Digital PCR: modern solution to parasite diagnostics and population trait genetics

Abstract

The use of polymerase chain reaction (PCR)-based diagnostic approaches has steadily increased in the field of parasitology in recent decades. The most recent large-scale technological modification of the PCR formula, also known as third-generation PCR, came in the form of digital PCR (dPCR). Currently, the most common form of dPCR on the market is digital droplet PCR (ddPCR). Unlike quantitative real-time PCR (qPCR), the digital format allows for highly sensitive, absolute quantification of nucleic acid targets and does not require external standards to be included in the developed assays. Dividing each sample into thousands of compartments and using statistical models also eliminates the need for technical replicates. With unprecedented sensitivity and enforcement of binary endpoint reactions, ddPCR not only allows the use of tiny sample volumes (especially important when working with limited amounts of DNA) but also minimises the impact of variations in amplification efficiency and the presence of inhibitors. As ddPCR is characterised by excellent features such as high throughput, sensitivity and robust quantification, it is widely used as a diagnostic tool in clinical microbiology. Due to recent advances, both the theoretical background and the practical, current applications related to the quantification of nucleic acids of eukaryotic parasites need to be updated. In this review, we present the basics of this technology (particularly useful for new users) and consolidate recent advances in the field with a focus on applications to the study of helminths and protozoan parasites. Graphical Abstract

Ascaridia galli - An old problem that requires new solutions

“…In addition, a loop-mediated isothermal amplification in conjunction with a lateral flow dipstick (LAMP-LFD) assay that recognises the ITS-2 was recently presented for the visual detection of A. galli eggs in faecal samples ( Panich et al, 2023 ). Although the performance of these tools has never been compared such as in Haemonchus contortus , it is reasonable to assume that ddPCR is the most sensitive molecular assay ( Ljungström et al, 2018 ; Baltrušis and Höglund, 2023 ). However, a general problem with molecular tools is the cost.…”

Current Applications of Digital PCR in Veterinary Parasitology: An Overview

“…High cost No reference samples required Inability to sequence the PCR amplic No standard curve required Better performance in the presence $$$$$$of inhibitors High sensitivity Among the different dPCR forms, the droplet dPCR (ddPCR) is the most comm used in veterinary parasitology [4,7,12]. The general principles and technical advan have been described in detail elsewhere and are beyond the scope of this review a [2,13,14]. Concisely, the PCR reaction is subdivided into thousands of small part (droplets), uniform in volume, by various available methods.…”

“…Digital PCR (dPCR) is an emerging technology that enables the absolute quantification of the targeted nucleic acids. It is regarded as a vastly improved version of conventional PCR and qPCR [2]. For the time being, most studies on parasitic diseases of veterinary importance involve developing and validating dPCR assays and evaluating their performance compared to qPCR or Next Generation Sequencing (NGS) platforms.…”

“…Parasitologia 2023, 3 270 This novel technology is based on endpoint quantification after sample division. It also provides information on the precise analyte concentrations in the original sample, expressed in DNA copies per microliter, along with confidence interval estimations [2]. The dPCR assays usually combine probe-based PCR (typically TaqMan probes) with a microfluidics analysis platform.…”

Development of a three-colour digital PCR for early and quantitative detection of benzimidazole resistance-associated single nucleotide polymorphisms in Haemonchus contortus

“…For dPCR the sample is divided in several partitiones. Most dPCR systems currently applied in veterinary parasitology are droplet digital PCR systems (ddPCR) where the reaction is conducted in water droplets separated by a continuous oil phase ( Baltrušis and Höglund, 2023 ). A second approach is the use of a chip where the droplets are physically isolated ( Madic et al, 2016 ).…”