Evaluation of a 5′ -Nuclease (TaqMan) Assay with the Thin Agar Layer Oxyrase Method for the Detection of Yersinia enterocolitica in Ground Pork Samples†

2004 ◽  
Vol 67 (2) ◽  
pp. 271-277 ◽  
Author(s):  
V. C. H. WU ◽  
D. Y. C. FUNG ◽  
R. D. OBERST
Keyword(s):  
Detection Limit ◽  
Yersinia Enterocolitica ◽  
Food Samples ◽  
Selective Medium ◽  
Taqman Assay ◽  
Ground Pork ◽  
Sensitivity Studies ◽  
Agar Layer ◽  
Pork Samples ◽  
Nuclease Assay

A 5′-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica. Sensitivity studies of a 5′-nuclease assay for detecting Y. enterocolitica O:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5′-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of “Oxyrase® for Agar” onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (−15°C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN (P < 0.05). The 5′-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.

Enrichment Procedures and Plating Media for Isolation of Yersinia enterocolitica†

2000 ◽  
Vol 63 (11) ◽  
pp. 1483-1486 ◽  
Author(s):  
G. C. JIANG ◽  
DONG-HYUN KANG ◽  
DANIEL Y. C. FUNG
Keyword(s):  
Yersinia Enterocolitica ◽  
Selective Medium ◽  
Ground Meat ◽  
Isolation Rate ◽  
Direct Plating ◽  
Morphologic Characteristics ◽  
Ground Pork ◽  
Meat Samples ◽  
Pork Samples ◽  
Combined Data

A shortened enrichment procedure (25°C for 24 h) was compared with cold enrichment procedures (4°C for 1 to 3 weeks) and direct plating for isolation of Yersinia enterocolitica from commercial ground meat samples. The combined data of all recovery procedures showed that this organism was isolated from 34% of the ground beef samples. The highest isolation rate was 32% for the 4°C/3-week enrichment, followed by 28% for the 4°C/2-week enrichment, 26% for the 25°C/24-h enrichment, 22% for the 4°C/1-week enrichment, and 10% for direct plating. No significant differences (P > 0.05) in isolation rate occurred between the 4°C/3-week, 4°C/2-week, 25°C/24-h, and 4°C/1-week enrichments. The combined data of all recovery procedures showed that Y. enterocolitica was isolated from 64% of ground pork samples. The highest isolation rate was 48% for the 4°C/3-week enrichment, followed by 40% for the 25°C/24-h enrichment, 34% for the 4°C/2-week enrichment, 24% for the 4°C/1-week enrichment, and 24% for direct plating. No significant differences (P > 0.05) in isolation rate occurred between the 4°C/3-week, 25°C/24-h, and 4°C/2-week enrichments. During the plating phase of the experiment, the efficiency of a dye-containing, Yersinia-selective medium (KV202) was compared with that of a commercially available cefsulodin-irgasan-novobiocin medium. Recovery rates were similar for both media. However, KV202 agar differentiated Y. enterocolitica from such contaminating bacteria as Enterobacter, Serratia, and Salmonella by colony morphologic characteristics and color.

Evaluation of a 5′-Nuclease (TaqMan) Assay for the Detection of Virulent Strains of Yersinia enterocolitica in Raw Meat and Tofu Samples†

2001 ◽  
Vol 64 (3) ◽  
pp. 355-360 ◽  
Author(s):  
A. VISHNUBHATLA ◽  
R. D. OBERST ◽  
D. Y. C. FUNG ◽  
W. WONGLUMSOM ◽  
M. P. HAYS ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Detection System ◽  
Detection Methods ◽  
Taqman Assay ◽  
Contamination Rate ◽  
Virulence Plasmid ◽  
Recovery Method ◽  
Ground Pork ◽  
Pure Cultures ◽  
Nuclease Assay

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5′-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with “gold standard” methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica. The 5′-nuclease assay was able to identify the organism in 100% of the repetitions when 102 CFU/ml or more organisms were present in pure cultures and 103 CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 105 CFU/ml or more organisms were present in pure culture and 106 CFU/g or more organisms were present in inoculated ground pork. The 5′-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5′-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5′-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.

scholarly journals Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

2000 ◽  
Vol 66 (9) ◽  
pp. 4131-4135 ◽  
Author(s):  
A. Vishnubhatla ◽  
D. Y. C. Fung ◽  
R. D. Oberst ◽  
M. P. Hays ◽  
T. G. Nagaraja ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Pcr Amplification ◽  
Enteric Bacteria ◽  
Taqman Assay ◽  
Virulent Strains ◽  
Field Samples ◽  
Ground Pork ◽  
Standard Culture ◽  
Pure Cultures ◽  
Nuclease Assay

