Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae)

Abstract

Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand’s borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

Resolving the confused identity of Frankliniella panamensis (Thysanoptera: Thripidae)

“…For DNA barcoding of the COI gene region from the thrips samples, LCO1490 and HCO2198 primers (Folmer et al 1994) or mtD-7.2F and mtD-9.2R (Brunner et al 2002) were used. For all the PCR reactions and sequencing were conducted as per Li et al (2015). The obtained DNA sequences were edited in Geneious Pro 10.0.6 (http:// www.geneious.com, Kearse et al 2012) and BLAST searched against the GenBank (Altschul et al 1990) and/or BOLD databases (Ratnasingham & Herbert 2007).…”

Real‐time PCR assays for rapid detection of Zeugodacus cucumis and Bactrocera jarvisi (Diptera: Tephritidae) for quarantine application

“…The amplicons were electrophoresed on 1.2% agarose (in 1× TAE buffer) gel stained with SYBR ® safe (Life Technologies™), and visualized using a Gel Doc Software system (Bio‐Rad, Hercules, CA, USA). Amplified products were sequenced bidirectionally using the amplification primers, and DNA sequences were analysed as previously described (Li et al, ; Li, Waite, Fan, et al, ; Li, Waite, Gunawardana, et al, ; Sooda, Gunawardana, Li, & Kumarasinghe, ). The DNA sequences were submitted to BOLD database under the project of “Barcode of Bactrocera Specimens” (BBS): BBS050‐18 to BBS055‐18 for Z. cucumis and BBS056‐18 to BBS060‐18 for B. jarvisi .…”

“…Plasmid DNA was extracted using the Wizard ® Plus SV Miniprep (Promega, Madison, WI, USA). The plasmid DNA was quantified using a µDrop plate in MultiSkan GO DNA quantification system (Thermo Scientific, USA) and normalized to a concentration of 10 7 copies/µl (Li et al, ; Li, Waite, Fan, et al, ; Li, Waite, Gunawardana, et al, ). A dilution series of the plasmid from 10 7 to 1 copies/µl was created in TE buffer containing 1 mM EDTA (Invitrogen).…”

“…The assays were tested in the singleplex and duplex formats, and with 18S ribosomal RNA (rRNA) gene internal control realtime PCR (Applied Biosystems, CA, USA). and normalized to a concentration of 10 7 copies/µl (Li et al, 2015;Li, Waite, Fan, et al, 2018;Li, Waite, Gunawardana, et al, 2018). A dilution series of the plasmid from 10 7 to 1 copies/µl was created in TE buffer containing 1 mM EDTA (Invitrogen).…”

Multiplex real‐time PCR assay for the detection of three invasive leafminer species: Liriomyza huidobrensis, L. sativae and L. trifolii (Diptera: Agromyzidae)

“…PCR cycling conditions were an initial denaturation step at 94 °C for 2 min followed by 5 cycles of denaturation step at 94 °C for 40 s, annealing step at 45 °C for 40 s and an elongation step at 72 °C for 60 s, which was followed by an additional 35 cycles of denaturation at 94 °C for 40 s, annealing at 51 °C for 40 s, and elongation at 72 °C for 60 s, followed by a final elongation step at 72 °C for 7 min. PCR products were analysed and sequenced as per Li et al (). The sequences were submitted into GenBank under the accession numbers KU244270–KU244272, KX3733669–KX373684; the details about the sequences are listed in Table .…”