A PCR-Based Intergenic Spacer Region-Capillary Gel Electrophoresis Typing Method for Identification and Subtyping of Nocardia Species

Abstract

c While 16S rRNA sequence-based identification of Nocardia species has become the gold standard, it is not without its limitations. We evaluated a novel approach encompassing the amplification of the Nocardia 16S-23S rRNA intergenic spacer (IGS) region followed by fragment analysis by capillary gel electrophoresis (CGE) of the amplified product for species identification of Nocardia. One hundred forty-five Nocardia isolates (19 species) and four non-Nocardia aerobic actinomycetes were studied. Reproducibility testing was performed in a subset (21%) of isolates. Ninety-five different electropherograms were identified, with heterogeneity within species being a general observation. Among common Nocardia species (e.g., Nocardia cyriacigeorgica, N. nova, N. farcinica), 2 or 3 dominant electropherogram subgroups were typical. While only a minority (8/19; 42%) of the different Nocardia species contained isolates displaying unique fragment sizes that were predictive of a particular species, virtually all isolates (142/145; 98%) could be assigned to the correct species using IGS-CGE typing based on the number and size of amplified fragments. The median number of fragments for each isolate was 2 (range, 1 to 5) with only a minority (17%) having a single fragment detected. The majority (93%) of amplified fragments were between 408 and 461 bp. The technique was also non-operator dependent, highly reproducible, and quicker and less expensive than 16S sequencing. In summary, PCR-based IGS-CGE typing is relatively simple, accurate, reproducible, and cost-effective and offers a potential alternative to 16S rRNA sequencing for identifying and subtyping Nocardia isolates.

Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

“…PCR fragment analysis was performed as before [ 20 ]. Generally, the ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA) was used employing a 96-capillary 50-cm POP-7 gel.…”

“…Proteomic approaches such as matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) also lends itself as an identification tool [ 19 ]. As a simple and less expensive alternative to ITS sequencing, another molecular identification method, sequencer-based capillary gel electrophoresis (SCGE) systems, has been reported to have good accuracy, reproducibility and inter-laboratory consistency, whilst retaining flexibility [ 20 , 21 ].…”

Merits and Pitfalls of Currently Used Diagnostic Tools in Mycetoma

“…This method was used to correctly identify 142 out of the 145 tested isolates to the correct species [58]. McTaggart developed a multilocus sequence analysis (MLSA) based on sequencing of 16S rRNA genes, the β subunit of type II DNA topoisomerase ( gyrB ) genes, the subunit A of SecA preprotein translocase ( secA1 ) genes, hsp65 genes, and RNA polymerase ( rpoB ) genes [59].…”

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

“…Nocardia species are ubiquitous Gram-positive bacilli causing various clinical syndromes, such as localized cutaneous infections, systemic infections, with a preference to infect the central nervous system. Thus, Wehrhahn et al [80] established a method based on PCR-CGE. After amplifying the Nocardia 16S-23S rRNA IGS region, PCR products were analyzed using CGE for identifying Nocardia species.…”