Beard SS, Scott IAW (2012) A rapid method for the detection and quantification of the vector-borne bacterium ‘Candidatus Liberibacter solanacearum’ in the tomato potato psyllid, Bactericera cockerelli. Entomologia Experimentalis et Applicata 147(2): e12056. https://doi.org/10.1111/eea.12056
The tomato potato psyllid (TPP), Bactericera cockerelli (Šulc) (Hemiptera: Psyllidae), is a serious pest of solanaceous crops and is widespread in areas of the USA, Mexico, Central America, and New Zealand (NZ). The species vectors the unculturable phloem‐limited bacterial pathogen ‘Candidatus Liberibacter solanacearum’ (Lso) which is accepted as the causal agent of economically important diseases in several crops including potato, tomato, capsicum, and tamarillo (Liefting et al., 2008, 2009; Munyaneza et al., 2008; Abad et al., 2009; Crosslin & Bester, 2009; Lin et al., 2009). Recently, Lso has been associated with disease in carrots affected by the psyllid Trioza apicalis Foerster in Finland, Sweden, Norway, and in carrots affected by psyllids in the genus Bactericera in mainland Spain and the Canary Islands (Alfaro‐Fernández et al., 2012a,b; Munyaneza et al., 2010, 2012a,b).
Effective management of vector-borne plant disease depends on rapid, specific, and sensitive detection of the pathogen in both the insect vector and the plant host. The Lso bacterium cannot be cultured and therefore PCR-based methods are required for Lso diagnostics. Here, two methods are compared, using CTAB and MicroGEM reagents (referenced here as ZyGEM), respectively, for the preparation of DNA for the detection and quantitation of Lso in the TPP host using qPCR. The MicroGEM method is a rapid and high-yielding method for the preparation of PCR-ready DNA from TPP in a single step using prepGEM reagents (e.g., see Ball & Armstrong, 2008).
The method involves proteolytic digestion of the insect sample followed by heat-inactivation of the proteinase. No sample transfer or clean-up steps are required. This protocol can be completed in just over 1 h, compared with ca. 6 h required for extraction via CTAB, and is easily adaptable for high-throughput processing. The effectiveness of the two methods was assessed by comparing the yield of TPP and Lso target DNA as measured by qPCR. In addition, a qPCR assay targeting the TPP Internal Transcribed Spacer region 2 (ITS2) is described for use as an internal control in conjunction with an Lso-specific qPCR assay for the quantitation and normalization of Lso titer in infective TPP.
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Suitable for extracting DNA from bacteria (Gram-positive and Gram-negative), protozoa, archaea, colonies and liquid cultures, biofilm and mucosal samples, swabs, and metagenomic DNA including soil, stool and water
Produces single-stranded DNA suitable for PCR, qPCR, targeted sequencing (amplicon sequencing) from metagenomic samples, 16S rDNA sequencing
Each kit contains prepGEM, lysozyme, GREEN+ buffer, enhancer and wash.