Nonaplex PCR using Cliffhanger primers to identify diarrhoeagenic <i>Escherichia coli</i> from crude lysates of human faecal samples

<div><p>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 <i>Escherichia coli</i> 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.</p></div

Detecting DEC pathogens in clinical samples.

<p>The samples are reported as being either positive by both methods or positive by only one of the methods. All samples that were positive only by the Cliffhanger method were verified by Sanger sequencing.</p

Routine multiplex primers as reported by Brandal et al, 2007.

<p>Routine multiplex primers as reported by Brandal et al, 2007.</p

Cliffhanger multiplex primers consisted of a 5’-positioned <i>ortho-</i>TINA molecule (Z), a custom-designed ssDNA overhang, an internally placed <i>ortho</i>-TINA molecule (Z) and a target-specific primer sequence.

<p>Reverse primers were labelled with af 5’-positioned biotin (Bio) and an <i>ortho-</i>TINA molecule (Z).</p

Workflows of the routine analysis method versus the Cliffhanger method.

<p>Workflows of the routine analysis method versus the Cliffhanger method.</p

Testing of 105 DEC strains from a reference collection by the routine method and by the Cliffhanger method.

<p>Testing of 105 DEC strains from a reference collection by the routine method and by the Cliffhanger method.</p

Positive findings using the routine method versus the Cliffhanger method.

<p>¹Samples were collected from Dec 1, 2012 to March 20, 2013. ²For retesting, the samples were diluted 8-fold and 20-fold prior to multiplex PCR. Pos, positive; neg, negative. The number of positives and the percentages of all samples that were positive are shown for each gene.</p

A Cliffhanger PCR primer with a Z-X-Z-S structure.

<p>Z is an <i>ortho</i>-TINA molecule, X is the custom-designed single-stranded nucleic acid overhang and S is the target-specific priming sequence. The 5’ <i>ortho</i>-TINA molecule protects the oligonucleotide against 5’ to 3’ exonuclease activity, whereas the internally placed <i>ortho</i>-TINA molecule blocks the DNA polymerase and thereby creates a single-stranded DNA overhang on the PCR amplicon.</p

Comparison of primers in samples spiked with human genomic DNA (gDNA).

<p>(a-c) unmodified DNA primers and (d-f) 5′-<i>o</i>-TINA modified primers. Each ○ on the efficiency curves represents one threshold cycle (Cq) determination on an amplification curve with a corresponding melting curve, reported as the first derivative. Cq determinations highlighted in red would normally have been excluded based on the amplification curve and melting curve evaluation. (a, d) Unspiked samples. (b, e) All samples and negative controls spiked with 10 ng gDNA. (c, f) All samples and negative controls spiked with 100 ng gDNA. A uniform primer concentration of 200 nM was used in all samples and negative controls. The annealing temperatures for unmodified and <i>o-</i>TINA primers were 60.0°C and 66.0°C, respectively.</p

Efficiency curves for unmodified and 5′-<i>o</i>-TINA modified primers.

<p>All experiments were conducted at an annealing temperature (T<i>a</i>) of 66.0°C and primer concentrations (C<i>primers</i>) of unmodified and <i>o</i>-TINA modified primers were compared on the same plate. The melting curves corresponding to the amplification curves used for efficiency curve determination are included for the lowest C<i>primers</i> that allows an efficiency of 100% for unmodified and 5′-<i>o</i>-TINA modified primers.</p