Use of Oligonucleotide Array for Identification of Six Foodborne Pathogens and Pseudomonas aeruginosa Grown on Selective Media

2005 ◽  
Vol 68 (11) ◽  
pp. 2278-2286 ◽  
Author(s):  
MIAO CHU LIN ◽  
AY HUEY HUANG ◽  
HAU YANG TSEN ◽  
HIN-CHUNG WONG ◽  
TSUNG CHAIN CHANG
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Foodborne Pathogens ◽  
Intergenic Spacer ◽  
Oligonucleotide Array ◽  
Universal Primers ◽  
Morphological Observation ◽  
Biochemical Tests ◽  
Nylon Membrane ◽  
Selective Media ◽  
Species Specific

Identification of presumptive foodborne pathogens grown on selective media may take one to several days and requires a different battery of biochemical tests for each microorganism. A molecular identification method was developed in which universal primers were used to amplify the 16S to 23S rDNA intergenic spacer of target microorganisms, and PCR products were hybridized to a panel of species-specific oligonucleotides that were immobilized on a nylon membrane. The seven target microorganisms were Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella, Staphylococcus aureus, and Vibrio parahaemolyticus. After testing a large collection of target bacteria (29 to 51 strains) and nontarget bacteria (>500 strains), the performances (sensitivity and specificity) of the oligonucleotide array were as follows: B. cereus (100 and 77%), E. coli (100 and 100%), L. monocytogenes (100 and 90%), P. aeruginosa (100 and 100%), Salmonella (100 and 100%), S. aureus (100 and 100%), and V. parahaemolyticus (100 and 94.2%). Other species in the B. cereus group cross-hybridized to the probes used for identification of B. cereus, and positive results should be confirmed by additional morphological observation of colonies. Listeria innocua cross-reacted with probes used to identify L. monocytogenes, but a simple hemolysis test was used to differentiate the two species. Some strains of Vibrio harveyi and Vibrio mimicus cross-hybridized with probes used for identification of V. parahaemolyticus and caused false-positive reactions. The advantage of the array is that a common protocol was used to identify the seven target microorganisms and multiple different microorganisms could be simultaneously identified on a single array.

scholarly journals Identification of non-fermenting Gram-negative bacteria of clinical importance by an oligonucleotide array

2009 ◽  
Vol 58 (5) ◽  
pp. 596-605 ◽  
Author(s):  
Siou Cing Su ◽  
Mario Vaneechoutte ◽  
Lenie Dijkshoorn ◽  
Yu Fang Wei ◽  
Ya Lei Chen ◽  
...  
Keyword(s):  
Clinical Importance ◽  
Intergenic Spacer ◽  
16S Rrna Genes ◽  
Oligonucleotide Array ◽  
Rrna Genes ◽  
Universal Primers ◽  
23S Rrna ◽  
Rrna Gene ◽  
Nylon Membrane ◽  
Gram Negative

Many species of non-fermenting Gram-negative bacilli (non-fermenters) are important opportunistic and nosocomial pathogens. Identification of most species of non-fermenters by phenotypic characteristics can be difficult. In this study, an oligonucleotide array was developed to identify 38 species of clinically relevant non-fermenters. The method consisted of PCR-based amplification of 16S–23S rRNA gene intergenic spacer (ITS) regions using bacterial universal primers, followed by hybridization of the digoxigenin-labelled PCR products with oligonucleotide probes immobilized on a nylon membrane. A total of 398 strains, comprising 276 target strains (i.e. strains belonging to the 38 species to be identified) and 122 non-target strains (i.e. strains not included in the array), were analysed by the array. Four target strains (three reference strains and one clinical isolate) produced discrepant identification by array hybridization. Three of the four discordant strains were found to be correctly identified by the array, as confirmed by sequencing of the ITS and 16S rRNA genes, with the remaining one being an unidentified species. The sensitivity and specificity of the array for identification of non-fermenters were 100 and 96.7 %, respectively. In summary, the oligonucleotide array described here offers a very reliable method for identification of clinically relevant non-fermenters, with results being available within one working day.

