Evaluation of Applied Biosystems MicroSEQ® Real-Time PCR System for Detection of Listeria spp. in Food and Environmental Samples

2012 ◽  
Vol 95 (4) ◽  
pp. 1074-1083 ◽  
Author(s):  
Olga V Petrauskene ◽  
Yanxiang Cao ◽  
Patrick Zoder ◽  
Lily Y Wong ◽  
Priya Balachandran ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Sample Preparation ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Method ◽  
Test Method ◽  
Stainless Steel Surface ◽  
Acid Extraction ◽  
Chi Square ◽  
Chi Square Analysis

Abstract A complete system for real-time PCR detection of Listeria species was validated in five food matrixes and five environmental surfaces, namely, hot dogs, roast beef, lox (smoked salmon), pasteurized whole cow's milk, dry infant formula, stainless steel, plastic cutting board, ceramic tile, rubber sheets, and sealed concrete. The system consists of the MicroSEQ®Listeria spp. Detection Kit, two sample preparation kits (PrepSEQ® Nucleic Acid Extraction Kit and PrepSEQ Rapid Spin Sample Preparation Kit), the Applied Biosystems 7500 Fast Real-Time PCR instrument, and the RapidFinder™ Express v1.1 Software for data analysis. The test method was compared to the ISO 11290-1 reference method using an unpaired study design. The MicroSEQ Listeria spp. Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all matrixes except hot dogs. For hot dogs, the MicroSEQ method detected more positives than the reference method for the low- and high-level inoculations, with all of the presumptive positives confirmed by the reference method. An independent validation study confirmed these findings on lox and stainless steel surface. The MicroSEQ kit detected all 50 Listeria strains tested and none of the 31 nontarget bacteria strains.

Validation of the Applied Biosystems MicroSEQ® Real-Time PCR System for Detection of E. coli O157:H7 in Food

2012 ◽  
Vol 95 (5) ◽  
pp. 1495-1504 ◽  
Author(s):  
Lily Y Wong ◽  
Yanxiang Cao ◽  
Priya Balachandran ◽  
Patrick Zoder ◽  
Manohar R Furtado ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Reference Method ◽  
Test Method ◽  
Acid Extraction ◽  
Culture Methods ◽  
Chi Square ◽  
E Coli

Abstract Modern molecular methods offer the advantages of simplicity and short time-to-results compared to traditional culture methods. We describe the validation of a new Real-Time PCR method to detect E. coli O157:H7 in five food matrixes. The complete system consists of the MicroSEQ®E. coli O157:H7 Detection Kit, sample preparation (two sample preparation methods, the PrepSEQ® Nucleic Acid Extraction Kit and the PrepSEQ Rapid Spin Sample Preparation Kit, were validated), the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder™ Express software. The test method was compared to the U.S. Department of Agriculture Microbiology Laboratory Guidebook 5.04 reference method for detecting E. coli O157:H7 in 25 g and 375 g ground beef and beef trim, and to the ISO 16654 reference method for detecting E. coli O157:H7 in 25 g spinach, orange juice, and apple juice. The MicroSEQ E. coli O157:H7 Detection Kit showed equivalent detection compared to the corresponding reference method based on Mantel-Haenszel Chi-square statistics for all matrixes tested. An independent validation confirmed these findings on ground beef. The MicroSEQ kit detected all 51 E. coli O157:H7 strains tested and showed good discrimination against an exclusivity panel of 30 strains.

scholarly journals Reveal®Salmonella 2.0 Test for Detection of Salmonella spp. in Foods and Environmental Samples

2011 ◽  
Vol 94 (5) ◽  
pp. 1467-1480
Author(s):  
Rebecca Hoerner ◽  
Jill Feldpausch ◽  
R Lucas Gray ◽  
Stephanie Curry ◽  
Zahidul Islam ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Irrigation Water ◽  
Environmental Samples ◽  
Steel Surface ◽  
Reference Method ◽  
Stainless Steel Surface ◽  
Salmonella Spp ◽  
Culture Methods ◽  
Chi Square Analysis ◽  
Positive Results

