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

Abstract

The spotted wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae), distributes in most soft‐skinned fruit areas in China, is an economically important pest of fresh cherries during Sino‐European trade and is considered a quarantine pest in A2 list by European and Mediterranean Plant Protection Organization. Loop‐mediated isothermal amplification (LAMP) is an accurate, quick and convenient molecular identification method, applied to distinguish D. suzukii from other fruit flies. This method can be used for inspection in the field, and at the points of entry (POEs), because its results can be detected with the naked eye due to colour changes. In our study, first, we reported a simple and fast LAMP colorimetric detection method for molecular identification of D. suzukii. We designed primer sets for D. suzukii based on mitochondrial cytochrome oxidase I (COI) sequences. The specificity and sensitivity of the primers were tested by using target and non‐target fruit flies in the family Drosophilidae and Tephritidae, usually intercepted during the Sino‐European cherry trade. Second, for detection in the field and at POEs, the adoption of a quick DNA extraction method could save the total time of LAMP identification to about 90 min. Taken together, this accurate, quick and convenient LAMP‐based colorimetric identification assay combined with a quick DNA extraction method could visually detect clearly with just one portable heating device, which will be useful for rapid on‐site identification and inspection for D. suzukii by the quarantine department.

Rapid detection of insect pest with CRISPR‐Cas13a‐Based Lateral Flow Strip: Using Locusta migratoria manilensis

“…Importantly, the results can be visualized with the naked eye without additional devices. When compared with the colorimetric method (Wang et al, 2021), this method is more intuitive and does not need to distinguish colour differences.…”

“…A rapid DNA extraction strategy for insects is required to enable simple and portable testing. For example, a sterile toothpick method was developed for the quick extraction of DNA from tephritid fruit flies and combined with LAMP, which is more impurity‐tolerant than PCR for DNA detection (Kitano & Takakura, 2020; Wang et al, 2021). Further studies combining isothermal amplification, rapid DNA extraction, and Cas13a‐LFD are needed to facilitate onsite pest detection in the field.…”

“…com] F I G U R E 5 Sensitivity test of PCR-CRISPR-Cas13 system by using different concentration of template DNA of L. migratoria manilensis (Lmi: L. migratoria manilensis; Cki: C. kiangsu; C: nuclease-free water as control). [Colour figure can be viewed at wileyonlinelibrary.com]& Takakura, 2020;Wang et al, 2021). Further studies combining isothermal amplification, rapid DNA extraction, and Cas13a-LFD are needed to facilitate onsite pest detection in the field.…”

Optimizing a rapid LAMP assay for discrimination of Drosophila suzukii (Diptera: Drosophilidae) from common drosophilids captured in monitoring traps from the Midwest, United States

“…An alternative to morphological-based identification are DNA-based tests, which have the potential to improve detection and reduce reliance on morphological training. In fact, a number of polymerase chain reaction (PCR)-based methods have been developed to identify D. suzukii : PCR restriction fragment length polymorphism (PCR-RFLP) (Kim et al 2014), high-resolution melt analysis, high-resolution melt real-time PCR (HRM-RTPCR) (Dhami and Kumarasinghe 2014), quantitative PCR(qPCR) (Renkema et al 2022), and loop-mediated isothermal amplification PCR (LAMP) (Kim et al 2016, Wang et al 2021). Although these approaches differ, each offers the benefits of high sensitivity and accuracy in target DNA detection.…”

“…Previous studies have demonstrated that the LAMP assay can successfully detect D. suzukii and discriminate D. suzukii from D. melanogaster (Kim et al 2016), as well as more distantly related tephritid flies (Wang et al 2021). For example, tephritid flies of economic importance to the Sino-European cherry trade have been screened to identify and help prevent international movement, including Bactrocera dorsalis (Hendel) and B. correcta (Bezzi) from China, Rhagoletis cerasi (Linnaeus) from Italy, R. pomonella (Walsh) from the United States, and Ceratitis capitata from Iran (Wang et al 2021). However, the ability of the LAMP assay to accurately discriminate between D. suzukii and closely related drosophilids native to the Midwestern United States, especially those that are commonly captured in D. suzukii monitoring traps, has yet to be tested.…”