scholarly journals Development of PCR-Based Assays for Detecting Xanthomonas campestris pv. carotae, the Carrot Bacterial Leaf Blight Pathogen, from Different Substrates

Plant Disease ◽  
2004 ◽  
Vol 88 (11) ◽  
pp. 1226-1234 ◽  
Author(s):  
X. Q. Meng ◽  
K. C. Umesh ◽  
R. M. Davis ◽  
R. L. Gilbertson
Keyword(s):  
Xanthomonas Campestris ◽  
Random Amplified Polymorphic Dna ◽  
Bacterial Species ◽  
Leaf Blight ◽  
Bacterial Leaf Blight ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Agar Media ◽  
Extensive Collection ◽  
Polymerase Chain

Detection of the carrot bacterial leaf blight pathogen, Xanthomonas campestris pv. carotae, was achieved using polymerase chain reaction (PCR) along with primer pairs developed from sequences of cloned random amplified polymorphic DNA (RAPD) fragments. Primer pairs 3S and 9B directed the amplification of ∼350-bp and ∼900-bp (or ∼2 kb) DNA fragments, respectively, from genomic DNA of all known X. campestris pv. carotae strains tested, but not from that of 13 other X. campestris pathovars or other bacterial species, including yellow non-xanthomonad bacteria isolated from carrot tissues and seeds. In tests conducted with an extensive collection of X. campestris pv. carotae-like strains isolated from different substrates from California, Idaho, Oregon, Washington, and Canada, the 3S primer pair directed the amplification of the ∼350-bp target fragment from all strains. These results indicated that the 3S primer pair is highly specific for X. campestris pv. carotae detection. Using the 3S primer pair, PCR assays were developed for detection of X. campestris pv. carotae from colonies on agar media, carrot leaf and stem tissues, and seeds. These tests could be performed in a single day. The PCR-based seed assay detected X. campestris pv. carotae from lots with contamination rates ranging from 2 × 102 to 2.3 × 108 CFU per gram of seed. This assay gave results similar to a seed-wash dilution plating assay and proved more sensitive than an enzyme-linked immunosorbent assay (ELISA)-based assay.

scholarly journals Characterization of Xanthomonas axonopodis pv. phaseoli isolates

2008 ◽  
Vol 34 (3) ◽  
pp. 228-231 ◽  
Author(s):  
Willian Mário de Carvalho Nunes ◽  
Maria Júlia Corazza ◽  
Silvana Aparecida Crestes Dias de Souza ◽  
Siu Mui Tsai ◽  
Eiko Eurya Kuramae
Keyword(s):  
Xanthomonas Campestris ◽  
Random Amplified Polymorphic Dna ◽  
Hypersensitive Reaction ◽  
Chain Reaction ◽  
Xanthomonas Axonopodis ◽  
Paulo State ◽  
Total Dna ◽  
Polymerase Chain ◽  
Bacterial Plant Pathogen

A simple, quick and easy protocol was standardized for extraction of total DNA of the bacteria Xanthomonas axonopodis pv. phaseoli. The DNA obtained by this method had high quality and the quantity was enough for the Random Amplified Polymorphic DNA (RAPD) reactions with random primers, and Polymerase Chain Reaction (PCR) with primers of the hypersensitivity and pathogenicity gene (hrp). The DNA obtained was free of contamination by proteins or carbohydrates. The ratio 260nm/380nm of the DNA extracted ranged from 1.7 to 1.8. The hrp gene cluster is required by bacterial plant pathogen to produce symptoms on susceptible hosts and hypersensitive reaction on resistant hosts. This gene has been found in different bacteria as well as in Xanthomonas campestris pv. vesicatoria (9). The primers RST21 and RST22 (9) were used to amplify the hrp gene of nine different isolates of Xanthomonas axonopodis pv. phaseoli from Botucatu, São Paulo State, Brazil, and one isolate, "Davis". PCR amplified products were obtained in all isolates pathogenic to beans.