Abstract Reveal Salmonella 2.0 is an improved version of the original Reveal Salmonella lateral flow immunoassay and is applicable to the detection of Salmonella enterica serogroups A–E in a variety of food and environmental samples. A Performance Tested MethodSM validation study was conducted to compare performance of the Reveal 2.0 method with that of the U.S. Department of Agriculture-Food Safety and Inspection Service or U.S. Food and Drug Administration/Bacteriological Analytical Manual reference culture methods for detection of Salmonella spp. in chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, raw shrimp, a ready-to-eat meal product, dry pet food, ice cream, spinach, cantaloupe, peanut butter, stainless steel surface, and sprout irrigation water. In a total of 17 trials performed internally and four trials performed in an independent laboratory, there were no statistically significant differences in performance of the Reveal 2.0 and reference culture procedures as determined by Chi-square analysis, with the exception of one trial with stainless steel surface and one trial with sprout irrigation water where there were significantly more positive results by the Reveal 2.0 method. Considering all data generated in testing food samples using enrichment procedures specifically designed for the Reveal method, overall sensitivity of the Reveal method relative to the reference culture methods was 99%. In testing environmental samples, sensitivity of the Reveal method relative to the reference culture method was 164%. For select foods, use of the Reveal test in conjunction with reference method enrichment resulted in overall sensitivity of 92%. There were no unconfirmed positive results on uninoculated control samples in any trials for specificity of 100%. In inclusivity testing, 102 different Salmonella serovars belonging to serogroups A–E were tested and 99 were consistently positive in the Reveal test. In exclusivity testing of 33 strains of non-salmonellae representing 14 genera, 32 were negative when tested with Reveal following nonselective enrichment, and the remaining strain was found to be substantially inhibited by the enrichment media used with the Reveal method. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device development time.

scholarly journals iQ-Check Salmonella II: Real-Time Polymerase Chain Reaction Test Kit

2009 ◽  
Vol 92 (6) ◽  
pp. 1865-1870 ◽  
Author(s):  
Wendy F Lauer ◽  
Caroline D Sidi ◽  
Jean-Philippe Tourniaire ◽  
Thomas Hammack
Keyword(s):  
Real Time ◽  
Peanut Butter ◽  
Reference Method ◽  
Polymerase Chain Reaction Test ◽  
Target Sequence ◽  
Chi Square ◽  
Test Kit ◽  
Significant Difference ◽  
Chi Square Analysis ◽  
Chain Reaction Test

Abstract iQ-Check Salmonella II is a real-time PCR kit for detection of Salmonella in foods. Specific oligonucleotide probes are used to detect target DNA during the amplification, by hybridizing to the amplicons. These probes are linked to a fluorophore, which fluoresces only when hybridized to the target sequence. As part of an Emergency Response Validation due to a massive outbreak and subsequent recall, peanut butter was tested to compare the performance of iQ-Check Salmonella II to the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) reference method for detection of Salmonella. A single enrichment in buffered peptone water was used for a reduced enrichment time of 21 1 h over the 48 h reference method. There was no significant difference in the performance of the iQ-Check kit when compared to the FDA-BAM method, as determined by Chi-square analysis. All samples identified as positive by iQ-Check were confirmed by reference method protocol.

Detection of Salmonella species in a Variety of Foods by the DuPont™ BAX® System Real-Time PCR Assay for Salmonella: First Action 2013.02

2014 ◽  
Vol 97 (3) ◽  
pp. 868-875 ◽  
Author(s):  
F Morgan Wallace ◽  
Bridget Andaloro ◽  
Dawn Fallon ◽  
Nisha Corrigan ◽  
Stephen Varkey ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Orange Juice ◽  
Collaborative Study ◽  
Reference Method ◽  
Probability Of Detection ◽  
Test Method ◽  
Pcr Assay ◽  
Test Portion ◽  
Significant Difference

Abstract A multilaboratory study was conducted to evaluate the ability of the DuPont™ BAX® System Real-Time PCR Assay for Salmonella to detect the target species in a variety of foods and environmental surfaces. Internal validation studies were performed by DuPont Nutrition & Health on 24 different sample types to demonstrate the reliability of the test method among a wide variety of sample types. Two of these matrixes—pork and turkey frankfurters and pasteurized, not-from-concentrate orange juice without pulp—were each evaluated in 14 independent laboratories as part of the collaborative study to demonstrate repeatability and reproducibility of the internal laboratory results independent of the end user. Frankfurter samples were evaluated against the U. S. Department of Agriculture, Food Safety and Inspection Service reference method as a paired study, while orange juice samples were evaluated against the U. S. Food and Drug Administration reference method as an unpaired study, using a proprietary media for the test method. Samples tested in this study were artificially inoculated with a Salmonella strain at levels expected to produce low (0.2–2.0 CFU/test portion) or high (5 CFU/test portion) spike levels on the day of analysis. For each matrix, the collaborative study failed to show a statistically significant difference between the candidate method and the reference method using the probability of detection statistical model.

