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

Abstract

The ‘Nucleic Acid Lateral Flow Immunoassay’ (NALFIA) using a generic ‘Lateral Flow Device’ (LFD), combined with PCR employing labelled primers (PCR-NALFIA), enables to circumvent the use of electrophoresis, making the diagnostic procedure more rapid and easier. If the specific amplicon is present in the sample, a coloured band, with an intensity proportional to the amplicon concentration, will develop on the LFD strip in addition to the control band.Species-specific primers for M. phaseolina based on the rDNA intergenic spacer (IGS) were developed and their specificity was checked and confirmed using 20 isolates of M. phaseolina and other 16 non-target fungi.A DNA extraction protocol based on a bead-beating technique using silica beads, skimmed milk and PVP was also developed.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.