Multiplex touchdown PCR assay to enhance specificity and sensitivity for concurrent detection of four foodborne pathogens in raw milk

2019 ◽  
Vol 127 (1) ◽  
pp. 262-273 ◽  
Author(s):  
P. Moezi ◽  
M. Kargar ◽  
A. Doosti ◽  
M. Khoshneviszadeh
Keyword(s):  
Foodborne Pathogens ◽  
Raw Milk ◽  
Pcr Assay ◽  
Specificity And Sensitivity ◽  
Touchdown Pcr

scholarly journals The pCS20 PCR assay for Ehrlichia ruminantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the United States of America

2004 ◽  
Vol 71 (2) ◽  
Author(s):  
S.M. Mahan ◽  
B.H. Simbi ◽  
M.J. Burridge
Keyword(s):  
United States ◽  
High Specificity ◽  
Pcr Primers ◽  
The United States ◽  
Rrna Gene ◽  
The Novel ◽  
Pcr Assay ◽  
Ehrlichia Ruminantium ◽  
Cowdria Ruminantium ◽  
Specificity And Sensitivity

White-tailed deer are susceptible to heartwater (Ehrlichia [Cowdria] ruminantium infection) and are likely to suffer high mortality if the disease spreads to the United States. It is vital, therefore, to validate a highly specific and sensitive detection method for E. ruminantium infection that can be reliably used in testing white-tailed deer, which are reservoirs of antigenically or genetically related agents such as Ehrlichia chaffeensis, Anaplasma (Ehrlichia) phagocytophilum (HGE agent) and Ehrlichia ewingii. Recently, a novel but as yet unnamed ehrlichial species, the white-tailed deer ehrlichia (WTDE), has been discovered in deer populations in the United States. Although the significance of WTDE as a pathogen is unknown at present, it can be distinguished from other Ehrlichia spp. based on 16S rRNA gene sequence analysis. In this study it was differentiated from E. ruminantium by the use of the pCS20 PCR assay which has high specificity and sensitivity for the detection of E. ruminantium. This assay did not amplify DNA from the WTDE DNA samples isolated from deer resident in Florida, Georgia and Missouri, but amplified the specific 279 bp fragment from E. ruminantium DNA. The specificity of the pCS20 PCR assay for E. ruminantium was confirmed by Southern hybridization. Similarly, the 16S PCR primers (nested) that amplify a specific 405-412 bp fragment from the WTDE DNA samples, did not amplify any product from E. ruminantium DNA. This result demonstrates that it would be possible to differentiate between E. ruminantium and the novel WTDE agent found in white tailed deer by applying the two respective PCR assays followed by Southern hybridizations. Since the pCS20 PCR assay also does not amplify any DNA products from E. chaffeensis or Ehrlichia canis DNA, it is therefore the method of choice for the detection of E. ruminantium in these deer and other animal hosts.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

Development of multiplex PCR assay for rapid detection Listeria monocytogenes in clinical samples

2021 ◽  
Vol 30 (4) ◽  
pp. 20-26
Author(s):  
Le Thanh Huong ◽  
Ha Thi Phuong Mai ◽  
Hoang Thi Thu Ha ◽  
Nguyen Dong Tu ◽  
Bui Tien Sy ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Rapid Detection ◽  
Pathogenic Bacteria ◽  
Vulnerable Groups ◽  
Clinical Samples ◽  
Specific Gene ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Specificity And Sensitivity

Listeria monocytogenes is widely present in the natural environment. This bacteria can cause infections in both humans and animals. In humans, the most vulnerable groups to be infected with L. monocytogenes are the elderly, people with an impaired immune system and chronically illness, pregnant women, and newborn babies. The aim of this study was to develop a multiplex PCR assay for the rapid detection of L. monocytogenes in mock clinical samples. A pair of primers were designed for detection of L. monocytogenes based on prs, a Listeria genus specific gene, and hly, a hemolysin gene. The specificity of the primers were tested by using different L. monocytogenes strains and other common pathogenic bacteria. The results showed that L. monocytogenes strains were positive in the detection and other tested strains were negative in mock (spiked) clinical samples. The sensitivity of multiplex PCR assay was 102 CFU/ml per reaction. The specificity and sensitivity of multiplex PCR technology for detecting L. monocytogenes in mock (spiked) clinical samples were high, and the assay could be completed within 1.5 hours. Therefore, this established multiplex PCR provides a rapid and reliable method and will be useful for the detection of L. monocytogenes in mock clinical samples.

