A Diagnostic Loop-Mediated Isothermal Amplification Method to Distinguish Helicoverpa armigera (Lepidoptera: Noctuidae) From Other Related Species in the New World

Abstract

Helicoverpa armigera (Hübner) is a notorious agricultural pest native to the Old World. Recently, its invasion into South and Central America has become a serious problem in the New World. The rapid detection of invasive pests is essential to eradicate them and prevent establishment. However, an extremely similar species, H. zea (Boddie) distributed in the New World makes identification difficult. Helicoverpa armigera and H. zea have only minor differences in male genitalia to separate them morphologically. Both species are attracted to the same pheromone lure, and it takes considerable time and effort to identify them from bulk samples obtained during trap monitoring. Although several molecular approaches based on PCR have been reported, these methods require expensive equipment and are unsuitable for onsite diagnostics. Here, we developed a rapid and convenient diagnostic method based on the loop-mediated isothermal amplification to distinguish H. armigera from related species: H. zea, H. assulta (Guenée), H. punctigera (Wallengren), and Chloridea virescens (Fabricius). The diagnostic method makes it possible to detect H. armigera within 90 min only using simple equipment. The method also worked with mixed DNA templates containing excess DNA from H. zea at the ratio of 1:999 (H. armigera:H. zea). This method can be an effective tool for onsite diagnostics during monitoring surveys for invasive H. armigera.

Keywords: Helicoverpa; loop-mediated isothermal amplification; molecular identification.

Fig. 1.

Universal primer PCR products separated in 2% agarose gel pres-tained with 0.01% GelRed. Lanes: DNA ladder marker (M); PCR products from Helicoverpa armigera (1), Helicoverpa zea (2), Helicoverpa assulta (3), Helicoverpa punctigera (4), Chloridea virescens (5), and the negative control (6).

Fig. 2.

The fluorescence observations of LAMP products obtained using different incubation times (30, 60, and 90 min). LAMP products from Helicoverpa armigera (1), Helicoverpa zea (2), and the negative control (3).

Fig. 3.

LAMP products obtained using different incubation times (30, 60, and 90 min) separated in 2% agarose gel pre-stained with 0.01% GelRed. Lane: DNA ladder marker (M); LAMP products from Helicoverpa armigera (1), Helicoverpa zea (2), and the negative control (3).

Fig. 4.

The fluorescence observation of LAMP products obtained from Helicoverpa armigera and four related species (a: under UV light, b: under room light). Each tube contains LAMP products from H. armigera (1), Helicoverpa zea (2), Helicoverpa assulta (3), Helicoverpa punctigera (4), Chloridea virescens (5), and the negative control (6).

Fig. 5.

The fluorescence observation of LAMP products obtained from mixed DNA templates. Each tube contains LAMP products amplified with DNA templates mixed at ratio of 1:1, 1:9, 1:99, 1:999, 1:0 (as a positive control), and 0:1 (as a negative control). Ratios of Helicoverpa armigera:Helicoverpa zea. Helicoverpa armigera was clearly detected as fluorescence at all ratios except in the negative control. Similar results were obtained in all 10 replications.

Fig. 6.

The specific primer of PCR products was separated in 2% agarose gel pre-stained with 0.01% GelRed. M, DNA ladder maker. Each ratio means PCR products amplified with DNA templates were mixed at ratio of 1:1, 1:9, 1:99, 1:999, 0:1 (as a negative control), and 1:0 (as a positive control). Ratios of Helicoverpa armigera:Helicoverpa zea. Helicoverpa armigera’s specific single bands of ≈150 bp were observed at all instances except in the negative control. Similar results were obtained in all 10 replications.