Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material

Abstract

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 microl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.

FIG. 1

Diversity of fragments obtained after amplification following the nested-PCR method in a single closed tube. The sizes vary, including the expected 447 bp (lanes 4 and 7), 391 bp (lanes 1, 2, 3, and 9), and intermediate values (lanes 5, 6, 8, 10, and 11). Numbered lanes contain samples from the NCPPB collection (strain number and country of origin are given in parentheses): lane 1, 2292 (United States); lane 2, 2293 (United States); lane 3, 2950 (United States); lane 4, 311 (Canada); lane 5, 683 (United Kingdom); lane 6, 1734 (Egypt); lane 7, 1819 (United States); lane 8, 2080 (New Zealand); lane 9, 2791 (United States); lane 10, 3159 (The Netherlands); lane 11, 3548 (Turkey); lane M, marker (100-bp DNA ladder; Gibco BRL).

FIG. 2

Location and extent of the deletion in the amplified fragments. The fragments amplified by the one-round PCR using primers designed by Bereswell et al. (2) from strains CFBP 1430 and PMV 6089 that gave a band of 391 bp by the nested PCR in a single closed tube were sequenced and compared to the corresponding sequence of strain CA11 (447 bp) using the program CLUSTAL W. For simplicity, only the sequence surrounding the 56-bp deletion present in strains CFBP 1430 and PMV 6089 is shown, since the rest was identical for the three strains. The 8-bp repeats are indicated by arrows.

FIG. 3

Specificity of the nested PCR in a single closed tube compared to that of other PCR methods. Samples were taken from naturally infected plants and analyzed by one-round PCR using the primers described by Bereswill et al. (2) (A), the primers described by McManus and Jones (27) (B), the nested PCR developed in this work (C). Note that the first two pairs of primers produce unspecific amplifications. Samples: 1, Pyrus communis 1892-b.1; 2, Pyracantha sp. 1952-b.5; 3, Pyracantha sp. 1952-b.9; 4, Pyracantha sp. 1952-b.11; 5, Pyrus communis 1961-d.1; 6, Pyrus communis 1961-e.2; 7, Pyrus communis 1961-e.3; 8, Pyrus communis 1961-f; 9, Pyrus communis 1961-g.3; 10, Malus domestica 1899-h. All the samples were positive except number 10. Sample number 6 gave a faint band. C+, positive control; M, marker (100 bp; New England Biolabs). The negative control is not shown in this figure.