Loop-Mediated Isothermal Amplification Combined with PCR for Rapid Identification of the Ethiopian Fruit Fly (Diptera: Tephritidae)

Abstract

Nowadays, with increasing trend of trans-boundary transportation of agricultural products and higher probability of introduction of many invasive species into new areas, fast and precise species diagnosis is of great significance particularly at the port of entry, where morphological identification often requires adult insect specimens especially with specialist insects. The cucumber fruit fly, Dacus ciliatus Loew (Diptera: Tephritidae), ranks as one of the most destructive agricultural pests attacking mainly fruits of Cucurbitaceae. This pest is also widespread and highly invasive; thus, it is a high priority for pest detection and quarantine programs. Although cucumber fruit fly adults can usually be identified and distinguished from the other species by morphological keys, it is often difficult or impossible to distinguish this species from the other tephritids that share host plants by using material from other stages of development. In such situations, using a quick and robust alternative species diagnostic tool would be valuable. In this study, we assessed a technique combining loop-mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for the rapid detection and discrimination of cucumber fruit fly DNA from some other common tephritid species attacking Cucurbitaceae, using material from different stages of development. The described method was species-specific and sensitive and provided a rapid diagnostic tool to detect D. ciliaus even by non-experts.

Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

“…Among the most versatile, sensitive and specific methods, loop-mediated isothermal amplification (LAMP) can be used as a fieldfriendly and cost-effective diagnostic tool (Notomi et al 2000(Notomi et al , 2015. Several LAMP tests have been used both in the field and in laboratories, in particular for human and animal diseases (Lucchi et al 2010), in food safety controls (Abdulmawjood et al 2014), as well as in identifying plant pathogens (Aglietti et al 2019;Luchi et al 2020;Blaser et al 2018) and invasive insect pests (Huang et al 2009;Hsieh et al 2012;Fekrat et al 2015;Przybylska et al 2015;Ide et al 2016a, b;Blaser et al 2018;Sabahi et al 2018;Rizzo et al 2020b). LAMP is a highly specific and robust identification method for species with previously known DNA or RNA sequences and suitable for on-site application because it can be performed in a laboratory-free environment after minimal training (Kogovšek et al 2015).…”

A rapid LAMP‐based colorimetric assay with quick DNA extraction for on‐site identification of Drosophila suzukii Matsumura

“…Regulated quarantine insect species Thrips palmi Karny (Thysanoptera: Thripidae) , Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) and several regulated fruit fly species in the genera Bactrocera and Zeugodacus as target pests for LAMP assay were identified successfully at POEs (Blaser et al, 2018). Some LAMP primers had been designed with success for Dacus ciliatus Loew (Diptera: Tephritidae) (Sabahi et al, 2018), Ze ugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) (Zhong et al., 2019), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) (Blacket et al., 2020) and Zeugodacus scutellatus Hendel (Diptera: Tephritidae) (Kitano & Takakura, 2020).…”

A LAMP assay for the detection of Bactrocera tryoni Queensland fruit fly (Diptera: Tephritidae)

“…1). This is the standard DNA barcoding gene, and has been employed in other recent fruit fly LAMP assays 5,15 . This region is highly variable, with enough resolution to differentiate between most insect species and consequently, has been sequenced from more fruit fly species than any other locus 4 .…”