Agronomically important thrips: development of species-specific primers in multiplex PCR and microarray assay using internal transcribed spacer 1 (ITS1) sequences for identification

Abstract

Thrips, the sole vector of plant Tospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.

A multiplex PCR assay for rapid identification of major tospovirus vectors reported in India

“…AG35F-AG36R, AG47F-AG48R, AG87F-AG88R, and AG79F-AG80R for T. palmi, S. dorsalis, T. tabaci, and F. schultzei used in the present investigation were not cross-reactive and used to carry out concurrent detection of thrips vectors by multiplex PCR. Multiplex PCR has been widely used by various researchers for rapid and simultaneous identification of thrips vectors [28,34,38,41]. However, the present study is unique in the sense that, this is the first effort in concurrent identification of all the major thrips vectors present in India.…”

“…The speed, reproducibility, and accuracy of molecular techniques have made them a valuable tool for identification of thrips vectors [13,[29][30][31][32][33][34][35][36]. The simultaneous identification of several species using multiplex PCR has become popular for its added advantages of saving time, money, and effort over other methods of diagnosis [37,38].…”

Rapid identification of the invasive fall armyworm Spodoptera frugiperda (Lepidoptera, Noctuidae) using species-specific primers in multiplex PCR

“…Among the PCR-based approaches for species identification, multiplex PCR using universal primers, namely 28S rDNA, has ensured the DNA quality of sampled individuals 35,36 , which was seldom used. In this study, a few specimens died and rotted rapidly during their delivery process to our laboratory.…”

“…Although the species-specific primers using mitochondrial genes are common in insects [37][38][39] , the more efficient nuclear ITS region with hypervariable sites, particularly the distinct inter-and intraspecific genetic variations of ITS sequences, is more suitable for developing species-specific primers for species identification 32,[34][35][36][40][41][42] . However, the application of species-specific primers based on ITS sequences in insect identification remains rare.…”

Prediction of potential barcoding sites on ITS1 by wavelet transform

“…A brief survey of available literature indicates the need to develop a more standardized design protocol. The emerging relevance of ITS1 in different biotechnological fields (Yeh, Tseng, Chang, Wu, & Tsai, 2014) encourages the development and application of more rational methods to design probes, especially in the case of a high compositional bias (Cross & Bird, 1995). Even though some cleavage sites, embedded in ITS1, have been experimentally identified, it is reasonable to consider the possibility that other recognition sites not yet validated are also be present.…”