Application of Random Amplified Polymorphic DNA Analysis for Detection of Salmonella spp. in Foods

1998 ◽  
Vol 61 (7) ◽  
pp. 785-791 ◽  
Author(s):  
TAKAHISA MIYAMOTO ◽  
HUAI ZE TIAN ◽  
TAKASHI OKABE ◽  
SUDSAI TREVANICH ◽  
KAORU ASOH ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Culture Method ◽  
Cell Number ◽  
Oligonucleotide Primer ◽  
Salmonella Spp ◽  
Initial Inoculum ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Raw Foods

The random amplified polymorphic DNA (RAPD) band patterns from 23 Salmonella spp. produced by use of an oligonucleotide primer (called du primer) designed on the basis of the N-terminal sequence of dulcitol 1-phosphate dehydrogenase (5'-GTGGTGACCCAGGATGGCCAGGTG-3') were different from those from 16 non-Salmonella spp. The bands at 460 and 700 bp were produced in all Salmonella strains tested. These RAPD fragments obtained from Salmonella typhimurium strongly hybridized with the corresponding RAPD bands from the other strains of Salmonella, but not with those from non-Salmonella spp. in Southern blot analysis. The RAPD bands were detected by ethidium bromide staining even when genomic DNA prepared from as few as 2.8 × 103 cells was used. The minimum detectable cell number in the initial inoculum of S. typhimurium was 4 × 10 −1 CFU/25 g of raw beef after the preenrichment in Enterobacteriaceae enrichment mannitol (EEM) broth for 6 h and the selective enrichment in dulcitol-magnesium chloride-pyridinesulfonic acid-brilliant green-novobiocin (DMPBN) medium for 18 h at 42°C. Seven raw foods inoculated with S. typhimurium at numbers from 4 × 10−1 to 2.6 × 102 CFU/25 g of food were positive in both the RAPD analysis and the conventional culture method.

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.

scholarly journals High Prevalence of yadA-Positive Yersinia enterocolitica in Pig Tongues and Minced Meat at the Retail Level in Finland

1999 ◽  
Vol 62 (2) ◽  
pp. 123-127 ◽  
Author(s):  
MARIA FREDRIKSSON-AHOMAA ◽  
SEBASTIAN HIELM ◽  
HANNU KORKEALA
Keyword(s):  
Yersinia Enterocolitica ◽  
Culture Method ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
High Prevalence ◽  
Retail Outlets ◽  
Polymerase Chain ◽  
Meat Samples ◽  
Minced Meat ◽  
Conventional Culture Method

Polymerase chain reaction (PCR) was used to determine the prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland and to confirm the yadA-positive Y. enterocolitica isolates recovered from the same samples using the conventional culture method. A total of 51 pig tongues purchased at 12 retail outlets and 255 minced meat samples purchased at 40 retail outlets in the Helsinki area were studied. The prevalence of Y. enterocolitica carrying the yadA gene was 92% in pig tongues and 25% in minced meat using PCR and 78% in tongues and 2% in minced meat with the culture method. The prevalence of yadA-positive tongues was higher (98%) when both PCR- and culture-positive results were included because Y. enterocolitica carrying the yadA gene could also be isolated in three PCR-negative tongue samples. In the minced meat samples, all PCR-negative samples were also culture-negative. With the culture method, 66 of 80 yadA-positive isolates in 38 tongues and all yadA-positive isolates (4) in four minced meat samples were recovered after selective enrichment. A total of 92 isolates of Y. enterocolitica bioserotype 4/O:3 in tongues and 5 isolates in minced meat were found, of which 13% in tongues and 20% in minced meat did not carry the yadA gene.

Rapid Detection of Salmonella spp. in Foods by Combination of a New Selective Enrichment and a Sandwich ELISA Using Two Monoclonal Antibodies against Dulcitol 1-Phosphate Dehydrogenase

1996 ◽  
Vol 59 (11) ◽  
pp. 1158-1163 ◽  
Author(s):  
HUAIZE TIAN ◽  
TAKAHISA MIYAMOTO ◽  
TAKASHI OKABE ◽  
YOICHIRO KURAMITSU ◽  
KEN-ICHI HONJOH ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Rapid Detection ◽  
Enrichment Culture ◽  
Sandwich Elisa ◽  
False Negative ◽  
Detection Sensitivity ◽  
Elisa Method ◽  
Salmonella Spp ◽  
Selective Enrichment ◽  
Raw Foods

