A Multiplex Real-Time Polymerase Chain Reaction Assay to Diagnose Epiphyas postvittana (Lepidoptera: Tortricidae)

Abstract

A molecular assay for diagnosis of light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), in North America is reported. The assay multiplexes two TaqMan real-time polymerase chain reaction (RT-PCR) probe systems that are designed to target DNA segments of the internal transcribed spacer region 2 (ITS2) and 18S rRNA gene. The RT-PCR probe designed for the 18S target recognizes a DNA sequence conserved in all of the moths included in the study and functions as a control in the assay. The second probe recognizes a segment of the ITS2 specifically found in E. postvittana and not found in the other moths included in the study, i.e., this segment is not conserved. Inclusion of the two markers in a single multiplex reaction did not affect assay performance. The assay was tested against 637 moths representing > 90 taxa in 15 tribes in all three subfamilies in the Tortricidae. The assay generated no false negatives based on analysis of 355 E. postvittana collected from California, Hawaii, England, New Zealand, and Australia. Analysis of a data set including 282 moths representing 41 genera generated no false positives. Only three inconclusive results were generated from the 637 samples. Spike experiments demonstrated that DNA contamination in the assay can affect samples differently. Contaminated samples analyzed with the ITS2 RT-PCR assay and DNA barcode methodology by using the cytochrome oxidase I gene can generate contradictory diagnoses.

A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World

“…Following Barr et al [ 22 , 27 ], the ITS2 locus was selected as a potential diagnostic marker and the 18S rDNA locus was selected as a control. Conventional PCR was used to test the Barr et al [ 22 ] 18S rDNA real-time PCR primers on four samples of H . armigera , four samples of H .…”

“…armigera , five samples of H . zea , and two Heliothis samples were used to sequence a region of 5.8S-ITS2-28S using the ITSF/ITSR primers [ 22 , 27 ], and sequences generated using these primers were loaded into Geneious. Primer3 [ 28 ] was used to design internal primers to amplify a smaller region of the ITS2 locus that would maximize differences between H .…”

“…zea probe was labeled with HEX (both with BHQ-1 as quencher). The 18S rDNA control probe was designed identical to that of Barr et al [ 22 ] with the exception of Quasar 670 (= Cy5; BHQ-2 as quencher) replacing CAL Red 610 for greater compatibility with real-time PCR machines that use the Red 610 channel for calibration. All probes were HPLC purified.…”

“…In addition, real-time PCR eliminates the need to process and sequence the final PCR product, the lengthiest step in DNA barcoding. We have previously developed a real-time PCR assay to diagnose economically important Tortricidae using the internal transcribed spacer region 2 (ITS2) locus as a diagnostic marker and the 18S rDNA locus as an internal control [ 22 ]. Here we apply a similar method to develop a real-time PCR assay for diagnosing and separating H .…”

Integrated management of tortricid pests of tree fruit

“…DNA barcoding via polymerase chain reaction (PCR), quantitative real-time PCR (qRT-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) amplification of mitochondrial cytochrome oxidase I (mCOI) have been utilized for this purpose (Barcenas et al 2009;Chen and Dorn 2009;Kwon et al 2018). qRT-PCR assay of ribosomal RNA has also been used for diagnostic identification of LBAM in North America (Barr et al 2011). Simple sequence repeat microsatellites are rapidly evolving genetic loci that are selectively neutral and vary in number of repeated units.…”