Molecular identification of Trogoderma granarium (Coleoptera: Dermestidae) using the 16s gene

Olson, Rachel; Farris, Roxanne E.; Barr, Norman B.; Cognato, Anthony I.

doi:10.1007/s10340-014-0621-3

Cited by 21 publications

(30 citation statements)

References 34 publications

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“…The prepared strip was placed in the Genie III machine, used as an incubator, for DNA extraction: 65 °C for 6 min, followed by 2 min at 98 °C. 23 2.3 Development of Khapra beetle LAMP assay 2.3.1 Khapra LAMP primer design A species-specific LAMP assay for the detection of Khapra beetle was developed from existing reference DNA sequences of the 16S locus 17 by targeting primer regions with low intraspecific variation and high interspecific variability in an alignment of T. granarium sequences with the 13 most closely related species available. 17 Six novel LAMP primers were designed manually to target eight DNA regions in the present study, synthesized by Sigma (Australia).…”

Section: Dna Extractionmentioning

confidence: 99%

“…23 2.3 Development of Khapra beetle LAMP assay 2.3.1 Khapra LAMP primer design A species-specific LAMP assay for the detection of Khapra beetle was developed from existing reference DNA sequences of the 16S locus 17 by targeting primer regions with low intraspecific variation and high interspecific variability in an alignment of T. granarium sequences with the 13 most closely related species available. 17 Six novel LAMP primers were designed manually to target eight DNA regions in the present study, synthesized by Sigma (Australia). For all primers the GC content (%), predicted melting temperature (Tm), and potential secondary structure (hairpins or dimers) were analysed using the integrated DNA technologies (IDTs) online OligoAnalyzer tool (https://sg.idtdna.com/ calc/analyzer), using the quantitative polymerase chain reaction (qPCR) parameter sets.…”

Section: Dna Extractionmentioning

confidence: 99%

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A LAMP (loop‐mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium)

Rako

Agarwal

Semeraro

et al. 2021

Pest Management Science

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BACKGROUND: Khapra beetle (Trogoderma granarium Everts) is a significant pest of food products around the world, causing great losses of stored grain and produce, with export restrictions imposed on countries with established beetle populations. Khapra beetle is a high-priority exotic invertebrate pest in many countries requiring a rapid quarantine/biosecurity response when incursions occur. To address this, we developed a novel Khapra LAMP (loop-mediated isothermal amplification) assay using a portable real-time fluorometer and an additional 18S ribosomal DNA (18S) insect control LAMP assay for confirmation of the presence of insect DNA. Both LAMP tests can be performed either in a portable real-time fluorometer or using simple, visual colorimetric technique.RESULTS: Both the Khapra and 18S LAMP tests amplify positive samples within ≤ 25 min, with an anneal derivative temperature of 77.7 ± 0.7 °C for Khapra LAMP test and 88.0 ± 1.0 °C for 18S. The new Khapra LAMP assay is sensitive to very low levels of DNA (1.02 × 10 −6 ng ∼L −1 ). Additionally, we developed a gBlock double stranded DNA fragment for use as positive Khapra control with a different anneal derivative of 80 °C. Both assays are simple to use in the field and are capable of amplifying DNA from target beetles, even when samples are partially degraded which is typically found during surveillance activities. By screening a broad panel of Dermestidae species we demonstrate that our new assay is species-specific, with no detections of false positives. Also, we evaluated multiple DNA extraction methods, with both QuickExtract and HotSHOT extraction methods proving suitable for in-field use. CONCLUSION:The novel Khapra and 18S LAMP assays should improve speed, accuracy and confidence of detection of Khapra beetle at incursion points and aid rapid biosecurity responses in any country affected, especially as the assays described here are portable and easy to implement in the field conditions where resources are limited.

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Section: Dna Extractionmentioning

confidence: 99%

Section: Dna Extractionmentioning

confidence: 99%

A LAMP (loop‐mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium)

Rako

Agarwal

Semeraro

et al. 2021

Pest Management Science

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“…Primers were replaced with H 2 O as a negative control and Harp49 ( H. axyridis ribosomal protein 49 gene, GenBank Accession No. AB552923) was used as an endogenous control or other reference genes ( Olson et al, 2014 ). The primers were as follows: Harp49-qF (5′-GCG ATC GCT ATG GAA AAC TC-3′) and Harp49-qR (5′-TAC GAT TTT GCA TCA ACA GT-3′) ( Osanai-Futahashi et al, 2012 ; Shi et al, 2016 ).…”

Section: Methodsmentioning

confidence: 99%

Effects of starvation on the carbohydrate metabolism in Harmonia axyridis (Pallas)

Shi

Wang

et al. 2017

Biology Open

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Trehalose plays an important role in energy storage, metabolism, and protection from extreme environmental conditions in insects. Trehalose is the main blood sugar in insects, and it can be rapidly used as an energy source in times of need. To elucidate the mechanisms of the starvation response, we observed the effects of starvation on trehalose and glycogen, trehalase activity, and the relative gene expression of genes in the trehalose and glycogen metabolic pathways in the invasive beetle Harmonia axyridis. Our results show that trehalose levels and the activities of two types of trehalases decreased significantly in the first 8 h of starvation, while the relative expression of HaTreh1-1 increased. While trehalose remained nearly constant at a relatively high level from 8 to 24 h, glycogen levels decreased significantly from 8 h to 24 h of starvation. Likewise, glycogen phosphorylase (HaGP) expression was significantly higher at 12 to 24 h starvation than the first 8 h, while the expression of glycogen synthase (HaGS) was relatively stable. Furthermore, trehalose decreased significantly from 24 h starvation to 72 h starvation, while trehalase activities and the relative expression of some HaTreh genes generally increased toward the end of the starvation period. The expression of trehalose-6-phosphate synthase (HaTPS) increased significantly, supporting the increase in trehalose synthesis. These results show that trehalose plays a key role in the energy provided during the starvation process through the molecular and biochemical regulation of trehalose and glycogen metabolism.

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“…The visual and microscopic identification of khapra beetle larvae and adults is a difficult task due to the morphological similarity with other Trogoderma species. Trogoderma anthrenoides and Trogoderma variabile are not considered as seriously as Trogoderma granarium, but they are also major pests that occasionally cause damage to grains and other food substances [22].…”

Section: Identification Of Invertebratesmentioning

confidence: 99%

A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes

Abeynayake

Fiorito

Dinsdale

et al. 2021

Genes

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The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border.

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Molecular identification of Trogoderma granarium (Coleoptera: Dermestidae) using the 16s gene

Cited by 21 publications

References 34 publications

A LAMP (loop‐mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium)

A LAMP (loop‐mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium)

Effects of starvation on the carbohydrate metabolism in Harmonia axyridis (Pallas)

A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes

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