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. 2022 Nov 30;11(23):3314.
doi: 10.3390/plants11233314.

Quantitative Real-Time PCR Based on SYBR Green Technology for the Identification of Philaenus italosignus Drosopoulos & Remane (Hemiptera Aphrophoridae)

Domenico Rizzo  1 , Matteo Bracalini  2 , Sara Campigli  2 , Anita Nencioni  2 , Francesco Porcelli  3 , Guido Marchi  2 , Daniele Da Lio  4 , Linda Bartolini  1 , Elisabetta Rossi  4 , Patrizia Sacchetti  2 , Tiziana Panzavolta  2
Affiliations

Quantitative Real-Time PCR Based on SYBR Green Technology for the Identification of Philaenus italosignus Drosopoulos & Remane (Hemiptera Aphrophoridae)

Domenico Rizzo et al. Plants (Basel). .

Abstract

The use of molecular tools to identify insect pests is a critical issue, especially when rapid and reliable tests are required. We proposed a protocol based on qPCR with SYBR Green technology to identify Philaenus italosignus (Hemiptera, Aphrophoridae). The species is one of the three spittlebugs able to transmit Xylella fastidiosa subsp. pauca ST53 in Italy, together with Philaenus spumarius and Neophilaenus campestris. Although less common than the other two species, its identification is key to verifying which role it can play when locally abundant. The proposed assay shows analytical specificity being inclusive with different populations of the target species and exclusive with non-target taxa, either taxonomically related or not. Moreover, it shows analytical sensibility, repeatability, and reproducibility, resulting in an excellent candidate for an official diagnostic method. The molecular test can discriminate P. italosignus from all non-target species, including the congeneric P. spumarius.

Keywords: Xylella fastidiosa vector; alpha taxonomic identification; molecular diagnostic tool; spittlebug.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Partial alignment of the intronic regions of nine EF1-α alleles cloned from two P. italosignus (Pi1 and Pi2) and two P. spumarius (Ps3 and Ps4) specimens collected during this study. The homologous sequences of the two alleles of P. spumarius PSPU_08 (GCA_018207615.1) and the only allele of P. italosignus (JF309079) currently available in the Genbank database were included for comparative purposes as PsRef1, PsRef2, and PiRef1, respectively. The positions of the P. italosignus-specific qPCR assay primers, Pital_348F and Pital_465R, are highlighted in black and gray, respectively (the complete alignment of Spit-Uni1/2 products is shown in supplemental material Figure S1). The “*” character indicates positions that are fully conserved.
Figure 2
Figure 2
Amplification curves (a) and melting peaks (b) of P. italosignus adults (red circles). Black triangles: adults of N. campestris and P. spumarius.
Figure 3
Figure 3
P. italosignus: serial dilutions 1:5 from 10 ng of adult DNA extracts, showing amplification (a) and titration curves (b).
See this image and copyright information in PMC

References

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