Detection, Enumeration, and Isolation of Vibrio parahaemolyticus and V. vulnificus from Seafood: Development of a Multidisciplinary Protocol

2017 ◽  
Vol 100 (2) ◽  
pp. 445-453 ◽  
Author(s):  
Swapan K Banerjee ◽  
Jeffrey M Farber
Keyword(s):  
Foodborne Pathogens ◽  
Vibrio Parahaemolyticus ◽  
Cost Effective ◽  
Molecular Methods ◽  
Biochemical Tests ◽  
Selective Media ◽  
Isolation And Characterization ◽  
Species Specific ◽  
Pcr Targeting ◽  
Vibrio Spp

Abstract Vibrio parahaemolyticus and V. vulnificus are bacterial foodborne pathogens that can cause illnesses in humans after ingestion or exposure to contaminated seafood or coastal waters. Aprocedure that combines microbiological, biochemical, and molecular methods was designed and optimized for thedetection, enumeration, isolation, and characterization of these clinically significant Vibrio spp. Initially, microbiological culturing is used to resuscitate and isolate presumptive Vibrio spp. from chilled seafood samples. Biochemical tests are then used to analyze and select presumptive isolates at the species level, and, lastly, molecular methods, such as PCR targeting species-specific hemolysin genes, are used to confirm identification and assess the potential pathogenicity of presumptive isolates. By using artificially contaminated molluscan homogenates with known numbers of V. parahaemolyticus,this method yielded, on average, 90% recovery on complete agar media and 88% recovery on selective media. For V. vulnificus, the recovery rates were 86% (complete media) and 84% (selective media). Linearity of recovery of Vibrio spp. from artificially contaminated seafood homogenates supportedthe applicability of this method. Overall, this performance-tested protocol is easy to use, cost-effective, and fit-for-purpose, with potential for routine use in basic microbiological facilities.

scholarly journals Identification of Some Oomycetes by Reverse Dot Blot Hybridization

1998 ◽  
Vol 88 (3) ◽  
pp. 213-222 ◽  
Author(s):  
C. André Lévesque ◽  
Colleen E. Harlton ◽  
Arthur W. A. M. de Cock
Keyword(s):  
Phytophthora Cinnamomi ◽  
Blot Hybridization ◽  
Its Region ◽  
Phytophthora Species ◽  
Universal Primers ◽  
Dot Blot ◽  
Nylon Membrane ◽  
Species Specific ◽  
Small Aliquot ◽  
Reverse Dot Blot

An assay was developed that can identify unknown isolates of Pythium or Phytophthora species in a single hybridization. This reverse dot blot system is based on arrays of species-specific amplified fragments or oligonucleotides derived from the internal transcribed spacer (ITS) region, which are blotted as dots on a nylon membrane. By using total DNA from a sample as the template, universal primers, and digoxigenin-dUTP, the ITS was amplified and labeled simultaneously by the polymerase chain reaction (PCR). A small aliquot of the resultant labeled and amplified product was used as a probe for hybridization to a dot blot membrane that contained the immobilized species-specific oligonucleotides or amplified PCR fragments. The reverse dot blot system based on arrays of oligonucleotides showed far fewer cross-hybridizations than one based on entire amplified ITS I fragments. Unknown species can be identified simply by visualizing the positive hybridization reaction between the DNA labeled directly from the sample and the immobilized specific oligonucleotide. Currently, the assay can be used to identify Pythium aphanidermatum, P. ultimum, P. acanthicum, and Phytophthora cinnamomi. An oligonucleotide that was originally designed to identify Phytophthora hybridized to 10 of the 14 Phytophthora species tested. Another oligonucleotide designed to identify oomycetes hybridized to the 68 species tested, which represented two of the four orders of this phylum.

scholarly journals Identification of Clinical Pseudomonas spp. by VITEK 2 Compact System and Species-specific Polymerase Chain Reaction Assay for Identification of Pseudomonas aeruginosa

2020 ◽  
Vol 10 (03) ◽  
pp. 383-388
Author(s):  
Layla Abdulhamed Said ◽  
Sawsan Saeed Hasan ◽  
Saad L. Hamed
Keyword(s):  
Polymerase Chain Reaction ◽  
Pseudomonas Aeruginosa ◽  
Universal Primers ◽  
Local Alignment ◽  
Sequencing Analysis ◽  
Chain Reaction ◽  
Pseudomonas Spp ◽  
Vitek 2 ◽  
Polymerase Chain ◽  
Species Specific

