scholarly journals Development of a Standard Curve to Account for Viable Loads of Bifidobacterium animalis subsp. lactis HN019 Using RNA by Real-Time PCR

2019 ◽  
Vol 102 (2) ◽  
pp. 532-538
Author(s):  
Md. A Ali ◽  
Wang Hui ◽  
Guangen Lin ◽  
Fang F Dang ◽  
Shuang Wu ◽  
...  
Keyword(s):  
Real Time ◽  
Growth Curve ◽  
Real Time Pcr ◽  
Plate Count ◽  
Standard Curve ◽  
Bifidobacterium Animalis ◽  
Ct Value ◽  
Culture Independent ◽  
Optimal Health ◽  
Y Cells

Abstract Background: Bifidobacterium animalis subsp. lactis HN019 is widely used as a probiotic in various dairy preparations. To ensure optimal health benefits, an adequate number of probiotics should be viable in products, but the culture-based methods require longer periods to account for the strain. Objective: Develop a standard curve to account viable loadof B. animalis subsp. lactis HN019 by real-time PCR. Methods: The growth curve was developed accordingto plate count method, and cycle threshold (CT) curve was prepared using CT values. These two curves were then combined to construct the standard curve. To validate the method, the strain was proliferated in whole milk, and viable loads were enumerated by plate counting and relativePCR counting followed by determining the SEM betweenthe two counts. Results: The growth curve and CT curve, respectively, showed the highest viable load (2.13 × 108 CFU/mL) and lowest CT value (18.29) after 18 h, and during the entire growth phase (0–18 h), viable loads are inversely proportional to the CT values. The standard curverevealed the model y = 2E + 18e−1.233x (y = cells/mL, x = CT value; R2 = 0.992). In validation, the highest SEM (± 0.70 × 108) was found between the plate count (1.15 × 108 cells/mL) and relative PCR count (1.29 × 108 cells/mL) after cultivating for 14 h. Conclusions: The method could be readilyused in dairy industries to quantify viable B. animalis subsp. lactis HN019 by a shorter period. Highlights: Culture-independent enumeration of viable B. animalis subsp. lactis HN019 by real-time PCR.

scholarly journals (179) Using Quantitative Real Time PCR to Predict Levels of Botrytis allii in Stored Onion

HortScience ◽  
2006 ◽  
Vol 41 (4) ◽  
pp. 1016D-1016
Author(s):  
Timothy Coolong ◽  
William Randle ◽  
Ronald Walcott
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dry Weight ◽  
The United States ◽  
Quantitative Real Time Pcr ◽  
Standard Curve ◽  
Ct Value ◽  
Botrytis Allii ◽  
Log Linear ◽  
A Minor

Onion (Allium cepa L.) is an economically important vegetable in the United States. Though considered a minor crop in terms of total acreage, onions have high value when compared to other crops and, nationally, their value approaches $800 million. Because harvested onions are routinely stored for long periods, disease can be a major obstacle to the industry. The primary disease reported in stored onions is botrytis neck rot caused by the fungus Botrytis allii (syn. B. aclada). Losses from neck rot can approach 35% of the stored crop. In order to accurately quantify the level of B. allii inoculum in bulbs at harvest to be able to predict potential botrytis neck rot in storage, a quantitative real-time PCR test to quantify levels of B. allii DNA present in onion bulb tissue has been developed. We have employed the TaqMan real time PCR assay and report log-linear (R2= 0.9915) relationship between B. allii DNA concentration and cycle threshold (Ct) value with a detection limit of 5 pico gram/microliter DNA. In addition, a log-linear standard curve plotting mycelial dry weight against Ct value has been developed to allow prediction of mycelial weight in onion tissue at harvest. Currently, the ability of this test to predict botrytis neck rot during storage is being tested.

scholarly journals Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC)

2020 ◽  
Vol 8 (11) ◽  
pp. 1801
Author(s):  
Michael Bording-Jorgensen ◽  
Brendon D. Parsons ◽  
Gillian A.M. Tarr ◽  
Binal Shah-Gandhi ◽  
Colin Lloyd ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Gene Detection ◽  
Culture Positivity ◽  
Hands On ◽  
Culture Independent ◽  
Chromogenic Agar ◽  
Stool Samples