ABSTRACT We have developed a rapid procedure for the detection of virulentYersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species ofYersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri,Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia,Salmonella, Citrobacter, andFlavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥102 CFU/ml in pure cultures and ≥103 CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked withY. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocoliticawith both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.

scholarly journals Detection ofYersinia enterocoliticain Retail Chicken Meat, Mashhad, Iran

2018 ◽  
Vol 2018 ◽  
pp. 1-4 ◽  
Author(s):  
Khadigeh Sirghani ◽  
Tayebeh Zeinali ◽  
Abdollah Jamshidi
Keyword(s):  
Yersinia Enterocolitica ◽  
Health Hazard ◽  
Culture Method ◽  
Good Alternative ◽  
Food Samples ◽  
Selective Medium ◽  
Chicken Meat ◽  
Poultry Meat ◽  
Rrna Gene ◽  
Pcr Assay

Poultry meat is one of the most important sources of infection ofYersiniaspp. for humans. The aim of the present study was to evaluate the incidence ofYersinia enterocoliticain chicken meat by using culture method on selective medium and confirmation by PCR assay. Also, biochemical methods were used for biotyping. A total of 100 chicken thigh meat samples were collected randomly from retail outlets in Mashhad, Iran. Samples were enriched in Peptone-Sorbitol-Bile (PSB) broth and then cultured on Cefsulodin-Irgasan-Novobiocin (CIN) agar containing antibiotics supplement. The DNA was extracted from suspected colonies ofYersiniaspp. and then PCR test using specific primers for 16S rRNA gene ofYersinia enterocoliticawas performed. In this study, 30% of chicken meat was contaminated withYersiniaspp. by culture method and 25% of chicken meat was contaminated withYersinia enterocolitica. Biotyping of isolated colonies showed that all of the isolates belonged to biotype 1A. Culture and detection ofYersiniaspp. from food samples traditionally take 4 days. Due to high accuracy and speed of PCR assay, it is a good alternative method for microbiological techniques. In conclusion, poultry meat can act as a source ofY. enterocoliticaand could be considered as a public health hazard.

scholarly journals Development of IMBs-qPCR Detection Method for Yersinia enterocolitica Based on the foxA Gene

2021 ◽  
Author(s):  
Jingxuan Shi ◽  
Heng Chi ◽  
Aiping Cao ◽  
Yinna Song ◽  
Min Zhu ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Culture Method ◽  
Food Samples ◽  
Pcr Detection ◽  
Immunomagnetic Beads ◽  
Foxa Gene ◽  
Qpcr Method ◽  
Virulence Factor Gene ◽  
Food Safety Risk ◽  
Pork Samples

Abstract Yersinia enterocolitica is an important zoonotic pathogen, which seriously endangers food safety risk. In this study, the recombinant outer membrane protein OmpF and its antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Y. enterocolitica in food samples, combining the quantitative PCR detection with primers of virulence factor gene fox A for Yersinia enterocolitica contamination. The results showed that the capture efficiency of approximately 80% using anti-OmpF antibody-immunomagnetic beads and linearly dependent capture under 10 1 -10 5 CFU/mL Y. enterocolitica . compare with less than 10% capture of other bacteria. The detection limit of 64 CFU/mL was obtained by based on fox A gene PCR detection combined with capture of the anti-OmpF antibody-immunomagnetic beads to detect Yersinia enterocolitica in artificially contaminated milk and pork samples. Comparing with the culture method, the developed IMBs-qPCR method has higher consistency, less time consuming, which providing an effective alternative method for rapid detection of Y. enterocolitica in food.

Comparison of Culture, Multiplex, and 5′ Nuclease Polymerase Chain Reaction Assays for the Rapid Detection of Yersinia enterocolitica in Swine and Pork Products†

2001 ◽  
Vol 64 (9) ◽  
pp. 1352-1361 ◽  
Author(s):  
SANDHYA BOYAPALLE ◽  
IRENE V. WESLEY ◽  
H. SCOTT HURD ◽  
P. GOPAL REDDY
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Culture Method ◽  
Taqman Assay ◽  
Chain Reaction ◽  
Pork Products ◽  
Ground Pork ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Pork Samples

