Real-time PCR assay for distinguishing<i>Frankliniella occidentalis</i>and<i>Thrips palmi</i>Arnika Przybylska, Żaneta Fiedler, Aleksandra Obrępalska-Stęplowska

Przybylska, Arnika; Fiedler, Z; Frąckowiak, Patryk; Obrępalska‐Stęplowska, Aleksandra

doi:10.1017/s0007485317000177

Cited by 7 publications

(9 citation statements)

References 37 publications

(47 reference statements)

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“…The RPA assay reported here was sensitive enough to amplify as little as 0.2 ag of template DNA, 10 9 -fold more sensitive than PCR using the same primers. The previously reported sensitivity of real-time PCR for discrimination of T. palmi was 1 pg (Przybylska et al 2017 ). The detection threshold for LAMP assay was 2 × 10 –10 parts of an adult T. palmi (Przybylska et al 2015 ), whereas the RPA assay could detect as low as 2.9 × 10 −13 parts of an adult considering 700 ng total DNA extracted from a single adult T. palmi .…”

Section: Discussionmentioning

confidence: 97%

“…No change in color with DNA of thrips collected from chilli, onion, tomato, and grape plants confirmed that different thrips species were present between the small-subunit ribosomal RNA (rRNA) and large-subunit rRNA genes. This hypervariable region has been preferred by several workers for discrimination of thrips at the species level because of a larger interspecific genetic distance than mtCoI (Glover et al 2010;Przybylska et al 2015Przybylska et al , 2017Ghosh et al 2020). Recent reports of several variants of mtCoI in T. palmi (Iftikhar et al 2016;Tyagi et al 2017;Ghosh et al 2020) were another reason to choose ITS2 over mtCoI in designing the RPA primers.…”

Section: Discussionmentioning

confidence: 99%

“…Rapid identification of T. palmi is crucial in pest and disease management and quarantine. Several methods have been reported to date for the molecular identification of T. palmi including PCR, RFLP, SCAR marker, multiplex PCR, real-time PCR, LAMP, and monoclonal antibodies (Paran and Michelmore 1993 ; Banks et al 1998 ; Walsh et al 2005 ; Yeh et al 2014 ; Nakahara and Minoura 2015 ; Przybylska et al 2015 , 2017 ; Jangra et al 2020 b). Most of these techniques cannot be done in a farmer’s field without access to a laboratory.…”

Section: Discussionmentioning

confidence: 99%

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A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses

et al. 2020

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Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, ornamentals, and legumes worldwide. Besides damage caused by feeding, it transmits several tospoviruses. Identification of T. palmi at an early stage is crucial in implementing appropriate pest management strategies. Morpho-taxonomic identification of T. palmi based on the adult stage is time-consuming and needs taxonomic expertise. Here, we report a rapid, on-site, field-based assay for identification of T. palmi based on recombinase polymerase amplification (RPA), its first application in insects. RPA primers designed based on 3′ polymorphisms of the Internal Transcribed Spacer 2 region efficiently discriminated T. palmi without any cross-reactivity to other predominant thrips species. RPA was performed with crude DNA, extracted from single T. palmi simply by crushing in sterile distilled water and could be completed within 20 min by holding the reaction tubes in the hand. The assay was further simplified by using fluorescent as well as colorimetric dyes thus eliminating the gel-electrophoresis steps. The presence of T. palmi was visualized by a change in color from dark blue to sky blue. The assay was validated with known thrips specimens and found to be effective in diagnosing the presence of T. palmi in natural vegetation. This on-site, rapid assay for diagnosis of T. palmi can be used by non-expert personnel in the field of quarantine and pest management. Keywords RPA • Melon thrips • Insect vector • Tospovirus transmission • Thrips diagnostics Key message • Thrips palmi an important pest of vegetables and ornamentals transmitting several tospoviruses • Morpho-taxonomic identification stage-specific, timeconsuming, and needs taxonomic expertise • Rapid, on-site, field-based assay for identification of T. palmi using recombinase polymerase amplification • Assay uses crude extract of T. palmi, completed within 20 min by holding the reaction tubes in hand without use of sophisticated laboratory instruments • The presence of T. palmi visualized by a change in reaction color • Useful for non-expert personnel in field-based identification, quarantine, and adopting suitable pest management strategies

