Rapid and specific detection of Tilletia indica using loop-mediated isothermal DNA amplification

Abstract

Karnal bunt in wheat is caused by the fungus Tilletia indica. It is a quarantine disease, and therefore its timely and specific detection is important. Current detection protocols involve DNA amplification by polymerase chain reaction (PCR). However, this technique has encountered specificity issues due to the high DNA homology of T. indica with other Tilletia species, in particular T. walkeri. Here, we report the specific and rapid detection of T. indica DNA using the loop-mediated isothermal amplification (LAMP) at 62°C. Alignment of the mitochondrial DNA of T. indica and T. walkeri has revealed four major unique regions in T. indica. Six LAMP primers designed in one of these unique regions were able to amplify T. indica DNA. The amplification could be completed in 30 min and was nearly as sensitive as conventional PCR. The amplification was found to be highly specific to T. indica DNA during the screening of 17 isolates of T. indica, T. walkeri, T. horrida, T. ehrhartae and T. caries. The use of the fluorescent chemical calcein has enabled endpoint detection with the naked eye. This method, with its specificity, rapidity and simple visualization, is well suited for the detection of this important disease in wheat.

Characterization of SNP and Structural Variations in the Mitochondrial Genomes of Tilletia indica and Its Closely Related Species Formed Basis for a Simple Diagnostic Assay

“…These isolates are thus predicted to give false negative results for the T . indica LAMP assay by Gao et al [37]. This has been confirmed for T .…”

“…This has been confirmed for T . indica isolates; Ps2, Ps7, Ps9, Ps12, Ps14, Ps17, WL1562 and Ps23 (Fig 6) which gave negative results for the assay by Gao et al [37]. A false negative will result in serious, costly consequences for the industry.…”

“…indica [37] used a target sequence that lies in the element, PAV5 and had screened only a very small set of five T . indica isolates.…”

“…A false negative will result in serious, costly consequences for the industry. The LAMP assay developed by Gao et al [37] is thus likely to be unreliable and should not be used for quarantine diagnosis.…”

“…indica isolates were tested ‘negative’ as predicted by the absence of PAV5 (Fig 3). The positive results obtained for isolates Ps6, Ps21 and MKTi9 with assay by [37] suggested the presence of PAV5 in a few copies of mt DNA in these isolates.…”