Development of a novel dual priming oligonucleotide system‐based PCR assay for specific detection of Salmonella from food samples

2020 ◽  
Vol 40 (3) ◽  
Author(s):  
Dan‐Dan Li ◽  
Chun‐Bo Hao ◽  
Zhong‐Mei Liu ◽  
Sui‐Jia Wang ◽  
Yu Wang ◽  
...  
Keyword(s):  
Food Samples ◽  
Specific Detection ◽  
Pcr Assay ◽  
Dual Priming Oligonucleotide

Development of Rapid Real-Time PCR and Most-Probable-Number Real-Time PCR Assays To Quantify Enterotoxigenic Strains of the Species in the Bacillus cereus Group

2007 ◽  
Vol 70 (12) ◽  
pp. 2774-2781 ◽  
Author(s):  
I-CHEN YANG ◽  
DANIEL YANG-CHIH SHIH ◽  
JAN-YI WANG ◽  
TZU-MING PAN
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Samples ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Bacillus Cereus Group ◽  
Probable Number ◽  
Cooked Rice ◽  
Group Detection

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (102 to 107 CFU/g for cooked rice and chicken, 103 to 107 CFU/ml for milk, and 104 to 107 CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 100 CFU/ml. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification.

Development of a hemi-nested PCR assay for the specific detection ofMelissococcus pluton

1998 ◽  
Vol 37 (3) ◽  
pp. 165-174 ◽  
Author(s):  
Steven P Djordjevic ◽  
Kerrie Noone ◽  
Lisa Smith ◽  
Michael A Z Hornitzky
Keyword(s):  
Nested Pcr ◽  
Specific Detection ◽  
Pcr Assay

Multiplex PCR Detection of Enterotoxin Genes in Aeromonas spp. from Suspect Food Samples in Northern Taiwan

2008 ◽  
Vol 71 (10) ◽  
pp. 2094-2099 ◽  
Author(s):  
YU-CHANG CHANG ◽  
JAN-YI WANG ◽  
AMMAIYAPPAN SELVAM ◽  
SHU-CHEN KAO ◽  
SHANG-SHYNG YANG ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Diarrheal Disease ◽  
Simultaneous Detection ◽  
Food Poisoning ◽  
Food Samples ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Enterotoxin Genes ◽  
Causative Agents ◽  
Northern Taiwan

Aeromonads possess an array of virulence factors and are causative agents of a number of human infections. Among them, genes of one cytotoxic (Act) and two cytotonic (Alt, Ast) enterotoxins are implicated in a human diarrheal disease. A rapid, specific, simultaneous detection of these enterotoxin genes in suspected food poisoning samples is not yet reported. Hence, a multiplex PCR assay was designed to amplify the cytotoxic (act), heat-labile cytotonic (alt), and heat-stable cytotonic (ast) enterotoxin genes of aeromonads. The PCR assay was tested with 133 Aeromonas spp. isolated from suspect food poisoning samples and retail samples of poultry and fish from wet markets in and around Taipei, Northern Taiwan. The Aeromonas spp. isolates were divided into six genotypes based on absence or presence of one or more enterotoxin genes. Of these 133 isolates, Aeromonas caviae (52.5%) and Aeromonas hydrophila (43.4%) were the most frequently isolated species from food poisoning samples and retail samples, respectively. Among the species, A. hydrophila had a significantly higher proportion for harboring three enterotoxin genes than had the others, whereas Aeromonas encheleia, considered a nonpathogen, was found harboring three enterotoxin genes. The multiplex PCR assays are rapid and specific, and provide a useful tool for the detection and genotyping of enterotoxin genes of aeromonads.

scholarly journals Detection of Small Numbers of Campylobacter jejuni and Campylobacter coli Cells in Environmental Water, Sewage, and Food Samples by a Seminested PCR Assay

1999 ◽  
Vol 65 (4) ◽  
pp. 1636-1643 ◽  
Author(s):  
Astrid S. Waage ◽  
Traute Vardund ◽  
Vidar Lund ◽  
Georg Kapperud
Keyword(s):  
Campylobacter Jejuni ◽  
Simple Procedure ◽  
Environmental Water ◽  
Food Samples ◽  
Proteinase K ◽  
Coliform Bacteria ◽  
Intergenic Sequence ◽  
Campylobacter Coli ◽  
Pcr Assay ◽  
Seminested Pcr

ABSTRACT A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA andflaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as ≤3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.

scholarly journals A TaqMan-based real-time PCR assay for specific detection of novel duck reovirus in China