Bacteriological culture was compared with multiplex and fluorogenic (TaqMan) polymerase chain reaction (PCR) assays for the detection of attachment invasion locus (ail)-bearing Yersinia enterocolitica in market weight swine, chitterlings, and ground pork. The TaqMan assay detected 1 pg of purified Y. enterocolitica DNA, whereas conventional gel-based PCR detected 1 ng of the same. The presence of ail-bearing Y. enterocolitica was tested in pork and feces artificially inoculated with Y. enterocolitica strain NADC 5561. The sensitivity limits of culture, multiplex, and TaqMan PCR assays were 4 × 103, 4 × 102, and 0.4 CFU/g, respectively, for the artificially inoculated pork. The sensitivity limits were 4 × 102, 4 × 102, and 0.4 CFU/g, respectively, for feces after a 48-h enrichment in a Yersinia selective broth. By the culture method, Y. enterocolitica was not detected in any of the swine specimens (n = 2,403) examined. By contrast, it was detected in 48 (2%) of the swine samples screened using the multiplex PCR and in 656 (27.2%) of these samples using the TaqMan assay. Using the culture method, Y. enterocolitica was detected in 8% of chitterling samples (n = 350) and in none of the ground pork samples (n = 350). It was identified in 27% of the chitterling samples using multiplex PCR and in 79% of these samples using the TaqMan assay. Ten percent of the ground pork samples contained Y. enterocolitica, as determined by the multiplex PCR, and 38% based on the TaqMan assay. The results suggest that pork products harbor more ail-bearing Y. enterocolitica than selected organs of freshly slaughtered hogs and that the TaqMan assay is more sensitive than either the multiplex PCR or traditional culture methods.

scholarly journals Identification and Characterization of Pathogenic Yersinia enterocolitica Isolates by PCR and Pulsed-Field Gel Electrophoresis

2005 ◽  
Vol 71 (7) ◽  
pp. 3674-3681 ◽  
Author(s):  
S. Thisted Lambertz ◽  
M.-L. Danielsson-Tham
Keyword(s):  
Multiplex Pcr ◽  
Yersinia Enterocolitica ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Virulence Plasmid ◽  
Pork Meat ◽  
Pulsed Field ◽  
Pork Products ◽  
Food Borne ◽  
Pork Samples

ABSTRACT Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n = 48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.

Simultaneous Enrichment of Shiga Toxin–Producing Escherichia coli O157 and O26 and Salmonella in Food Samples Using Universal Preenrichment Broth

2009 ◽  
Vol 72 (10) ◽  
pp. 2065-2070 ◽  
Author(s):  
MASASHI KANKI ◽  
KAZUKO SETO ◽  
JUNKO SAKATA ◽  
TETSUYA HARADA ◽  
YUKO KUMEDA
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Food Samples ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Significant Difference ◽  
Peptone Water ◽  
Radish Sprouts ◽  
Pork Samples ◽  
Better Than

Universal preenrichment broth (UPB) was compared with modified Escherichia coli broth with novobiocin (mEC+n) for enrichment of Shiga toxin–producing E. coli O157 and O26, and with buffered peptone water (BPW) for preenrichment of Salmonella enterica. Ten strains each of the three pathogens were inoculated into beef and radish sprouts following thermal, freezing, or no treatment. With regard to O157 and O26, UPB incubated at 42°C recovered significantly more cells from inoculated beef than UPB at 35°C and from radish sprout samples than UPB at 35°C and mEC+n. With regard to Salmonella, UPB incubated at 42°C was as effective as UPB at 35°C and BPW at recovering cells from beef and radish sprout samples. No significant difference was noted between the effectiveness of UPB at 42°C and UPB at 35°C or BPW in the recovery of Salmonella from 205 naturally contaminated poultry samples. By using UPB at 42°C, one O157:H7 strain was isolated from the mixed offal of 53 beef samples, 6 cattle offal samples, and 50 pork samples all contaminated naturally, with no pathogen inoculation. The present study found that UPB incubated at 42°C was as effective as, or better than, mEC+n for enrichment of O157 and O26 and comparable to BPW for preenrichment of Salmonella. These findings suggest that a great deal of labor, time, samples, and space may be saved if O157, O26, and Salmonella are enriched simultaneously with UPB at 42°C.

A Novel Electrochemical Sensor for the Analysis of Salbutamol in Pork Samples by Using NiFe2O4 Nanoparticles Modified Glassy Carbon Electrode

2013 ◽  
Vol 850-851 ◽  
pp. 1279-1282 ◽  
Author(s):  
Su Xing Luo ◽  
Yuan Hui Wu ◽  
Hua Gou ◽  
Yan Liu
Keyword(s):  
Detection Limit ◽  
Electrochemical Sensor ◽  
Glassy Carbon Electrode ◽  
Glassy Carbon ◽  
Electrochemical Method ◽  
Carbon Electrode ◽  
Modified Glassy Carbon Electrode ◽  
Peak Current ◽  
Anodic Peak Current ◽  
Pork Samples

In this work, a simple and sensitive electrochemical method sensor was developed to determine salbutamol based on magnetic NiFe2O4nanoparticles modified glassy carbon electrode. It was found the anodic peak current of salbutamol was linear with the concentration of salbutamol from 2.0 μM to 60 μM with a detection limit of 1.0 μM (S/N=3). The developed method was successfully applied to determine salbutamol content in pork samples with satisfactory results.

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