Abstract
BackgroundAt present, pig industry in China is faced with the complex situation of mixed infection caused by multiple pathogens. It is urgent to develop some new high-throughput molecular diagnosis assays to simultaneously detect pathogens or antibodies. Biochip array technology has made it possible to screen thousands of samples simultaneously; it has been twice named as one of the top 10 scientific and technological breakthroughs. Studies have reported encouraging results using protein biochips for detecting antibodies against avian infectious bronchitis virus and ruminant bluetongue virus, but the research of this technology for the diagnosis of swine diseases is still sparse.ResultsIn this study, a novel protein chip was developed that can simultaneously detect the antibodies of four important swine viruses as follow, classical swine fever virus (CSFV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine reproductive and respiratory syndrome virus (PRRSV). Four prokaryotic expression plasmids pET-32a-E2 of CSFV, −VP2 of PPV, −EDIII of JEV, and -N of PRRSV were induced by IPTG (Isopropyl β-D-1-Thiogalactopyranoside) and overexpressed in E.coli, respectively. The purified proteins were identified by Western blotting and then printed on epoxy-coated glass slides. The optimized parameters of this diagnostic chip showed that the spotting concentrations of E2、VP2、EDIII、N proteins were 0.2, 0.4, 0.4, and 0.4 mg/mL. The optimal primary and secondary antibody dilutions were 1:50 and 1: 600. Compared with the commercial ELISA (Enzyme-linked immunosorbent assay) kits, the positive and negative coincidence rates of this chip were 95.8% ~ 100 and 86.2% ~ 100%, as well as, no cross-reaction.ConclusionThis protein chip provided a fast, specific, and sensitive method for simultaneous detection of antibodies in clinical serum samples. Compared with traditional methods, this protein chip can monitor very small amount of serum.
Highlights
At present, pig industry in China is faced with the complex situation of mixed infection caused by multiple pathogens
The protein biochip is a novel application of the sandwich-type antibody-capture assays, such as Enzyme-linked immunosorbent assay (ELISA), the fundamental difference is that the capture proteins are covalently attached to the surface of the biochip in an ordered array
Expression and purification of fusion proteins The four gene fragments were successfully amplified by PCR (Polymerase chain reaction), double-digested, and cloned into pET-32a vectors
Summary
Pig industry in China is faced with the complex situation of mixed infection caused by multiple pathogens. Under pressure from vaccination and antibiotic treatment, new strains of familiar pathogens, as well as new pathogens, are leading to large scale outbreaks [5,6,7] These conditions make it necessary to develop diagnostic assays that can quickly and reliably screen large numbers of clinical samples. This will allow a savings of clinical manpower and material resources, as well as reduce the mortality and morbidity of pigs [8,9,10]. The development of multi-level biochips will enable high-throughput quantitative and qualitative diagnosis of important pig diseases and will help solve current diagnostic needs for large number of animal diseases [15].
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