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Shuai Zhang ◽  
Weihua Li ◽  
Xiaodong Liu ◽  
Xudong Li ◽  
Bin Gao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Specific Detection ◽  
Pcr Assay

Simultaneous norovirus outbreak in three Portuguese army bases in the Lisbon region, December 2017

2019 ◽  
pp. jramc-2019-001242
Author(s):  
António Lopes-João ◽  
J R Mesquita ◽  
R de Sousa ◽  
M Oleastro ◽  
C Penha-Gonçalves ◽  
...  
Keyword(s):  
Armed Forces ◽  
Food Samples ◽  
Common Source ◽  
Microbiological Analysis ◽  
Pcr Assay ◽  
Institutional Settings ◽  
Enteropathogenic Bacteria ◽  
Source Of Infection ◽  
Positive Stool ◽  
Stool Specimens

IntroductionNorovirus outbreaks frequently occur in communities and institutional settings acquiring a particular significance in armed forces where prompt reporting is critical. Here we describe the epidemiological, clinical and laboratorial investigation of a multicentre gastroenteritis outbreak that was detected simultaneously in three Portuguese army units with a common food supplier, Lisbon region, between 5 and 6 December 2017.MethodsQuestionnaires were distributed to all soldiers stationed in the three affected army units, and stool specimens were collected from soldiers with acute gastrointestinal illness. Stool specimens were tested for common enteropathogenic bacteria by standard methods and screened for a panel of enteric viruses using a multiplex real-time PCR assay. Food samples were also collected for microbiological analysis. Positive stool specimens for norovirus were further genotyped.ResultsThe three simultaneous acute gastroenteritis outbreaks affected a 31 (3.5%) soldiers from a total of 874 stationed at the three units and lasted for 2 days. No secondary cases were reported. Stool specimens (N=11) were negative for all studied enteropathogenic agents but tested positive for norovirus. The recombinant norovirus GII.P16-GII.4 Sydney was identified in all positive samples with 100% identity.ConclusionsThe results are suggestive of a common source of infection plausibly related to the food supplying chain. Although centralisation of food supplying in the army has economic advantages, it may contribute to the multifocal occurrence of outbreaks. A rapid intervention is key in the mitigation of outbreak consequences and in reducing secondary transmission.

Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction.

1994 ◽  
Vol 12 (4) ◽  
pp. 725-729 ◽  
Author(s):  
M Gerhard ◽  
H Juhl ◽  
H Kalthoff ◽  
H W Schreiber ◽  
C Wagener ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
Tumor Cells ◽  
Normal Bone Marrow ◽  
Specific Detection ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Normal Bone ◽  
Polymerase Chain

PURPOSE To establish a sensitive assay for the specific detection of carcinoembryonic antigen (CEA)-expressing tumor cells in the bone marrow of patients with colorectal cancer and other CEA-positive carcinomas. PATIENTS AND METHODS A CEA-specific nested reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed and optimized using limiting dilutions of a CEA-positive cancer cell line mixed with normal bone marrow cell specimens. The optimized test was then used to examine bone marrow samples obtained from 15 patients with abdominal carcinomas (colorectal, n = 10; pancreatic, n = 3; gastric, n = 2) and six patients with breast cancer. Specificity was assessed by examination of 56 negative controls (malignant hematologic disease, n = 28; nonmalignant disease conditions, n = 5; healthy bone marrow donors, n = 8; normal peripheral-blood samples, n = 15). For 11 patients with abdominal carcinomas, immunostaining evaluations were performed using an anti-CEA and an anticytokeratin antibody, and the results compared with the nested PCR assay. RESULTS In the sensitivity calibration system, single CEA-expressing tumor cells were detected in 2 to 5 x 10(7) normal bone marrow cells. All 56 control samples failed to amplify. This demonstrates that mRNAs coding for highly homologous CEA-related antigens expressed by various lineages of blood cells do not interfere. Bone marrow samples from 10 of 15 patients with abdominal cancers and four of six breast cancer patients scored positive, indicating micrometastatic bone disease. Four of 11 samples from the gastrointestinal cancer patients were found to be positive by the PCR method, but were negative with the immunocytology method. CONCLUSION Since approximately 30% of the colorectal carcinoma patients that score negative in immunocytology staining of bone marrow samples have been reported to relapse, earlier diagnosis of the presence of malignant cells is needed. Our result that samples scoring positive in the described CEA-specific PCR test remained negative by two immunostaining methods suggests a higher sensitivity. We conclude that PCR amplification of CEA mRNA may lead to an earlier diagnosis of micrometastatic bone disease in patients with CEA-expressing carcinomas.

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