Development Of A Real-time PCR Assay For Detection And Quantification Of The Microorganisms On Porphyra

In this study, the bacteria strain NPyS3was separated from the health Porphyra yezoensis.,which had a strong antibacterial activity. Based on the analysis of morphological characteristics,physiological, biochemical characterization and the homology comparison of16S rRNA genesequence, the strain NPyS3was identified as Pseudoalteromonas porphyrae.A real-time PCR assay was designed and evaluated for rapid detection and quantification ofthe Porphyra microorganisms. Two pairs of specific primers were designed from the sequence of16S rDNA region of P. porphyrae. One pairs were designed for Pseudoalteromonas, the otherswere for total bacteria, and the PCR specificity was examined compared with the other twenty-fivePorphyra microorganisms. For Pseudoalteromonas specific primers the PCR amplification weredetected only from samples which contained Pseudoalteromonas, and specific signals were notdetected from any other bacteria. All the twenty-five strains of bacteria were amplified by thespecific primers for total bacteria.Made the retrieved purified PCR amplification DNA fragmentsof16S rDNA of P.porphyrae for the standard. The results show that the determination ranges ofPseudoalteromonas and total bacteria are7.74×10~1~7.74×10~5gene copies/μL (R2=0.999) and7.74×10~1~7.74×10~6gene copies/μL (R2=0.998). DNAs from Porphyra microorganisms that werecollected from field samples were assayed in the run. Using the developed standard curves tocreate a new kind of relative quantitative method to establish the growth curve of P.porphyrae. Theresults were not in agreement with the traditional method by testing OD600nm absorbtion lightvalue. Using the developed standard curves Porphyra bacteria could be quantified under thecondition of laboratory. The real-time quantitative PCR technology could detect the dynamicschanges of microorganisms on seaweed. It is believed that compared to the traditional method,RTQ-PCR method were rapid, efficient and accurate in detecting the Pseudoalteromonas inenvironment samples, could detect the bacteria which was uncultured or have not found, and coulddetect the abundance of Pseudoalteromonas in environment samples quantitatively. The real-timequantitative PCR method has the high potential value in detecting of the microorganisms onseaweed.