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes’ amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3–5 days until a sample is negative by culture.

scholarly journals Direct detection of microorganisms responsible for the sexually transmitted infection on the mobile real-time PCR device without treating in advance

2021 ◽  
Author(s):  
Masaaki Muraoka ◽  
Kazunori Sohma ◽  
Osamu Kawaguchi ◽  
Mikio Mizukoshi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Treponema Pallidum ◽  
Nucleic Acid Amplification ◽  
Direct Pcr ◽  
Transmitted Infection ◽  
Sexually Transmitted ◽  
Ct Value

ABSTRACTAs WHO reported, four curable STIs-chlamydia, gonorrhoea, syphilis and trichomoniasis occur more than 1 million per each day globally almond 2016. For this reason, it is important to control these STIs, one of which is “to detect”. The general methods in order to detect STIs are nucleic acid amplification tests (NAATs). One of the reasons why NAATs are utilized in many tests is that it is possibly to be more sensitive than other test. However, there needs to treat extraction of nucleic acids in advance and amplify specific regions by NAATs, and hence it must take much labour and much time. In this work, for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP) which is each etiological agent of chlamydia, gonorrhoea and syphilis, we evaluate and propose “quicker and simpler” NAATs. Specifically, utilizing mobile real-time PCR device “PCR1100” and PCR reagent kit “KAPA3G Plant PCR Kit”, it was considered whether real-time direct PCR could be performed or not without treating DNA extraction in advance so-called “direct”.As a result, firstly, we established that real-time direct PCR could be performed in all of CT, NG, and TP, and moreover, each Ct value correlated with the concentration of each organism similarly to detection of genome DNA (each correlation coefficient R2 > 0.95). Moreover, each assay demonstrated a limit of detection (LOD) of the follows; CT was 10^0.86 = 7.24 IFU/reaction, NG was 10^-0.19 = 0.65 CFU/reaction, and TP was 10^1.4 = 25.1 organisms/reaction. However, it appeared the sensitivity was a little low, especially for CT and TP.Secondly, we found that even as without treating sample in advance, the time of detection was required more less 15 minutes at any of case, which was very quick compared with other current methods for real-time PCR. Additionally, compared with other commercial devices, it was easier to operate the PCR1100 device, for example, start, analysis of Ct value.In conclusion, the present study has demonstrated that it is possible for real-time direct PCR to perform with combination of the PCR1100 device and the PCR reagent kit in 3 kinds of microorganisms-CT, NG and TP. Furthermore, we propose “quicker and simpler” methods for NAATs, which it would not take labour and time. Further studies are needed in order to contribute to control STIs.

Detection and Enumeration of Salmonella Enteritidis in Homemade Ice Cream Associated with an Outbreak: Comparison of Conventional and Real-Time PCR Methods

2006 ◽  
Vol 69 (3) ◽  
pp. 639-643 ◽  
Author(s):  
K. H. SEO ◽  
I. E. VALENTIN-BON ◽  
R. E. BRACKETT
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Direct Detection ◽  
Ice Cream ◽  
Plate Count ◽  
Pcr Assay ◽  
The Real ◽  
Real Time Quantitative Pcr

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

scholarly journals A SYBR green I real-time polymerase chain reaction (PCR) assay for detection and quantification of Trichomonas gallinae

2020 ◽  
Vol 119 (11) ◽  
pp. 3909-3913
Author(s):  
Zaida Rentería-Solís ◽  
Tran Nguyen-Ho-Bao ◽  
Shahinaz Taha ◽  
Arwid Daugschies
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Small Subunit ◽  
Sybr Green ◽  
Pcr Assay ◽  
Standard Curve ◽  
Trichomonas Gallinae ◽  
Host Interaction