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses

et al. 2020

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“…Morphometric key-based identification of thrips species is timetaking, labor-intensive, requires expertise, and dependent on developmental stage as only adults can be identified. 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].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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Background: To date, four thrips vectors have been reported to transmit five different tospoviruses in India. Their identification at an early stage is crucial in formulating appropriate pest management strategies. Since morphometric key-based thrips identification based on the adult stage is time-consuming, there is a need to develop diagnostic tools which are rapid, accurate, and independent of developmental stages. Here, we report a multiplex PCR assay to identify four major thrips vectors viz. Thrips palmi, T. tabaci, Scirtothrips dorsalis, and Frankliniella schultzei present in India. Results: Cytochrome oxidase subunit III and internal transcribed spacer region 2 were utilized to design speciesspecific primers. Of 38 pairs of primers tested, primer pairs AG35F-AG36R, AG47F-AG48R, AG87F-AG88R, and AG79F-AG80R amplified 568 bp, 713 bp, 388 bp, and 200 bp products from the DNA templates of T. palmi, S. dorsalis, T. tabaci, and F. schultzei, respectively at same PCR conditions. The specificity of the primer pairs was validated with a large number of known specimens and no cross-reactivity was observed with other thrips species. The multiplex PCR assay with a cocktail of all the four primer pairs detected four thrips vectors efficiently and could discriminate all of them concurrently in a single reaction. Conclusion: The multiplex PCR reported in this study could identify the major thrips vectors reported in India. The assay will be useful in ascertaining distribution profile of major thrips vectors, disease epidemiology, screening large samples, and quarantine.

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“…The efficiency of this qPCR is 95%, and the sensitivity is 0.1 pg µL −1 . Przybylska and colleagues [ 184 ] have developed a duplex qPCR based on the 5.8S ITS2 ribosomal region to discriminate between T. palmi and F. occidentalis . This sensitive assay can detect 1 pg of template DNA.…”

Section: Pcr-based Identification Of Thrips Using Molecular Markersmentioning

confidence: 99%

Frontiers Approaches to the Diagnosis of Thrips (Thysanoptera): How Effective Are the Molecular and Electronic Detection Platforms?

Ghosh

Jangra

Dietzgen

et al. 2021

Insects

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Thrips are insect pests of economically important agricultural, horticultural, and forest crops. They cause damage by sucking plant sap and by transmitting several tospoviruses, ilarviruses, carmoviruses, sobemoviruses, and machlomoviruses. Accurate and timely identification is the key to successful management of thrips species. However, their small size, cryptic nature, presence of color and reproductive morphs, and intraspecies genetic variability make the identification of thrips species challenging. The use of molecular and electronic detection platforms has made thrips identification rapid, precise, sensitive, high throughput, and independent of developmental stages. Multi-locus phylogeny based on mitochondrial, nuclear, and other markers has resolved ambiguities in morphologically indistinguishable thrips species. Microsatellite, RFLP, RAPD, AFLP, and CAPS markers have helped to explain population structure, gene flow, and intraspecies heterogeneity. Recent techniques such as LAMP and RPA have been employed for sensitive and on-site identification of thrips. Artificial neural networks and high throughput diagnostics facilitate automated identification. This review also discusses the potential of pyrosequencing, microarrays, high throughput sequencing, and electronic sensors in delimiting thrips species.

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Real-time PCR assay for distinguishingFrankliniella occidentalisandThrips palmiArnika Przybylska, Żaneta Fiedler, Aleksandra Obrępalska-Stęplowska

Cited by 7 publications

References 37 publications

A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses

A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses

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

Frontiers Approaches to the Diagnosis of Thrips (Thysanoptera): How Effective Are the Molecular and Electronic Detection Platforms?

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