Selection of DNA aptamers to cervical intraepithelial neoplasia by SELEX / 生物工程学报

An in vitro synthesized random ssDNA library was subjected to 12 rounds of selection against anti-screening cells and sieving cells by SELEX. Normal and inflammatory cervical exfoliation cells were selected as anti-screening cells, and the cervical exfoliation cells of low-grade squamous intraepithelial lesion (CIN1), high-grade squamous intraepithelial lesion (CIN2, CIN3) and cervical carcinoma were selected as sieving cells during the screening process. Then, the highly specific aptamer CIN-Ap4 was established by the analysis of the specificity, affinity and cell immunofluorescence, which can be used as biomarker for Cervical Intraepithelial Neoplasia. Prime Premier 5.0 was applied to design a random ssDNA library. According to the fixed sequence at both ends of the library, a pair of primers were designed and synthesized. At the same time, the optimal annealing temperature, cycle times and primer concentration ratio of PCR procedure were selected. The results under the optimal condition are shown as follows. In the 50 μL reaction system, the optimum reaction conditions of symmetry PCR are as follows: annealing temperature is 49.5 ℃, number of cycles is 15. The optimal reaction conditions of indirect asymmetric PCR are as follows: the primer concentration ratio is 80:1, and the number of cycles is 35. The experiment proves that the oligonucleotide library is constructed successfully, and the highly specific dsDNA and ssDNA can be obtained under optimal PCR conditions with good repeatability, which establishes the foundation for the further exploration and experimentation.

Modified Method of Multilocus Sequence Typing (MLST) for Serotyping in Salmonella Species

Multilocus sequence typing (MLST) of Salmonella is useful method for replacing serotyping using antisera but is limited by difficulties associated with in polymerase chain reaction (PCR). We optimized the PCR reaction, especially annealing temperature and extension time (94degrees C for 2 min; 40 cycles at 94degrees C for 30 sec, 56.8degrees C for 1 min, 72degrees C for 2 min; and 72degrees C for 10 min). The degradation of PCR product by thermostable nucleases was inhibited by using template DNAs treated proteinase K or purified by a commercialized preparation kit. The resulting modified MLST was used as accurate and fast typing method.