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Method for detecting Macrophomina phaseolina

Molecular diagnosisNALFIA assayPCRPhytopathogenic fungiPlant diseases

Introduction

The invention relates to a molecular diagnostic method for the detection of the phytopathogenic fungus Macrophomina phaseolina in infected soil, seeds and plants samples. M. phaseolina is a fungal pathogen that has a global distribution and can infect over 500 plant species. The symptoms caused by the fungus are often very similar to those caused by other pathogens and to confirm its presence, a laboratory diagnosis consisting of conventional culture-based morphological techniques is needed. These assays are time-consuming, not very sensitive and labor-intensive. Therefore, the early diagnosis of M. phaseolina requires a simple, rapid, sensitive and inexpensive test to be performed directly on infected biological samples.

Technical features

The invention involves the use of a species-specific primers pair for the phytopathogenic fungus Macrophomina phaseolina to detect its presence, by a PCR reaction, in infected soil, seeds and plants samples. The patented primers, labelled at 5′ end with biotin and FITC, amplify a target fungus DNA portion by PCR and these amplified portions, amplicons, are identified by NALFIA assay with a simple immunochromatographic stick.
The assay is feasible to be directly performed outside on infected plant or soil samples thanks to a small portable laboratory of physiopathologic diagnosis, able to execute DNA assays in a sensitive, rapid and cheap way (LAB IN A BAG). This technique was recently adopted for the early stages of diagnostic kit development, validated by means of samples of laboratory and of field.

Possible Applications

  • Diagnostic field: public and private diagnostic laboratories and the laboratories of the Phytosanitary Inspection Services which process a very large number of samples;
  • Human clinical pathology laboratories as the fungus is an opportunistic human pathogen too;
  • Companies that produce diagnostic kits.

Advantages

  • Simple, rapid, and cheap diagnostic technique exportable outside the research laboratories, as it does not require the use of sophisticated equipment;
  • The NALFIA assay reduces the detection time of the amplicons maintaining a high sensitivity of the test.