Detection of Salmonella Enteritidis in asymptomatic carrier animals: comparison of quantitative real-time PCR and bacteriological culture methods

2011 ◽  
Vol 10 (4) ◽  
pp. 2578-2588 ◽  
Author(s):  
B.M. Maciel ◽  
J.C.T. Dias ◽  
C.C. Romano ◽  
N. Sriranganathan ◽  
M. Brendel ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Asymptomatic Carrier ◽  
Quantitative Real Time Pcr ◽  
Culture Methods ◽  
Bacteriological Culture

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 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.

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 ofLegionella pneumophilacontamination in Italian hotel water systems by quantitative real-time PCR and culture methods

2010 ◽  
Vol 108 (5) ◽  
pp. 1576-1583 ◽  
Author(s):  
Sa. Bonetta ◽  
Si. Bonetta ◽  
E. Ferretti ◽  
F. Balocco ◽  
E. Carraro
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Water Systems ◽  
Quantitative Real Time Pcr ◽  
Culture Methods

A real-time PCR regimen for testing environmental samples for Salmonella enterica subsp. enterica serovars of concern to the poultry industry, with special focus on Salmonella Enteritidis

2019 ◽  
Vol 65 (2) ◽  
pp. 162-173 ◽  
Author(s):  
S. Nadin-Davis ◽  
L. Pope ◽  
D. Ogunremi ◽  
B. Brooks ◽  
J. Devenish
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Culture Collection ◽  
Special Focus ◽  
Culture Methods ◽  
Poultry Facilities ◽  
Culture Process

A real-time PCR (qPCR) regimen, using up to six genetic targets, was developed to rapidly detect Salmonella and in particular identify Salmonella Enteritidis. The test regimen was first evaluated using a reference culture collection of Salmonella to confirm the appropriateness of the selected targets, which included up to three genetic markers for discrimination of Salmonella Enteritidis from other Salmonella serovars commonly found in poultry facilities. The qPCR procedure was then compared with culture methods used to detect Salmonella using a collection of enrichment broths previously generated from 239 environmental samples collected from a large number of hatchery facilities across Canada over several years. The qPCR regimen facilitated specific detection of Salmonella Enteritidis, and on a sample basis, it showed excellent agreement with the culture methods. Moreover, in many cases, qPCR detected Salmonella earlier in the culture process than did the culture method. Application of this method will significantly shorten test times and allow more timely identification of infected poultry premises, thereby improving present programmes aimed at controlling Salmonella Enteritidis at the environmental source.

Salmonella detection in poultry samples

2012 ◽  
Vol 40 (06) ◽  
pp. 383-389 ◽  
Author(s):  
D. Sommer ◽  
D. Enderlein ◽  
A. Antakli ◽  
H. Schönenbrücher ◽  
J. Slaghuis ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Detection System ◽  
Cell Damage ◽  
Culture Method ◽  
Reference Laboratory ◽  
Salmonella Spp ◽  
Culture Methods ◽  
Fecal Samples

Summary Study: The efficiency of two commercial PCR methods based on real-time technology, the foodproof® Salmonella detection system and the BAX® PCR Assay Salmonella system was compared to standardized culture methods (EN ISO 6579:2002 – Annex D) for the detection of Salmonella spp. in poultry samples. Material and methods: Four sample matrices (feed, dust, boot swabs, feces) obtained directly from poultry flocks, as well as artificially spiked samples of the same matrices, were used. All samples were tested for Salmonella spp. using culture methods first as the gold standard. In addition samples spiked with Salmonella Enteridis were tested to evaluate the sensitivity of both PCR methods. Furthermore all methods were evaluated in an annual ring-trial of the National Salmonella Reference Laboratory of Germany. Results: Salmonella detection in the matrices feed, dust and boot swabs were comparable in both PCR systems whereas the results from feces differed markedly. The quality, especially the freshness, of the fecal samples had an influence on the sensitivity of the real-time PCR and the results of the culture methods. In fresh fecal samples an initial spiking level of 100 cfu/25 g Salmonella Enteritidis was detected. Two-days-dried fecal samples allowed the detection of 14 cfu/25 g. Both real-time PCR protocols appear to be suitable for the detection of Salmonella spp. in all four matrices. The foodproof® system detected eight samples more to be positive compared to the BAX® system, but had a potential false positive result in one case. In 7-days-dried samples none of the methods was able to detect Salmonella likely through letal cell damage. Clinical relevance: In general the advantage of PCR analyses over the culture method is the reduction of working time from 4–5 days to only 2 days. However, especially for the analysis of fecal samples official validation should be conducted according to the requirement of EN ISO 6579:2002 – Annex D.

scholarly journals KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yi Wang ◽  
Hongjuan Liao ◽  
Yueheng Wang ◽  
Jinlin Zhou ◽  
Feng Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Mtor Signaling ◽  
Heart Weight ◽  
Cardiomyocyte Hypertrophy ◽  
Quantitative Real Time Pcr

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs

2007 ◽  
Vol 70 (2) ◽  
pp. 245-251 ◽  
Author(s):  
Burkhard Malorny ◽  
Cornelia Bunge ◽  
Reiner Helmuth
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Poultry Meat

Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

2008 ◽  
Vol 375 (1) ◽  
pp. 150-152 ◽  
Author(s):  
Cheng Xin Yi ◽  
Jun Zhang ◽  
Ka Man Chan ◽  
Xiao Kun Liu ◽  
Yan Hong
Keyword(s):  
Gossypium Hirsutum ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Transgene Copy Number
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