Low Incidence of Foodborne Pathogens of Concern in Raw Milk Utilized for Farmstead Cheese Production

2008 ◽  
Vol 71 (8) ◽  
pp. 1580-1589 ◽  
Author(s):  
DENNIS J. D'AMICO ◽  
ERROL GROVES ◽  
CATHERINE W. DONNELLY
Keyword(s):  
Foodborne Pathogens ◽  
Microbiological Quality ◽  
Goat Milk ◽  
Raw Milk ◽  
Pasteurized Milk ◽  
Milk Samples ◽  
Cell Counts ◽  
Standard Plate ◽  
Low Incidence ◽  
Raw Milk Cheese

Overall milk quality and prevalence of four target pathogens in raw milk destined for farmstead cheesemaking was examined. Raw milk samples were collected weekly from June to September 2006 from 11 farmstead cheese operations manufacturing raw milk cheese from cow's, goat's, and sheep's milk. Samples were screened for Listeria monocytogenes, Staphylococcus aureus, Salmonella, and Escherichia coli O157:H7 both quantitatively (direct plating) and qualitatively (PCR). Overall, 96.8% of samples had standard plate counts of <100,000 CFU/ml, 42.7% of which were <1,000 CFU/ml. Although no federal standards exist for coliforms in raw milk, 61% of samples tested conformed to pasteurized milk standards under the U.S. Pasteurized Milk Ordinance (PMO) at <10 CFU/ml. All cow and sheep milk samples and 93.8% of goat milk samples were within the limits dictated by the PMO for somatic cell counts. Of the 11 farms, 8 (73%) produced samples that were positive for S. aureus, which was detected in 34.6% (46 of 133) of milk samples. L. monocytogenes was isolated from three milk samples (2.3%), two of which were from the same farm. E. coli O157:H7 was recovered from one sample of goat's milk for an overall incidence of 0.75%. Salmonella was not recovered from any of the 133 samples. The findings of this study suggest that most raw milk intended for farmstead cheesemaking is of high microbiological quality with a low incidence of pathogens. These data will help inform risk assessments associated with the microbiological safety of farmstead cheeses, particularly those manufactured from raw milk.

Simultaneous Detection of Multiple Salmonella Serovars from Milk and Chicken Meat by Real-Time PCR Using Unique Genomic Target Regions

2017 ◽  
Vol 80 (11) ◽  
pp. 1944-1957 ◽  
Author(s):  
Sayma Afroj ◽  
Khaled Aldahami ◽  
Gopal Reddy ◽  
Jean Guard ◽  
Abiodun Adesiyun ◽  
...  
Keyword(s):  
Salmonella Enteritidis ◽  
Simultaneous Detection ◽  
Taqman Assay ◽  
Comparative Genomic ◽  
Pcr Assay ◽  
Salmonella Dublin ◽  
Milk Samples ◽  
Specificity And Sensitivity ◽  
Salmonella Serovars ◽  
Salmonella Heidelberg

