scholarly journals Performance characteristics of a quantitative PCR assay on repository stool specimens and smeared filter-paper cards

2020 ◽  
Author(s):  
Michele Tisdale ◽  
Indrani Mitra ◽  
Andrea J McCoy ◽  
Mark P Simons ◽  
Nathanael D Reynolds ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Quantitative Pcr ◽  
Pathogen Detection ◽  
Prolonged Storage ◽  
Storage Duration ◽  
Acid Yield ◽  
Stool Specimens ◽  
Pcr Assays ◽  
The Impact ◽  
Archival Specimens

Abstract Objective Stool repositories are a valuable resource for retrospective analyses including quantitative PCR assays to distinguish between asymptomatic shedding and clinical disease. The suitability of archival specimens for this purpose is unclear and requires assessment. We conducted a pilot study to evaluate pathogen detection by TaqMan Array Card (TAC) in travelers’ diarrhea (TD) stool specimens stored for 1-13 years, as well as the impact of transporting specimens on Whatman FTA Elute cards (FTA Cards) on detection. Results The positive percent agreement (PPA) for TAC on stool vs. microbiologic testing was lower than our a priori PPA estimate of 80% for most pathogens: Shigella spp. (100% [95%CI: 69-100%]), enterotoxigenic E coli (ETEC) (63% [95%CI: 49-75%]), Campylobacter spp. (66% [95%CI: 43-85%]) and Norovirus (37% [95%CI: 16-61%]). Use of the FTA card resulted in a further reduction of PPA. Our findings suggest that archival specimens may lead to insensitive detection on quantitative PCR assays due to degradation of nucleic acid with prolonged storage, although our limited sample size precluded us from evaluating the impact of storage duration on nucleic acid yield. Additional studies are needed to understand the impact of storage duration on quantitative PCR data.

scholarly journals Performance characteristics of a quantitative PCR assay on repository stool specimens and smeared filter-paper cards

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Michele D. Tisdale ◽  
Indrani Mitra ◽  
Andrea J. McCoy ◽  
Mark P. Simons ◽  
Nathanael D. Reynolds ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Quantitative Pcr ◽  
Pathogen Detection ◽  
Prolonged Storage ◽  
Storage Duration ◽  
Acid Yield ◽  
Stool Specimens ◽  
Pcr Assays ◽  
The Impact ◽  
Archival Specimens

Abstract Objective Stool repositories are a valuable resource for retrospective analyses including quantitative PCR assays to distinguish between asymptomatic shedding and clinical disease. The suitability of archival specimens for this purpose is unclear and requires assessment. We conducted a pilot study to evaluate pathogen detection by TaqMan Array Card (TAC) in travelers’ diarrhea (TD) stool specimens stored for 1–13 years, as well as the impact of transporting specimens on Whatman FTA Elute cards (FTA Cards) on detection. Results The positive percent agreement (PPA) for TAC on stool vs. microbiologic testing was lower than our a priori PPA estimate of 80% for most pathogens: Shigella spp. (100% [95%CI 69–100%]), enterotoxigenic E coli (ETEC) (63% [95%CI 49–75%]), Campylobacter spp. (66% [95%CI 43–85%]) and Norovirus (37% [95%CI 16–61%]). Use of the FTA card resulted in a further reduction of PPA. Our findings suggest that archival specimens may lead to insensitive detection on quantitative PCR assays due to degradation of nucleic acid with prolonged storage, although our limited sample size precluded us from evaluating the impact of storage duration on nucleic acid yield. Additional studies are needed to understand the impact of storage duration on quantitative PCR data.

scholarly journals Performance characteristics of a quantitative PCR assay on repository stool specimens and smeared filter-paper cards

2020 ◽  
Author(s):  
Michele Tisdale ◽  
Indrani Mitra ◽  
Andrea J McCoy ◽  
Mark P Simons ◽  
Nathanael D Reynolds ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Quantitative Pcr ◽  
Pathogen Detection ◽  
Prolonged Storage ◽  
Storage Duration ◽  
Acid Yield ◽  
Stool Specimens ◽  
Pcr Assays ◽  
The Impact ◽  
Archival Specimens

