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Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification

2 Jul 2008

Marcia Triunfol

Source: PubMed Central (see original article)

Natural disasters in warm climates are often followed by seafood- and water-borne outbreaks of diarrhoea. These are mostly caused by Cholera toxin (CT), the major virulence determinant of Vibrio cholerae. The rapid detection of CT-producing strains of V. cholerae is of major epidemiologic importance because it allows the rapid identification of infected areas. These areas can then be isolated and their populations treated more aggressively, to avoid the appearance of life-threatening diarrhoea.

The traditional methods for identifying CT-producing strains rely on culture-based assays that can take more than 3 days to generate useful information. A molecular diagnosis method that is based on PCR (either conventional PCR or real time) requires the use of expensive thermocyclers that might not be available in remote or poor areas.

In this paper, the authors test a technique named LAMP (Loop-mediated isothermal amplification) and show that LAMP is a faster and cheaper substitute for PCR detection of CT-producing V. cholerae strains in stool samples. The LAMP technique requires a single constant temperature and a simple water-bath, instead of an expensive thermocycler. Also, the test can be performed within 60 minutes, which is a major benefit since effective measures for outbreak containment depends on how fast the infectious agent is identified.

Basically, LAMP uses a set of 6 primers that align to specific and closely located areas in the target sequence. This generates a high number of copies of the target DNA, but it also produces high amounts of pyrophosphates, which is a by-product of this type of amplification reaction. To read the results, scientists look for the presence of these pyrophosphates in the reaction tube, which can be done by using simple colorimetric methods that apply metal indicators that change the colour of the solution according to its content. LAMP results can even be detected by the naked eye because the production of high number of copies of the target DNA turn an initially translucid reaction solution into a hazy mixture. Finally, the study shows that LAMP is more sensitive than PCR at detecting CT-producing strains in both pure cultures and in spiked human faeces.

Any technical breakthrough that improves diagnosing of human and animal diseases helps health agencies get a head start when outbreaks occur. This study shows a simpler, faster and cheaper technique for the identification of CT-producing bacteria. No doubt it is a technique worth testing in risk areas.