Novel Methods For The Detection Of Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) is associated with many diseases especially postweaning multisystemic wasting syndrome (PMWS), which has brought huge economic loss to the swine industry worldwide. The mainly used methods for detection of PCV2 are various kinds of biology methods. But these methods have some limits. It has great significance to develop and establish new methods for early detection of PCV2. This paper combines the chemiluminescence immunoassay, latex agglutination technique and fluorescence microscopy, using gold nanoparticle-monoclonal antibody conjugate, latex-antibody compound and quantum dot fluorescent probe for the research of detection methods and mechanism of PCV2. The main concents and results are listed as following:1. Gold nanoparticles and gold nanoparticle-monoclonal antibody conjugate were synthetized and characterized them using UV-vis absorption spectra and TEM image. We developed a facile, rapid and ultrasensitive method for detection of PCV2 based on gold(III) enhanced chemiluminescence immunoassay (CLIA). The limit of detection was as low as 2.67×102 copy/mL. Compared with conventional polymerase chain reaction (PCR), the proposed method has good sensitivity and reliability in 36 serum samples analysis, and showed great potential in virus assay.2. Ternary polymer latex particles with sulfonic acid group were synthesized, then we connected the monoclonal antibody of PCV2 with latex particles to obtain latex-antibody compound using electrostatic interactions. The specific reaction of monoclonal antibody and virus showed visible agglutination phenomenon in the presence of a certain concentration of virus. Latex-antibody compound didn't react with deionized water, phosphoric acid buffer, porcine circovirus type 1, porcine reproductive and respiratory syndrome virus, porcine parvovirus and porcine transmissible gastroenteritis virus. Under the optimized experimental conditions, the detection range of PCV2 was from 3.1×103 copy/mL to 8.00×107 copy/mL, and the detection limit was 1.0x105 copy/mL. Compared with PCR, the proposed method has good efficiency, sensitivity and specificity in 34 serum samples analysis. Results show that it can be used in the detection of the clinical samples and provided a great promising future for detection of PCV2 in clinical analysis.3. CdTe quantum dots and CdTe-PCV2 monoclonal antibody fluorescent probe were synthesized and characterized them using UV-vis absorption spectra, fluorescence spectroscopy, dynamic light scattering, agarose gel electrophoresis and fluorescence microscopy. The results show that CdTe-PCV2 monoclonal antibody fluorescent probe can recognize PCV2.In conclusion, nanoprobers were used for detection methods of PCV2. The new methods hold promising potential for sensitive, simple, rapid and visual detection of PCV2, also offers important references for the development of the detection kit and the application on the diagnostics and determination for other major animal desease of nanoprobers.