scholarly journals Detection of soybean by real-time PCR in the samples subjected to deep technological processing

2019 ◽  
Vol 4 (4) ◽  
pp. 23-27
Author(s):  
K. A. Kurbakov ◽  
E. A. Konorov ◽  
V. N. Zhulinkova ◽  
M. Yu. Minaev
Keyword(s):  
Glycine Max ◽  
Real Time ◽  
Real Time Pcr ◽  
High Sensitivity ◽  
Food Product ◽  
Single Copy ◽  
Probe System ◽  
Canned Foods ◽  
Technological Processing ◽  
The Difference

During deep technological processing, DNA of food product components (specifically, in canned foods) is subjected to strong degradation, which makes the PCR-based food components identification more difficult. In this work, a primer-probe system is proposed, which was selected for the multi-copy region of long terminal repeat (LTR) of soybean (Glycine max). We confirmed its high sensitivity and specificity for soybean detection by real-time PCR. Using the selected system, we successfully analyzed the samples of meat-and-plant canned foods and other food products subjected to deep technological processing — tofu, preserved tofu, soy sauces, confectionary products containing soy lecithin. To compare with these samples, real-time PCR was carried out using the primer-probe system selected for the single-copy le1 gene. In terms of sensitivity, the use of the primer-probe system specific to the single-copy region was significantly inferior to the primer-probe system specific to the LTR region. The difference in the rate of degradation of these genomic DNA regions of Glycine max was found.

scholarly journals Single-copy detection of somatic variants from solid and liquid biopsy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana-Luisa Silva ◽  
Paulina Klaudyna Powalowska ◽  
Magdalena Stolarek ◽  
Eleanor Ruth Gray ◽  
Rebecca Natalie Palmer ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Clinical Decision Making ◽  
High Sensitivity ◽  
Clinical Decision ◽  
Single Copy ◽  
Turnaround Time ◽  
Copy Detection ◽  
Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

scholarly journals Real-Time PCR for Molecular Detection of Zoonotic and Non-Zoonotic Giardia spp. in Wild Rodents

2021 ◽  
Vol 9 (8) ◽  
pp. 1610
Author(s):  
Christian Klotz ◽  
Elke Radam ◽  
Sebastian Rausch ◽  
Petra Gosten-Heinrich ◽  
Toni Aebischer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gastrointestinal Disease ◽  
Single Copy ◽  
Marker Genes ◽  
Small Rodent ◽  
Analytical Sensitivity ◽  
Single Copy Gene ◽  
Wild Rodents ◽  
Copy Gene

Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents.

Exploring a phage-based real-time PCR assay for diagnosing Acinetobacter baumannii bloodstream infections with high sensitivity

2018 ◽  
Vol 1044 ◽  
pp. 147-153 ◽  
Author(s):  
Jun Luo ◽  
Mengwei Jiang ◽  
Jin Xiong ◽  
Junhua Li ◽  
Xiaoxu Zhang ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Real Time ◽  
Real Time Pcr ◽  
Bloodstream Infections ◽  
High Sensitivity ◽  
Pcr Assay

scholarly journals A Set of Conventional and Multiplex Real-Time PCR Assays for Direct Detection of Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis in Citrus Fruits

Plant Disease ◽  
2019 ◽  
Vol 103 (2) ◽  
pp. 345-356 ◽  
Author(s):  
Yosra Ahmed ◽  
Jacqueline Hubert ◽  
Céline Fourrier-Jeandel ◽  
Megan M. Dewdney ◽  
Jaime Aguayo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Media ◽  
Direct Detection ◽  
Elongation Factor ◽  
Single Copy ◽  
The United States ◽  
Performance Criteria ◽  
In Planta ◽  
Conventional Pcr

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg μl−1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) μl−1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

scholarly journals Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment

2017 ◽  
Vol 55 (10) ◽  
pp. 3104-3112 ◽  
Author(s):  
Heather L. Wilson ◽  
Thomas Tran ◽  
Julian Druce ◽  
Myrielle Dupont-Rouzeyrol ◽  
Michael Catton
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Sensitivity ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Health Concern ◽  
Confirmatory Test ◽  
Virus Neutralization Assay ◽  
History Of ◽  
Infective Complications

ABSTRACTThe global spread and infective complications of Zika virus (ZKV) and dengue virus (DENV) have made them flaviviruses of public health concern. Serological diagnosis can be challenging due to antibody cross-reactivity, particularly in secondary flavivirus infections or when there is a history of flavivirus vaccination. The virus neutralization assay is considered to be the most specific assay for measurement of anti-flavivirus antibodies. This study describes an assay where the neutralization endpoint is measured by real-time PCR, providing results within 72 h. It demonstrated 100% sensitivity (24/24 ZKV and 15/15 DENV) and 100% specificity (11/11 specimens) when testing well-characterized sera. In addition, the assay was able to determine the correct DENV serotype in 91.7% of cases. The high sensitivity and specificity of the real-time PCR neutralization assay makes it suitable to use as a confirmatory test for sera that are reactive in commercial IgM/IgG enzyme immunoassays. Results are objective and the PCR-based measurement of the neutralization endpoint lends itself to automation so that throughput may be increased in times of high demand.

scholarly journals Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

Toxins ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 757
Author(s):  
Sara Franco Ortega ◽  
Ilenia Siciliano ◽  
Simona Prencipe ◽  
Maria Lodovica Gullino ◽  
Davide Spadaro
Keyword(s):  
Food Safety ◽  
Real Time ◽  
Aspergillus Flavus ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Screening Method ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Isothermal Amplification

