|ZHANG, QIAN - Shanghai Jiaotong University|
|DAI, MIN - Shanghai Jiaotong University|
|ZHOU, MIN - Shanghai Jiaotong University|
|SHI, XIANMING - Shanghai Jiaotong University|
|WANG, DAPENG - Shanghai Jiaotong University|
Submitted to: Journal of Microbial and Biochemical Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2014
Publication Date: 5/6/2014
Citation: Zhang, Q., Dai, M., Liu, Y., Zhou, M., Shi, X., Wang, D. 2014. Comparison of different methods for isolation of bacterial DNA from retail oyster tissues. Journal of Microbial and Biochemical Technology. 6(4):212-216.
Interpretive Summary: Oysters filter large volumes of water during feeding and are able to bio-accumulate bacteria including food-borne pathogens from the surrounding water, therefore, oysters may be an important vehicle for dissemination of food-borne pathogens. The fact that pathogens could be maintained in oysters for at least one month imposes a huge risk for human health. Detection and identification of pathogens in oysters have relied on growing the bacteria on agar media, which is time-consuming and labor intensive. To improve efficiency, more rapid assays were developed for detection of these pathogens in oysters using methods based on detecting regions of the DNA that are specific for the pathogens. Efficient amounts of sample DNA of high quality is of critical importance for molecular assays. To obtain high quality genomic DNA, four methods were compared for bacterial DNA isolation from retail oyster tissues for detection of the pathogens. The quality of the DNA was evaluated to determine a good candidate method for DNA extraction. Our work enhances the ability to detect pathogens in seafood and may benefit the seafood industry and consumers.
Technical Abstract: Oysters are filter-feeders that bio-accumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic DNA (Allrun, Shanghai, China), and the MiniBEST Bacterial Genomic DNA Extraction kits (Takara, Dalian, China), and the phenol-chloroform and boiling lysis methods. The concentration of the genomic DNA was measured using a spectrophotometer. The purity of the genomic DNA was evaluated by PCR amplification of 16S rDNA followed by determining the cloning efficiency of the amplicon into the pMD19-T vector. Furthermore, the bacterial DNA quality was also evaluated by PCR assays using a pair of species-specific primers for Vibrio parahaemolyticus. Our results showed that the two commercial kits produced the highest purity of DNA, but with the lowest yields. The phenol-chloroform method produced the highest yield although it was time-consuming. The boiling lysis method was simple and cost effective; however, it was only suitable to isolate genomic DNA from bacteria present in retail samples following an enrichment step. The two commercial kits were good candidates for genomic DNA extraction from retail oyster tissues without enrichment.