Abstract Trichomonas gallinae are parasitic flagellates of importance in wild and domestic birds. The parasite is worldwide distributed, and Columbine birds are its main host. Current research focuses mostly on epidemiological and phylogenetic studies. However, there is still a lack of knowledge regarding parasite-host interaction or therapy development. Real-time PCR is a useful tool for diagnostic and quantification of gene copies in a determined sample. By amplification of a 113-bp region of the 18S small subunit ribosomal RNA gene, a SYBR green-based real-time PCR assay was developed. A standard curve was prepared for quantification analysis. Assay efficiency, linearity, and dissociation analysis were successfully performed. Specificity, sensibility, and reproducibility analysis were tested. This assay could be a useful tool not only for diagnostic purposes but also for future in vivo and in vitro T. gallinae studies.

scholarly journals Detection of Bifidobacterium animalis subsp. lactis (Bb12) in the Intestine after Feeding of Sows and Their Piglets

2008 ◽  
Vol 74 (20) ◽  
pp. 6338-6347 ◽  
Author(s):  
Gloria Solano-Aguilar ◽  
Harry Dawson ◽  
Marta Restrepo ◽  
Kate Andrews ◽  
Bryan Vinyard ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Protein Biosynthesis ◽  
Bifidobacterium Longum ◽  
Elongation Factor ◽  
Single Copy ◽  
Bifidobacterium Animalis ◽  
Gene Copies ◽  
Intestinal Contents ◽  
High Level

ABSTRACT A real-time PCR method has been developed to distinguish Bifidobacterium animalis subspecies in the gastrointestinal tracts of pigs. Identification of a highly conserved single-copy tuf gene encoding the elongation factor Tu involved in bacterial protein biosynthesis was used as a marker to differentiate homologous Bifidobacterium animalis subsp. lactis (strain Bb12) from Bifidobacterium animalis subsp. animalis, as well as Bifidobacterium suis, Bifidobacterium breve, Bifidobacterium longum, several species of Lactobacillus, and Enterococcus faecium. Real-time PCR detection of serially diluted DNA extracted from a pure culture of Bb12 was linear for bacterial numbers ranging from 10 to 10,000 tuf gene copies per PCR (r 2 = 0.99). Relative differences in Bb12 bacterial numbers in pigs fed daily with Bb12 were determined after detection of Bb12 tuf gene copies in DNA extracted from the intestinal contents. Piglets treated with Bb12 immediately after birth maintained a high level of Bb12 in their large intestines with continuous daily administration of Bb12. Piglets born to Bb12-treated sows during the last third of their gestation and also treated with Bb12 at birth (T/T group) had a higher number of Bb12 organisms per gram of intestinal contents compared to placebo-treated piglets born to placebo-treated sows (C/C group), Bb12-treated sows (T/C group), or piglets born to placebo sows but treated with Bb12 immediately after birth (C/T group). In addition, there was a significant increase in gene expression for Toll-like receptor 9 (TLR9) in piglets from the T/T group, with no change in TLR2 and TLR4. These findings suggest that the tuf gene represents a specific and functional marker for detecting Bifidobacterium animalis subsp. lactis strain Bb12 within the microbiota of the intestine.

scholarly journals Using the TxtAB Operon to Quantify Pathogenic Streptomyces in Potato Tubers and Soil

2008 ◽  
Vol 98 (4) ◽  
pp. 405-412 ◽  
Author(s):  
Xinshun Qu ◽  
Leslie A. Wanner ◽  
Barbara J. Christ
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Common Scab ◽  
Cost Effective ◽  
Potato Tubers ◽  
Pcr Assay ◽  
Streptomyces Species ◽  
Standard Curve ◽  
Target Dna

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R2 = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 101 to 106 pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 103 to 106 per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.

scholarly journals Establishment of a simpler method for measuring HDL-microRNAs

2018 ◽  
Vol 56 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Hiroaki Ishikawa ◽  
Hiroya Yamada ◽  
Kanako Kondo ◽  
Takeru Ota ◽  
Mirai Yamazaki ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Phosphotungstic Acid ◽  
Density Lipoprotein ◽  
Clinical Applications ◽  
Processing Capacity ◽  
Quantitative Real Time Pcr ◽  
Simple Method ◽  
Standard Curve ◽  
Purification Methods

