Development of a rapid PCR-Nucleic Acid Lateral Flow Immunoassay (PCR-NALFIA) based on rDNA IGS sequence analysis for the detection of Macrophomina phaseolina in soil

The M. phaseolina specific primers MP102F/MP102R, 5′ labelled with biotin and FITC respectively, were used in the PCR-NALFIA assay to identify the pathogen starting from mycelium or microsclerotia. Microsclerotia of M. phaseolina (1, 10, 100 and 200) were manipulated under a stereomicroscope and their DNA was extracted using microsclerotia alone or mixed with different types of soil. The resulting DNA, used for the PCR-NALFIA assay, provided positive results for all the samples tested. A semi-quantitative grey-scale reference card based on the PCR-NALFIA assay using intervals corresponding to microsclerotia soil number was developed. For this purpose, the normalized pixel grey volumes obtained after a densitometric analysis of the test line intensity generated by the LFD dipsticks were used.