Procedures and time to perform 16S rRNA gene sequence analysis for bacterial identification in a routine clinical microbiology laboratorya

StepProcedureTime (hands-on)Waiting time (machine time)b
1Harvest. Organism can be harvested from any plate or broth if it is a pure culture, e.g., antibiotic susceptibility plate. The age of the culture is not important. One or two 0.01 loops full is sufficient. Save for processing as a batch.3-5 min each
2Extraction of DNA.0.5 h10 min and 3 min
3PCR amplification.0.5 h2.0 h
4Analysis of the PCR product. Loading, running, and examining gel.20 min1 h
5Purification of PCR products.1 h
6Cycle sequencing.30 min3.0 h
7Purification of PCR products.1 h
8Sequencing of the 16S rRNA gene. Load capillary tray; allow to run during time away, e.g., overnight or while doing something else.1 h2.5 h
9Analysis time. It takes 5 min or less to edit the sequence if the operator, software, and runs are good.5-15 min/sample
10Assignment of a name. If the organism is in the database, it takes 1 min; if it is a novel organism and several databases must be searched and sequences compared in detail, 15-30 min. At this point, correlation with phenotypic characteristics and clinical presentation is also done.Not counted for this analysis
11Reporting of results.30 min
Total labor time, based on integrating and completing three runs of 20 samples per wk.60 samples/40 h1 sample/40 min
  • a Modified from Clarridge et al., Abstr. 101st Gen. Meet. Am. Soc. Microbiol. 2001, with subsequent contributions by Kristina Hulten.

  • b Based on an ABI 3100 instrument.