A rapid-detection method was developed for food-borne dulcitol-positive Salmonella spp. in foods that involves a new preenrichment and selective enrichment system and a sandwich ELISA using two monoclonal antibodies against dulcitol 1-phosphate dehydrogenase. Preenrichment and selective enrichment were in Enterobacteriaceae enrichment mannitol (EEM) broth at 42°C for 6 h and in a new dulcitol-magnesium chloride-pyridinesulfonic acid brilliant green-novobiocin (DMPBN) medium at 42°C for 27 h, respectively. The cells were collected from the selective enrichment culture and suspended in 0.1 ml of 1 N NaOH for 2 min. The solution was neutralized with 0.1 ml of 2 M Tris-HCl buffer (pH 7.5) and the mixture was used as a sample for ELISA. The detection sensitivity of the ELISA was 105 CFU of Salmonella spp. per ml of culture. Competing non-Salmonella organisms in raw food did not interfere with the detection of Salmonella cells even when present at 107: 1 (non-Salmonella: Salmonella ratio) in food. Nonmotile Salmonella gallinarum was detected by the ELISA. The minimum detectable number of initial inoculum of Salmonella typhimurium was 0.69 CFU/25 g of raw chicken after the preenrichment in EEM broth and the selective enrichment in DMPBN medium. The present ELISA method required a total analysis time of 36 h including the preenrichment and selective enrichment periods. The ELISA method was compared with a conventional cultural method for the detection of Salmonella cells in 130 samples of raw foods. Of the samples tested, 16 were Salmonella-positive and 114 samples were negative by both methods. False-positive and false-negative results were not encountered.

Comparison of Salmonella Bio-EnzaBead Immunoassay Method and Conventional Culture Procedure for Detection of Salmonella in Foods

1987 ◽  
Vol 50 (5) ◽  
pp. 379-385 ◽  
Author(s):  
KARL F. ECKNER ◽  
RUSSELL S. FLOWERS ◽  
BARBARA J. ROBISON ◽  
JEROME A. MATTINGLY ◽  
DAMIEN A. GABIS ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Enzyme Immunoassay ◽  
Culture Method ◽  
Food Samples ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Significant Difference ◽  
Standard Culture ◽  
Raw Shrimp ◽  
Culture Procedure

A rapid enzyme immunoassay screening procedure (EIA) utilizing two monoclonal antibodies specific for salmonellac was compared to the standard culture method (BAM/AOAC) on 1,289 samples representing 26 food types. The samples consisted of 760 artificially inoculated, 150 naturally contaminated, and 379 uninoculated food samples. There were 594 samples positive by the EIA (optical densities greater than 0.2 at 405 nm), of which 568 were confirmed culturally from M-broth. A total of 570 samples was positive by the BAM/AOAC procedure. Of the foods tested, there was no significant difference between the two methods, with the exception of cake mix and raw shrimp. The EIA was significantly better for detecting Salmonella in cake mix, while the culture procedure was more productive for shrimp. The method employed a 24 ± 2-h preen-richment, an 18-h selective enrichment, and a 6-h M-broth post-enrichment. The EIA assay required an additional 2 h for a total of 48 h, compared to a minimum of 4 d by BAM/AOAC.

Prevalence of Salmonella species from poultry eggs of local stores in Duhok

2017 ◽  
Vol 5 (6) ◽  
pp. 2468 ◽  
Author(s):  
Anas I. Zubair ◽  
Mohammad Ismail Al-Berfkani ◽  
Araz Ramadhan Issa
Keyword(s):  
Salmonella Enteritidis ◽  
Public Health Problem ◽  
Culture Method ◽  
Salmonella Typhi ◽  
Major Public Health Problem ◽  
Salmonella Spp ◽  
Isolation And Identification ◽  
Conventional Culture ◽  
Poultry Eggs ◽  
Local Store

Background: Salmonellosis is one of the foodborne illness acquired by consumption of infected raw or undercooked eggs and causes major public health problem. The aim of this study was isolation and identification of Salmonella spp. from the eggshells and the egg contents samples.Methods: In this study, a total 350 eggs were randomly collected from five local stores in Duhok and Zakho city over a period of 6 months in summer of 2016. Eggs from each local store were collected and transferred to the microbiology laboratory. The conventional culture method used for detection of Salmonella spp.Results: Out of the 350 eggs, seventeen (4.85%) samples of eggshells contaminated with Salmonella spp. and none of the egg content samples were contaminated with Salmonella genus. Out of 17 positive eggs, three different Salmonella serotypes were identified including; Salmonella enteritidis (10 strains), Salmonella typhimurium (5 strains), Salmonella typhi (2 strains).Conclusions: The results of the present study provide the recent dataset of the prevalence of Salmonella spp. in eggs sold at local stores in the city. All isolates showed resistant to tetracycline, oxacillin and sulphadimethoxazole due to the indiscriminate use of these antibiotics in chicken at sub-therapeutic level or prophylactic doses which promotes selection of antimicrobial resistant strains and also increases the human health risks associated with consumption of contaminated quail eggs. To the best of our knowledge, this is the first study in Zakho- Duhok city, investigating the occurrence of Salmonella spp. in eggshell and content egg sold at local stores.

