A HRM Real-Time PCR Assay for Rapid and Specific Identification of the Emerging Pest Spotted-Wing Drosophila (Drosophila suzukii)

Dhami, Manpreet K.; Kumarasinghe, Lalith

doi:10.1371/journal.pone.0098934

Cited by 29 publications

(27 citation statements)

References 32 publications

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“…The efficiency of amplification of all primer pairs ranged from 95-105% and r 2 >0.99, which is in accordance with established criteria for real-time RT-PCR (Bio-Rad Laboratories, 2006; Dhami and Kumarasinghe, 2014;Ma et al, 2013). In order to determine the most stable reference genes, expression levels of transcripts from these six reference genes were determined in Huay Bong 60 and Hanatee, which are significantly different in CN (Whankaew et al, 2011) at 6, 9 and 12 MAP.…”

Section: Validation Of Reference Gene Transcriptssupporting

confidence: 68%

Validation of a reference gene for transcript analysis in cassava (Manihot esculenta Crantz) and its application in analysis of linamarase and -hydroxynitrile lyase expression at different growth stages

Whankaew¹,

Sraphet²,

Thaikert³

et al. 2015

Afr. J. Biotechnol.

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Relative real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a wellestablished method for the precise quantification of gene expression. For accurate relative real-time RTqPCR analysis, validation of the expression of an appropriate reference gene is required. In this study, the expression of six commonly used reference genes, namely 40S ribosomal protein (40S), actin (ACT), cyclophilin C (CYCC), EF-1 alpha (EF1), TATA box binding protein (TBP) and polyubiquitin (UBI) was investigated in leaf and root samples of cassava obtained at 6, 9 and 12 months after planting (MAP). A transcript stability analysis was undertaken in two different varieties of cassava, namely Huay Bong 60 which has high cyanogenic potential (CN) and Hanatee which has low CN. The results reveal that TBP was the most stable reference gene for expression studies. This information was applied to an analysis of linamarase and α-hydroxynitrile lyase gene expression in samples from six low and six high CN cassava plants collected at 6, 9 and 12 MAP. The results indicate that at 6 MAP, the linamarase transcript from leaf of the high CN group was significantly increased, and the α-hydroxynitrile lyase transcript was significantly increased at 12 MAP.

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Section: Validation Of Reference Gene Transcriptssupporting

confidence: 68%

Validation of a reference gene for transcript analysis in cassava (Manihot esculenta Crantz) and its application in analysis of linamarase and -hydroxynitrile lyase expression at different growth stages

Whankaew¹,

Sraphet²,

Thaikert³

et al. 2015

Afr. J. Biotechnol.

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“…There are a number of assays one can employ for SNP genotyping to facilitate species identification, e.g. TaqMan real-time PCR (Dhami et al 2016;Zhang et al 2016;Linck et al 2017), High-Resolution Melt (HRM) real-time PCR (Dhami et al 2014;Ajamma et al 2016), species-specific PCR (Sint et al 2016), KASP genotyping (Middlesex, UK), and SNP microarrays. We chose to adopt the Agena MassARRAY platform in combination with iPLEX chemistry (Gabriel et al 2009) to maximize the number of SNP markers we can multiplex in a single assay to reduce false positive rate and increase rigor of species identification.…”

Section: Discussionmentioning

confidence: 99%

Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification

et al. 2019

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Tuta absoluta is one of the most devastating pests of fresh market and processing tomatoes. Native to South America, its detection was confined to that continent until 2006 when it was identified in Spain. It has now spread to almost every continent, threatening countries whose economies rely heavily on tomatoes. This insect causes damage to all developmental stages of its host plant, leading to crop losses as high as 80 to 100%. Although T. absoluta has yet to be found in the U.S. and China, which makes up a large portion of the tomato production in the world, computer models project a high likelihood of invasion. To halt the continued spread of T. absoluta and limit economic loss associated with tomato supply chain, it is necessary to develop accurate and efficient methods to identify T. absoluta and strengthen surveillance programs. Current identification of T. absoluta relies on examination of morphology and assessment of host plant damage, which are difficult to differentiate from that of native tomato pests. To address this need, we sequenced the genomes of T. absoluta and two closely related Gelechiidae, Keiferia lycopersicella, and Phthorimaea operculella, and developed a bioinformatic pipeline to design a panel of 21 SNP markers for species identification. The accuracy of the SNP panel was validated in a multiplex format using the iPLEX chemistry of Agena MassARRAY system. Finally, the new T. absoluta genomic resources we generated can be leveraged to study T. absoluta biology and develop species-specific management strategies. Key Message  Surveillance and identification of Tuta absoluta are challenging because it is morphologically similar to closely related species, e.g. Keiferia lycopersicella and Phthorimaea operculella.  We generated new genomic sequences for these three species and identified single nucleotide polymorphisms (SNPs) to facilitate species identification.  We validated a multiplex genotyping panel of 21 SNPs using the iPLEX MassARRAY platform and confirmed its accuracy for species identification.  We generated new molecular tools and genome resources to aid Tuta absoluta management. leaves, where the larvae will emerge from eggs and begin mining host tissues. Larvae can also enter the stems through the buds, and feed within the tomato fruit, leaving them unmarketable. Its distribution was largely confined to South America until it was first detected in Spain in 2006 (Desneux et al. 2010; Guillemaud et al. 2015). Since then T. absoluta has spread rapidly and is now

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“…PCR cycling with a 2 min initial denaturation at 94°C, was followed by 30 cycles of 15 s at 94°C, 30 s at 52°C and 45 s at 72°C, followed by a final extension step of 7 min at 72°C. The approximately 700 bp amplicons were visualized via gel electrophoreses as described in Dhami and Kumarasinghe ( 2014 ). PCR products were sequenced bi-directionally using the amplification primers, by EcoGene (Auckland, New Zealand).…”

Section: Methodsmentioning

confidence: 99%

Real-Time PCR Assay for the Identification of the Brown Marmorated Stink Bug (Halyomorpha halys)

Dhami

Dsouza

Waite

et al. 2016

Front. Mol. Biosci.

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The brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), is a gregarious crop pest that has rapidly spread across the world in the last two decades. It is an excellent hitchhiker species, especially as an over-wintering adult. During this period it is often associated with non-biological commodities such as shipping containers and machinery that travel long distances. Inadequate identification keys and similarity to common species has assisted its spread across Europe, while accurate identification from immature stages or eggs is not possible. We developed a real-time TaqMan PCR assay for the accurate and sensitive detection of the brown marmorated stink bug from all life stages. The assay performance against required diagnostic criterion and within a quarantine framework are described.

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A HRM Real-Time PCR Assay for Rapid and Specific Identification of the Emerging Pest Spotted-Wing Drosophila (Drosophila suzukii)

Cited by 29 publications

References 32 publications

Validation of a reference gene for transcript analysis in cassava (Manihot esculenta Crantz) and its application in analysis of linamarase and -hydroxynitrile lyase expression at different growth stages

Validation of a reference gene for transcript analysis in cassava (Manihot esculenta Crantz) and its application in analysis of linamarase and -hydroxynitrile lyase expression at different growth stages

Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification

Real-Time PCR Assay for the Identification of the Brown Marmorated Stink Bug (Halyomorpha halys)

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