Assessment of Psyllid Handling and DNA Extraction Methods in the Detection of ‘Candidatus Liberibacter Solanacearum’ by qPCR

  1. Quintana, María
  2. de-León, Leandro
  3. Cubero, Jaime
  4. Siverio, Felipe
  1. 1 Universidad de La Laguna
    info

    Universidad de La Laguna

    San Cristobal de La Laguna, España

    ROR https://ror.org/01r9z8p25

Revista:
Microorganisms

ISSN: 2076-2607

Año de publicación: 2022

Volumen: 10

Número: 6

Páginas: 1104

Tipo: Artículo

DOI: 10.3390/MICROORGANISMS10061104 GOOGLE SCHOLAR lock_openAcceso abierto editor

Otras publicaciones en: Microorganisms

Resumen

‘Candidatus Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in Solanaceae and Apiaceae crops. CaLsol detection in psyllids often requires insect destruction, preventing a subsequent morphological identification. In this work, we have assessed the influence on the detection of CaLsol by PCR in Bactericera trigonica (Hemiptera: Psyllidae), of four specimen preparations (entire body, ground, cut-off head, and punctured abdomen) and seven DNA extraction methods (PBS suspension, squashing on membrane, CTAB, Chelex, TRIsureTM, HotSHOT, and DNeasy®). DNA yield and purity ratios, time consumption, cost, and residues generated were also evaluated. Optimum results were obtained through grinding, but it is suggested that destructive procedures are not essential in order to detect CaLsol. Although CaLsol was detected by qPCR with DNA obtained by the different procedures, HotSHOT was the most sensitive method. In terms of time consumption and cost, squashed on membrane, HotSHOT, and PBS were the fastest, while HotSHOT and PBS were the cheapest. In summary, HotSHOT was accurate, fast, simple, and sufficiently sensitive to detect this bacterium within the vector. Additionally, cross-contamination with CaLsol was assessed in the ethanol solutions where B. trigonica specimens were usually collected and preserved. CaLsol-free psyllids were CaLsol-positive after incubation with CaLsol-positive specimens. This work provides a valuable guide when choosing a method to detect CaLsol in vectors according to the purpose of the study.

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Referencias bibliográficas

  • 10.1099/00207713-44-3-379
  • 10.1128/AEM.01268-08
  • 10.1094/PDIS-92-10-1474A
  • 10.1099/ijs.0.007377-0
  • 10.1007/s12230-012-9262-3
  • 10.1111/aab.12149
  • 10.1094/PHYTO-07-13-0182-R
  • 10.1007/s10658-010-9737-3
  • 10.1007/s10658-012-0121-3
  • 10.1094/PHYTO-12-17-0410-R
  • 10.1094/PDIS-06-18-0937-RE
  • 10.1007/s10658-019-01890-0
  • 10.1111/epp.12611
  • 10.1603/EC10027
  • Munyaneza, (2011), J. Plant Pathol., 93, pp. 697
  • 10.1007/s12230-015-9448-6
  • 10.1094/PDIS-10-11-0878-PDN
  • 10.1094/PDIS-11-11-0918-PDN
  • 10.1094/PDIS-08-13-0900-PDN
  • 10.1094/PDIS-05-14-0509-PDN
  • 10.1094/PDIS-03-17-0419-PDN
  • 10.1007/s42161-018-0045-7
  • 10.1094/PHYTO-10-17-0348-R
  • 10.1007/s11540-017-9350-3
  • (2019)
  • 10.3390/microorganisms8091446
  • 10.1038/s41598-019-45975-6
  • 10.1007/s10482-019-01362-9
  • 10.1038/s41598-020-73382-9
  • 10.1038/srep45534
  • 10.5424/sjar/2017154-10762
  • EPPO Global Database: Bactericera Trigonica https://gd.eppo.int/taxon/BCTCTR
  • 10.1371/journal.pone.0138946
  • 10.5424/sjar/2015131-6551
  • 10.3390/insects10090287
  • 10.5197/j.2044-0588.2017.036.004
  • 10.3157/021.123.0403
  • 10.1111/j.1365-294X.1993.tb00100.x
  • 10.1093/jee/101.2.523
  • 10.3390/ijms11125056
  • 10.1371/journal.pone.0011963
  • Oliveira, (2011), Ciênc. Rural, 419419, pp. 1563
  • 10.3791/3281
  • 10.4238/2014.October.27.19
  • 10.1093/aesa/88.3.281
  • 10.1371/journal.pone.0000272
  • 10.1016/j.aspen.2010.04.003
  • 10.1007/s13355-017-0481-4
  • Non-destructive DNA extraction from Psyllids. POnTE, Pest Organisms Threatening Europe https://www.ponteproject.eu/protocols-calsol/non-destructive-dna-extraction-psyllids/
  • 10.4236/ae.2015.34016
  • 10.1016/j.mimet.2009.04.009
  • 10.1093/nar/8.19.4321
  • 10.1111/j.1755-0998.2011.03073.x
  • 10.1111/ppa.12197
  • 10.1094/PHYTO-96-0485
  • Truett, (2014), J. Am. Coll. Cardiol., 64, pp. 1602, 10.1016/j.jacc.2014.05.073
  • Bioline. Meridian Bioscience. TRIsureTM https://www.bioline.com/trisure.html
  • 10.1094/PDIS-05-11-0386
  • 10.1371/journal.pone.0226818
  • 10.1098/rstb.2005.1727
  • 10.1111/j.1755-0998.2011.03074.x
  • 10.1186/1756-0500-5-668
  • 10.1007/s10493-006-9042-1
  • 10.1016/j.ab.2009.10.041
  • Stephen, (2015), Sci. Justice, 55, pp. 200, 10.1016/j.scijus.2015.01.005
  • 10.1016/j.mimet.2015.06.005
  • Sambrook, (2006)
  • Fang, (1992), Biotechniques, 13, pp. 52
  • 10.1007/s13205-020-2051-7
  • Supplementary Protocol: Purification of Total DNA from Insects Using the DNeasy® Blood &Tissue Kit https://www.qiagen.com/br/resources/faq?id=58ddc6c7-d1f4-45d8-9156-8174fed15bf3&lang=en
  • Safety Data Sheet: Hexadecyltrimethylammonium Bromide Version 6.0 https://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=ES&language=es&productNumber=H9151&brand=SIGMA&PageToGoToURL=https%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fh9151%3Flang%3Des
  • 10.1139/gen-2016-0024
  • 10.1099/ijs.0.63677-0
  • Capoor, (1967), Indian Agric. Res. Inst., 376, pp. 572
  • McClean, (1965), S. Afr. J. Agric. Sci., 81, pp. 297
  • 10.1094/PHYTO-98-4-0387
  • 10.1093/jee/tov038