Background MicroRNAs are present not only in exosomes but also in high-density lipoprotein (HDL) and have the potential as biomarkers for various diseases. Various purification methods have been developed to quantify HDL-miRNAs; however, they are unsuitable for clinical applications. Therefore, we aimed to establish a simpler analytical method to quantify HDL-miRNAs for clinical applications. Methods We purified HDL fraction from pooled plasma using a three-step protocol consisting of ultracentrifugation, phosphotungstic acid/MgCl2 precipitation and desalting/buffer exchange followed by the quantification of HDL-miRNAs by quantitative real-time PCR. In order to establish a method to quantify HDL-miRNAs by quantitative real-time PCR, we prepared standard curves for miR-223 and miR-92. The HDL-miRNAs of 10 volunteers were assessed. Results Exosomes and LDL were not detected in the purified HDL fraction. Furthermore, we confirmed that only HDL was purified and that the HDL recovery rate of our method was at least approximately 50%. The threshold cycle values of miR-223, miR-92, miR-146a and miR-150 in the same subject were 32.11 ± 0.58, 32.50 ± 0.35, 34.30 ± 0.70 and 34.91 ± 0.77, respectively ( n = 10). The coefficient of variation values for these miRNAs were 1.08–2.21%. In addition, the standard curve for the quantitative analysis of miRNAs showed high linearity (30–30,000 copies/ μL) with a correlation coefficient of >0.99. The concentrations of HDL-miR-223 and HDL-miR-92 in the plasma of 10 subjects were 1.98 ± 0.32 and 0.90 ± 0.14 copies/mL (×104). Conclusions We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.

scholarly journals Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of novel-goose parvovirus in vivo

2021 ◽  
Author(s):  
Haibin Ma ◽  
Yahui Li ◽  
Junzheng Yang
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Real Time Pcr ◽  
Bursa Of Fabricius ◽  
Pcr Assay ◽  
Standard Curve ◽  
Goose Parvovirus ◽  
Detection And Quantification

Objectives: To develop a sensitive, highly specific fluorescent quantitative real-time PCR assay for accurate detection and quantification of novel-goose parvovirus (N-GPV) in vitro and in vivo. Methods: Specific primers was designed based on N-GPV inverted terminal repeats region; virus RNA (DFV, NDV, AIV, DHV-1, DHV-3) and virus DNA (MDPV, GPV, N-GPV) were extracted, cDNA (DFV, NDV, AIV, DHV-1, DHV-3) were prepared from viral RNAs using M-MLV Reverse Transcriptase, and prepared cDNA (DFV, NDV, AIV, DHV-1, DHV-3) and DNA (MDPV, GPV, N-GPV) amplified by real-time PCR; the sensitivity, specificity and reproducibility of established real-time PCR methods were evaluated, and finally we validated the reliability of real-time PCR methods in ducklings models in vivo. Results: The standard curve of established real-time PCR had a good linearity (slope was -0.3098, Y-intercept was 37.865, efficiency of standard curve was 0.995); the detection limit of established real-time PCR for N-GPV was 10 copies/reaction. The sensitivity of real-time PCR was 10 copies/uL, which was 1000 times higher than conventional gel-based PCR assay. The results of intra-assay CVs (0.04-0.74%) and inter-assay CVs (0.16-0.53%) showed that the real-time PCR assay had an excellent repeatability. This method also could efficiently detect viral load in heart, liver, spleen, lung, kidney, pancreas, bursa of Fabricius, brain, blood and excrement from ducklings models after N-GPV infection from 6h to 28 days, which could provided us a dynamic distribution observation of N-GPV viral load using this real-time PCR assay in vivo. Conclusion: In the study, we developed a high sensitive, specific and reproducible real-time PCR assay for N-GPV detection. The established real-time PCR assay was suitable for parvovirus detection and quantification simultaneously, no matter sample obtained from blood, internal organs or ileac contents; the present work may provide insight into the pathogenesis of N-GPV and will contributes to better understanding of this newly emerged novel GPV related virus in cherry valley ducks.

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