Abstract
Conventional Sanger sequencing remains time-consuming and laborious. In this study, we developed a rapid improved sequencing protocol of 16S rRNA for pathogens identification by using a new combination of SYBR Green I real-time PCR and Sanger sequencing with FTA® cards. To compare the sequencing quality of this method with conventional Sanger sequencing, 12 strains, including three kinds of strains (1 reference strain and 3 clinical strains, which were previously identified by biochemical tests), which have 4 Pseudomonas aeruginosa, 4 Staphyloccocus aureus and 4 Escherichia coli, were targeted. Additionally, to validate the sequencing results and bacteria identification, expanded specimens with 90 clinical strains, also comprised of the three kinds of strains which included 30 samples respectively, were performed as just described. The results showed that although statistical differences (P<0.05) were found in sequencing quality between the two methods, their identification results were all correct and consistent. The workload, the time consumption and the cost per batch were respectively light versus heavy, 8 h versus 11 h and $420 versus $400. In the 90 clinical strains, all of the Pseudomonas aeruginosa and Staphyloccocus aureus strains were correctly identified, but only 26.7% of the Escherichia coli strains were recognized as Escherichia coli, while 33.3% as Shigella sonnei and 40% as Shigella dysenteriae. The protocol described here is a rapid, reliable, stable and convenient method for 16S rRNA sequencing, and can be used for Pseudomonas aeruginosa and Staphyloccocus aureus identification, yet it is not completely suitable for discriminating Escherichia coli and Shigella strains.
Highlights
To date, conventional Sanger sequencing technology is sometimes used in a few diagnostic laboratories, it remains time-consuming and laborious
We have improved the conventional Sanger sequencing and validated it for detecting and genotyping the most common pathogens, including Pseudomonas aeruginosa, Staphyloccocus aureus and Escherichia coli. We presented this protocol and it described a new combination of SYBR Green I real-time polymerase chain reaction (PCR) and Sanger sequencing of DNA collected and extracted through Whatman FTAH cards
The turnaround time required for real-time PCR, Sanger sequencing, capillary electrophoresis reaction and data analysis
Summary
Conventional Sanger sequencing technology is sometimes used in a few diagnostic laboratories, it remains time-consuming and laborious. We have improved the conventional Sanger sequencing and validated it for detecting and genotyping the most common pathogens, including Pseudomonas aeruginosa, Staphyloccocus aureus and Escherichia coli We presented this protocol and it described a new combination of SYBR Green I real-time polymerase chain reaction (PCR) and Sanger sequencing of DNA collected and extracted through Whatman FTAH cards. Whatman FTAH paper is a commercial product that provides a remarkably easy way to collect, preserve and purify genomic DNA from bacteria, consisting of filter paper impregnated with a proprietary mix of chemicals that serve to lyse cells, prevent the growth of bacteria, protect the DNA in the sample, and can be stored at room temperature for even as long as 50 years [5] Though it has been widely used for PCR, few researches reported its utility of pathogens sequencing typing, we would give a confirmation here. We optimized all hands-on instrument steps by using modern reagents, by means of sequencing 16S rRNA gene of reference and clinical pathogenic strains, we validated the applicability and found the shortcomings of 16S rRNA gene sequencing method for identification
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