A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples

Abstract

Loop-mediated isothermal amplification (LAMP) method was designed for clinical diagnosis of Vibrio cholerae carrying the ctxA gene. The detection limits of the method were 5 fg of purified genomic DNA/reaction and 0.54 CFU/reaction. The method was applied to rectal swab samples from cholera patients and healthy volunteers (19 subjects each) and yielded the same results as the “gold standard” culture method, while the polymerase chain reaction-based method failed to detect V. cholerae in 8 of the positive samples. Direct application of this LAMP method without precultivation enabled the rapid detection of 5 asymptomatic carriers from rectal swabs of 21 household contacts of cholera patients. This LAMP method could be a sensitive, specific, inexpensive, and rapid detection tool for V. cholerae carrying the ctxA gene in the clinical laboratory and in the field.

Introduction

Toxigenic Vibrio cholerae strains belonging to the O1 and O139 serogroups cause cholera, an acute, diarrheal illness induced by cholera toxin (CT), in humans. The infection can be severe, causing profuse watery diarrhea and vomiting, potentially resulting in dehydration and death. Cholera is a highly communicable disease and has been categorized as an emerging and reemerging infection that threatens many developing countries (Satcher, 1995). Cases involving severe diarrhea are identified more readily than those that are mild or asymptomatic, but such cases should also be diagnosed quickly, because they can contribute to the dissemination of cholera infection (Kaper et al., 1995).

CT, encoded by the ctxAB genes, is the major virulence factor of V. cholerae. Toxigenic V. cholerae harbors lysogens of a filamentous bacteriophage designated CTXΦ, which carry the ctxAB genes, and the propagation of CTXΦ may be associated with the origin of novel strains of toxigenic V. cholerae from nontoxigenic progenitors (Faruque et al., 1998).

The current “gold standard” for identifying toxigenic V. cholerae O1 or O139 from clinical samples is the culture method, but it is laborious and time consuming. It takes several days to confirm and complete the identification of suspicious colonies by biochemical and serologic tests. The development of a rapid, sensitive, and cost-effective method is required, not only for the quick and appropriate treatment of cholera patients, but also for the prompt control of cholera outbreaks and prevention of the disease.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technique that relies on autocycling strand-displacement DNA synthesis performed by the Bst DNA polymerase large fragment (Tomita et al., 2008). In contrast to polymerase chain reaction (PCR), LAMP occurs continuously under isothermal conditions (60–64 °C) for about 60 min, which, in a positive sample, eventually produces a visible, white precipitate composed of magnesium pyrophosphate. The presence or absence of the white precipitate allows the easy assessment of samples with the naked eye (Mori et al., 2001, Tomita et al., 2008). The LAMP method amplifies DNA with high efficiency and without significant influence from coexisting nontargeted or contaminating DNA. The detection limit is a few copies of DNA, comparable to that of PCR (Hara-Kudo et al., 2005, Notomi et al., 2000).

Recently, Yamazaki et al. (2008) reported a LAMP method for detecting V. cholerae. These authors demonstrated the specificity of their method using bacterial cultures; however, they did not examine its sensitivity or its specificity using clinical specimens obtained from cholera patients. We report here another LAMP method for the sensitive and specific detection of toxigenic V. cholerae. Moreover, we performed a field study using our method that demonstrated that it reliably identified V. cholerae in clinical samples obtained during cholera outbreaks in Thailand.