The objective of this study was to isolate, identify, and diagnose Pseudomonas spp. from different clinical sources in Baghdad, Iraq. VITEK 2 compact system identification gram-negative bacteria (ID gNB) cards were used to confirm the identification. Polymerase chain reaction (PCR) technique and sequencing were used for recognition of the 16S rDNA gene, by two pairs of primers, universal primers (930 bp fragments) for recognition of Pseudomonas spp., and Pseudomonas aeruginosa specific species (PASS) primers (956 bp fragments) for differentiation of P. aeruginosa from other species. Amplified PCR products of PASS primers were sent for DNA Sanger sequencing to Macrogen Company, Seoul, Korea; data were compared with the database using the Basic Local Alignment Search Tool (BLAST). Ninety-two Pseudomonas spp., including 86 isolates of P. aeruginosa and 1, 2, 3 isolates of Pseudomonas luteola, Pseudomonas putida, and Pseudomonas fluorescens, respectively, were obtained using VITEK 2 compact system ID gNB cards with a percentage of identification ranging from 91 to 99%. Gene amplification and sequencing results confirm identification ranging from 99 to 100%. After sequencing analysis, eleventh of the P. aeruginosa isolates had been submitted in GenBank National Centre for Biotechnology Information (NCBI) under accession numbers MN630696–MN630707. It was observed that phenotypic tests supported by PCR techniques have enabled to conduct a detailed characterization of Pseudomonas bacteria isolates.

scholarly journals Heteroduplex Panel Analysis, a Novel Method for Genetic Identification of Aspergillus SectionFlavi Strains

2001 ◽  
Vol 67 (9) ◽  
pp. 4084-4090 ◽  
Author(s):  
Yuko Kumeda ◽  
Tsutomu Asao
Keyword(s):  
Airborne Fungi ◽  
Genetic Identification ◽  
Universal Primers ◽  
Morphological Observation ◽  
Rrna Gene ◽  
Panel Analysis ◽  
Polymer Gel ◽  
Lower Mobility ◽  
Novel Method ◽  
Species Specific

ABSTRACT For genetic identification of Aspergillus SectionFlavi isolates and detection of intraspecific variation, we developed a novel method for heteroduplex panel analysis (HPA) utilizing fragments of the internal transcribed spacer (ITS) regions (ITS1-5.8S-ITS2) of the rRNA gene that was PCR amplified with universal primers. The method involves formation of heteroduplexes with a set of reference fragments amplified from Aspergillus flavus, A. parasiticus, A. tamarii, and A. nomius and subsequent minislab vinyl polymer gel electrophoresis. The test panel is compared with species-specific standard panels (F-1, P-1, T-1, and N-1) generated by pairwise reannealing among four reference fragments. Of 90 test panels, 89 succeeded in identifying the species and 74 were identical to one of the four standard panels. Of the 16 new panels, 11A. flavus/A. oryzae panels were identical and typed as F-2 and 4 of 5 A. nomius panels were typed as N-2 or N-3. The other strain, A. nomius IMI 358749, was unable to identify the species because no single bands were formed with any of the four reference strains. DNA sequencing revealed that our HPA method has the highest sensitivity available and is able to detect as little as one nucleotide of diversity within the species. WhenPenicillium or non-Section Flavi Aspergilluswas subjected to HPA, the resulting bands of heteroduplexes showed apparently lower mobility and poor heteroduplex formation. This indicates that HPA is a useful identification method without morphological observation and is suitable for rapid and inexpensive screening of large numbers of isolates. The HPA typing coincided with the taxonomy of Section Flavi and is therefore applicable as an alternative to the conventional methods (Samson, R. A., E. S. Hoekstra, J. C. Frisvad, and O. Filtenborg, p. 64–97, in Introduction to Food- and Airborne Fungi, 6th ed., 2000).

