A loop-mediated isothermal amplification assay to detect Bacteroidales and assess risk of fecal contamination

Wang, Jiangshan; Ranjbaran, Mohsen; Ault, Aaron; Verma, Mohit S.

doi:10.1016/j.fm.2022.104173

Cited by 11 publications

(24 citation statements)

References 52 publications

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“…To generate a fecal contamination risk evaluation map, we converted the Ct value of each qPCR reaction to log 10 (copies/cm 2 ) via a linear fit to log-transformed concentrations (Wang, Ranjbaran, Ault, et al, 2023). The qPCR results showed that all 96 samples had a Bacteroidales concentration of 0 – 2.24 copies/cm 2 (Figure 3C).…”

Section: Resultsmentioning

confidence: 99%

“…The µPADs were separately sealed inside resealable polypropylene bags (Uline, S-17954) and heated at 65 °C for 60 min in a water bath (Anova, ANTC01). Time-lapse video of the µPADs was taken from 0 to 60 min using a HERO8 Black digital camera (GoPro, SPJB1) (Pascual-Garrigos et al, 2021; Wang, Ranjbaran, Ault, et al, 2023). We also scanned the µPADs at 60 min using a flatbed scanner (Epson, B11B223201).…”

Section: Methodsmentioning

confidence: 99%

“…Following our previous results (Wang, Ranjbaran, Ault, et al, 2023), we decided to use collection flags (made from plastic sheets) to collect microbial samples in the field. These collection flags provided more consistent and reproducible LAMP results compared to lettuce leaves with grooves (Wang, Ranjbaran, Ault, et al, 2023). The collection flags were assembled using bamboo skewers (29.8 cm), transparent film (Apollo, VPP100CE), a stapler, and a paper cutter.…”

Section: Methodsmentioning

confidence: 99%

“…As a result, the qPCR assay is used with culture-based enrichment methods, which can run into the problem of pathogens entering a viable-but-nonculturable (VBNC) state (FDA, 2021). Aside from determining the presence or absence of pathogens, an alternative is to quantify the abundance of fecal indicator bacteria (FIB)—microorganisms selected as indicators of fecal contamination (Brauwere et al, 2014; Wang, Ranjbaran, Ault, et al, 2023). FIB such as Escherichia coli , Enterococcus faecalis , and Bacteroidales , have been used to assess possible fecal contamination in fresh produce (Denis et al, 2016; Drozd et al, 2013; Harris et al, 2017; Ordaz et al, 2019; Wang, Ranjbaran, & Verma, 2023).…”

Section: Introductionmentioning

confidence: 99%

“…While none of the previous works have focused on enabling LAMP testing for on-farm applications, our group made several efforts in this direction (Pascual-Garrigos et al, 2021; Wang, Ranjbaran, Ault, et al, 2023). We have shown that LAMP has the potential for on-farm NAAT and have established paper-based assays as a method that can partly decrease the number of steps for performing in-situ tests (Davidson et al, 2021; Pascual-Garrigos et al, 2021; Wang et al, 2021; Wang, Ranjbaran, Ault, et al, 2023). We also developed and tested a micro-drop generator as another effort to enhance user-friendliness (Ranjbaran et al, 2023).…”

Section: Introductionmentioning

confidence: 99%

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A portable, easy-to-use paper-based biosensor for rapid in-field detection of fecal contamination on fresh produce farms

Wang,

Kaur,

Kayabasi

et al. 2023

Preprint

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Laboratory-based nucleic acid amplification tests (NAATs) are highly sensitive and specific, but they require the transportation of samples to centralized testing facilities and have long turnaround times. During the Coronavirus Disease 2019 (COVID-19) pandemic, substantial advancement has been achieved with the development of paper-based point-of-care (POC) NAATs, offering features such as low cost, being easy to use, and providing rapid sample-to-answer times. Although most of the POC NAATs innovations target clinical settings, we have developed a portable, paper-based loop-mediated isothermal amplification (LAMP) testing platform for on-farm applications, capable of detectingBacteroidalesas a fecal contamination biomarker. Our integrated platform includes a drop generator, a heating and imaging unit, and paper-based biosensors, providing sensitive results (limit of detection 3 copies ofBacteroidalesper cm2) within an hour of sample collection. We evaluated this integrated platform on a commercial lettuce farm with a concordance of 100% when compared to lab-based tests. Our integrated paper-based LAMP testing platform holds great promise as a reliable and convenient tool for on-site NAATs. We expect that this innovation will encourage the fresh produce industry to adopt NAATs as a complementary tool for decision-making in growing and harvesting. We also hope that our work can stimulate further research in the development of on-farm diagnostic tools for other agricultural applications, leading to improved food safety and technology innovation.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%