9K and then introduced into strain GS115 of the yeast Pichia pastoris. The recombinant AChE protein was then expressed in P. pastoris strain GS115 by methanol induction. Site-directed mutagenesis of the A262G, Y327F, Y327D and I374D residues, either singly or in combination, was performed by reverse PCR. These mutants improved the catalytic activity and sensitivity to the organophosphate and carbamate insecticides. Although the sensitivity of other mutants was slightly increased, the results still showed that the sensitivity of triple mutant, GDD (A262G/Y327D/I374D), enhanced remarkably as much as 16 times for methomyl, 14 times for both carbofuran and chlorpyrifos, and ten times for parathion-methyl, compared to that of the wild-type. The results strongly suggested that these residues are the key structural elements controlling AChE enzyme catalytic activity and sensitivity to inhibition by insecticides. The AChE enzyme obtained by this method could be used to detect the organophosphate and carbamate insecticide residues in fruits and vegetables, a characteristic of great potential research and industrial application.