Identification Pseudomonas aeruginosa by OprD Gene for Differentiation from Other Pseudomonas Species that Isolated from Clinical Samples

2019 ◽  
Vol 9 (01) ◽  
pp. 46-50
Author(s):  
Ashwak B Al-Hashimy ◽  
Huda S Alagely ◽  
Akeel K Albuaji ◽  
Khalid R Majeed
Keyword(s):  
Pseudomonas Aeruginosa ◽  
General Hospital ◽  
Culture Media ◽  
Final Diagnosis ◽  
Clinical Samples ◽  
Bacterial Isolates ◽  
Molecular Method ◽  
Biochemical Tests ◽  
Pseudomonas Species ◽  
Specific Sequences

The present study included the collection of 100 samples from various clinical sources for investigating the presence of P. aeruginosa in those sources, the samples have been collected from some hospitals in Baghdad and Hillah city (Al-qassim General Hospital, ,Al-hillah teaching hospital,and Al-hashimya General hospital ) which included wounds, burns, ear and sputum infections. The study was carried out through October 2017 till the end of March 2018. The samples were identified based on the morphological and microscopically characteristics of the colonies when they were culturing or number of culture media as well as biochemical tests, molecular identification were also used as a final diagnostic test for isolates that were positive as they belong to P.aeruginosa bacteria during previous tests based on the OprD gene which has specific sequences for P.aeruginosa bacteria as a detection gene and also consider as virulence factor so it have a synonyms mechanism to antibiotic resistance . The results of the final diagnosis showed that 38 isolates belong to target bacteria were distributed as 18 of burns, 11 isolates of wounds, 6 isolates of ear infection and 3 isolates of sputum, The examination of the sensitivity of all bacterial isolates was done for elected 38 isolation towards the 9 antibiotic by a Bauer - Kirby and the isolates were resistant for a number of antibiotics used such as Ciprofloxacin 65.7%, Norflaxacin 71%, Imipenem 63.1% Meropenem 68.4%, Gentamicin 65.7%, Amikacin 26.3%, Cefepime 68.4%, Ceftazidime 65.7% and Piperacillin 57.8%.Molecular method , All isolates (38) of P. aeruginosa positive for the diagnostic special gene (OprD) genes (100%).

scholarly journals Trichothecene Genotypes of Fusarium graminearum Populations Isolated from Winter Wheat Crops in Serbia

Toxins ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 460 ◽  
Author(s):  
Vesna Krnjaja ◽  
Slavica Stanković ◽  
Ana Obradović ◽  
Tanja Petrović ◽  
Violeta Mandić ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Gene Clusters ◽  
Sensu Stricto ◽  
Molecular Techniques ◽  
Weather Data ◽  
Morphological Observation ◽  
Specific Primer ◽  
Head Blight ◽  
As Species ◽  
Species Specific

Fusarium graminearum as the main causal agent of Fusarium head blight (FHB) and its ability to produce trichothecenes was investigated by molecular techniques. A total of 37 strains isolated from the wheat, harvested in Serbia in 2005, 2008 and 2015, and previously designated by morphological observation as F. graminearum, were used for trichothecene genotypes characterization. The strains were identified using the species-specific primer set FG16R/FG16F while genotypic characterization was done using specific TRI13 and TRI3 sequences of the trichothecene gene clusters. The PCR assays identified all strains as species of F. graminearum sensu stricto with the DON/15-ADON genotype. The quantification of the mycotoxin (DON) was performed using the biochemical assay. The high levels of DON (>20,000 µg kg−1) were recorded in all of the strains from 2005, four strains from 2008 and two strains from 2015. Weather data of the investigated seasons, showed that the optimal temperature, frequent rains and high relative humidity (RH) was very favourable for the development of F. graminearum, affecting the DON biosynthesis.

scholarly journals MOLECULAR DETECTION AND CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS ISOLATED FROM RAW MILK SOLD IN DIFFERENT MARKETS OF BANGLADESH

2017 ◽  
Vol 14 (2) ◽  
pp. 277-282 ◽  
Author(s):  
M. A. Islam ◽  
S. M. L. Kabir ◽  
M. T. Rahman
Keyword(s):  
Molecular Detection ◽  
Culture Media ◽  
Raw Milk ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Selective Media ◽  
Milk Samples ◽  
Antimicrobial Sensitivity ◽  
Antimicrobial Sensitivity Test

