Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5′-end. In qPCR, the 5′-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5′-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5′-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5′-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5′-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5′-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions

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

Workflows of the routine analysis method versus the Cliffhanger method.

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

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

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

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

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