PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings

Abstract

Molecular diagnostics are revolutionising the clinical practice of infectious disease. Their effects will be significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes. PCR is the most well-developed molecular technique up to now, and has a wide range of already fulfilled, and potential, clinical applications, including specific or broad-spectrum pathogen detection, evaluation of emerging novel infections, surveillance, early detection of biothreat agents, and antimicrobial resistance profiling. PCR-based methods may also be cost effective relative to traditional testing procedures. Further advancement of technology is needed to improve automation, optimise detection sensitivity and specificity, and expand the capacity to detect multiple targets simultaneously (multiplexing). This review provides an up-to-date look at the general principles, diagnostic value, and limitations of the most current PCR-based platforms as they evolve from bench to bedside.

Figure 1

Schematic of PCR. The PCR reaction takes place in a thermocycler. Each PCR cycle consists of three major steps: (1) denaturation of template DNA into single-stranded DNA; (2) primers annealing to their complementary target sequences; and (3) extension of primers via DNA polymerisation to generate new copy of the target DNA. At the end of each cycle the newly synthesised DNA act as new targets for the next cycle. Subsequently, by repeating the cycle multiple times, logarithmic amplification of the target DNA occurs.

Figure 2

Real-time PCR using Taqman probe. Taqman probe is a single-stranded oligonucleotide that is labelled with two different fluorescent dyes. On the 5′ terminus is a reporter dye and on the 3′ terminus is a quenching dye. This oligonucleotide probe sequence is homologous to an internal target sequence present in the PCR amplified product. When the probe is intact, the proximity of the two fluorescent dyes results in quenching of the reporter dye emission by the quencher dye. During the extension phase of PCR the probe is cleaved by 5′ exonuclease activity of Taq polymerase thereby releasing the reporter from the quencher and producing an increase in reporter emission intensity which can detected and quantified. As amplification continues, the amount of reporter dye signal measured is proportional to the amount of PCR product made.

Figure 3

Ligation-dependent PCR (LD-PCR). LD-PCR is a process in which non-amplifiable hemiprobes for each target will be constructed as follows: (A) For the 5′ hemiprobe the 5′ end will be a generic primer sequence shared by all 5′ hemiprobes, and the 3′ end will be target-specific. The 3′ hemiprobe will have a mirror symmetrical arrangement with an intervening stuffer sequence of variable length. (B) In the presence of target sequences the hemiprobes are juxtaposed to each other as they hybridise to their targets. (C) A single PCR primer set based on the generic sequences on the hemiprobes will be used for amplification of any ligated functional probes. The amplified product of each ligated functional probe has a unique length that can be separated by electrophoresis.

Figure 4

Padlock probe with rolling circle amplification. (A) Padlock probe hybridises to the target sequence. (B) The padlock probe can be converted to a circle by ligation only if ends of the probe are matched to their target. (C) Rolling circle amplification begins when primer 1 (P1) anneals to circularised probe and polymerase copies probe sequence, eventually copyin the entire circle. The polymerase begins displacing the previously synthesised product, opening up single-stranded primer 2 (P2) binding site. (D) Each turn of displacement synthesis around the circle exposes another P2 binding site, and the resulting synthesis from the annealed P2 primers results in the displacement of downstream primers and products. Displacement of the downstream primers and products also opens up additional P1 binding sites, and the process continues in an exponential cascade. The amplification process occurs in an isothermal reaction. Adapted from reference 138.

Figure 5

MALDI-TOF mass spectrometry. Sample molecules (ie, amplified DNA products) are co-crystallised with matrix and then subjected to desorption and ionisation by an incident laser pulse. An applied electric field accelerates the resultant ionised sample molecules across the time-of-flight (TOF) drift tube in vacuum, and a detector at the end of the tube accurately measures the flight time from the ion source to the detector. Typically, ions with larger mass-to-charge (M/z) ratios travel more slowly than those with smaller m/z. The data are recorded as a “spectra” that displays ion intensity vs m/z value.