From laboratory to point of entry: development and implementation of a loop‐mediated isothermal amplification (LAMP)‐based genetic identification system to prevent introduction of quarantine insect species

Blaser, Simon; Diem, Hanspeter; Felten, Andreas von; Gueuning, Morgan; Andreou, Michael; Boonham, Neil; Tomlinson, J. A.; Müller, Pie; Utzinger, Jürg; Frey, Jürg E.; Bühlmann, Andreas

doi:10.1002/ps.4866

Cited by 64 publications

(94 citation statements)

References 38 publications

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“…The diagnostic specificity was therefore 100% for all the protocols we developed (simplex). The verification of the diagnostic specificity [ 21 , 33 ] was 100%, too. All target specimens were correctly identified using the specific tests and no false positives (within the reference cut-off values for qPCR assays) were obtained with non-target organisms, resulting in 100% diagnostic sensitivity and specificity for all the tests.…”

Section: Optimization Of the Probe-based Real-time Pcr Assay Conditiomentioning

confidence: 99%

A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis

Rizzo¹,

Lio

Bartolini³

et al. 2020

PLoS ONE

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The cultivation of walnuts ( Juglans sp.) in Europe retains high economic, social, and environmental value. The recent reporting of the Thousand Cankers Disease (TCD) fungus, Geosmithia morbida , and of its vector, Pityophthorus juglandis , in walnut trees in Italy is alarming the whole of Europe. Although Italy is at present the only foothold of the disease outside North America, given the difficulties inherent in traditional identification of both members of this beetle/fungus complex, a rapid and effective protocol for the early detection and identification of TCD organisms is an absolute priority for Europe. Here we report the development of an effective and sensitive molecular tool based on simplex/duplex qPCR assays for the rapid, accurate and highly specific detection of both the bionectriaceous fungal pathogen and its bark-beetle vector. Our assay performed excellently, detecting minute amounts of target DNA without any non-specific amplification. Detection limits from various and heterogeneous matrices were lower than other reported assays. Our molecular protocol could assist in TCD organism interception at entry points, territory monitoring for the early identification and eradication of outbreaks, delineation of quarantine areas, and tracing back TCD entry and dispersal pathways.

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Section: Optimization Of the Probe-based Real-time Pcr Assay Conditiomentioning

confidence: 99%

A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis

Rizzo¹,

Lio

Bartolini³

et al. 2020

PLoS ONE

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show abstract

“…15 Those advantages render the LAMP technique a broad perspective of many usages, especially in the rapid genetic on-site identification. 16 It has the most potential to be an important tool at points of entry such as sea-and airports. It could be used to prevent introduction and spread of economically harmful pest species that are unintentionally transported by the global trade.…”

Section: Discussionmentioning

confidence: 99%

Species identification of felis and vulpes by a novel loop-mediated isothermal amplification assay in fur products

et al. 2019

Journal of Engineered Fibers and Fabrics

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In the chaotic market of fur goods, genetic distinction is increasingly important for identifying species. A vast diversity of species identification methods has been proposed, while little is developed, particularly those easy, fast, and costeffective ones. In this study, a simple and reliable novel loop-mediated isothermal amplification method for identifying cytochrome c oxidase I of felis and vulpes was established. It saves laborious post-polymerase chain reaction procedures and shortens the time for high-fidelity gene amplification. The sensitivity of this method for felis and vulpes identification, which is well matched to quantitative polymerase chain reaction, could be 10 or 1.0 pg, respectively. Predominantly, the sensitivity of loop-mediated isothermal amplification is more tolerant to those polymerase chain reaction inhibitors such as pigments, dyes, or other fur ingredients, compared to quantitative polymerase chain reaction. Even without costly specialized equipment, a water bath is sufficient for genetic distinction. Our approach is a new technique with broad application perspective, such as on-site species identity tests, commercial fraud, and wildlife crimes.

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“…However, these molecular tools are expensive, time consuming, and depend on specialized equipment. A simpler technique, termed loop-mediated isothermal amplification assay (LAMP), is also widely used for the rapid identification of pest species (Blaser et al, 2018;Blaser., 2018;Hsieh et al, 2012;Choi et al, 2016). The LAMP assay was developed for rapid, simple, effective and specific amplification of DNA.…”

Section: Introductionmentioning

confidence: 99%

Development of a simple and accurate molecular tool forSpodoptera frugiperdaspecies identification using LAMP

Kim

Nam

Kwon

et al. 2020

Preprint

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The fall armyworm, Spodoptera frugiperda is a native species in the Americas. However, nowadays it is one of the most serious invasive lepidopteran pests in African and Asian countries. S. frugiperda has been spread very quickly after the first outbreak was reported in many countries. Based on mt genome sequence alignment, S. frugiperda specific sequence region was identified in tRNAs coding region between NADH dehydrogenase, ND3 and ND5.By using this unique region, species diagnostic primers were designed and applied in LAMP (lamp loop mediated isothermal amplification) assay as well as conventional PCR to identify the field-collected samples of S. frugiperda. Optimal incubation condition of LAMP assay was 61℃ for 90 minutes with 4 LAMP primers, and additional loop primer increased the amplification efficiency. Also, wide range of DNA concentration responded in LAMP assay and minimum detectable DNA concentration was 10 pg. This LAMP assay was also applied in DNA releasing technique from larval and adult sample, without DNA extraction, 95℃ incubation for five minutes of the tissue sample. This new molecular diagnostic method is easy to use and accurate. It possibly applied in intensive field monitoring of S. frugiperda and its ecological studies.

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From laboratory to point of entry: development and implementation of a loop‐mediated isothermal amplification (LAMP)‐based genetic identification system to prevent introduction of quarantine insect species

Cited by 64 publications

References 38 publications

A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis

A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis

Species identification of felis and vulpes by a novel loop-mediated isothermal amplification assay in fur products

Development of a simple and accurate molecular tool forSpodoptera frugiperdaspecies identification using LAMP

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