Abstract Objective Stool repositories are a valuable resource for retrospective analyses including quantitative PCR assays to distinguish between asymptomatic shedding and clinical disease. The suitability of archival specimens for this purpose is unclear and requires assessment. We conducted a pilot study to evaluate pathogen detection by TaqMan Array Card (TAC) in travelers’ diarrhea (TD) stool specimens stored for 1-13 years, as well as the impact of transporting specimens on Whatman FTA Elute cards (FTA Cards) on detection. Results The positive percent agreement (PPA) for TAC on stool vs. microbiologic testing was lower than our a priori PPA estimate of 80% for most pathogens: Shigella spp. (100% [95%CI: 69-100%]), enterotoxigenic E coli (ETEC) (63% [95%CI: 49-75%]), Campylobacter spp. (66% [95%CI: 43-85%]) and Norovirus (37% [95%CI: 16-61%]). Use of the FTA card resulted in a further reduction of PPA. Our findings suggest that archival specimens may lead to insensitive detection on quantitative PCR assays due to degradation of nucleic acid with prolonged storage, although our limited sample size precluded us from evaluating the impact of storage duration on nucleic acid yield. Additional studies are needed to understand the impact of storage duration on quantitative PCR data.

scholarly journals Performance Characteristics of the TaqMan Array PCR on Repository Stool Specimens and Smears on Whatman® FTA Elute Cards

2020 ◽  
Author(s):  
Michele Tisdale ◽  
Indrani Mitra ◽  
Andrea J McCoy ◽  
Mark P Simons ◽  
Nathanael D Reynolds ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Quantitative Pcr ◽  
Prolonged Storage ◽  
Storage Duration ◽  
Acid Yield ◽  
Stool Specimens ◽  
Pcr Assays ◽  
Taqman Array ◽  
The Impact ◽  
Archival Specimens

Abstract Objective Stool repositories are a valuable resource for retrospective analyses including quantitative PCR assays to distinguish between asymptomatic shedding and clinical disease. The suitability of archival specimens for this purpose is unclear and requires assessment. We conducted a pilot study to evaluate pathogen detection by TaqMan Array Card (TAC) in travelers’ diarrhea (TD) stool specimens stored for 1–13 years, as well as, the impact of transporting specimens on Whatman FTA Elute cards (FTA Cards) on detection. Results The positive percent agreement (PPA) for TAC on stool vs. microbiologic testing was lower than our a priori PPA estimate of 80% for most pathogens: Shigella spp. (100% [95%CI: 69–100%]), enterotoxigenic E coli (ETEC) (63 [49–75%]), Campylobacter spp. (66 [43–85%]) and Norovirus (37 [16–61%]). Use of the FTA card resulted in a further reduction of PPA. Our findings suggest that archival specimens may lead to insensitive detection on quantitative PCR assays due to degradation of nucleic acid with prolonged storage, although our limited sample size precluded us from evaluating the impact of storage duration on nucleic acid yield. Additional studies are needed to understand the impact of storage duration on quantitative PCR data.

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 Application of Quantitative-PCR to Monitor Netpen Sites in British Columbia (Canada) for Tenacibaculum Species

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 414
Author(s):  
Joseph P. Nowlan ◽  
Scott R. Britney ◽  
John S. Lumsden ◽  
Spencer Russell
Keyword(s):  
Atlantic Salmon ◽  
Quantitative Pcr ◽  
Salmo Salar ◽  
Bacterial Load ◽  
Temporal Distribution ◽  
Quality Parameters ◽  
Antimicrobial Use ◽  
Live Fish ◽  
Dead Fish ◽  
Pcr Assays

