Detection and Identification of Salmonella Typhimurium in Bovine Diarrhoeic Fecal Samples by Immunomagnetic Separation and Multiplex PCR Assay

ABSTRACT In the present work we develop a multiplex PCR assay for the detection and identification of the fish pathogen Vibrio vulnificus biotype 2 with discriminating potential for zoonotic strains (serovar E). The PCR assay allowed the identification of two new biotype 2 serovar E human isolates from culture collections. Finally, the multiplex was successfully applied to both diagnosis and carrier detection in field samples.

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Direct Detection and Identification of Arcobacter Species by Multiplex PCR in Chicken and Wastewater Samples from Spain

Journal of Food Protection ◽

10.4315/0362-028x-70.2.341 ◽

2007 ◽

Vol 70 (2) ◽

pp. 341-347 ◽

Cited By ~ 37

Author(s):

A. GONZÁLEZ ◽

S. BOTELLA ◽

R. M. MONTES ◽

Y. MORENO ◽

M. A. FERRÚS

Keyword(s):

Multiplex Pcr ◽

Water Samples ◽

Direct Detection ◽

Pcr Amplification ◽

Pcr Assay ◽

Direct Pcr ◽

Culture Methods ◽

Multiplex Pcr Assay ◽

Detection And Identification ◽

Wastewater Samples

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30°C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37°C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.

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Large-volume immunomagnetic separation combined with multiplex PCR assay for simultaneous detection of Listeria monocytogenes and Listeria ivanovii in lettuce

Food Control ◽

10.1016/j.foodcont.2015.06.048 ◽

2016 ◽

Vol 59 ◽

pp. 601-608 ◽

Cited By ~ 53

Author(s):

Yan Mao ◽

Xiaolin Huang ◽

Sicheng Xiong ◽

Hengyi Xu ◽

Zoraida P. Aguilar ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Multiplex Pcr ◽

Large Volume ◽

Simultaneous Detection ◽

Immunomagnetic Separation ◽

Pcr Assay ◽

Listeria Ivanovii ◽

Multiplex Pcr Assay

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Evaluation of a Multiplex PCR Assay for the Identification of Salmonella Serovars Enteritidis and Typhimurium Using Retail and Abattoir Samples

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-167 ◽

2017 ◽

Vol 80 (2) ◽

pp. 295-301 ◽

Cited By ~ 12

Author(s):

Dele Ogunremi ◽

Susan Nadin-Davis ◽

Andrée Ann Dupras ◽

Imelda Gálvan Márquez ◽

Katayoun Omidi ◽

...

Keyword(s):

Salmonella Typhimurium ◽

Multiplex Pcr ◽

Internal Control ◽

Broiler Chickens ◽

Salmonella Enteritidis ◽

Bacterial Species ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Retail Outlets ◽

Dna Fragment

ABSTRACT A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, namely, serovars Enteritidis and Typhimurium. The PCR was designed to amplify DNA fragments from four Salmonella genes, namely, invA gene (211-bp fragment), iroB gene (309-bp fragment), Typhimurium STM 4497 (523-bp fragment), and Enteritidis SE147228 (612-bp fragment). In addition, a 1,026-bp ribosomal DNA (rDNA) fragment universally present in bacterial species was included in the assay as an internal control fragment. The detection rate of the PCR was 100% among Salmonella Enteritidis (n = 92) and Salmonella Typhimurium (n = 33) isolates. All tested Salmonella isolates (n = 194) were successfully identified based on the amplification of at least one Salmonella-specific DNA fragment. None of the four Salmonella DNA amplicons were detected in any of the non-Salmonella isolates (n = 126), indicating an exclusivity rate of 100%. When applied to crude extracts of 2,001 field isolates of Salmonella obtained during the course of a national microbiological baseline study in broiler chickens and chicken products sampled from abattoir and retail outlets, 163 isolates, or 8.1%, tested positive for Salmonella Enteritidis and another 80 isolates, or 4.0%, tested as Salmonella Typhimurium. All isolates identified by serological testing as Salmonella Enteritidis in the microbiological study were also identified by using the multiplex PCR. The new test can be used to identify or confirm pure isolates of the two serovars and is also amenable for integration into existing culture procedures for accurate detection of Salmonella colonies.

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Detection and Identification ofActinobacillus pleuropneumoniae Serotype 5 by Multiplex PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.36.6.1704-1710.1998 ◽

1998 ◽

Vol 36 (6) ◽

pp. 1704-1710 ◽

Cited By ~ 38

Author(s):

Terry M. Lo ◽

Christine K. Ward ◽

Thomas J. Inzana

Keyword(s):

Early Detection ◽

Multiplex Pcr ◽

Sensitivity And Specificity ◽

Dna Sequences ◽

Capsular Polysaccharide ◽

Actinobacillus Pleuropneumoniae ◽

Pcr Assay ◽

Multiplex Pcr Assay ◽

Detection And Identification ◽

Serotype 5

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniaeserotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 102 CFU. The A. pleuropneumoniaeserotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.

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