The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently availabl...
The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 100 fg/µl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.
The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 100 fg/µl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.
The genomic DNA was isolated from the Acidovorax strains and the other bacterial strains using a Genomic DNA Prep Kit (SolGent, Korea) according to the manufacturer’s protocol. The quantity and purity of the bacterial genomic DNA was evaluated by measuring its absorbance using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
이론/모형
5×TAE buffer. The SYBR Green-based real-time PCR assay was conducted using a total volume of 20 µl of a reaction mixture containing 10 µl of SYBR Premix Ex Taq (Takara Bio, Inc., Japan), 5 pM of each AC158F/R primers, and 5 ng of purified DNA. The SYBR Green-based real-time PCR assay was performed using a CFX96 real-time PCR system (Bio-Rad Laboratories, Inc.
성능/효과
In conclusion, as a result of the high sensitivity and specificity of the SYBR Green real-time PCR assay with its relatively rapid and simple procedure, this method is rapid and less cumbersome than other diagnostic methods for the identification of A. avenae subsp.
The YDrepeat proteins (Rhs repertoires) of the Enterobacteriaceae are reported to be highly dynamic, which has been attributed to repeated gains and losses of genes. In contrast, the key structures of the rhs genes have been evolutionarily conserved, indicating that the sequence diversity of these genes is driven not by rapid mutation, but by the slow evolution of novel core/tip combinations. Comparison of the C-terminal tips and dissociated fragments of YD-repeat protein genes indicated that although C-termini diverged greatly within a locus, each distinct sequence was conserved in the related strains and, indeed, in related species and genera.
avenae subsp. citrulli in infested samples is a fast, accurate, and sensitive surveillance tool, and more sensitive and reliable than previous results (Table 3).
후속연구
avenae subsp. citrulli from seed and plant samples without the use of selective media, additional biochemical tests, or DNA extraction.
참고문헌 (32)
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