Interlaboratory Validation for a Real-Time PCR Salmonella Detection Method Using the ABI 7500 FAST Real-Time PCR System

Sixteen FERN (Food Emergency Response Network) member laboratories collaborated in this study to verify extension of the real-time PCR Salmonella detection method originally designed for the single-tube Cepheid SmartCycler II and validated against the Salmonella method of the U.S. Food and Drug Administration Bacteriological Analytical Manual to the Applied Biosystems (ABI) 7500 FAST Real-Time PCR system multiwell plate platform. Four foods were selected for this study: chili powder, soft cheese, fish, and tomatoes; these foods represent products that are commonly analyzed for the presence of Salmonella for regulatory purposes. Each food consisted of six uninoculated control samples, six samples inoculated with low Salmonella levels (target 1 to 5 CFU/25 g), and six samples inoculated with high levels (target 10 to 50 CFU/25 g). All samples were tested for Salmonella using the 24-h quantitative PCR (qPCR) method for detecting Salmonella, which utilizes modified buffered peptone water as the sole enrichment medium and an internal control for the qPCR. Each of these 18 samples was individually analyzed for Salmonella by the collaborating laboratories using both the ABI 7500 FAST system (alternative method) and the SmartCycler II system (reference method). Statistical analysis of the data revealed no significant difference (P ≥ 0.05) between these two qPCR platforms except for the chili powder samples. The differences noted with chili powder (P = 0.0455) were attributed to the enhanced sensitivity of the ABI 7500 FAST system compared with the SmartCycler II system. The detection limit of both qPCR methods was 0.02 to 0.15 CFU/g. These results provide a solid basis for extending the 24-h qPCR Salmonella method to the ABI 7500 FAST system for high-throughput detection of Salmonella in foods.

Download Full-text

TLR, RAGE, and HMGB1 in the Inflammatory Response in Ph(-) Myeloproliferative Neoplasm

Blood ◽

10.1182/blood-2020-134988 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Guanfang Shi ◽

Kiron Nair ◽

Preethi Ramachandran ◽

Chi Chen ◽

Ching Wong ◽

...

Keyword(s):

Inflammatory Response ◽

Real Time ◽

Inflammatory Cytokines ◽

Internal Control ◽

Real Time Pcr ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Philadelphia Chromosome ◽

Myeloproliferative Neoplasm ◽

Significant Difference

Recent evidence of increased constitutional symptoms and inflammatory cytokines in Philadelphia chromosome negative (Ph (-)) MPN suggests that an inflammatory response is important in the pathogenesis of Ph (-) MPN. Toll-like receptors (TLR), Receptor for Advanced Glycation End products (RAGE) and High mobility group protein B1 (HMGB1) are the important pathways for the inflammatory response. All these three important pathway proteins were studied in MPN diseases in the current studies. Materials and Methods: TLR assay. TLR 2,3, 4, 7, 9 quantification was performed by immuno-staining of 1×106 mononuclear cells (peripheral blood) which were incubated with fluorescence-conjugated anti-TLR-2,3, 4, 7, 9 antibodies and assayed by flow cytometry. HMGB1assay:HMGB1 ELISA kit from Immuno-Biological Laboratories, Inc. (IBL-America) were used. The plasma samples were diluted four times with the provided sample dilution buffer, and assayed in duplicate according to the manufacturer's suggestion. RAGE (RT-PCR) Assay: Total RNA was extracted from normal control or patient mononuclear cells. Predesigned primers for RAGE, and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of RAGE were compared with each internal control. Average of three was used to calculate the ratio of final patient to normal Results: Total of 97 patients with MPN were studied 1) TLR: TLR 3,7,9 was not significantly different from controls. But TLR 2 was significantly increased in both PV, as well as in the MPN group when PV, ET and MF were grouped together as MPN (Fig A). TLR 4 was not significantly increased in PV, ET, MF individually but was found to be significantly increased than the controls, when they are grouped together as MPN (Fig B). 2) RAGE: No significant difference was found between ET, PV, MF individually or when they were grouped together as MPN than the controls (Fig C). 3) HMGB1: No significant difference was seen between ET, PV, MF or when they were grouped as MPN (Fig D). Conclusion: Current study suggests that TLR pathway especially TLR2, and to a lesser extent TLR4 are the important pathways for inflammatory response with increased inflammatory cytokines in MPN, while HMGB1 and RAGE pathways were not different from controls. Figure Disclosures No relevant conflicts of interest to declare.

