Loop-Mediated Isothermal Amplification-Lateral-Flow Dipstick (LAMP-LFD) to detect Toxoplasma gondii oocyst in ready to eat salad

Authors: LALLE, MARCO - Istituto Superiore Di Sanita; POSSENTI, ALESSIA - Istituto Superiore Di Sanita; Dubey, Jitender; POZIO, EDOARDO - Istituto Superiore Di Sanita

Submitted to: Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/6/2017
Publication Date: 4/1/2018
Citation: Lalle, M., Possenti, A., Dubey, J.P., Pozio, E. 2018. Loop-Mediated Isothermal Amplification-Lateral-Flow Dipstick (LAMP-LFD) to detect Toxoplasma gondii oocyst in ready to eat salad. Food Microbiology. 70:137-142. https://doi.org/10.1016/j.fm.2017.10.001
DOI: https://doi.org/10.1016/j.fm.2017.10.001

Interpretive Summary: Toxoplasmosis, caused by the single celled parasite, Toxoplasma gondii, continues to be a public health problem worldwide. This parasite infects all warm-blooded hosts, including humans. It causes mental retardation and loss of vision in children, and abortion in livestock. The ingestion of food and water contaminated with resistant stage of the parasite, the oocyst, is a major mode of transmission of this parasite. Of all the hosts infected, only cats are known to excrete oocysts in feces. Cats can excrete millions of oocysts after eating an infected prey, such as a mouse or a bird. Oocysts can survive outdoors for months. The number of oocysts in municipal waters is low and difficult to detect by microscopy of centrifuged water samples. In the present paper the authors report on the success of a Loop-Mediated Isothermal Amplification-PCR method in conjuction with an easy read out method (Lateral-Flow Dipstick) for its efficacy to detect Toxoplasma DNA in green salad intentionally contaminated with T. gondii oocysts. These results will be of interest to biologists, parasitologists, and epidemiologists.

Technical Abstract: The apicomplexan parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a foodborne zoonosis with a global distribution and estimated to cause up to 20% of the total foodborne disease burden in Europe. Association between T. gondii infection and the consumption of unwashed raw fruits and vegetables contaminated with oocysts has been reported and the increasing habit to eat prewashed ready-to-eat salads poses a new potential risk for consumers. It is therefore important to trace the occurrence of potential contamination with this parasite to guarantee the safety of ready-to-eat vegetables. Detection of T. gondii in vegetable by molecular techniques has been achieved but low sensitivity (PCR) or expensive equipments (qPCR) limit routine applicability. Here, we describe the development and validation of a sensitive and robust method relying on a LAMP assay, targeting the 529 bp locus, to detect T. gondii oocysts down to 25 oocysts/50g in ready-to-eat baby lettuce. The LAMP has been also adapted for a faster visualization of the result by a lateral flow dipstick chromatographic detection method.