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

scholarly journals Molecular approach for insect detection in feed and food: the case of Gryllodes sigillatus

2020 ◽  
Vol 246 (12) ◽  
pp. 2373-2381
Author(s):  
Enrico Daniso ◽  
Francesca Tulli ◽  
Gloriana Cardinaletti ◽  
Roberto Cerri ◽  
Emilio Tibaldi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Agricultural Industry ◽  
Mitochondrial Cytochrome B ◽  
The Matrix ◽  
Pcr Products ◽  
Technological Processing ◽  
Pcr Method ◽  
Gryllodes Sigillatus ◽  
Novel Protein

AbstractThe production of insects on an industrial scale has attracted the attention of the research and agricultural industry as novel protein sources. To detect the presence of Gryllodes sigillatus (GS) in feed and food, a real-time PCR method based on the mitochondrial cytochrome b (CYB) gene is proposed by this study. Forty DNA samples of animal and plant origin were used to confirm the specificity of the qPCR system. The detection method’s performance was evaluated on different processed GS matrices including native GS (UnGS) and different commercial products: crunchy roasted samples (RoGS), insect meal mixtures (ACGS) and energetic snacks containing GS (GSS). Data on sequencing were aligned with the reference gene to confirm the PCR products. The regression curve (y = −3.394 x + 42.521; R2 = 0.994, d.f. 14) between Ct values and Log DNA concentrations of Gryllodes sigillatus resulted in an efficiency of 96.4%. The severity of the technological processing treatments and the matrix structure affected the intensity of the PCR signal with the same amount of insect DNA as observed by different y-intercepts of the three-regression lines for RoGS, ACGS, and GSS. The real-time PCR method resulted in robust and sensitive outcomes able to detect low amounts of GS DNA (5 g/100 g) in a complex matrix, making it suitable for detecting the presence or absence of labeled Gryllodes sigillatus material both in feed and food.

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.

Quantitative detection of donkey hide gelatin (colla corii asini) adulteration by real-time PCR based on single-copy nuclear genes

2020 ◽  
Author(s):  
Rui Liu ◽  
Luyao Zhao ◽  
Zhiying Wang ◽  
Tong Li ◽  
Ailiang Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Single Copy ◽  
High Price ◽  
Quantitative Detection ◽  
Potential Candidate ◽  
Animal Products ◽  
Medicinal Value ◽  
System Establishment ◽  
Colla Corii Asini

Donkey hide gelatin (colla corii asini) is highly recognized for its high nutritional value, especially the medicinal value. However, it is also a potential candidate for adulteration because of its low yield and high price. In order to quantitatively detect adulterated donkey-hide gelatin with all possible adulterated animal species, a real-time PCR approach based on single-copy housekeeping nuclear reference primers was proposed in this study. For the system establishment, mixtures containing designated contents of pig hide with donkey-hide were employed to generate a calibration curve based on the ratio of Ct (Specificity/Reference) with reasonable linearity (5%-100%). Then, a set of experiments were performed on commercially available samples. The proposed PCR approach could specifically identify donkey-hide from mixed animal products and quantify the content of donkey-hide gelatin, thus facilitating the control over this novel form of donkey hide gelatin adulteration.

scholarly journals Real-time evaluation of an optimized real-time PCR assay versus Brilliance chromogenic MRSA agar for the detection of meticillin-resistant Staphylococcus aureus from clinical specimens

2011 ◽  
Vol 60 (3) ◽  
pp. 323-328 ◽  
Author(s):  
J. Danial ◽  
M. Noel ◽  
K. E. Templeton ◽  
F. Cameron ◽  
F. Mathewson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Test Performance ◽  
High Sensitivity ◽  
Turnaround Time ◽  
Homology Search ◽  
Pcr Assay ◽  
Indirect Measure ◽  
The Mean

A total of 1204 meticillin-resistant Staphylococcus aureus (MRSA) screens (3340 individual swabs) were tested to evaluate a staphylococcal cassette chromosome mec (SCCmec) real-time PCR. In total, 148 (12.3 %) of the screens were MRSA-positive, where 146 (12.1 %) were MRSA-positive by the SCCmec real-time PCR assay. In contrast, 128 (10.6 %) screens were MRSA-positive by culture. One hundred and twenty-six (10.5 %) of the screens were positive by both culture and PCR. Twenty of the 1204 screens (1.66 %) were negative by culture but positive by PCR; these samples were sequenced. In 14 of the cases, a homology search confirmed the sequence as SCCmec, indicating that these samples could be considered true positives. Two of the 1204 (0.2 %) screens were positive by culture and negative by PCR. The mean turnaround time (TAT) for PCR-negative swabs was 6 h 12 min and for PCR-positive swabs was 6 h 48 min. In comparison, for culture-negative swabs the mean TAT was 29 h 30 min and for culture-positive swabs was 69 h. The cost per swab for routine culture was £0.41 (€0.48) and that of the real-time PCR assay was £2.35 (€2.75). This optimized, in-house, inexpensive, real-time PCR test maintained a very high sensitivity and specificity when evaluated under real-time laboratory conditions. The TAT of this real-time PCR assay was substantially lower than that of chromogenic culture. It was also maintained throughout the entire process, which can be taken as an indirect measure of test performance. This study showed that implementation of a molecular test can be achieved with limited resources in a standard microbiology laboratory.

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