Tenacibaculum are frequently detected from fish with tenacibaculosis at aquaculture sites; however, information on the ecology of these bacteria is sparse. Quantitative-PCR assays were used to detect T. maritimum and T. dicentrarchi at commercial Atlantic salmon (Salmo salar) netpen sites throughout several tenacibaculosis outbreaks. T. dicentrarchi and T. maritimum were identified in live fish, dead fish, other organisms associated with netpens, water samples and on inanimate substrates, which indicates a ubiquitous distribution around stocked netpen sites. Before an outbreak, T. dicentrarchi was found throughout the environment and from fish, and T. maritimum was infrequently identified. During an outbreak, increases in the bacterial load in were recorded and no differences were recorded after an outbreak supporting the observed recrudescence of mouthrot. More bacteria were recorded in the summer months, with more mortality events and antibiotic treatments, indicating that seasonality may influence tenacibaculosis; however, outbreaks occurred in both seasons. Relationships were identified between fish mortalities and antimicrobial use to water quality parameters (temperature, salinity, dissolved oxygen) (p < 0.05), but with low R2 values (<0.25), other variables are also involved. Furthermore, Tenacibaculum species appear to have a ubiquitous spatial and temporal distribution around stocked netpen sites, and with the potential to induce disease in Atlantic salmon, continued research is needed.

Multifunctional Printable Micropattern Array for Digital Nucleic Acid Assay for Microbial Pathogen Detection

2021 ◽  
Vol 13 (2) ◽  
pp. 3098-3108
Author(s):  
Yunho Choi ◽  
Younseong Song ◽  
Yong Tae Kim ◽  
Seok Jae Lee ◽  
Kyoung G. Lee ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Pathogen Detection ◽  
Microbial Pathogen ◽  
Nucleic Acid Assay

The Effects of Storage Duration on Biodiesel Derived from Waste Cooking Oil

2014 ◽  
Vol 660 ◽  
pp. 386-390 ◽  
Author(s):  
Norazwan Azman ◽  
Mirnah Suardi ◽  
Amir Khalid
Keyword(s):  
Diesel Fuel ◽  
Fossil Fuels ◽  
Acid Value ◽  
Waste Cooking Oil ◽  
Biodiesel Fuel ◽  
Storage Duration ◽  
Fuel Prices ◽  
Cooking Oil ◽  
Impact Reduction ◽  
The Impact

The use of fossil fuels as energy sources has grown to significantly be likely to have a major environmental impact. Reduction of world oil reserves and increasing environmental concerns have prompted alternative is found and renewable source of energy called biodiesel. Biodiesel fuel from vegetable oil is considered as the best candidates for diesel fuel replacement in diesel engines because of its closer. Fuel prices are going up day by day in the world. Thus, the means and methods have been trying for years to get fuel alternative outcomes. This study investigated the effects of different storage periods used in quality biodiesel blends (B5, B10, B15) of waste cooking oil and diesel fuel under low temperature and the temperature of the environment. Biodiesel samples were stored in glass containers under indoor conditions, and outdoor conditions for 10 weeks in total. These samples were monitored on a weekly basis through the test properties. The experimental density, viscosity, acid value, water content and flash point discussed in detail. Biodiesel storage at low temperatures is suitable and more advantageous because the impact on the physical properties is minimal and beneficial to slow down the degradation of biodiesel and storage.

scholarly journals Nucleic acid amplification-based pathogen detection in the blood of severe sepsis patients

Critical Care ◽  
2008 ◽  
Vol 12 (Suppl 5) ◽  
pp. P43 ◽  
Author(s):  
Frank Bloos ◽  
Svea Sachse ◽  
Karl-Herrmann Schmidt ◽  
Mark Lehmann ◽  
Roland Schmitz ◽  
...  
Keyword(s):  
Severe Sepsis ◽  
Nucleic Acid ◽  
Pathogen Detection ◽  
Nucleic Acid Amplification

scholarly journals ASSESSMENT OF SHELF LIFE AND BACTERIAL LOAD OF VIABLE EGGS OBTAINED AT THE POINT OF LAY FROM ROOM AND REFRIGERATOR STORAGE TEMPERATURES

