Determination of analytical specificity of PCR methods for Acidovorax citrulli identification

Acidovorax citrulli (Schaad et al.), the causative agent of bacterial fruit blotch of cucurbits is a significant object in plant quarantine. The main stage in the detection of the phytopathogen in the framework of laboratory diagnostics is using molecular-genetic methods, like PCR, and it is the results of the application of these methods that are decisive. In this regard, the used PCR-methods impose strict requirements for assessing their applicability. Applicability criteria such as analytical specificity, showing the ability of a test to reliably distinguish a target organism from a non-target organism, is an integral component of the assessment applicability of a diagnostic method. Analytical specificity is evaluated based on the values of parameters such as inclusivity and exclusivity. Inclusivity shows the ability of the method to identify strains of the target bacterium, representing all genetic diversity, different geographical origin and host plants. Exclusivity shows the ability of the method to distinguish non-target bacteria, especially those that can be in the DNA of host plants. This study was conducted to determine the analytical specificity of the PCR-methods for identification of A. citrulli. We detemined analytical specificity with 5 A. citrulli strains, 19 bacterial strains of other species from international collections, 28 other bacterial isolates (See Table 1) and 106 DNA samples isolated from various varieties and species of Cucurbitaceae plants. 30 samples were plant debris, and 76 were seed samples (See Table 2). The process of preparing analytical samples consisted in the extraction of microbiota from samples into phosphate-saline buffer and subsequent concentration using high-speed centrifuge. Then DNA was extracted from the samples. PCR for each sample was performed in triplicate for each of the following tests: qPCR Acit 1 F/R and Acit 1-probe according to Woudt et al [2009], commercial kits for qPCR by “Syntol” and “AgroDiagnostika” (Russia), PCR with primers AC158F/AC158R according to Cho et al. [2015], PCR SEQ ID 3/4 according to Schaad et al. [2000], PCR PL1/PL2 according to Zhong et al., 2015 and PCR G12AcFwd/G12AcRev according to Zivanovic & Walcott [2017]. The value of inclusivity for each PCR test was determined as the ratio of reliable positive results of the target to the total number of reactions, expressed as a percentage. The value of exclusivity was determined as the ratio of reliable negative results to the total number of reactions of non-target, expressed as a percentage. An assessment of analytical specificity was assigned to each of the test based on an analysis of the inclusivity and exclusivity parameters. The conclusion about the specificity of the tests was made with the values of inclusivity and exclusivity of 99% or higher. Isolation of bacteria was carried out from analytical samples of cucurbit crops that showed at least one positive result when tested by any of the above PCR methods (See Table 3). DNA from single colonies was used for PCR with primers 8UA/519B. For identification, we used the Sanger method for determining the nucleotide sequence, which were, then, compared with the sequences of bacterial genomes in the NCBI database. The process of diagnosing phytopathogens is a combination of sequentially applied methods and tests, the final stage of each of which is the interpretation of the data. In the case of obtaining positive results of the applied tests, it is important to establish the exact cause of these results. During the study, PCR tests showed positive results with samples known to be free of the A. citrulli DNA. Agarose gel amplicon detection also revealed the presence of reaction products characteristic of A. citrulli (See Fig. 1). Also, isolated 89 bacterial cultures whose colonies were morphologically different. These isolates were selected for testing by the Acit 1 F/R, Acit 1-probe in order to detect those isolates that can lead to positive results. 14 isolates showed positive results. These isolates were used for PCR 8UA/519B. For 4 of the 14 nucleotide sequences, matches in the NCBI were detected, and isolates identified as Microbacterium phyllosphaerae, Arthrobacter sp. (2 isolates) and Sphingomonas paucimobilis. These isolates showed a positive reaction when tested using Acit 1 F/R, Acit 1-probe, “Acidovorax citrulli-RV” “Synthol” and Acidovorax citrulli-Rt “AgroDiagnostika”. When tested using the AC158F/AC158R method, these isolates were also positive (See Fig. 1). In addition, the isolate identified as Microbacterium phyllosphaerae showed a positive reaction when tested by methods of SEQ ID 3/4 and PL1/PL2 (See Fig. 2). Thus, bacteria that are potentially sources of false-positive PCR tests were isolated and identified. Testing of other bacteria not belonging to the species A. citrulli showed negative results. The results of testing strains A. citrulli by Acit 1 F/R, Acit 1-probe, “Acidovorax citrulli-RV” “Synthol”, Acidovorax citrulli-Rt “AgroDiagnostika”, AC158F/AC158R, SEQ ID 3/4, PL1/PL2 and G12AcFwd/G12AcRev were used to determine the value of inclusivity (See Table 4). PCR methods showed a positive reaction with each of the 5 tested A. citrulli strains (100% inclusivity). Test results of bacterial strains not belonging to the species A. citrulli and DNA samples isolated from different varieties and species of Cucurbitaceae were used to determine the value of exclusivity (See Table 3). We found that the exclusivity values of the Acit 1 F/R, Acit 1-probe, “Acidovorax citrulli-RV” “Synthol”, Acidovorax citrulli-Rt “AgroDiagnostika” and AC158F/AC158R methods are below 99%, established as a criterion for recognizing the method as specific; therefore, these methods cannot be considered specific, despite 100% inclusivity. At the same time, the methods SEQ ID 3/4, PL1/PL2 and G12AcFwd/G12AcRev are specific (the value of exclusivity is more than 99%, the value of inclusivity is 100%). The results of the analytical specificity assessment allow us to conclude that Acit 1 F/R, Acit 1-probe, “Acidovorax citrulli-RV” “Synthol”, Acidovorax citrulli-Rt “AgroDiagnostika”, AC158F/AC158R can be used in diagnosing A. citrulli only as screening methods. Based on the positive results of testing the samples with the indicated methods, it is impossible to draw a conclusion about the presence of the pathogen in the sample. At the same time, PCR SEQ ID 3/4, PL1/PL2 and G12AcFwd/G12AcRev can be used as confirmatory diagnostic methods for A. citrulli. Thus, as a result of the study, we evaluated the analytical specificity of existing molecular-genetic methods for identifying A. citrulli. Such a full-scale assessment of the analytical specificity of diagnostic PCR methods for A. citrulli was carried out for the first time. The obtained data will allow testing laboratories to more reliably identify A. citrulli. The paper contains 2 Figures, 3 Tables and 24 References. The Authors declare no conflict of interest.

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