Abstract
BackgroundAt present, the process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port. This process had the risks of degradation of biological samples and generation of pathogenic microorganisms and did not meet the rapid on-site detection demand because it took a rather long time. Therefore, it is urgently needed to develop a rapid and high-throughput detection assay of pathogenic microorganisms at the customs port. The aim of this study was to develop a microfluidic chip to rapidly detect swine pathogenic microorganisms with high-throughput and higher accuracy. Moreover, this chip will decrease the risk of spreading infection during transportation.ResultsA series of experiments were performed to establish a microfluidic chip. The resulting data showed that the positive nucleic acid of four swine viruses were detected by using a portable and rapid microfluidic PCR system, which could achieve a on-site real-time quantitative PCR detection. Furthermore, the detection results of eight clinical samples were obtained within an hour. The lowest concentration that amplified of this microfluidic PCR detection system was as low as 1 copies/μL. The results showed that the high specificity of this chip system in disease detection played an important role in customs inspection and quarantine during customs clearance.ConclusionThe microfluidic PCR detection system established in this study could meet the requirement for rapid detection of samples at the customs port. This chip could avoid the risky process of transporting the samples from the sampling site to the testing lab, and drastically reduce the inspection cycle. Moreover, it would enable parallel inspections on one chip, which greatly raised the efficiency of inspection.
Highlights
At present, the process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port
Microfluidic chip assay We chose four principal swine viruses, including PRRSV, Porcine epidemic diarrhea virus (PEDV), Pseudorabies virus (PRV) and PCV2, which are often found in related inspection at the customs, and we synthesized the plasmids of these viruses as positive references
The concentrations of the primers and probes, and the reaction temperatures were optimized by means of the plasmid amplifications on the Polymerase chain reaction (PCR) microfluidic chips
Summary
The process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port This process had the risks of degradation of biological samples and generation of pathogenic microorganisms and did not meet the rapid on-site detection demand because it took a rather long time. The virion has no envelope, and contains a covalently closed single-strand circular negative-strand DNA It often causes weaning piglets with multiple system failure syndrome (PMWS) and was tested using quantitative PCR and antigen capture ELISA [16]. Genome and pathogenicity of the virus, PRRSV can be divided into two types, namely European type and American type It mainly causes reproductive disorders in pigs and first isolated in China in 1996 [17]. Some researchers have designed primers for gE and gB genes to establish gene chip detection methods to distinguish between field and vaccine virus [19]
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