An Inter-Laboratory Comparative Study on the Influence of Reagents to Perform the Identification of the Xylella fastidiosa Subspecies Using Tetraplex Real Time PCR-Reference-Cited by-同舟云学术

An Inter-Laboratory Comparative Study on the Influence of Reagents to Perform the Identification of the Xylella fastidiosa Subspecies Using Tetraplex Real Time PCR

Published:2023-09-19 Issue:9 Volume:9 Page:1053
ISSN:2311-7524
Container-title:Horticulturae
language:en
Short-container-title:Horticulturae

Author:

Pucci Nicoletta¹^ORCID,Scala Valeria¹^ORCID,Cesari Erica¹^ORCID,Crosara Valeria¹²,Fiorani Riccardo¹,L’Aurora Alessia¹,Lucchesi Simone¹,Tatulli Giuseppe¹,Barra Eleonora³,Ciarroni Serena⁴,De Amicis Francesca⁵,Fascella Salvatore⁶,Giacobbi Francesca⁷,Gaffuri Francesca⁸,Gualandri Valeria⁹,Landi Lucia¹⁰^ORCID,Loconsole Giuliana¹¹^ORCID,Molinatto Giulia¹²^ORCID,Pollastro Stefania¹³^ORCID,Raimondo Maria Luisa¹⁴^ORCID,Rizzo Domenico¹⁵,Roggia Chiara¹⁶,Silletti Maria Rosaria¹⁷,Talevi Simona¹⁸,Testa Marco¹⁹,Loreti Stefania¹^ORCID

Affiliation:

1. Council for Agricultural Research and Economics, Research Centre for Plant Protection and Certification (CREA-DC), 00156 Rome, Italy

2. Department of Environmental Biology, Sapienza University of Rome, 00185 Rome, Italy

3. Phytopathological Laboratory, Campania Region, 80141 Napoli, Italy

4. Phytoparasites Diagnostics (Phy.Dia.) s.r.l., 01100 Viterbo, Italy

5. Regional Agency for Agricultural Development—Plant Protection Service of Friuli Venezia Giulia Region, 33050 Pozzuolo del Friuli, Italy

6. Plant Health Protection Service, Liguria Region, 16129 Genova, Italy

7. Regional Plant Protection Service, 40128 Bologna, Italy

8. Laboratory of Plant Health Service, 22070 Vertemate con Minoprio, Italy

9. Technology Transfer Centre, Fondazione Edmund Mach, 38010 San Michele all’Adige Trento, Italy

10. Department of Agricultural, Food and Environmental Sciences, Marche Polytechnic University, 60131 Ancona, Italy

11. National Research Council, Institute for Sustainable Plant Protection (IPSP)—National Research Council, 70126 Bari, Italy

12. Phytosanitary Laboratory of the Phytosanitary Sector and Technical, Scientific Services of the Piedmont Region, 10144 Torino, Italy

13. Department of Soil, Plant and Food Sciences (DISSPA), University of Bari, 70126 Bari, Italy

14. Department of Agricultural Sciences, Food, Natural Resources and Engineering, University of Foggia, 71122 Foggia, Italy

15. Laboratory of Phytopathological Diagnostics and Molecular Biology, Plant Protection Service of Tuscany, 51100 Pistoia, Italy

16. Laboratory Enocontrol Scarl Agri-Food Research Analysis Center, 12051 Alba, Italy

17. Research, Experimentation and Education Centre in Agriculture “Basile-Caramia”, Locorotondo, 70010 Bari, Italy

18. Plant Protection Service Marche Region—AMAP, 60027 Osimo, Italy

19. Agris Sardegna, Agricultural Research Agency of Sardinia, 09020 Sassari, Italy

Abstract

In 2022, a test performance study (TPS) assessing the influence of different master mixes on the performance of the tetraplex real-time PCR (TqPCR) assay was organized. TqPCR allows for the specific detection and identification of Xylella fastidiosa (Xf) subspecies in a single reaction. Eighteen official laboratories of the Italian National Plant Protection Organization received a panel of 12 blind samples, controls, primers, probes, and different master mixes to participate in the TPS. Furthermore, the Research Centre for Plant Protection and Certification of the Council for Agricultural Research and Economics performed an intra-laboratory study (ITS) on spiked plant matrices to evaluate the analytical sensitivity of TqPCR employing the selected master mixes with the best performance. Naturally infected samples were analyzed for subspecies identification via TqPCR compared with the official multilocus-sequence-typing (MLST) method. The best results in this comparative study were obtained using Fast Universal PCR Master Mix (Applied Biosystems) and Brilliant multiplex QPCR Master Mix (Agilent), and they confirmed that the TqPCR test is reliable, offering the advantage of identifying this subspecies at the same time, thus saving time and resources. The TqPCR assay is suggested among the tests to be used by laboratories performing the official diagnosis of Xf to support the activities of official monitoring.

Funder

MIPAAF

Publisher

MDPI AG

Subject

Horticulture,Plant Science

Link

https://www.mdpi.com/2311-7524/9/9/1053/pdf

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