Isolation and Identification of Campylobacter spp. from Poultry and Poultry By-Products in Tunisia by Conventional Culture Method and Multiplex Real-Time PCR

2017 ◽  
Vol 80 (10) ◽  
pp. 1623-1627 ◽  
Author(s):  
Hela Jribi ◽  
Hanen Sellami ◽  
Siala Mariam ◽  
Salma Smaoui ◽  
Asma Ghorbel ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Poultry Meat ◽  
Culture Methods ◽  
Isolation And Identification ◽  
Conventional Culture ◽  
Multiplex Pcr Assay ◽  
Thermophilic Campylobacter ◽  
By Products ◽  
Campylobacter Spp

ABSTRACT Thermophilic Campylobacter spp. are one of the primary causes of bacterial human diarrhea. The consumption of poultry meats, by-products, or both is suspected to be a major cause of human campylobacteriosis. The aims of this study were to determine the prevalence of thermophilic Campylobacter spp. in fresh poultry meat and poultry by-products by conventional culture methods and to confirm Campylobacter jejuni and Campylobacter coli isolates by using the multiplex PCR assay. Two hundred fifty fresh poultry samples were collected from a variety of supermarkets and slaughterhouses located in Sfax, Tunisia, including chicken (n =149) and turkey (n =101). The samples were analyzed using conventional microbiological examinations according to the 2006 International Organization for Standardization method (ISO 10272-1) for Campylobacter spp. Concurrently, a real-time PCR was used for identification of C. jejuni and C. coli. Of the 250 samples of poultry meat and poultry by-products, 25.6% (n = 64) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (26.8%) followed by turkey meat (23.7%). Among the different products, poultry breasts showed the highest contamination (36.6%) followed by poultry by-products (30%), poultry wings (28%) and poultry legs (26%) showed the lowest contamination, and no contamination was found on neck skin. Of the 64 thermophilic Campylobacter isolates, C. jejuni (59.7%) was the most frequently isolated species and 10.9% of the isolates were identified as C. coli. All of the 64 Campylobacter isolates identified by the conventional culture methods were further confirmed by PCR. The seasonal peak of Campylobacter spp. contamination was in the warm seasons (spring and summer). The study concluded that high proportions of poultry meat and poultry by-products marketed in Tunisia are contaminated by Campylobacter spp. Furthermore, to ensure food safety, poultry meats must be properly cooked before consuming.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

Rapid, Specific Detection of Salmonella Enteritidis in Pooled Eggs by Real-Time PCR

2004 ◽  
Vol 67 (5) ◽  
pp. 864-869 ◽  
Author(s):  
K. H. SEO ◽  
I. E. VALENTIN-BON ◽  
R. E. BRACKETT ◽  
P. S. HOLT
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Specific Detection ◽  
Culture Methods ◽  
Conventional Culture ◽  
Low Concentrations ◽  
Pcr Method ◽  
Group D

An assay was developed for the specific detection of Salmonella Enteritidis in eggs with the use of an application of the fluorogenic 5′ nuclease assay (TaqMan). In this assay, a segment of the gene sefA specific to Salmonella group D strains such as Salmonella Enteritidis was used. The amplification of the target gene products was monitored in real-time by incorporating a fluorescent dye–labeled gene-specific probe in the PCR reaction. This method correctly detected and distinguished Salmonella Enteritidis from nearly 50 of non–group D Salmonella and other non- Salmonella strains. Detection of the sefA gene was linear for DNA extracted from approximately 102 to 109 CFU/ml in phosphate-buffered saline and 103 to 108 CFU/ml in raw egg. In two trials, when applied to detection of Salmonella Enteritidis in homogenized egg pools and compared with conventional culture methods, the newly developed PCR method yielded a 100% correlation with results obtained by a conventional culture method. However, the PCR method required only 2 days, compared to the 5 days required by the culture method. The sensitivity of this assay was approximately less than 1 CFU/600 g of egg pool. The real-time PCR assay proved to be a rapid, highly sensitive test for detection and quantification of low concentrations of Salmonella Enteritidis in egg samples.

scholarly journals Evaluation of the Cobas TaqMan MTB real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory specimens