ABSTRACT A novel genomic and plasmid target-based PCR platform was developed for the detection of Salmonella serovars Heidelberg, Dublin, Hadar, Kentucky, and Enteritidis. Unique genome loci were obtained through extensive genome mining of protein databases and comparative genomic analysis of these serovars. Assays targeting Salmonella serovars Hadar, Heidelberg, Kentucky, and Dublin had 100% specificity and sensitivity, whereas those for Salmonella Enteritidis had 97% specificity and 88% sensitivity. The limits of detection for Salmonella serovars Heidelberg, Kentucky, Hadar, Enteritidis, and Dublin were 12, 9, 40, 13, and 5,280 CFU, respectively. A sensitivity assay was also performed by using milk artificially inoculated with pooled Salmonella serovars, yielding a detection limit of 1 to10 CFU/25 mL of milk samples after enrichment. The minimum DNA detected using the multiplexed TaqMan assay was 75.8 fg (1.53 × 101 genomic equivalents [GE]) for Salmonella Heidelberg, 140.8 fg (2.8 × 101 GE) for Salmonella Enteritidis, and 3.48 pg (6.96 × 102 GE) for Salmonella Dublin. PCR efficiencies were 89.8% for Salmonella Heidelberg, 94.5% for Salmonella Enteritidis, and 75.5% for Salmonella Dublin. Four types of 30 pasteurized milk samples were tested negative by culture techniques and with a genus-specific Salmonella invA gene PCR assay. Among 30 chicken samples similarly tested, 12 (40%) were positive by both culture and the invA PCR. Testing of these 12 samples with the serovar-specific PCR assay detected single and mixed contamination with Salmonella Kentucky, Salmonella Enteritidis, and Salmonella Heidelberg. Five unique primers were designed and tested by multiplex conventional PCR in conjunction with the use of the multiplex TaqMan assay with three of the primers. The diagnostic assays developed in this study could be used as tools for routine detection of these five Salmonella serovars and for epidemiological investigations of foodborne disease outbreaks.

scholarly journals Applications of Nanotechnology in Sensor-Based Detection of Foodborne Pathogens

Sensors ◽  
2020 ◽  
Vol 20 (7) ◽  
pp. 1966 ◽  
Author(s):  
Harsh Kumar ◽  
Kamil Kuča ◽  
Shashi Kant Bhatia ◽  
Kritika Saini ◽  
Ankur Kaushal ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Pathogen Detection ◽  
Foodborne Pathogen ◽  
Health Issues ◽  
Detection And Identification ◽  
Specificity And Sensitivity ◽  
Food Borne ◽  
Contaminated Food ◽  
Working Principle ◽  
Effective Use

The intake of microbial-contaminated food poses severe health issues due to the outbreaks of stern food-borne diseases. Therefore, there is a need for precise detection and identification of pathogenic microbes and toxins in food to prevent these concerns. Thus, understanding the concept of biosensing has enabled researchers to develop nanobiosensors with different nanomaterials and composites to improve the sensitivity as well as the specificity of pathogen detection. The application of nanomaterials has enabled researchers to use advanced technologies in biosensors for the transfer of signals to enhance their efficiency and sensitivity. Nanomaterials like carbon nanotubes, magnetic and gold, dendrimers, graphene nanomaterials and quantum dots are predominantly used for developing biosensors with improved specificity and sensitivity of detection due to their exclusive chemical, magnetic, mechanical, optical and physical properties. All nanoparticles and new composites used in biosensors need to be classified and categorized for their enhanced performance, quick detection, and unobtrusive and effective use in foodborne analysis. Hence, this review intends to summarize the different sensing methods used in foodborne pathogen detection, their design, working principle and advances in sensing systems.