Download Full-text

Validation of iQ-Check E. coli O157:H7 Real-Time PCR Test Kit for Detection of Escherichia coli O157:H7 in Selected Foods

Journal of AOAC International ◽

10.1093/jaoac/92.4.1095 ◽

2009 ◽

Vol 92 (4) ◽

pp. 1095-1104 ◽

Cited By ~ 1

Author(s):

Wendy F Lauer ◽

Sylvie Tymciu ◽

Caroline D Sidi ◽

Pierre Sonigo

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Ground Beef ◽

Reference Method ◽

Target Sequence ◽

Apple Cider ◽

E Coli ◽

Test Kit ◽

Significant Difference ◽

The U.S

Abstract iQ-Check E. coli O157:H7 (Bio-Rad Laboratories, Hercules, CA) is a real-time PCR kit for detection of E. coli O157:H7 from selected foods. Specific fluorescent oligonucleotide probes are used to detect target DNA during the amplification, by hybridizing to the amplicons. These fluorescent probes are linked to a fluorophore which fluoresces only when hybridized to the target sequence. Three foods (ground beef, apple cider, fresh spinach) were selected to compare the performance of iQ-Check E. coli O157:H7 to the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) reference method for ground beef and the U.S. Food and Drug Administration/Bacteriological Analytical Manual reference method for apple cider and fresh spinach. Three protocols were tested in this study: a shortened 8 h primary enrichment in buffered peptone water (BPW), a 24 h enrichment in BPW, and an enrichment in appropriate reference method enrichment broth. The iQ-Check E. coli O157:H7 method was able to identify more true/confirmed positive samples than the reference method. Inclusivity and exclusivity rates of the method were 100. iQ-Check E. coli O157:H7 performed as expected when minor procedural variations were introduced, validating the ruggedness of the method. There was no significant difference observed in performance over the shelf life of the kit.

Download Full-text

Evaluation of Applied Biosystems MicroSEQ® Real-Time PCR System for Detection of Salmonella spp. in Food

Journal of AOAC International ◽

10.1093/jaoac/94.4.1106 ◽

2011 ◽

Vol 94 (4) ◽

pp. 1106-1116 ◽

Cited By ~ 9

Author(s):

Priya Balachandran ◽

Yanxiang Cao ◽

Lily Wong ◽

Manohar R Furtado ◽

Olga V Petrauskene ◽

...

Keyword(s):

Real Time ◽

Study Design ◽

Real Time Pcr ◽

Detection System ◽

Peanut Butter ◽

Reference Method ◽

Black Pepper ◽

Salmonella Spp ◽

Culture Methods ◽

Significant Difference

Abstract Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time-to-results compared to traditional culture methods. In this study, the MicroSEQ® real-time PCR system was evaluated for detection of Salmonella spp. in 10 different food matrixes following the AOAC Research Institute's Performance Tested MethodSM validation program. In addition, the performance of the MicroSEQ system was evaluated for the detection of Salmonella in peanut butter as a part of the Emergency Response Validation Program sponsored by the AOAC Research Institute. The system was compared to the ISO 6579 reference method using a paired-study design for detecting Salmonella spp. in raw ground beef, raw chicken, raw shrimp, Brie cheese, shell eggs, cantaloupe, chocolate, black pepper, dry infant formula, and dry pet food. For the peanut butter study, the system was compared to the U.S. Food and Drug Administration's Bacteriological Analytical Manual procedures using an unpaired-study design. No significant difference in performance was observed between the MicroSEQ Salmonella spp. detection system and the corresponding reference methods for all 11 food matrixes. The MicroSEQ system detected all Salmonella strains tested, while showing good discrimination against detection of an exclusivity panel of 30 strains, with high accuracy.