2013 ◽  
Vol 62 (8) ◽  
pp. 1160-1164 ◽  
Author(s):  
Meng-Rui Lee ◽  
Kuei-Pin Chung ◽  
Hao-Chien Wang ◽  
Chih-Bin Lin ◽  
Chong-Jen Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Reference Method ◽  
Rapid Identification ◽  
Culture Methods ◽  
Cobas Taqman ◽  
Conventional Culture ◽  
Respiratory Specimens

The Cobas TaqMan MTB assay is a real-time PCR (qPCR) kit for rapid detection of Mycobacterium tuberculosis from clinical specimens. There are, however, limited studies validating its performance. We performed a prospective study in two hospitals in Taiwan on 586 respiratory specimens. By using culture as the reference method, the sensitivity and specificity of the Cobas TaqMan MTB assay were found to be 82.7 and 96.5 %, respectively. The sensitivity of the Cobas TaqMan MTB assay in acid-fast stain-negative respiratory specimens was only 34.9 %. Five specimens from five patients were positive for M. tuberculosis by the Cobas TaqMan MTB assay but were negative for M. tuberculosis by conventional culture methods. A diagnosis of pulmonary tuberculosis (TB) was made based on clinical and radiological findings as well as the response to anti-TB treatment in these five patients. Addition of data from these five specimens with discrepant results (PCR vs culture) from patients with symptoms clinically compatible with TB increased the sensitivity of the Cobas TaqMan MTB assay to 83.1 %. The Cobas TaqMan MTB assay is a rapid identification tool with a high degree of specificity for the direct detection of M. tuberculosis in respiratory specimens. The sensitivity for detecting acid-fast smear-negative respiratory specimens, however, is low.

An Evaluation of Conventional Culture, invA PCR, and the Real-Time PCR iQ-Check Kit as Detection Tools for Salmonella in Naturally Contaminated Premarket and Retail Turkey

2008 ◽  
Vol 71 (2) ◽  
pp. 386-391 ◽  
Author(s):  
CHANTAL W. NDE ◽  
MOHAMED K. FAKHR ◽  
CURT DOETKOTT ◽  
CATHERINE M. LOGUE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
False Positives ◽  
Poultry Meat ◽  
Processing Plant ◽  
False Negatives ◽  
Conventional Culture ◽  
Total Agreement ◽  
Conventional Culture Method

This study was aimed at comparing the ability of conventional culture, the iQ-Check real-time PCR kit, and invA PCR to detect Salmonella in naturally contaminated premarket and retail turkey parts. Premarket (n = 120) turkey parts collected from a commercial turkey processing plant, and retail turkey parts (n = 138) were examined. Both PCR methods detected a significantly greater (P < 0.05) number of positive samples when compared with the conventional culture method for the premarket turkey parts. The indices of total agreement between the conventional culture method and the iQ-Check kit for the premarket and retail parts were 79.2% (95% CI: 70.8, 86) and 90.6% (95% CI: 84.4, 94.9), respectively. When the conventional culture method was compared with invA PCR for Salmonella detection in the premarket and retail parts, the indices of total agreement were 75.8% (95% CI: 67.2, 83.2) and 84.1% (95% CI: 76.9, 89.7), respectively. The rates of false positives (premarket: 31.9%, retail: 9.7%) and false negatives (premarket: 5.9%, retail: 9.7%) were determined between the culture method and the iQ-Check kit. When invA PCR was compared with the culture method, the rates of false positives (premarket: 37.7%, retail: 11.1%) and false negatives (premarket: 5.9%, retail: 18.3%) were obtained. The higher total agreement and the lower rates of both false positives and false negatives for the iQ-Check kit compared with invA PCR for both premarket and retail turkey parts corroborates the use of the iQ-Check kit as a screening tool for Salmonella in poultry meat.

scholarly journals Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam

2016 ◽  
Vol 15 (1) ◽  
pp. 155-162 ◽  
Author(s):  
Pierangeli G. Vital ◽  
Nguyen Thi Van Ha ◽  
Le Thi Hong Tuyet ◽  
Kenneth W. Widmer
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Surface Waters ◽  
Real Time Pcr ◽  
Membrane Filtration ◽  
Quantitative Real Time Pcr ◽  
Wet Season ◽  
Culture Methods ◽  
Fecal Indicator ◽  
Filtration Method

Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

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