Development Of Multiplex PCR And Real-Time PCR For The Detection Of4Bacterial Pathogens In Cultured Turbot (Scophthalmus Maximus)

Turbot （Scophthalmus maximus） was firstly introduced into China in1992.With success in artificial breeding since1999, the commercial culture hasspread rapidly along the coast of North China and led the4thmarine culturewave in China. However, with the rapid development of high-intensivecultivation and improper management of turbot aquaculture, several diseaseshave been identified and caused high mortality and economic losses. Thediseases have become one of the important bottle-neck for the sustainabledevelopment of turbot culture. The epidemiological investigation revealed that mostof the diseases were caused by the bacteria, among which Vibrio harveyi, Vibrioanguillarum, Vibro splendidus and Edwardsiella tarda, were the important pathogens.Thus, it’s of great importance to establish rapid, practical, and sensitive pathogendetection methods to control the disease and reduce economic losses. In this research,we developed the Multiplex PCR and Real-Time PCR technology to detect the fourbacterial pathogens.1. Establishment of Multiplex PCR detection for Vibrio harveyi, Vibrioanguillarum, Vibro splendidus and Edwardsiella tardaThe vhhp2gene sequences of Vibrio harveyi and the metalloproteinase genesequences of the Vibrio anguillarum were downloaded from GenBank, and thespecific primers for the two genes were designed respectively. Partial gyrB gene ofVibro splendidus and Edwardsiella tarda were cloned according to the gyrB universalprimers and sequenced. Based on the sequence alignment of several important marinebacteria’s gyrB gene sequence, the specific primers for Vibro splendidus andEdwardsiella tarda were also designed. Cross-species amplification among different bacteria and inter-species amplification among different strains of the same speciesshowed that the four pairs of primer have good specificity and commonality. The PCRproducts for the four genes are151bp (Vibro splendidus),251bp (Vibro splendidus),412bp (Vibrio anguillarum) and582bp (Edwardsiella tarda), respectively, and can besperated clearly by agarose electrophoresis. Multiplex PCR amplification conditionwas established and the amplification result showed that the four pathogens can bedetected in one PCR reaction. The detection limit for multiplex PCR of the fourpathogens were3.9×10~4CFU/reaction (354.9pg DNA) of Vibrio harveyi,6.8×10~4CFU/reaction (201.5pg DNA) of Vibrio anguillarum,5.8×10~4CFU/reaction (204.7pg DNA) of Vibro splendidus and3.2×10~5CFU/reaction (967pg DNA) ofEdwardsiella tarda, respectively.2. Establishment of Real-Time PCR detection for Vibrio harveyiThe real-time PCR primers were designed according to vhhP2gene of Vibrioharveyi. The PCR product of vhhP2gene was inserted into pMD18-T vector toconstruct the recombinant plasmid. The resulted plasmid was serially diluted and usedas standards for quantitative analysis. The amplification curved used3replicates ofdifferent dilution and different storage time showed that the standard had goodrepeatability and stability. The relationship between plasmid copy number(10x)and Ctvalue (y) was described as a standard curve: y=-3.331x+37.48(with correlationcoefficient0.998and the amplification efficiency is1. The detection limit of the assaywas7copies per reaction.3. Establishment of Real-Time PCR detection for Vibro splendidusAccording to the sequenced gyrB gene sequence, the species specific Real-TimePCR primers for Vibro splendidus were designed. The PCR product using thedesigned primers was inserted into pMD18-T vector to construct the recombinantplasmid. The resulted plasmid was serially diluted and used as standards forquantitative analysis. The relationship between plasmid copy number (10x) and Ctvalue（y）was described as a standard curve: y=-3.338x+37.67(with correlation coefficient0.999and the amplification efficiency was0.99). The detection limit of theassay was20copies per reaction.4. Establishment of Real-Time PCR detection for Edwardsiella tardaAccording to the sequenced gyrB gene sequence, the species specific real-timePCR primers for Edwardsiella tarda were designed. The PCR product using thedesigned primers was inserted into pMD18-T vector to construct the recombinantplasmid. The resulted plasmid was serially diluted and used as standards forquantitative analysis. The relationship between plasmid copy number (10x) and Ctvalue（y）was described as a standard curve: y=-3.32x+39.38(with correlationcoefficient0.998and the amplification efficiency was1). And the amplificationreactions have good repeatability between and within groups. The detection limit ofthe assay was60copies per reaction.5. Application of the Fast Detection Technique for bacterial pathogensThe infected turbots were collected, the pathogen was identified as Edwardsiellatarda using traditional bacterial culture and16S rDNA analysis. The pathogendetection was conducted using the established Multiplex PCR and Real-Time PCR,which also revealed Edwardsiella tarda as the pathogen and the bacteria content was1.42×10~8CFU/g. The result showed that the newly developed detection techniqueswere accurate and efficient.Overall, the multiplex PCR and Real-Time PCR detection techniques wereestablished for bacterial pathogens of turbot in this study. These pathogens can bedetected qualitatively in one PCR reaction by Multiplex PCR and quality analysisdetected by Real-Time PCR. Thus, the new developed detection technologies aresimple, rapid, specific and sensitive. It will be helpful in disease control and healthymanagement of turbot culture.