1. Nonaplex PCR using Cliffhanger primers to identify diarrhoeagenic Escherichia coli from crude lysates of human faecal samples.
- Author
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Schneider UV, Mikkelsen ND, Scheutz F, Friis-Møller A, and Lisby G
- Subjects
- Escherichia coli classification, Female, Humans, Male, DNA Primers, Diarrhea genetics, Diarrhea microbiology, Escherichia coli genetics, Feces microbiology, Multiplex Polymerase Chain Reaction methods
- Abstract
Sensitive, probe-based detection of multiple DNA targets is limited by the competitive reannealing of the antiparallel duplex DNA helix with the complementary DNA strand. To address this, we developed Cliffhanger primers, which create single-stranded DNA overhangs on PCR amplicons while simultaneously increasing the multiplex PCR efficacy and allowing PCR amplification using crude lysates of human faecal samples. A multiplex PCR that targeted eight genes from diarrhoeagenic Escherichia coli plus an internal control was performed and compared to a routine method that consisted of culture followed by multiplex PCR with fragment length separation. A total of 2515 clinical faecal samples from patients with diarrhoea were tested using both methods, and there was a significant increase in clinical sensitivity and negative predictive value with the Cliffhanger method for seven out of eight genes. All Cliffhanger-only positive samples were confirmed by Sanger sequencing of the PCR amplicon. Notably, the Cliffhanger method reduced the total sample turn-around time in the laboratory from 20 hours to 6 hours. Hence, use of Cliffhanger primers increased assay robustness, decreased turn-around time and increased PCR efficacy. This increased the overall clinical sensitivity without the loss of specificity for a heavily multiplexed PCR assay., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: UVS financial competing interests: Current ownership of stocks, previous paid employment and previous consulting for Anapa Biotech A/S. Inventor of WO 2011/137911 “Method for generating a double stranded nucleic acid with a single stranded overhang” which is fully entrusted to Anapa Biotech A/S. Non-financial competing interests: None. NDM financial competing interests: Previous paid employment for Anapa Biotech A/S. Non-financial competing interests: None. FS financial and non-financial competing interests: None. AF-M financial and non-financial competing interests: None. GL financial competing interests: Previous paid employment and previous consulting for Anapa Biotech A/S. Inventor of WO 2009/112032 “Target amplification and sequencing with primers comprising triplex forming monomer units” and WO 2011/137911, which are fully entrusted to Anapa Biotech A/S. Non-financial competing interests: None. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
- Published
- 2018
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