Appeared to be devoid of some additional discernible bases, with a QV larger than 20, high-quality sequences were still acquired, and matches were still obtained when submitted to the Genbank blast system, supporting the report that some interference within products was not completely Tubacin eliminated or impacted by primer formation. From the identification results of pathogenic strains, we learn that partial 16S rRNA gene sequencing is a suitable tool for Staphylococcus aureus and Pseudomonas aeruginosa identification, which have produced consistent results with conventional culture methods as others have done. However, 30 Escherichia coli specimens generated 3 blast results of Shigella sonnei, Shigella dysenteriae and Escherichia coli, and the 16S rDNA-based phylogenetic tree suggested that it was difficult to distinguish each of them. It has been demonstrated by other researchers that there are many similarities in many respects between some Shigella and Escherichia coli, such as clinical symptoms, biochemical characteristics and antigens. In fact, previous study showed that a few Escherichia coli have been assigned to a different genus, based primarily on their distinct clinical presentation and their importance as human pathogens. A research by Pupo et al., analyzing sequence variation in housekeeping genes, also showed that most Shigella serotypes fall into three clusters within Escherichia coli, proving that, it is indeed difficult to distinguish Shigella from Escherichia coli. So the false identification results in some Escherichia coli of our specimens may attribute to the false classification of Escherichia coli sequences, which were virtually Shigella sequences submitted to GenBank by other researchers. Compared with conventional Sanger sequencing, our improved protocol has emerged as a faster and more convenient method to identify those common bacteria. However, it also should be applied cautiously. Firstly, although sequencing is particularly helpful in situations where organisms are difficult to characterize by using conventional culture methods, but 1 to 14% of the isolates remain unidentified after testing. Secondly, the variable regions, as a foundation for discriminating bacteria, only distributing V1–V3 in the first 500 bp area, is one third of full-length of 16S gene. This system uses universal primers to amplify and sequence a 500 bp fragment from the 59-terminus of the 16S rRNA gene, but only a mean of 404 bp is read, because the first approximately 100 bp had to be manually discarded owing to residual SYBR Green?left over from PCR products, and was difficult to be removed by purification kit. Consequently the V1, distributed in the first 104 bp, have to be discarded and hence slightly impaired the discrimination ability of the sequencing chromatogram. Lastly, though SYBR Green?does not require specific probes to be developed, as is the case for some other detection chemistries.