Download Full-text

iQ-Check Listeria spp. Real-Time PCR Test Kit

Journal of AOAC International ◽

10.5740/jaoacint.govval06 ◽

2013 ◽

Vol 96 (3) ◽

pp. 508-515

Author(s):

Wendy F Lauer ◽

Jean-Philippe Tourniaire

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Reference Method ◽

Sampling Area ◽

Fermented Sausage ◽

Low Level ◽

Test Kit ◽

Significant Difference ◽

High Level ◽

Health Canada

Abstract A comparative evaluation study of the Bio-Rad® iQ-Check™Listeria species Kit (Bio-Rad Laboratories, Hercules, CA) was conducted at Q Laboratories, Inc., Cincinnati, OH. iQ-Check is a rapid method based on real-time PCR amplification and detection of all species of Listeria, including L. grayi, in food and environmental samples. The iQ-Check method was compared to the Health Canada MFHPB-30 reference method for the analysis of five ready-to-eat meats—deli turkey, hot dogs, liver paté, raw fermented sausage, and deli ham—and one stainless steel surface. Each food matrix was analyzed at two contamination levels: a low level at 0.2–2 CFU/25 g and a high level at 2–5 CFU/25 g. The environmental surfaces were analyzed at a low level of 0.2–2 CFU/5 cm2 sampling area and a high level of 2–5 CFU/5 cm2 sampling area. There were 20 replicates per contamination level and five control replicates at 0 CFU/25 g or 0 CFU/5 cm2 sampling area (uninoculated). All samples that were detected by iQ-Check were subsequently confirmed by reference method protocol. There was no significant difference in the number of positive samples detected by the iQ-Check Listeria spp. Kit in comparison to the Health Canada MFHPB-30 method for all matrixes tested.

Download Full-text

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

Journal of AOAC International ◽

10.5740/jaoacint.13-407 ◽

2014 ◽

Vol 97 (3) ◽

pp. 868-875 ◽

Cited By ~ 1

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.

Download Full-text

Evaluation of the Biofire FilmArray BioThreat-E Test (v2.5) for Rapid Identification of Ebola Virus Disease in Heat-Treated Blood Samples Obtained in Sierra Leone and the United Kingdom

Journal of Clinical Microbiology ◽

10.1128/jcm.02287-15 ◽

2015 ◽

Vol 54 (1) ◽

pp. 114-119 ◽

Cited By ~ 27

Author(s):

Simon A. Weller ◽

Daniel Bailey ◽

Steven Matthews ◽

Sarah Lumley ◽

Angela Sweed ◽

...

Keyword(s):

United Kingdom ◽

Real Time ◽

Sierra Leone ◽

Real Time Pcr ◽

Ebola Virus ◽

Reference Method ◽

Blood Samples ◽

The United Kingdom ◽

Heat Treated ◽

Significant Difference

Rapid Ebola virus (EBOV) detection is crucial for appropriate patient management and care. The performance of the FilmArray BioThreat-E test (v2.5) using whole-blood samples was evaluated in Sierra Leone and the United Kingdom and was compared with results generated by a real-time Ebola Zaire PCR reference method. Samples were tested in diagnostic laboratories upon availability, included successive samples from individual patients, and were heat treated to facilitate EBOV inactivation prior to PCR. The BioThreat-E test had a sensitivity of 84% (confidence interval [CI], 64% to 95%) and a specificity of 89% (CI, 73% to 97%) in Sierra Leone (n= 60; 44 patients) and a sensitivity of 75% (CI, 19% to 99%) and a specificity of 100% (CI, 97% to 100%) in the United Kingdom (n= 108; 70 patients) compared to the reference real-time PCR. Statistical analysis (Fisher's exact test) indicated there was no significant difference between the methods at the 99% confidence level in either country. In 9 discrepant results (5 real-time PCR positives and BioThreat-E test negatives and 4 real-time PCR negatives and BioThreat-E test positives), the majority (n= 8) were obtained from samples with an observed or probable low viral load. The FilmArray BioThreat-E test (v2.5) therefore provides an attractive option for laboratories (either in austere field settings or in countries with an advanced technological infrastructure) which do not routinely offer an EBOV diagnostic capability.

