Development of a droplet digital polymerase chain reaction tool for the detection of Toxoplasma gondii in meat samples

Mancusi, Andrea; Giordano, Angela; Bosco, Antonio; Girardi, Santa; Proroga, Yolande T.R.; Morena, Luigi; Pinto, Renato; Sarnelli, Paolo; Cringoli, Giuseppe; Rinaldi, Laura; Capuano, Federico; Maurelli, Maria Paola

doi:10.1007/s00436-022-07477-9

Cited by 9 publications

(15 citation statements)

References 53 publications

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“…When comparing the ddPCR and qPCR results, it was found that the latter underestimated the exposure pressure. Similarly, a uniplex Toxoplasma gondii detection assay performed better than qPCR when testing the diaphragm tissue samples from farm animals [ 21 ].…”

Section: Resultsmentioning

confidence: 99%

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

Baltrušis

Höglund

2023

Parasites Vectors

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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

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Section: Resultsmentioning

confidence: 99%

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

Baltrušis

Höglund

2023

Parasites Vectors

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“…ddPCR outperformed qPCR in identifying pathogen DNA in E. coli , C. jejuni , CMV , C. trachomatis , Salmonella, and HIV [ 23 , 24 , 25 , 26 , 27 ]. In the case of parasites such as Plasmodium , ddPCR was shown to be more sensitive and accurate than qPCR; however, in Toxoplasma, Cryptosporidium, Leishmania , Trypanosoma , Theileria equi, Theileria cervi , Babesia, Bartonella , and Borrelia species, ddPCR was found to be equivalent to qPCR [ 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ].…”

Section: Discussionmentioning

confidence: 99%

“…Developing an assay with higher performance at low doses without the requirement for a reference standard curve should increase the detection of infection, hence facilitating case identification and disease monitoring. ddPCR is a breakthrough DNA quantification technique that avoids dependence on the reference standard curve and is effective in diagnosing and quantifying various human and animal pathogens, including vector-borne infections [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. With more consistent findings across technical replicates and higher sensitivity than qPCR, ddPCR has been gaining traction as a viable pathogen identification and quantification option.…”

Section: Introductionmentioning

confidence: 99%

Multiplex ddPCR: A Promising Diagnostic Assay for Early Detection and Drug Monitoring in Bovine Theileriosis

et al. 2023

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Accurate quantification based on nucleic acid amplification is necessary to avoid the spread of pathogens, making early diagnosis essential. Droplet digital PCR (ddPCR) stands out for absolute parasite quantification because it combines microfluidics with the TaqMan test. This helps deliver maximum accuracy without needing a reference curve. This study assessed the efficacy of ddPCR as a detection tool for the bovine theileriosis (BT) caused by Theileria parasites. We developed and validated a duplex ddPCR method that detects and quantifies the Theileria genus (18S rRNA) and identifies clinically significant Theileria annulata parasites (TaSP) in experimental and clinical samples. ddPCR was shown to be as effective as qPCR throughout a 10-fold sample dilution range. However, ddPCR was more sensitive than qPCR at lower parasite DNA concentrations and reliably assessed up to 8.5 copies/µL of the TaSP gene in the infected DNA (0.01 ng) samples. The ddPCR was very accurate and reproducible, and it could follow therapeutic success in clinical cases of theileriosis. In conclusion, our ddPCR assays were highly sensitive and precise, providing a valuable resource for the study of absolute parasite quantification, drug treatment monitoring, epidemiological research, large-scale screening, and the identification of asymptomatic parasite reservoirs in the pursuit of BT eradication.

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“…The use of undercooked/raw meat with viable tissue cysts (Plaza et al 2020;Mancusi et al 2023) is thought to be a significant cause of infection due to T. gondii's propensity to spread to a wide range of animals and remain in their structures for years (Tenter et al 2000;Kuruca et al 2023). Human toxoplasmosis has been documented following the intake of raw and uncooked flesh and organs, and live parasites have been identified from fresh, frozen, and cured meat (Liu et al 2015;Kuruca et al 2023).…”

Section: Transmission Of T Gondii With Different Kinds Of Meatmentioning

confidence: 99%

Control and Detection of Toxoplasma Gondii in Meat Chain

2023

IJAB

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Toxoplasmosis caused by infection with the Toxoplasma gondii which can infect all warm-blooded hosts including humans is estimated to be present in one-third of the world population. Human infection with T. gondii has different routes by ingesting sporulated oocysts present in water, soil, or vegetables. Foodborne toxoplasmosis in humans may result from exposure to different stages of T. gondii, especially from the ingestion of tissue cysts or tachyzoites contained in meat, offal, and meat-derived products of many different livestock animals. Although the prevalence of viable T. gondii in retail meat was very low, consumers can acquire T. gondii infection from ingestion of undercooked meat. Thus, raw or undercooked meat containing viable cysts has been suggested to be a major source of T. gondii infection in humans. Prevention of toxoplasmosis transmission in humans depends on meat safety strategies. Most realistic solutions to control T. gondii in the meat chain come in the form of a risk-based meat safety assurance system that incorporates risk-categorization of farms and slaughterhouses, as well as education of all interested parties. The difficulty of toxoplasmosis monitoring in farm animals is due to the asymptomatic and chronic expression of the disease. Vaccine development and vaccination of farm animals against T. gondii are traditionally being implemented. On the other hand, carcass surveys present several advantages and control plans to detect and take measures for this pathogen. After systematic meat inspection, some treatments must be applied to the meat found to contain cysts, such as freezing, cooking, irradiation, or high pressure. Laboratory analyses to detect T. gondii are very important in terms of protecting public health. The gold standard for detecting T. gondii in meat samples is a bioassay using either mice or cats. Molecular methods are used as fast and realiable in addition to conventional serological methods for the diagnosis of toxoplasmosis.

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Development of a droplet digital polymerase chain reaction tool for the detection of Toxoplasma gondii in meat samples

Cited by 9 publications

References 53 publications

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

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

Multiplex ddPCR: A Promising Diagnostic Assay for Early Detection and Drug Monitoring in Bovine Theileriosis

Control and Detection of Toxoplasma Gondii in Meat Chain

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