The study was intended for molecular detection of S. aureus isolated from raw cow’s milk. A total of 20 milk samples were collected from different upazila markets of Jamalpur, Tangail, Kishoreganj and Netrokona districts of Bangladesh. Milk samples were cultured onto various culture media for the isolation of bacteria. The isolated bacteria were identified by studying cultural properties on different selective media, biochemical tests, and finally by PCR. Out of 20 samples, 15 (75%) milk samples were found to be positive for S. aureus. S. aureus specific 16S rRNA gene was amplified from all isolates and identified as S. aureus. Antimicrobial sensitivity test was carried out to ascertain the susceptibility of the organism to various antibiotics. Its results showed that the S. aureus isolates were resistant to amoxicillin (100%), erythromycin (73.33%) and tetracycline (73.33%) but sensitive to azithromycin (93.33%), ciprofloxacin (93.33%), gentamicin (100%), norfloxacin (86.67%) and streptomycin (86.67%).

scholarly journals Pseudomonas aeruginosa as a Potential Contaminant of Packed Fresh-Cut Lettuce in a Controlled Atmosphere. The Role of Phenotypes muc+/muc–

2020 ◽  
Vol 11 (2) ◽  
pp. 8716-8724
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Real Time ◽  
Foodborne Pathogens ◽  
Rrna Gene ◽  
Modified Atmosphere ◽  
Fresh Cut ◽  
Time Specific ◽  
Sanitation Systems ◽  
Biofilm Development

In order to shed light on contamination risks along the ready-to-eat chain of fresh commodities by emerging foodborne pathogens, we investigated the biofilm development in vitro of two Pseudomonas aeruginosa strains on fresh-cut lettuce (Lactuca sativa L. var. Iceberg). The experiment was performed employing a floating bioreactor system where modified atmosphere package conditions were mimicked, and fresh-cut lettuce disks of 2 cm2 were put into contact with a 106 CFU/mL of a phenotypic mucoid P. aeruginosa phenotype (muc+) or a non-mucoid one (muc-). Following a simulated 2-day refrigerated-shelf quantitative Real-Time PCR, designed on a target gene region of the 16S rRNA gene, defined the different muc phenotypes behavior on biofilm in lettuce phyllo-plane. Between the two strains, a development difference of nearly 1.0 log CFU/cm2 occurred, with the muc+ phenotype being the most settled and adherent. This result clearly showed a distinct contamination risk according to P. aeruginosa phenotype and the need to develop real-time, specific, fast, and easy to use detection protocols along with specific sanitation systems for modified atmosphere package ready-to-eat commodities.

scholarly journals Isolation, Identification And Prevalence Of Pseudomonas Aeruginosa Isolates From Clinical And Environmental Sources In Onitsha Metropolis, Anambra State

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
C. O. Ezeador ◽  
P. C. Ejikeugwu ◽  
S. N. Ushie ◽  
N. R. Agbakoba
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Prevalence Rate ◽  
Nasal Swab ◽  
Blood Agar ◽  
Biochemical Tests ◽  
Anambra State ◽  
Mueller Hinton ◽  
Bluish Green ◽  
Mueller Hinton Agar

This study was aimed to isolate and identify Pseudomonas aeruginosa and to determine the prevalence rate of isolated P. aeruginosa in Hospitals in Onitsha. Isolates of P. aeruginosa were recovered from both clinical and environmental sources using Cetrimide agar, Blood agar, Mueller-Hinton agar and MacConkey agar.  All the inoculated plates were incubated at 37°C for 24-48 hours and growth was evaluated on these media. Isolates were identified on the basis of standard bacteriological methods like morphology, colonial characteristics, smell in culture, haemolysis, as well as pigment production on these media. All suspected isolates were further characterized and identified by many biochemical reactions. Results revealed that only 22 (18.3%) isolates were P. aeruginosa, while other 98 (81.7%) represented other bacterial genera. The 22 isolates included 19 (86.4%) environmental isolates and 3 (13.6%) clinical isolates. Pseudomonas aeruginosa was most commonly isolated from sink (13.6%), then mops and cleaning buckets (9.1%) and least from theatre bed, nasal swab, floor, disinfectant, ear and wound swab (4.5%). The pigment varied from bluish-green to yellowish-green with a grape-like odor. All isolates were Gram negative, produced β-hemolysis on blood agar and were motile. The biochemical tests showed all the isolates to be strongly positive for catalase, oxidase, citrate, and casein hydrolysis. The prevalence rate of P. aeruginosa is relatively high and its isolation from sources like sinks and theatre bed could be suggestive of the role of this pathogen in nosocomial infections.

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