Pyrosequencing with di-base addition for the identification of pathogenic bacteria

Dan Pu,Kunxian Shu,T Zhu,M Anpo,A Sharifi

doi:10.1051/e3sconf/202127103010

Dan Pu, Kunxian Shu + Show 3 more

Open Access

https://doi.org/10.1051/e3sconf/202127103010

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Abstract

We proposed a novel method for the identification of pathogenic bacteria, in which a partial amplicon of the molecular markers was targeted by using pyrosequencing with di-base addition (PDBA). PDBA was conducted by synchronously adding di-base instead of one base into a reaction, and a set of highly sequence-specific encodings containing the type and the number of incorporated nucleotide(s) (peak height) were obtained. By comparing the encoding sizes of each isolate and the number of incorporated nucleotide(s) in each cycle, moving from first to last, various kinds of bacteria could be unambiguously identified. To verify its feasibility, we simulated PDBA results from thirteen isolates of Mycobacterium species and compared their encoding sizes and the number of incorporated nucleotide(s) in each cycle with those predicted by a homemade software. The thirteen isolates were successfully differentiated. We also targeted partial RNase P RNA gene (rnpB) of cultured M. paratubercuosis and M. celatum to differentiate the two isolates. By comparing the encoding size of each isolate and the number of incorporated nucleotide(s) in each cycle, the two Mycobacterium isolates were successfully differentiated. In conclusion, PDBA enabled to reliably identify pathogenic bacteria.

Highlights

It is important to identify pathogenic bacteria in the field of microbiological diagnostics and the demand for simple, sensitive, specific, and robust tests is increasing
When the novel sequencing technique is combined with traditional pyrosequencing, we call it pyrosequencing with di-base addition (PDBA)
3.1 The principle for identification of pathogenic bacteria using PDBA We have proposed a real-time decoding sequencing strategy in which a template is cyclically interrogated in two parallel sequencing runs with two orders of di-base dispensation

Summary

Introduction

It is important to identify pathogenic bacteria in the field of microbiological diagnostics and the demand for simple, sensitive, specific, and robust tests is increasing. For the identification of pathogenic bacteria, both biochemical testing and molecular methods are developed. Molecular methods provide microbiologists with additional tools that may supplement biochemical testing for bacterial pathogen identification ([2]). Pyrosequencing is a sequencing-by-synthesis based method, which has been widely used to detect pathogenic bacteria by targeting molecular markers, such as rnpB ([3]), rpoB ([4]), 16s rRNA ([5]), gyrB ([6]). Due to the addition of di-base into the reaction, PDBA enables to increase signal intensity (peak height) in each sequencing run and to reduce the number of the sequencing cycles (A sequencing cycle represented a sequencing reaction) required to read though the target sequences. By comparing the encoding sizes and the peak height of species-specific nucleotide sequences with those predicted by a homemade software, the isolates can be unambiguously differentiated

Methods

Results

Conclusion