Multiplex Ligase Detection Reaction (LDR)-PCR And Microarray Hybrdization System For Co-infection Swine Viruses' Detection And Discrimination

With the fast development of global economy, world trade of animals is becoming increasingly frequent, and its scale is more and more huge. Traditional methods such as serology and single PCR cannot completely adapt to the fast, precise and high-throughput animal detection and quarantine. Simultaneous detection and identification of multiple pathogenic microorganisms are urgently required in many diagnostic fields. In this study a method based on ligase detection reaction (LDR) - polymerase chain reaction (PCR) and microarray hybridization was established to detect several species of swine viruses simultaneously and be applied to clinical samples detection.LDR uses a thermostable DNA ligase that ligates two adjacent oligonucleotides which are perfectly matched at the junction. This method has subsequently been used in detecting mutations, insertions, and deletions in functional genes. While DNA microarray are genomic tools originally developed to monitor gene expression, also applied for the detection of specific mutations in DNA sequences, and lately employed in the parallel detection and identification of microorganisms in environmental or clinical samples. DNA microarray technology has revolutionized molecular diagnostic techniques for pathogen detection not only enhancing assay capability of DNA microarray but also allowing for high-throughput detection. Although there are many advantages of DNA microarray technology, some limitations remain to be overcome, especially the limitation of its specificity and sensitivity.In the present study, an alternative system based on LDR-PCR and microarray hybridization was established to detect above-described six swine viruses simultaneously. The LDR-PCR method was used to amplify swine viruses for sensitivity and specificity, while microarray hybridization was mainly selected for multiplexing. The sequences of the viruses were obtained from NCBI and aligned by using the software to identify highly conserved regions. LDR probes were designed within these highly conserved regions. A LDR probe pairs contained from left-to-right an upstream universal sequence, an upstream virus-specific probe, a downstream virus-specific probe, a Zip-code and a downstream universal sequence. The ligase detection reaction was performed with the cDNA/DNA as templates, and the ligated products may be amplified and labeled by universal primers. Subsequently the target-specific amplicons were detected by a universal Zip-code microarray. After optimization of the single and multiplex LDR-PCR with standard plasmids, the multiplex LDR-PCR based microarray for detection of swine viruses was developed.Using this strategy we successfully detected and discriminated six swine viruses simultaneously. The assay proved to be specific, only yielding positive signal in the target site. This method can detect as low as 10 copies genome equivalents. We have also compared labeling methods by PCR amplification between the Cy5-dCTP incorporation and the Cy5 modified primer. The hybridization results showed that the method by Cy5-dCTP showed a better performance and was adopted in our study. Of the 47 clinical samples tested, the newly developed method observed higher infection rate (78.7%) compared to real-time PCR (70.2%), showing a specificity and sensitivity of 95.7-100% and 100%, respectively. Furthermore, this assay can also be feasibly and flexibly applied to other pathogens'detection.