Download Full-text

Genome-wide screening of m6A-modified transcript in the Wilms’ tumor

10.21203/rs.3.rs-197376/v1 ◽

2021 ◽

Author(s):

Zhuo Liu ◽

Feng He ◽

Jing Liu ◽

Shengrong OuYang ◽

Zexi Li ◽

...

Keyword(s):

Gene Ontology ◽

Real Time ◽

Real Time Pcr ◽

Wilms Tumor ◽

Expression Patterns ◽

Mrna Levels ◽

Gene Ontology Analysis ◽

Quantitative Real Time Pcr ◽

Related Factors ◽

Significant Difference

Abstract Background Wilms’ tumor, also called nephroblastoma, is the most common pediatric renal malignancy. The pathogenesis of Wilms’ tumor has been attributed to several genetic and epigenetic factors. However, the most pervasive internal mRNA modification that affects almost every process of RNA metabolism, RNA N6-Methyladenosine (m6A) methylation, has not been characterized in Wilms’ tumor. Methods Wilms’ tumor (WT) and adjacent non-cancerous (NC) tissue samples were obtained from 23 children with nephroblastoma, and the global m6A levels were measured by mass spectrometry. Analyses by m6A-mRNA epitranscriptomic microarray and mRNA microarray were performed, and m6A-related mRNAs were validated by quantitative real-time PCR for input and m6A-immunoprecipitated RNA samples from WT and NC tissues. Gene ontology analysis and KEGG pathway analysis were performed for differentially expressed genes, and expression of RNA methylation-related factors was measured by quantitative real-time PCR. Results The total m6A methylation levels in total RNA of WT samples and NC samples were (0.21 ± 0.01)% and (0.22 ± 0.01)%, respectively, with no statistically significant difference. Fifty-nine transcripts were differentially m6A-methylated between the WT and NC groups, which showed distinct m6A modification patterns. Gene ontology analysis indicated that m6A-modified genes were enriched in cancer-associated pathways, including the mTOR pathway, and conjoint analysis of the unique methylation and gene expression patterns in WT samples suggested an association with metabolic pathways.The mRNA levels of the m6A-related “reader” genes, YTHDF1, YTHDF2 and IGF2BP3, were statistically higher in WT samples than in NC samples. Conclusion This is the first study to determine the m6A modification profiles in Wilms’ tumor. Our data provide novel information regarding patterns of m6A modification that correlate with carcinogenesis in Wilms’ tumor.

Download Full-text

The association of male infertility with telomere length: A case control study

Indian Journal of Clinical Anatomy and Physiology ◽

10.18231/j.ijcap.2021.070 ◽

2021 ◽

Vol 8 (4) ◽

pp. 325-332

Author(s):

Kate Deepali Rajesh ◽

Puranam Vatsalaswamy ◽

Manvikar Purshotam Rao

Keyword(s):