A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species

2001 ◽  
Vol 22 (6) ◽  
pp. 1086-1089 ◽  
Author(s):  
Raúl Rivas ◽  
Encarna Velázquez ◽  
Angel Valverde ◽  
Pedro F. Mateos ◽  
Eustoquio Martínez-Molina
Keyword(s):  
Polymerase Chain Reaction ◽  
Random Amplified Polymorphic Dna ◽  
Bacterial Species ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Diversity of Xanthomonas campestris Isolates from Symptomatic Crucifers in New York State

2016 ◽  
Vol 106 (2) ◽  
pp. 113-122 ◽  
Author(s):  
H. W. Lange ◽  
M. A. Tancos ◽  
M. O. Carlson ◽  
C. D. Smart
Keyword(s):  
New York ◽  
Xanthomonas Campestris ◽  
Repetitive Element ◽  
New York State ◽  
The State ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sample Population ◽  
Chain Reaction ◽  
Pure Cultures ◽  
Polymerase Chain

To assess the diversity of Xanthomonas campestris spp. infecting crucifers in New York, 154 isolates were collected over 10 years across the state. The goal was to determine if isolates of the pathogen were overwintering in New York and serving as primary inoculum in subsequent years, or if novel isolates were entering the state each year. Pure cultures of isolates were characterized using multilocus sequence analysis (MLSA), a greenhouse pathogenicity assay, repetitive element-polymerase chain reaction (Rep-PCR) using the BOX-A1R primer, and enzyme-linked immunosorbent assay. The MLSA scheme proved to be more efficient than Rep-PCR for a large sample population and for comparison with global isolates. X. campestris isolated from crucifers in New York comprised of X. campestris pv. campestris and X. campestris pv. raphani, with X. campestris pv. raphani being predominately isolated from transplants. Evidence for unique haplotypes persisting on the same farm for several years due to improper seedbed rotations was documented in addition to novel haplotypes being spread throughout states through infected transplants and seed. Rep-PCR confirmed the high diversity of X. campestris and was used to generate 15 unique fingerprint patterns from isolates collected in the first 5 years. A worldwide comparison of isolates suggests that the X. campestris pv. campestris population appears to be very homogenous with dominant haplotypes persisting for extended periods and being globally disseminated.

scholarly journals First Report of Bacterial Leaf Blight of Coriander Caused by Xanthomonas campestris pv. coriandri in Taiwan

Plant Disease ◽  
2004 ◽  
Vol 88 (8) ◽  
pp. 910-910 ◽  
Author(s):  
Y.-A. Lee ◽  
Y.-H. Liu ◽  
H.-L. Liu
Keyword(s):  
16S Rdna ◽  
Xanthomonas Campestris ◽  
Pathogenic Bacteria ◽  
Leaf Blight ◽  
Bacterial Leaf Blight ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
First Occurrence ◽  
Plastic Bag ◽  
Agar Media

Leaf blight symptoms on coriander (Coriandrum sativum) were observed during the summers of 2000 to 2002 in fields at the Beidou and Sijhou townships, Changhua County, Taiwan. Symptoms first appeared as small spots on the lower sides of leaves and stems. The centers of the spots quickly turned brown and were surrounded by whitish yellow halos. The brown spots and halos enlarged rapidly and coalesced into irregular, yellowish or brownish dry dead areas on the leaf. V-shaped and chlorotic blotch symptoms were also found at the margins of leaves. Isolations from diseased leaves consistently yielded bacterial colonies that were yellow and glistening on nutrient and potato dextrose agar media. Five representative strains were chosen for further characterization. All strains were gram-negative rods, aerobic, and produced yellow, nonwater soluble, xanthomonadin pigments identified by thin-layer chromatography (1). The strains were positive for catalase and β-galactosidase and negative for oxidase, nitrate reductase, urease, and tryptophanase (indole production) and hydrolyzed starch, gelatin, and esculin. Hydrogen sulfide was produced from cysteine. L-asparagine was not sufficient as a sole carbon source for growth. In Dye's medium C, acids were produced from metabolizing arabinose, glucose, and sucrose but not from rhamnose, cellobiose, lactose, dulcitol, mannitol, and sorbitol. The bacterium was identified as Xanthomonas campestris. Almost complete 16S rDNA sequence of strain TC3 (1,502 bp; GenBank Accession No. AY604178) was determined and compared with available 16S rDNA sequences in GenBank. The sequence was highly identical (99%) to those of Xanthomonas campestris pathovars. Coriander plants were inoculated by spraying bacterial suspensions (108 CFU/ml) on leaves, enclosed in a plastic bag to maintain high humidity for 2 days, and kept in a growth chamber at 28°C. Typical symptoms were observed in 2 to 6 days in all four inoculated plants and appeared to be identical to those observed in the fields. Control plants were inoculated with sterile distilled water and showed no symptoms. The bacterium was readily reisolated from diseased leaves. Bacterial leaf blight of coriander was first reported in India, and the pathogen was identified as X. campestris pv. coriandri (2). To our knowledge, this is the first occurrence of this bacterium on coriander in Taiwan. References: (1) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (2) M. C. Srinivasan et al. Proc. Indian Acad. Sci. Sect. B 53:298, 1961.

