Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.

Effect of Universal Primers on the Tensile Bond Strength between Zirconia and Resin Composites

This study aimed to evaluate the effects of universal primers on the tensile bond strength between zirconia and resin composites.Zirconia specimens were divided into five groups based on the surface treatment with the following primers:MP(Monobond Plus),SU(ScotchBond Universal),AZ(AZ Primer),BM(Beauty bond Multi),and BL(Bondmer Lightless).After priming,stainless steel rods were bonded to the zirconia specimens with composite resin.The tensile bond strength test was performed:stored at room temperature for 1 day;stored in distilled water at 37°C for 7 days;and underwent thermal cycling.The BL group demonstrated a significantly higher tensile bond strength than other groups when stored at room temperature for 1 day(p<0.05).The primer that acted via chemical polymerization appeared to be most effective in improving the bond strength between the two materials in this study.

Simultaneous detection of enteroviruses from surface waters by real-time RT-PCR with universal primers

In order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcriptionpolymerase chain reaction （real-time RT-PCR） method was developed with universal primer pairs designed based on the highly conserved non-coding region sequences of genome targeting poliovirus, coxsackievirus and enterovirus 71. The recombinant plasmid was constructed as enterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector. The real-time RT-PCR method utilizing SYBR Green I was optimized. As a result of a series of examinations, the detection limit of the method was found to be 2.31 genome equivalent copy （GEC）/μL, the intraand inter-assay variations were lower than 2% and 5%, respectively, and enteroviruses were well distinguished from other microorganisms. There was a good linear relationship （r 2 = 0.997） between the logarithm of viral density and cycle threshold in a wide range of 2.31 × 10 0 to 2.31 × 10 9 GEC/μL. The validity of the method was further proved by its application for the detection of enteroviruses from various practical water samples.

Development and Application of the Nested PCR Assay for Duck Flavivirus

[ Objective] Aim to establish a kind of efficient detection method for duck flavivirus（DFV）. E Method] The method of nested PCR based on flavivirus universal primers which were designed according to the GeneBank flavivirus gene sequence. [ Result] The degenerate universal prim ers Flav P1 -Flav P4 were designed by genome comparison to target NS5 gene conserved area. The system could only amplify flavivirus purpose gene and the sensitivity was 90 copies/iJL, higher than the ordinary PCR 1 000 times. Homology and evolutionary analysis showed that duck flavivirus belonged to mosquito-born flavivirus, NTAV group, similar with Tembusu and BYD virus. [ Conclusion] Primers of the nested PCR system had good universality and specificity and method had high sensitivity. This system successfully detected flavivirus and clarified the evolution station of DFV.