Development Of Multiplex Pcr For Simultaneous Microarray Detection Of Six Virulence Factors Of Vibrio Anguillarum And Primary Exploration Of Innovative Detection Technologies

Vibriosis mainly caused by the Vibrio anguillarum, Vibrio splendidus, Vibrio vulnificus, Vibrio alginolyticus and Vibrio harveyi, is a fish disease infecting freshwater fish, seawater fish, and other farmed animals, responsible for considerable economic hardship to mariculture operations worldwide. Vibrio anguillarum is widely prevalent and its nosogenesis in seawater is a complex mechanism formed by a serry of virulent genes including plasmid encoded iron-uptake system, extracellular hemolysin, cell flagellum protein, extracellular protein enzyme and cell surface components. Lots of method are reported for the detection of pathogenic Vibrio anguillarum, for example, fluorescent antibody technique, nucleic acid hybridization. et al. However, immunoassay requires preparation for antiserum, and the sensitivity of nucleic acid hybridization only achieves 150 pg. With the growing wealth of aquiculture pathogen data, the diagnostic technology of aquiculture disease is inevitably trending toward high-throughput techniques. DNA microarray, which has been developing since the end of last century, is a novel molecular biotechnology with high-throughput, parallelism and low-cost. Huge potential of microarray generates considerable interest for its utility as a diagnostic tool for genes. Therefore, it is significant to utilize microarray for earlydiagnosis of farmed animal disease in aquiculture industry. Multiplex PCR is a kind of PCR technique which simultaneously amplifies multiple target DNA fragments by adding multiple primers in a single tube. Multiplex PCR saves time and cost, supports parallel and high-troughput microarray diagnosis.In this study, a multiplex PCR, in a single tube, that supports simultaneous microarray detaction of 6 virulence genes of Vibrio anguillarum-vah1, virC, flaA, empA, virA and angM is established, by optimizing the PCR reaction mixture and PCR cycle conditions including Mg2+ concentration, primer concentration, Taq polymerase, annealing temperature and cycle number. Genomic DNA is extracted from bacterial colonies using a modified CTAB (cetyltrimethyl ammonium bromide) protocol that allows for phenol/ chloroform extraction followed by ethanol precipitation. Primers and Probes are designed under common reacting conditions avoiding mispairing, hairpin, dimmers and crossing reaction. During the optimization of multiplex PCR, different gradients of Mg2+ concentration (1.6 mM-5.2 mM), primer concentration (0.1μM-1.0μM), annealing temprature (50℃-65℃), PCR cycles (20c,25c,30c,35c,40c,45c) and different combinations of Taq polimerase are set. The optimized Multiplex PCR system is as follows:Mg2+ 3.6 mM, primer pairs-angM 0.1μM, vah1 0.2μM, flaA 0.2μM, empA 0.2μM, virC 0.4μM and virA 1.0 pM, annealing temprature 55.5℃and 35 PCR cycles. Hot Start Taq polymerase with hot-start PCR step gives the best target gene bands yield and lowest non-specific dimmers yield. The amplification limit measured by agrose electrophoresis for the target template is estimated at 4 copies of genomic DNA template. Digoxigenin (DIG)-labeled target genes are amplified by gene specific primers and DIG-labeled dUTP. Oligonuleotides probes specific for each sequence are synthesized and appled to nylon membranes.The multiplex PCR products are hybridized to the microchip. Hybridization of simultaneous amplification products of 6 virulence genes of Vbrio anguillarum are observed to be specific by NBT/ BCIP color development, indicating that the multiplex PCR method for microarray detection is a reliable method to detecting Vibrio anguillarum in the strains under investigation. At present, innovative nucleic acid amplification technologies have been widely used in life science and its correlative fields, and play an important role in the development of life science. Some of the new nucleic acid amplification technologies are:LAMP (loop-mediated isothermal amplification), HDA (helicase-dependent isothermal DNA amplification), Solid Phase PCR, SELEX (systematic evolution of ligands by exponential enrichment), SOLEXA high throughput sequencing and Taqman Real-Time PCR, are summarized in this article. Finally, in this article, primary exploration of utilizing innovative nucliec acid amplification including Taqman Real-Time PCR, Helicase-Dependent isothermal DNA Amplification (HDA), Adapter-linked primer and Solid Phase PCR for multiplex PCR and DNA microarray is introduced.