Detection And Quantification Of Thalassiosira Rotula With Double Specific Probe Assay

To purify, identify, detect, and quantify phytoplankton is the main work of the study of phytoplankton. Morphologic analysis is the traditional method to study phytoplankton, and with the rapid development of science and technology, many modern technologies are introduced into this field, such as nucleic acid hybridization assay, molecular immunoassay, real time PCR, flow cytometry and HPLC et al. One cell can be detected by molecular technique now. Using nucleic acid probe assay, especially fluorescence in situ hybridization and sandwich hybridization, to detect and enumerate phytoplankton is the hotspot in the field of phytoplankton study.A brand-now technology named double specific probe assay was introduced in this study to detect and quantify Thalassiosira rotula veraciously and steadily. The detection procedure is as follows, separate and purify the genome DNA first, then amplify the rDNA of Thalassiosira rotula, then clone and do sequence analysis of this fragment, then design a set of probes including capture probe, S1 probe, linkage probe and signal probe specific to Thalassiosira rotula, follow on is to develop a steady assay based on double specific probe assay to detect Thalassiosira rotula through test to optimize the concentration of probes, comparison of different method to label signal probes and effect of temperature of S1 nuclease reaction on signal and specificity of the assay, to validate the specific of the set of probes, to get the standard curve about the detection of Thalassiosira rotula and to enumerate the sample whose concentration is unknown. Besides, the affect of different way to conservecells on detection signal and the variety of the signal during the whole cell cycle are also concerned in this paper.The result is as follows, the fragment of 18SrRNA gene of Thalassiosira rotula amplified is 1787bp.The set of probes are tested sufficiently specific to Thalassiosira rotula by the specificity assay, and can differentiate different species of the same genus. It is known that it is mostly rRNA that hybridize with the oligonucleotide probe through an experiment that developed as follow, deal the cell lysate with RNase or DNase or RNase&DNase before detect the signal, then get the discrimination of the result. The result showed that the signals obtained from the samples digested by RNase and RNase&DNase depresses sufficiently, by contraries, the group which is digested by DNase is almost equivalent with that is not digested with anything. The comparison of extracted total RNA and crude cell lysate as sample material in hybridization assay displays that using crude cell lysate as sample is more fast, simple and steady, so this way is selected as our procedure to do the next experiment.The standard curve is drawn by diluting the sample known concentration. The standard equation is 'y = 0. 0019x - 0. 0284' , and R2= 0. 995, x stands for number of target cells and y stands for OD values .The standard deviation is low enough, R2shows good correlation and the assay is sensitive to detect 88 cells. As to the test to detect natural samples, the result of double specific probe assay confirms independently what get using light microscopy. It is said that large quantities of nontarget species and detritus found in natural samples might obscure the species-specific double specific probe assay signals. As a first step toward determining if this background material is indeed a problem, the same density of Thalassiosira rotula is added to f/2 medium and natural sample containing a complex background of microorganisms and organic material. The differences between the responses of the double specificprobe assay of the f/2 medium and the natural field background are minimal, indicating that a complex field background does not necessarily affect the efficacy and sensitivity of the assay. Different method is used to preserve samples, and the result showed that the signal of fresh sample was the highest, and the next is the sample preserved after hybridized with the oligonucleotide probe, and next is the sample preserved as the state of lysate, and the lowest is to conserve the sample as dead cells, so the way to conserve samples after the hybridization with the oligonucleotide probe is used as the standard procedure when the natural samples are detected on the sea. The signal generated by cells through a growth cycle was examined. The cultures is collected per day and the sample hybridized with the oligonucleotide is stored at -80°C and once the grow-out is complete, all the samples are analyzed using the double specific probe assay. The result of this test showed that the signal obtained from cells in the early stationary phase of growth is indeed higher than that obtained in the late stationary phase, and combined with the growth curve. The result shows that when the growth rate is high, the signal is accordingly high.