Application of two isothermal amplification methods in pathogen identification using the next generation sequencing

Abstract

Objective This study evaluated two isothermal amplification methods for their ability to identify targeted virus in the clinical samples, which provided a reference for other researchers using NGS in clinical diagnostics. Methods Nasopharyngeal swabs from two patients with severe pneumonia were amplified with Single Primer Isothermal Amplification (SPIA) method and Multiple Displacement Amplification (MDA) approaches and further proceeded to NGS and metagenomics analysis to identify targeted virus. Results The SPIA produced shorter fragments than MDA, and had a lower amplification efficiency. The SPIA successfully identified targeted viruses in the two clinical samples tested, while MDA failed to detect that in one of the samples. Conclusions Both SPIA and MDA could be applied to pathogen identification using NGS in the clinical diagnostics. SPIA possessed lower amplification efficiency and higher economic cost, but had higher capacity to discover the causing agents in the clinical sample. Key words: Isothermal amplification; Next generation sequencing; Pathogen identification