scholarly journals Existence of two geographically-linked clonal lineages in the bacterial fish pathogen Photobacterium damselae subsp. piscicida evidenced by random amplified polymorphic DNA analysis

2000 ◽  
Vol 125 (1) ◽  
pp. 213-219 ◽  
Author(s):  
B. MAGARIÑOS ◽  
A. E. TORANZO ◽  
J. L. BARJA ◽  
J. L. ROMALDE
Keyword(s):  
Dna Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Epidemiological Studies ◽  
Pcr Analysis ◽  
Oligonucleotide Primer ◽  
Fish Pathogen ◽  
Dice Coefficient ◽  
Host Fish ◽  
Rapd Pcr ◽  
Photobacterium Damselae

In this work, we applied the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida), an important pathogen for different marine fish. Regardless of the oligonucleotide primer employed, the 29 isolates of Ph. damselae subsp. piscicida tested were separated into two groups, the RAPD-PCR analysis differentiated the European strains from the Japanese strains. The similarity between both groups estimated on the basis of the Dice coefficient was 75–80%. These results show that European and Japanese isolates of Ph. damselae subsp. piscicida, regardless of their host fish species, belong to two different clonal lineages. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.

Evaluation of TA10 Broth for Recovery of Listeria monocytogenes from Ground Beef

2017 ◽  
Vol 100 (2) ◽  
pp. 470-473
Author(s):  
Naoko Kamisaki-Horikoshi ◽  
Yukio Okada ◽  
Kazuko Takeshita ◽  
Makoto Takada ◽  
Shinichi Kawamoto ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Ground Beef ◽  
Culture Method ◽  
Most Probable Number ◽  
Salmonella Spp ◽  
Probable Number ◽  
Enrichment Broth ◽  
Conventional Culture ◽  
Significant Difference ◽  
Peptone Water

Abstract In 2009, the enrichment broth TA10 was released for simultaneous recovery of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7. This medium was compared with other Salmonella enrichment broths [lactose (LAC) broth, buffered peptone water (BPW), and universal pre-enrichment (UP) broth] for the recovery of heat- and freeze-injured Salmonella spp. in beef by the conventional culture method. There was a significant difference between TA10 and LAC enrichment broths for detecting injured Salmonella spp. In this study, the International Organization for Standardization Listeria pre-enrichment broth (Half-Fraser/Fraser) was compared with TA10 broth for the recovery of L. monocytogenes from ground beef. Ground beef samples were contaminated with single Listeria serovars at levels of 0.096 to 0.001 most probable number/g. Twenty 25 g test portions of the contaminated ground beef were pre-enriched in each broth, and the ISO-11290-1 Listeria official isolation protocol was used thereafter. There was a significant difference between TA10 broth (48 h) and Half-Fraser/Fraser broth (72 h) in the recovery of L. monocytogenes. In addition, the incubation time for TA10 broth was shorter than for Half-Fraser/Fraser broth. The results indicate that TA10 broth should be used instead of Half-Fraser/Fraser broth for analysis of beef that may be contaminated with very low levels of L. monocytogenes.

scholarly journals Bacteriological assessments of foodborne pathogens in poultry meat at different super shops in Dhaka, Bangladesh

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Jalal Uddin ◽  
Khaled Hossain ◽  
Saddam Hossain ◽  
Karabi Saha ◽  
Fatema Tuz Jubyda ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Culture Method ◽  
Poultry Meat ◽  
Salmonella Spp ◽  
E Coli ◽  
Poultry Products ◽  
Enteropathogenic Bacteria ◽  
Conventional Culture ◽  
Shigella Spp ◽  
Meat Samples

Poultry is now considered as a major fast-growing source of meat in the world. The consumers demand safe and hygienic products without contamination with pathogenic microorganisms when the production and consumption of poultry meat is gradually increasing. The present study was conducted to assess the bacterial contamination of dressed chicken collected from different supershops in Dhaka, Bangladesh. The chicken samples from S1, S2, M1, M2 and A supershops were analyzed to determine the enteropathogenic bacteria in poultry meat. Three genera of bacteria were isolated from all of the chicken meat samples. These enteropathogens from various organs of dressing chickens were also enumerated. The isolates were presumptively identified as E. coli, Salmonella spp., and Shigella spp. by conventional culture method. The three enteropathogens were subjected to PCR assay for their confirmation as virulent enteropathogens. Only E. coli isolates were confirmed as pathogenic E. coli (Enterotoxigenic), other isolates were not confirmed as virulent Salmonella spp., Shigella spp.. Results of this study demonstrated that more cautions are recommended for personnel hygiene in processing and handling of poultry and poultry products to prevent occurrence of enterotoxigenic E. coli in dressed poultry meat sold by the supershops in Bangladesh.

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