2020 ◽  
Vol 3 (3) ◽  
pp. 62-65
Author(s):  
Obhioze Augustine Akpoka
Keyword(s):  
Shelf Life ◽  
Proteus Mirabilis ◽  
Room Temperature ◽  
Bacterial Load ◽  
Viable Count ◽  
Pseudomonas Sp ◽  
Storage Duration ◽  
Egg Storage ◽  
Salmonella Pullorum ◽  
The Impact

It is well established that storing hatching eggs over a longer period of time affects its quality. The current study evaluated the impact of egg storage duration in-relation to two different temperature conditions (room and refrigerator) to determine the bacterial load and shelf life of viable eggs. One hundred and twenty eggs were used for this study, 60 were boiled and 60 were raw. Thirty of the boiled eggs were stored at room temperature and the other 30 eggs were kept in the refrigerator. Similarly, 30 raw eggs were each stored at room and optimal refrigeration temperatures for eggs (< 7 oC) respectively, while the egg weight, viability and sensory tests were performed daily on the eggs. However, the eggs kept in the refrigerator were viable for longer and relatively maintained higher physical appearance and sensory quality compared to eggs kept at room temperature. In the investigation of bacterial load, the total viable count ranged from 6.0× 103 to 11.9 × 103 coliform forming unit per millilitre (cfu/ml) and 1.0 × 103 to 6.5 × 103 cfu/ml for the boiled eggs kept at room and refrigeration temperatures (BRT and BFT) respectively. More so, the bacterial counts in raw eggs obtained at room and refrigerator storage ranged from 4.8 × 103 to 6.5 × 103 cfu/ml. Subsequently, the characterization and identification of bacterial isolates indicated the presence of Salmonella pullorum, Proteus mirabilis and Pseudomonas sp. The Salmonella pullorum was isolated from all the egg samples (BRT, BFT, RRT and RFT). The Proteus mirabilis was isolated from boiled eggs kept in both room and refrigerator temperatures (BRT and BFT) while Pseudomonas sp. was obtained only from raw eggs stored in the refrigerator (RFT). In addition, the boiled eggs at room temperature started deterioration on Day 9, while its counterpart in the refrigerator began spoilage or decrease in quality from Day 16. The weight of the viable eggs in relation to the non-viable ones was statistically significant (P < 0.05). The refrigeration of eggs increases its longevity while proper hygiene and adequate boiling of eggs reduces the risk of acquiring infections through bacterial contamination.

scholarly journals Contamination-resistant, rapid emulsion-based isothermal nucleic acid amplification with Mie-scatter inspired light scatter analysis for bacterial identification

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander S. Day ◽  
Tiffany-Heather Ulep ◽  
Elizabeth Budiman ◽  
Laurel Dieckhaus ◽  
Babak Safavinia ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Detection ◽  
Light Scatter ◽  
Emulsion Droplets ◽  
Assay Performance ◽  
Bacterial Adsorption ◽  
Scatter Intensity ◽  
The Impact ◽  
Theory Light

AbstractAn emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to reduce the impact that contamination has on assay performance. Ongoing LAMP reactions within the emulsion droplets cause a decrease in interfacial tension, causing a decrease in droplet size, which results in decreased light scatter intensity due to Mie theory. Light scatter intensity was monitored via spectrophotometers and fiber optic cables placed at 30° and 60°. Light scatter intensities collected at 3 min, 30° were able to statistically differentiate 103 and 106 CFU/µL initial Escherichia coli O157:H7 concentrations compared to NTC (0 CFU/µL), while the intensity at 60° were able to statistically differentiate 106 CFU/µL initial concentrations and NTC. Control experiments were conducted to validate nucleic acid detection versus bacterial adsorption, finding that the light scatter intensities change is due specifically to ongoing LAMP amplification. After inducing contamination of bulk LAMP reagents, specificity lowered to 0% with conventional LAMP, while the eLAMP platform showed 87.5% specificity. We have demonstrated the use of angle-dependent light scatter intensity as a means of real-time monitoring of an emulsion LAMP platform and fabricated a smartphone-based monitoring system that showed similar trends as spectrophotometer light scatter data, validating the technology for a field deployable platform.

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