Microbiological Baseline Study of Broiler Chickens at Swedish Slaughterhouses

2006 ◽  
Vol 69 (12) ◽  
pp. 2875-2882 ◽  
Author(s):  
M. LINDBLAD ◽  
H. LINDMARK ◽  
S. THISTED LAMBERTZ ◽  
R. LINDQVIST
Keyword(s):  
Foodborne Pathogens ◽  
Broiler Chickens ◽  
Pathogenic Bacteria ◽  
Indicator Bacteria ◽  
Pcr Assay ◽  
Management Actions ◽  
Prevalence Estimates ◽  
Low Prevalence ◽  
Coagulase Positive Staphylococci ◽  
Pathogenic Yersinia

This 1-year study was conducted to estimate the prevalence and concentrations of pathogenic and indicator bacteria on Swedish broiler chickens. A total of 636 chilled carcasses were collected from 10 slaughterhouses and sent to the National Food Administration for analyses of carcass rinses. No carcasses were positive for Salmonella. Campylobacter, predominantly Campylobacter jejuni, were detected on 15% (by enrichment) or 14% (by direct plating) of the carcasses. With one exception, all samples from late December through April were Campylobacter negative. The 10th and 90th percentiles of Campylobacter numbers per carcasses were 3.0 and 5.0 log CFU, respectively, and the maximum was 7.1 log CFU. Coagulase-positive staphylococci were detected on 68% of the carcasses, with a maximum of 3.5 log CFU/cm2. The 10th and 90th percentiles were 3.4 and 4.4 log CFU/cm2 for total aerobic microorganisms, 1.8 and 3.3 log CFU/cm2 for Enterobacteriaceae, and 2.0 and 3.6 log CFU/cm2 for Escherichia coli. No correlation was found between numbers of any indicator bacteria and numbers of pathogenic bacteria. Subsets of the samples were analyzed for Listeria monocytogenes, Clostridium perfringens, pathogenic Yersinia enterocolitica, and Enterococcus, resulting in prevalence estimates of 29, 18, 9 (as determined by a PCR assay), and 97%, respectively. L. monocytogenes was most common at slaughterhouses with a low prevalence of coagulase-positive staphylococci, and vice versa. These results will improve the ability of researchers to assess the importance of chicken as a source of foodborne pathogens and can serve as a basis for risk management actions.

scholarly journals Evaluation of Copan FecalSwab as Specimen Type for Use in Xpert C. difficile Assay

2017 ◽  
Vol 55 (10) ◽  
pp. 3123-3129 ◽  
Author(s):  
Michael J. Mashock ◽  
Matthew L. Faron ◽  
Blake W. Buchan ◽  
Nathan A. Ledeboer
Keyword(s):  
Test Performance ◽  
Pcr Assay ◽  
Content Type ◽  
Molecular Assays ◽  
Specificity And Sensitivity ◽  
Collection Device ◽  
Stool Samples ◽  
Stool Specimens ◽  
Xpert Assay ◽  
Preservation Medium

ABSTRACT Liquid-based microbiology (LBM) devices incorporating flocked swabs and preservation medium ease transport of specimens and improve specimen yield compared to traditional fiber wound swabs; however, the performance of LBM collection devices has not been evaluated in many molecular assays. It is unclear how the differences in matrix and specimen loading with an LBM device will affect test performance compared to traditional collection devices. The purpose of this study was to evaluate the performance of specimens collected in FecalSwab transport medium (Copan Diagnostics, Murrieta, CA) compared to unpreserved stool using the Cepheid Xpert C. difficile assay (Cepheid, Sunnyvale, CA). Results equivalent to unpreserved stool samples were obtained when 400 μl of FecalSwab-preserved stool was employed in the Xpert assay. The positive and negative percent agreement of specimens inoculated with FecalSwab medium ( n = 281) was 97.0% (95% confidence interval [CI], 90.9 to 96.4%) and 99.4% (95% CI, 96.4 to 99.9%), respectively, compared to reference results obtained using unpreserved stool. Throughout this study, only four discrepant results occurred when comparing preserved specimens to unpreserved stool specimens in the Xpert C. difficile PCR assay. Post discrepant analysis, using the BD MAX Cdiff assay, the specificity and sensitivity both increased to 100%. The high positive and negative percent agreements observed in this study suggest that stool preserved in FecalSwab media yields equivalent results to using unpreserved stool when tested on the Xpert C. difficile assay, allowing laboratories to adopt this liquid-based microbiology collection device.

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