Development and validation of a rapid field test to detect the chytrid fungus Batrachochytrium dendrobatidis at a high specificity and sensitivity, for use in surveys to determine the distribution of Chytridiomycosis

About the report

The purpose of this study was to develop and validate a rapid, accurate and user-friendly test to apply to environmental samples to identify potential pathogens, such as the chytrid fungus Batrachochytrium dendrobatidis, that may be present in low concentrations.

This waterborne fungus is highly pathogenic to some amphibians and has been identified as a significant cause of amphibian population declines and species extinctions [DEH 2006, Kirshtein et al. 2007, Skerratt et al. 2007]. Extensive research into the impacts of this fungus within Australia has lead to the organism being identified as a key process threatening our amphibian biodiversity [DEH 2006].

A deoxyribonucleic acid (DNA) amplification technology, known as Loop-Mediated Isothermal Amplification (LAMP) has been proposed to have many advantages over polymerase chain reaction (PCR) while retaining a high level of sensitivity. LAMP amplification occurs at a single temperature and therefore can be done in an easily transported heating block. LAMP technology uses a strand displacing DNA polymerase in combination with target specific primers to produce large quantities of target DNA. In so doing, pyrophosphate ions are released that react to form a white precipitate of magnesium pyrophosphate. Positive samples can then be identified with a turbidity meter or, potentially, with the naked eye [10]. LAMP technology has the added advantage in that it is not impacted by inhibitors the same way that the DNA polymerases used in PCR reactions are. Thus, relatively simple ways of releasing DNA (e. g., boiling) can be used to prepare samples for testing with LAMP.

In this present study we have developed a rapid test to detect the chytrid fungus B. dendrobatidis at a high specificity and sensitivity and have demonstrated that this test appears to be more sensitive at detecting the fungus in environmental water samples compared with conventional species-specific PCR techniques. The benefits of LAMP include that it is less prone to the presence of irrelevant DNA than PCR, it amplifies DNA with a high efficiency, is highly specific for the target sequence and is quick and easy to perform using only a heat block or a water bath (Notomi et al., 2000). Published claims that the LAMP test can be applied in the field as DNA products are visible with the naked eye were not repeatable within this study.

Additional studies are underway to further validate and optimise this LAMP test.