Method Description

The test utilizes DNA extraction from EDTA whole blood and polymerase chain reaction (PCR) of a highly variable fragment of the 16S ribosomal RNA (rRNA) gene. If present, the amplified DNA is sequenced to obtain identification of the source organism from the patient sample. Specimens undergo total nucleic acid extraction, and the purified eluate is produced by the MP96 (Roche Diagnostics). The DNA eluate is concentrated two-fold from the specimen input volume. PCR is performed on a LightCycler 480II instrument (Roche Diagnostics) to amplify approximately 530 base pairs (bp) of the bacterial 16S rRNA gene (V1-V3, exact length varies by species) with SYBR Green DNA detection.

Amplification inhibition is detected with a second PCR reaction performed using the extracted specimen spiked with a low concentration of positive control DNA. Due to high crossing point values reflecting the low abundance of pathogens, all patient samples for this test will undergo next-generation sequencing (NGS).

Samples are sequenced using NGS on a MiSeq sequencing platform (Illumina). The NGS process generates 500 bases of sequencing reads (250 bp in the forward and reverse directions). Due to the size of amplicon generated, there is a gap in coverage between forward and reverse sequences. Verification results and in silico analysis have demonstrated that forward sequence results (≥210 bp) offer the same bacterial identity as full-length amplicon sequence for all organisms that can be reported by this assay. Quality filtering and results generation from NGS files is performed with Pathogenomix cloud-based software (RipSeq NGS).

High-quality sequence (Q ≥30) of 210 bp or more in length are sorted into clusters based on the observed sequence and used for identification. Positive and negative controls are used throughout all processes to ensure assay performance. Forward and reverse sequences are analyzed to determine bacterial identity using the cloud-based analysis program RipSeq NGS (Pathogenomix).(Rodino KG, Wolf MJ, Sheldon S, et al. Detection of tick-borne bacteria from whole blood using 16S ribosomal RNA gene PCR followed by next-generation sequencing. J Clin Microbiol. 2021;59(5):e03129-20. doi:10.1128/JCM.03129-20)