Gilligan Todd M, Tembrock Luke R, Farris Roxanne E, Barr Norman B, van der Straten Marja J, van de Vossenberg Bart T L H, Metz-Verschure Eveline
USDA-APHIS-PPQ-Science & Technology, Identification Technology Program, Fort Collins, Colorado, United States of America.
Department of Biology, Colorado State University, Fort Collins, Colorado, United States of America.
PLoS One. 2015 Nov 11;10(11):e0142912. doi: 10.1371/journal.pone.0142912. eCollection 2015.
The Old World bollworm, Helicoverpa armigera (Hübner), and the corn earworm, H. zea (Boddie), are two of the most important agricultural pests in the world. Diagnosing these two species is difficult-adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP) analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion) to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2) amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae.
棉铃虫（Helicoverpa armigera (Hübner)）和玉米穗虫（H. zea (Boddie)）是世界上最重要的两种农业害虫。区分这两个物种很困难——成虫只能通过复杂的解剖来区分，而幼虫无法通过形态学鉴定到物种，因此在大多数情况下需要根据地理来源进行鉴定。随着在新大陆发现棉铃虫，基于来源鉴定未成熟的棉铃虫属已不再可行，因为在发现棉铃虫的所有地理区域也都有玉米穗虫。已在出版物中报道了DNA条形码和限制性片段长度多态性（RFLP）分析来区分这些物种，但这些方法都需要PCR后处理（即DNA测序或限制性消化）才能完成。我们报告了第一种基于两种水解探针的实时PCR检测方法，这两种探针与使用单一引物对扩增的内部转录间隔区2（ITS2）的一段结合。一种探针靶向棉铃虫，第二种探针靶向玉米穗虫，第三种靶向18S rDNA保守片段的探针用作DNA质量的对照。使用分离的DNA时，该检测可在50分钟内完成，并已在入境口岸截获的幼虫和国内调查中捕获的成虫上成功测试。我们证明该检测可以三重进行，对灵敏度没有负面影响，可以使用替代的实时PCR试剂和仪器进行，并且不会与其他新大陆夜蛾科昆虫发生交叉反应。