Male Infertility ◽

Telomere Length ◽

Real Time ◽

Real Time Pcr ◽

Case Control Study ◽

Case Control ◽

Control Group ◽

Significant Difference ◽

Control Study ◽

Sperm Telomere Length

To study the relevance of sperm telomere length and infertility in men. : Our case-control study included twenty-five males in couple with sub-fertility/infertility (test group) and twenty five healthy males (control group) with proven paternity in the age group 25 to 35 years. The Absolute Sperm Telomere length (aSTL) was measured by real-time PCR. We investigated whether any significant difference in the aSTL value existed between the groups and analysed the relationship between aSTL and other sperm parameters.The mean (SE) aSTL recorded in the infertile cases was significantly shorter than for the control group being 140.60 (6.66) Kb/genome and 239.63 (12.32) Kb/genome respectively (p <0.001) A weak correlation was eminent between aSTL kb/genome and the total sperm count mil/ml (rho= 0.04, p - 0.86), progressive sperm motility (rho= - 0.02, p=0.934) and sperm viability (rho= - 0.07 p=0.741) in the infertile group. The measurement of aSTL by real-time PCR is a simple and rapid method that offers further paramount information with respective to the quality of sperm. It is befitted for epidemiological studies, hence opening new perspectives in the evaluation of male infertility. Limitations - Our study was confined to men aged between 25 and 35 years. Further comparative studies are needed to explore the significance of STL and infertility in older males. Additional studies will help illumine the significance of aSTL as a prognostic biomarker in assisted reproduction.

Download Full-text

Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

10.21203/rs.3.rs-45487/v2 ◽

2021 ◽

Author(s):

Stephen Tukwasibwe ◽

James A. Traherne ◽

Olympe Chazara ◽

Jyothi Jayaraman ◽

John Trowsdale ◽

...

Keyword(s):

Infectious Diseases ◽

Malaria Transmission ◽

Real Time ◽

High Throughput ◽

Real Time Pcr ◽

Association Studies ◽

Transmission Intensity ◽

Malaria Transmission Intensity ◽

Significant Difference ◽

Pcr Method

Abstract Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches.Methods: High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method developed will be useful in disease association studies involving large cohorts.

Download Full-text

Investigation of the effect of Prunus Amygdalus Amara on the expression of some genes of apoptosis and immortality in breast cancer cells (MCF-7)

Current Drug Research Reviews ◽

10.2174/2589977513666211202094433 ◽

2021 ◽

Vol 13 ◽

Author(s):

Maryam Abdolahi-Majd ◽

Gholamhossein Hassanshahi ◽

Mahboubeh Vatanparast ◽

Mojgan Noroozi Karimabad

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Analysis ◽

Mrna Levels ◽

Prunus Amygdalus ◽

Mcf7 Cells ◽

Significant Difference ◽

Anti Cancer ◽

Bitter Almond ◽

Mcf 7

Background: Anti-cancer effects of almond nuts or oil have been approved, but there are a few pieces of research that have evaluated, in detail, almond and other seeds' effects on cancer. Therefore, in the present project, the aim was to explore the regulatory effect of the bitter almond extract (Prunus amygdalus Batsch) on the apoptotic and anti-cancer potency of MCF-7 cells. Objectives: In the current experimental research, the Almond effect on MCF7 cells was evaluated by investigating the expression and the balance between Bcl-2, Bax genes to unmark the potential molecular mechanism. Methods: For 24 and 48h, the MCF7 cells were treated with the bitter almond extract (187.5-3000 µg/mL). MTT assay was used to assess the viability, and Real-time-PCR was applied to determine the expression of Bax and Bcl-2, facing β-actin. Results: Our results revealed a significant difference between different extract concentrations on the viability of MCF7 cell lines in 24 and 48 h; cell viability decreased time-dependently (P < 0.05). After 24 and 48h of extract facing MCF7 cells, the evaluated IC50 value was 3000 and 1500 µg/mL, respectively. Based on Real Time-PCR analysis, after 24 and 48 h, the mRNA levels of BCL-2 decreased by the extract, whereas BAX was in the MCF-7 cell line. Conclusion: From the results, it can be concluded that bitter almond extract has anti-cancer properties that may influence the apoptotic pathways by regulating relative gene expression.

Download Full-text