Duplex Real-Time PCR Assay for Rapid Detection of Ampicillin-Resistant Enterococcus faecium

ABSTRACT Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the d-Ala-d-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.

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Development of a novel real‐time PCR assay targeting p54 gene for rapid detection of African swine fever virus (ASFV) strains circulating in Vietnam

Veterinary Medicine and Science ◽

10.1002/vms3.605 ◽

2021 ◽

Author(s):

Thi Bich Ngoc Trinh ◽

Thang Truong ◽

Van Tam Nguyen ◽

Xuan Dang Vu ◽

Le Anh Dao ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Pcr Assay

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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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-297 ◽

2012 ◽

Vol 75 (4) ◽

pp. 743-747 ◽

Cited By ~ 23

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.

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Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

PLoS ONE ◽

10.1371/journal.pone.0143444 ◽

2015 ◽

Vol 10 (11) ◽

pp. e0143444 ◽

Cited By ~ 6

Author(s):

Yong Zhao ◽

Guilian Li ◽

Chongyun Sun ◽

Chao Li ◽

Xiaochen Wang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Locked Nucleic Acid ◽

Pcr Assay ◽

Locked Nucleic Acid Probe

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A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Most Relevant Members of the B. pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis

PLoS ONE ◽

10.1371/journal.pone.0164006 ◽

2016 ◽

Vol 11 (10) ◽

pp. e0164006 ◽

Cited By ~ 12

Author(s):

Chinn-Woan Lowe ◽

Benjamin A. Satterfield ◽

Daniel B. Nelson ◽

Joseph D. Thiriot ◽

Michael J. Heder ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Pcr Assay

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Detection of Peanut Allergen Traces with a Real Time PCR Assay - The Challenge to Protect Food-Allergic Consumers

Journal of Food Research ◽

10.5539/jfr.v7n1p32 ◽

2017 ◽

Vol 7 (1) ◽

pp. 32 ◽

Cited By ~ 2

Author(s):

Dimitra Houhoula ◽

Stamatios Koussissis ◽

Vladimiros Lougovois ◽

John Tsaknis ◽

Dimitra Kassavita ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Food Products ◽

Molecular Techniques ◽

Food Allergens ◽

Pcr Assay ◽

Peanut Allergen ◽

Pcr Method ◽

Detection And Quantification

The aim of the present study was the implementation of molecular techniques in the detection and quantification of allergic substances of peanut in various kinds of food products, e.g., breakfast cereals, chocolates and biscuits that are frequently related to allergies. In some cases, the presence of peanuts can be due to contamination during production and are not declared on the label. A total of 152 samples were collected from supermarkets and were analysed by a Real Time PCR method. The results indicated that 125 samples (83,3%) were found positive in peanut traces but the most important finding is that from the 84 samples that had no allergen declaration for peanuts, 48 (57,1%) of them were found positive. In conclusion, Real Time PCR can be a very important tool for the rapid detection and quantification of food allergens.

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

Journal of Food Protection ◽

10.4315/0362-028x-70.5.1080 ◽

2007 ◽

Vol 70 (5) ◽

pp. 1080-1087 ◽

Cited By ~ 63

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.

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