A novel protocol to isolate, detect and differentiate taeniid eggs in leafy greens and berries using real-time PCR with melting curve analysis

Abstract

BackgroundZoonotic taeniid cestodes are amongst the most important food-borne parasites affecting human health worldwide. Contamination of fresh produce with the eggs of Echinococcus granulosus (s.l.), Echinococcus multilocularis, and some Taenia species pose a potential food safety risk. However, very few studies have attempted to investigate the potential contamination of fresh produce with taeniid eggs and the available methods are not standardized for this purpose. Established protocols do exist for testing leafy greens and berries for contamination with protozoan parasites and are used in national surveillance programmes. This methodology could be suitable for the detection of taeniids. The objective of this project was to develop and standardize a sensitive and reliable method to detect contamination of leafy greens and berries with eggs of zoonotic taeniids and to differentiate between E. multilocularis, E. granulosus (s.l.) and Taenia spp.MethodsWe compared the efficacy of different wash solutions to remove Taenia spp. eggs from spiked produce, assessed two DNA extraction kits for their performance on Taenia spp. eggs, and adapted a published conventional multiplex PCR into a real-time PCR with fluorescence melting curve analysis (MCA) that was optimized for use on produce washes. Analytical specificity of this protocol was assessed using non-spiked produce washes as well as a variety of other potentially contaminating parasites.ResultsThe protocol as established in this study had an analytical sensitivity of detecting five eggs per spiked sample for both romaine lettuce and strawberries. Unequivocal identification of E. multilocularis, E. granulosus (s.l.) and Taenia spp. was possible through MCA. Amplicon sequencing allowed identification of Taenia to the species level. The real-time PCR also amplified DNA from Dicrocoelium sp., but with a clearly discernable melting curve profile.ConclusionThe new protocol for screening produce for taeniid contamination was highly sensitive. Melting curve analysis and the possibility of amplicon sequencing made this assay very specific. Once further validated, this method could be employed for surveillance of produce for contamination with taeniid parasites to assess potential risks for consumers.

A Sensitive, One-Way Sequential Sieving Method to Isolate Helminths’ Eggs and Protozoal Oocysts from Lettuce for Genetic Identification

“…The established method is able to identify as few as two embryonated Toxocara eggs and one-two E. multilocularis eggs in samples consisting of 300 g of lettuce. The method reported by Frey et al [32], which is based on a centrifugation step of the washing water containing 0.1% alconox or glycine followed by a real-time PCR version of the multiplex PCR used in the present study [26], had a detection limit of five taeniid eggs (T. pisiformis) per 35 g of lettuce. However, the feasibility of the method was not tested with a field study.…”

“…multilocularis eggs in samples consisting of 300 g of lettuce. The method reported by Frey et al [ 32 ], which is based on a centrifugation step of the washing water containing 0.1% alconox or glycine followed by a real-time PCR version of the multiplex PCR used in the present study [ 26 ], had a detection limit of five taeniid eggs ( T . pisiformis ) per 35 g of lettuce.…”

Diagnostic tools for the detection of taeniid eggs in different environmental matrices: A systematic review.

“…and fruits (apple and pear) Quantitative N.M. N.M. 11 N.M. Switzerland Federer et al, 2016 Washing (Alconox ®, glycine or sodium pyrophosphate), filtration and real-time qPCR with MCA E. multilocularis ( nad1 ), E. granulosus (12S rRNA) and Taenia spp. (12S rRNA) Leafy greens (romaine lettuce) and berries (strawberries) Quantitative LOD: 5 eggs/35 g lettuce or 55 g berries N.M. 16 16 h Canada Frey et al, 2019 s.g. = specific gravity, PCR = Polymerase chain reaction, DW = distilled water, LOD = limit of detection, nad1 or 2 = NADH-dehydrogenase subunit 1 or 2, N.A. = not applicable, N.M. = not mentioned, RT = room temperature, MCA = melting curve analysis.…”

“…In the multiplex end-point PCR used by Federer et al (2016) , the LOD, sensitivity, and specificity were not mentioned, although an extra alkaline lysis step was added prior to DNA-extraction with a QIAamp DNA mini kit (Qiagen). In a final study, a published multiplex end-point PCR was converted to a real-time PCR (probe-based) with melting curve analysis for use on berries and lettuce washes ( Frey et al, 2019 ). Here, an LOD for both the washing and PCR was calculated and defined as 5 eggs per 35 g lettuce or 55 g berries.…”

“…However, proteinase K digestion was the most implemented. Frey et al (2019) also discovered that a DNA extraction kit for soil (FastDNA Spin Kit) was superior to a DNA extraction kit for stool (QIAmp DNA Stool mini-Kit) when extracting taeniid DNA from food matrices. Similarly, it has been shown that for other helminths, such as soil-transmitted helminths, the choice of DNA extraction kit significantly influences the outcome of the result ( Ayana et al, 2019 ).…”