A PCR procedure for the detection of Giardia intestinalis cysts and Escherichia coli in lettuce

Abstract

Giardia intestinalis is a pathogen associated with foodborne outbreaks and Escherichia coli is commonly used as a marker of faecal contamination. Implementation of routine identification methods of G. intestinalis is difficult for the analysis of vegetables and the microbiological detection of E. coli requires several days. This study proposes a PCR-based assay for the detection of E. coli and G. intestinalis cysts using crude DNA isolated from artificially contaminated lettuce. The G. intestinalis and E. coli PCR assays targeted the β-giardin and uidA genes, respectively, and were 100% specific. Forty lettuces from local markets were analysed by both PCR and light microscopy and no cysts were detected, the calculated detection limit was 20 cysts per gram of lettuce; however, by PCR, E. coli was detected in eight of ten randomly selected samples of lettuce. These data highlight the need to validate procedures for routine quality assurance. These PCR-based assays can be employed as alternative methods for the detection of G. intestinalis and E. coli and have the potential to allow for the automation and simultaneous detection of protozoa and bacterial pathogens in multiple samples. Significance and impact of the study: There are few studies for Giardia intestinalis detection in food because methods for its identification are difficult for routine implementation. Here, we developed a PCR-based method as an alternative to the direct observation of cysts in lettuce by light microscopy. Additionally, Escherichia coli was detected by PCR and the sanitary quality of lettuce was evaluated using molecular and standard microbiological methods. Using PCR, the detection probability of Giardia cysts inoculated onto samples of lettuce was improved compared to light microscopy, with the advantage of easy automation. These methods may be employed to perform timely and affordable detection of foodborne pathogens.

Detection methods and prevalence of transmission stages of Toxoplasma gondii, Giardia duodenalis and Cryptosporidium spp. in fresh vegetables: a review

“…Whereas compared to other methods, they were suboptimal for the detection of parasites. Indeed, Ramirez-Martinez et al, (2015) reported a recovery efficiency of 20±4.3% and a detection limit of 20 cysts of Giardia/g of lettuce, using light microscopy, while PCR targeting the β-giardin gene was 10-fold more sensitive. Another method is the IFA, which was used for the detection of parasites (oo)cysts in various vegetable matrices, it is in fact the method recommended by the 18744 method (ISO, 2016).…”