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

scholarly journals Development of a Genoserotyping Method for Salmonella Infantis Detection on the Basis of Pangenome Analysis

2020 ◽  
Vol 9 (1) ◽  
pp. 67
Author(s):  
Seung-Min Yang ◽  
Jiwon Baek ◽  
Eiseul Kim ◽  
Hyeon-Be Kim ◽  
Seyoung Ko ◽  
...  
Keyword(s):  
Public Health ◽  
Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Cost Effective ◽  
The Other ◽  
Gene Marker ◽  
Diagnostic Assay ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Substantial Economic Burden

In recent years, Salmonella Infantis has become a predominant serovariant in clinical and poultry isolates, thereby imposing a substantial economic burden on both public health and the livestock industry. With the aim of coping with the steep increase in serovar Infantis prevalence, a polymerase chain reaction (PCR)-based rapid and accurate diagnostic assay was developed in this study through pangenome profiling of 60 Salmonella serovars. A gene marker, SIN_02055, was identified, which is present in the S. Infantis genome but not in the pangenome of the other serovars. Primers specific to SIN_02055 were used to accurately detect serovar Infantis, and to successfully differentiate Infantis from the other 59 serovars in real-time PCR with a R2 of 0.999 and an efficiency of 95.76%. The developed method was applied to 54 Salmonella strains belonging to eight dominant serovars, and distinguished Infantis from the other seven serovars with an accuracy of 100%. The diagnostic primer set also did not show false positive amplification with 32 strains from eight non-Salmonella bacterial species. This cost-effective and rapid method can be considered an alternative to the classic serotyping using antisera.

scholarly journals Occurrence of abortions induced by Neospora caninum in dairy cattle from Santa Catarina, southern Brazil

2017 ◽  
Vol 26 (3) ◽  
pp. 292-298 ◽  
Author(s):  
Cesar Augusto Barbosa de Macedo ◽  
Madlaine Frigo Silveira Barbosa de Macedo ◽  
Ana Carolina Miura ◽  
Alessandra Taroda ◽  
Sergio Tosi Cardim ◽  
...  
Keyword(s):  
Dairy Cattle ◽  
Dairy Cows ◽  
High Frequency ◽  
Neospora Caninum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Southern Brazil ◽  
Chain Reaction ◽  
Santa Catarina ◽  
Tissue Samples ◽  
Polymerase Chain

Abstract The aim of the present study was to investigate the occurrence of N. caninum associated with abortions of dairy cattle from Santa Catarina state, southern Brazil by using enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and polymerase chain reaction (PCR). Blood from dairy cows that aborted along with intrathoracic fluid and tissue samples (brain, heart, liver, and lung) from their fetuses were collected and used for serology; PCR, histopathological, and immunohistochemistry (IHC) evaluations were also conducted. Twenty-one cows (51.2%) out of 41, and eight fetuses (26.7%) out of 30 were ELISA (HerdCheck, IDEXX) positive for N. caninum. Dams > 36 months of age had a higher risk of being serum positive than younger animals. PCR and IHC revealed that 38.8% (14/36) and 25.0% (9/36) of the fetuses were positive for N. caninum, respectively for each of the tests. Seropositive cows had a higher frequency of fetuses that were also positive by either intrathoracic fluid, PCR, or IHC. In summary, the present study observed a high frequency of N. caninum in abortions from dairy cows from southern Brazil, with a higher N. caninum prevalence found in cows that were older than 36 months. In addition, serology, PCR, and IHC should be used all together for better diagnosis of neosporosis in cattle.

scholarly journals Development of Quantitative Real-Time Polymerase Chain Reaction for Detection of and Discrimination between Erysipelothrix Rhusiopathiae and Other Erysipelothrix Species

2009 ◽  
Vol 21 (5) ◽  
pp. 701-706 ◽  
Author(s):  
Ho To ◽  
Tomohiro Koyama ◽  
Shinya Nagai ◽  
Kotaro Tuchiya ◽  
Tetsuo Nunoya
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Practical Applications ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

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