scholarly journals Evaluation of Applied Biosystems MicroSEQ® Real-Time PCR System for Detection of Listeria monocytogenes in Food

2011 ◽  
Vol 94 (5) ◽  
pp. 1481-1489 ◽  
Author(s):  
Robert S Tebbs ◽  
Priya Balachandran ◽  
Lily Y Wong ◽  
Patrick Zoder ◽  
Manohar R Furtado ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Sample Preparation ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Method ◽  
Cow’S Milk ◽  
Preparation Methods ◽  
Cow's Milk ◽  
Smoked Salmon ◽  
Sample Preparation Methods

Abstract Increasingly, more food companies are relying on molecular methods, such as PCR, for pathogen detection due to their improved simplicity, sensitivity, and rapid time to results. this report describes the validation of a new Real-time PCR method to detect Listeria monocytogenes in nine different food matrixes. the complete system consists of the MicroSEQ®L. monocytogenes Detection Kit, sample preparation, the Applied Biosystems 7500 Fast Real-time PCR instrument, and RapidFinder™ Express software. two sample preparation methods were validated: the PrepSEQ® Nucleic Acid extraction kit and the PrepSEQ Rapid Spin sample preparation kit. the test method was compared to the ISO 11290-1 reference method using an unpaired-study design to detect L. monocytogenes in roast beef, cured bacon, lox (smoked salmon), lettuce, whole cow's milk, dry infant formula, ice cream, salad dressing, and mayonnaise. the MicroSEQ L. monocytogenes Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all food matrixes when the samples were prepared using either of the two sample preparation methods. An independent validation confirmed these findings on smoked salmon and whole cow's milk. the MicroSEQ kit detected all 50 L. monocytogenes strains tested, and none of the 30 nontargeted bacteria strains.

VIDAS® Listeria species Xpress (LSX)

2013 ◽  
Vol 96 (2) ◽  
pp. 242-245 ◽  
Author(s):  
Ronald Johnson ◽  
John Mills
Keyword(s):  
Stainless Steel ◽  
Reference Method ◽  
Culture Method ◽  
Culture Methods ◽  
Chi Square ◽  
Listeria Species ◽  
Standard Culture ◽  
Inspection Service ◽  
Chi Square Analysis ◽  
Positive Results

Abstract The AOAC GovVal study compared the VIDAS®Listeria species Xpress (LSX) to the Health Products and Food Branch MFHPB-30 reference method for detection of Listeria on stainless steel. The LSX method utilizes a novel and proprietary enrichment media, Listeria Xpress broth, enabling detection of Listeria species in environmental samples with the automated VIDAS in a minimum of 26 h. The LSX method also includes the use of the chromogenic media, chromID™ Ottaviani Agosti Agar (OAA) and chromID™ Lmono for confirmation of LSX presumptive results. In previous AOAC validation studies comparing VIDAS LSX to the U. S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) and the U. S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) reference methods, the LSX method was approved as AOAC Official Method2010.02 for the detection of Listeria species in dairy products, vegetables, seafood, raw meats and poultry, and processed meats and poultry, and as AOAC Performance Tested Method 100501 in a variety of foods and on environmental surfaces. The GovVal comparative study included 20 replicate test portions each at two contamination levels for stainless steel where fractionally positive results (5–15 positive results/20 replicate portions tested) were obtained by at least one method at one level. Five uncontaminated controls were included. In the stainless steel artificially contaminated surface study, there were 25 confirmed positives by the VIDAS LSX assay and 22 confirmed positives by the standard culture methods. Chi-square analysis indicated no statistical differences between the VIDAS LSX method and the MFHPB-30 standard methods at the 5% level of significance. Confirmation of presumptive LSX results with the chromogenic OAA and Lmono media was shown to be equivalent to the appropriate reference method agars. The data in this study demonstrate that the VIDAS LSX method is an acceptable alternative method to the MFHPB-30 standard culture method for the detection of Listeria species on stainless steel.

scholarly journals Detection of Salmonella enterica in Meat in Less than 5 Hours by a Low-Cost and Noncomplex Sample Preparation Method

