A loop-mediated isothermal amplification (LAMP) assay for the consensus detection of human pathogenic Campylobacter species

Highlights

• LAMP is widely used for the detection of thermotolerant Campylobacter species

• Select thermosensitive Campylobacter species are emerging human pathogens

• LAMP was developed for detecting both thermo-tolerant and sensitive Campylobacter

• This method uses naked-eye observation and does not require expensive instruments

• This method is sensitive and was tested in spiked and aged spinach leaves.

Abstract

Most rapid identification methods for Campylobacter are designed to detect thermotolerant Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli). A growing number of thermosensitive Campylobacter species are now gaining recognition as emerging human pathogens. Methods are lacking for the rapid screening of these emerging species. Loop-mediated Isothermal Amplification (LAMP) is a nucleic acid amplification method that allows for the rapid and cost-effective detection of bacteria. Degenerate primers against the 16S rRNA sequences for C. jejuni, C. coli, C. lari, C. upsaliensis, C. ureolyticus, C. fetus, C. gracilis, C. rectus, and C. concisus were designed. Isothermal amplification was conducted using ATCC reference strains at 68 °C for 30 min using WarmStart® Colorimetric LAMP reagents. Positive reactions were indicated by a color change from pink to yellow; specificity to Campylobacter was confirmed using a restriction enzyme digest (RsaI). The developed LAMP reaction was specific for the reference strains, which was confirmed against an exclusivity panel that consisted of other enteric pathogens, including E. coli, Salmonella, Shigella, Helicobacter, and Arcobacter. This method was also evaluated for the detection of C. jejuni, C. coli, and C. lari in primary enrichment media from artificially contaminated fresh spinach samples. The LAMP method provides an option to rapidly screen for the presence of pathogenic Campylobacter spp. in field surveillance and trace-back analysis.

Introduction

Among the foodborne bacterial pathogens, Campylobacter species are the leading cause of human gastroenteritis. The annual incidence of foodborne campylobacteriosis has increased world-wide, with 19.5 and 64.9 cases per 100,000 population in the US [Interagency Food Safety Analytics Collaboration (IFSAC), 2018; (Tack et al., 2019)] and the EU/EEA [European Union/European Economic Area; (European Centre for Disease Prevention and Control (ECDC), 2019)], respectively. Further, the widespread prevalence of Campylobacter in low- and middle-income countries, and the substantial risk to human health cannot be underscored enough (Delahoy et al., 2018). Campylobacteriosis is usually sporadic and self-limiting, however antimicrobial treatment is recommended when patients are infants, immunocompromised or have other co-morbidities. The infectious dose of Campylobacter, as determined by human volunteer studies, can be as low as 500 bacteria (Glass et al., 1983). Campylobacter species colonize the mucosal surfaces of the gastrointestinal tracts of humans and a wide variety of wild and domesticated birds and mammals, including food animals. They have also been isolated from contaminated soil, surface water, and groundwater (Lastovica et al., 2014). For US outbreaks from 1998 through 2017; raw milk, poultry, and seafood were the most frequently attributed sources of foodborne human Campylobacteriosis. Further, vegetable row crops and seeded vegetables are also food categories that have been linked to Campylobacter illness (Interagency Food Safety Analytics Collaboration (IFSAC), 2018).

To date, the genus Campylobacter consists of 32 species and 9 subspecies (Costa and Iraola, 2019) and most of the human infections are attributed to Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli). However, human Campylobacteriosis may also be caused by the “emerging Campylobacter spp.,” which include C. concisus, C. lari, C. upsaliensis and C. ureolyticus (Kaakoush et al., 2015; Man, 2011), as well as other species that are frequently associated with clinical presentations including gastroenteritis and/or bacteremia (Costa and Iraola, 2019).

The increased trend in the detection of Campylobacter species in clinical samples is partially due to the use of rapid diagnostics—either nucleic acid or protein (matrix-assisted laser desorption/ionization time-of-flight)-based tests (Hsieh et al., 2018; Tack et al., 2019), as well as to the clinical recognition of the importance of emerging Campylobacter species. However, a key disadvantage of these methods is the requirement of a non-portable and expensive laboratory setup. Hence, the development of rapid, reliable, on-site detection protocols with minimal laboratory handling and training of personnel is pivotal for pathogen detection and subsequent prevention or reduction of foodborne outbreaks and illness. These rapid methods could be used for testing food products, environmental monitoring, or at the point-of-field applications. Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test, which has been developed and optimized for microbiology diagnostics and the rapid detection of various bacterial, fungal, parasitic, and viral agents (Li et al., 2017; Niessen et al., 2013; Notomi et al., 2015). LAMP is highly specific as this method uses an efficient DNA polymerase (Bst DNA polymerase) and six primers that recognize eight distinct DNA regions. Because of the isothermal nature and robustness against inhibitors, the LAMP assay can be performed in a simple and rapid manner in a laboratory-free environment (Notomi et al., 2000; Tanner and Evans, 2014). Furthermore, detection can be simplified using visual observation of color change within 30–60 min (Hara-Kudo et al., 2005; Tanner et al., 2015). A few LAMP-based assays for the detection of Campylobacter have been developed in recent years. However, they are all specific for the detection of the frequently-studied thermotolerant Campylobacter species, namely C. jejuni, C. coli, C. lari, and have targeted either the 16S rRNA gene or used conventional gene and species-specific targets (Romero and Cook, 2018; Yamazaki, 2013).

To increase the range of available tests for the rapid diagnosis of human pathogenic Campylobacter species, we designed a LAMP assay that incorporates degenerate 16S rRNA gene LAMP primers against pure cultures and evaluated with spiked spinach leaves—a matrix representing ready-to-eat fresh leafy greens. This is the first reported LAMP assay to use degenerate primers to achieve consensus detection of Campylobacter species.