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
M. S. R. Fachmann ◽  
C. Löfström ◽  
J. Hoorfar ◽  
F. Hansen ◽  
J. Christensen ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Real Time ◽  
Rapid Method ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Preparation Method ◽  
Reference Method ◽  
Pork Meat ◽  
Sample Preparation Method ◽  
Content Type

ABSTRACT Salmonella is recognized as one of the most important foodborne bacteria and has wide health and socioeconomic impacts worldwide. Fresh pork meat is one of the main sources of Salmonella, and efficient and fast methods for detection are therefore necessary. Current methods for Salmonella detection in fresh meat usually include >16 h of culture enrichment, in a few cases <12 h, thus requiring at least two working shifts. Here, we report a rapid (<5 h) and high-throughput method for screening of Salmonella in samples from fresh pork meat, consisting of a 3-h enrichment in standard buffered peptone water and a real-time PCR-compatible sample preparation method based on filtration, centrifugation, and enzymatic digestion, followed by fast-cycling real-time PCR detection. The method was validated in an unpaired comparative study against the Nordic Committee on Food Analysis (NMKL) reference culture method 187. Pork meat samples (n = 140) were either artificially contaminated with Salmonella at 0, 1 to 10, or 10 to 100 CFU/25 g of meat or naturally contaminated. Cohen's kappa for the degree of agreement between the rapid method and the reference was 0.64, and the relative accuracy, sensitivity, and specificity for the rapid method were 81.4, 95.1, and 97.9%, respectively. The 50% limit of detections (LOD50s) were 8.8 CFU/25 g for the rapid method and 7.7 CFU/25 g for the reference method. Implementation of this method will enable faster release of Salmonella low-risk meat, providing savings for meat producers, and it will help contribute to improved food safety. IMPORTANCE While the cost of analysis and hands-on time of the presented rapid method were comparable to those of reference culture methods, the fast product release by this method can provide the meat industry with a competitive advantage. Not only will the abattoirs save costs for work hours and cold storage, but consumers and retailers will also benefit from fresher meat with a longer shelf life. Furthermore, the presented sample preparation might be adjusted for application in the detection of other pathogenic bacteria in different sample types.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals A two minute liquid based sample preparation for rapid SARS-CoV2 real-time PCR screening: a multicentre evaluation

2020 ◽  
pp. 104720
Author(s):  
Eric C.J. Claas ◽  
Pieter W. Smit ◽  
Mario J.A.W.M. van Bussel ◽  
Harold Verbakel ◽  
Mohammed Taouil ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Screening

Molecular Detection of Adenovirus in Tap Water from Coastal Areas Of Karachi Pakistan:The Real-Time PCR Assay

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S140-S140
Author(s):  
A Kalam
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Low Income ◽  
Water Samples ◽  
Real Time Pcr ◽  
Tap Water ◽  
Watery Diarrhea ◽  
Acid Extraction ◽  
Pcr Assay ◽  
Viral Contamination

Abstract Introduction/Objective Diarrhea is a major source of morbidity and mortality in low-income and middle-income countries. In underdeveloped countries, diseases caused by viruses identified in environmental samples cause major health problems. Little knowledge about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. Adenovirus which causes watery diarrhea, particular has been recognized as important causal pathogen. Adenovirus remains a global threat to public health and an indicator of inequity and lack of social development. Tap water samples from coastal sites in Karachi between 2019 and 2020 over a period of 11 months. The total of 40 tap water sample was examined for infectious Adenovirus by a real time polymerase chain reaction (PCR) amplification. Methods/Case Report This Pilot study is conducted on tap water samples from Karachi Pakistan, n=40 are processed. Extraction of nucleic acid from all filtered water samples collected with Sterivex filter units by using Qiagen DNeasy Power Water Sterivex Kit. As per the manufacturer’s instruction. Phocine herpesvirus(PhHV) is added as an external positive control to monitor the efficiency of nucleic acid extraction and amplification. TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) is being used in probe based real time PCR assay,the below 35 Ct value is considered as a positive sample. Results (if a Case Study enter NA) Results showed the total of 37.7% of the sources were positive for adenovirus.The level of viral contamination was moderate to high. Conclusion The results has been showed that no seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Further the Real time PCR assay increases the sensitivity and provides the high resolution of pathogen detection.

Sign in / Sign up

Export Citation Format

Share Document