Molecular Tracing of the Origin of Six Different Plant Species in Bee Honey Using Real-Time PCR

2017 ◽  
Vol 100 (3) ◽  
pp. 744-752 ◽  
Author(s):  
Yajun Wu ◽  
Yange Yang ◽  
Mingchang Liu ◽  
Bin Wang ◽  
Meige Li ◽  
...  
Keyword(s):  
Real Time ◽  
Plant Species ◽  
Real Time Pcr ◽  
Sensitivity Testing ◽  
Foreign Pollen ◽  
Geographic Regions ◽  
Fair Market ◽  
Chaste Tree ◽  
Parallel Tests

Abstract The quality of honey is significantly influenced by floralorigin. Mislabeling floral species occurs frequently in bee honey products. To protect consumers from economic fraud and maintain a fair market environment, methods to identify floralspecies in honey are necessary. In our study, real-time PCRs were established, targeting six honey types mainly produced in China (canola, Chinese milkvetch, Chinese chaste tree, locust tree, litchi, and longan). Sensitivity testing on DNA fromplant tissues exhibited LODs of about 0.5–5 pg/μL. For DNA extracts of pollen sediments from different honeyspecies, LODs ranged from 13.6 to 403.2 pg/μL. In an experiment to determine the practical LODs of honey in which adulterant honey was spiked in the genuine honey, adulterant honey as low as about 0.1–0.5% was detected in 90–100% in 10 parallel tests. Additionally, pollen was spiked in the honey and stored under various conditions to investigate the migration of pollen DNA into the honey supernatant. Finally, the efficiency of our method was investigated by testing honey samples of unknown compositions from different geographic regions. Of the 159 honey samples that were supposed tobe monofloral that had been collected in five provinces, a small portion were found to be contaminated with foreign pollen(7%). The methods proved to be specific, sensitive, and reliable in identifying the six plant species in honey, which would be a useful tool during the market supervision and QC of honey products.

Quality of UHT goat's milk in Poland evaluated by real-time PCR

2010 ◽  
Vol 94 (1-3) ◽  
pp. 32-37 ◽  
Author(s):  
A. Dąbrowska ◽  
E. Wałecka ◽  
J. Bania ◽  
M. Żelazko ◽  
M. Szołtysik ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Goat’S Milk ◽  
Goat's Milk

scholarly journals TaqMan Real-Time PCR Assays To Assess Arbuscular Mycorrhizal Responses to Field Manipulation of Grassland Biodiversity: Effects of Soil Characteristics, Plant Species Richness, and Functional Traits

2010 ◽  
Vol 76 (12) ◽  
pp. 3765-3775 ◽  
Author(s):  
Stephan König ◽  
Tesfaye Wubet ◽  
Carsten F. Dormann ◽  
Stefan Hempel ◽  
Carsten Renker ◽  
...  
Keyword(s):  
Species Richness ◽  
Real Time ◽  
Plant Species ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Large Scale ◽  
Arbuscular Mycorrhizal ◽  
Sequence Type ◽  
Plant Biodiversity ◽  
Pcr Assays

ABSTRACT Large-scale (temporal and/or spatial) molecular investigations of the diversity and distribution of arbuscular mycorrhizal fungi (AMF) require considerable sampling efforts and high-throughput analysis. To facilitate such efforts, we have developed a TaqMan real-time PCR assay to detect and identify AMF in environmental samples. First, we screened the diversity in clone libraries, generated by nested PCR, of the nuclear ribosomal DNA internal transcribed spacer (ITS) of AMF in environmental samples. We then generated probes and forward primers based on the detected sequences, enabling AMF sequence type-specific detection in TaqMan multiplex real-time PCR assays. In comparisons to conventional clone library screening and Sanger sequencing, the TaqMan assay approach provided similar accuracy but higher sensitivity with cost and time savings. The TaqMan assays were applied to analyze the AMF community composition within plots of a large-scale plant biodiversity manipulation experiment, the Jena Experiment, primarily designed to investigate the interactive effects of plant biodiversity on element cycling and trophic interactions. The results show that environmental variables hierarchically shape AMF communities and that the sequence type spectrum is strongly affected by previous land use and disturbance, which appears to favor disturbance-tolerant members of the genus Glomus. The AMF species richness of disturbance-associated communities can be largely explained by richness of plant species and plant functional groups, while plant productivity and soil parameters appear to have only weak effects on the AMF community.

scholarly journals Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia

2020 ◽  
Author(s):  
AG Stewart ◽  
EP Price ◽  
K Schabacker ◽  
M Birikmen ◽  
PNA Harris ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Real Time Pcr ◽  
Generation Cephalosporin ◽  
Whole Genome ◽  
Third Generation ◽  
Sensitivity Testing

AbstractThird-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 258 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using a rapid (~40 min) pentaplex real-time PCR assay targeting the most common extended spectrum β-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). Additionally, AmpC CMY β-lactamase prevalence was examined using a singleplex PCR. A subset of isolates, including all 3GC-R isolates obtained from the intensive care unit, were subjected to whole-genome sequencing (WGS) to assess transmission dynamics, the presence of unidentified resistance determinants, and genotyping accuracy. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance was identified in 97% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, the majority (98.3%) of isolates harbored at least one β-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. WGS of β-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. No evidence of transmission among intensive care unit patients was identified. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

scholarly journals Development of qualitative methods of bifidobacterium bifidum in probiotic with Real-time PCR method

2018 ◽  
Vol 1 (1) ◽  
pp. 13-17
Author(s):  
Trong Pham Nhu ◽  
Long Le Thanh ◽  
Trung Nguyen Thanh ◽  
Yen Ta Thi ◽  
Loan Pham Thi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dairy Products ◽  
Food Additives ◽  
Bifidobacterium Bifidum ◽  
Pcr Assay ◽  
Accurate Identification ◽  
Powdered Milk ◽  
Pcr Method

Bifidobacterium strains with probiotic effects have been widely used in dairy products, food additives and pharmaceuticals. Especially, Bifidobacterium bifidum (B. bifidum) is usually presented into food products such as functional food. However, it is difficult to detect, and quantify B. bifidum in a sample with a combination of different probiotics. In Vietnam, there is no official standard method to identify and quantify B. bifidum in the sample with the mix of probiotic species. To fulfil the requirements of a robust quality management, we have developed a quantitative real-time PCR assay based on groEL gene for accurate identification and quantification of Bifidobacterium bifidum. The developed assay allows an unambiguous speciesspecific detection. We built the real-time PCR method to detect and identify B. bifidum in functional and supplemented food with specific up to 100% and reproducibility (SR<0.25) suitable with Annex F AOAC: 2016. This real-time PCR method is rapidly and effectively than conventional method. It takes only 24 hours to detect and identify B. bifidum in compare with at least a period of 3-5 days for conventional methods. The low quantitative limit is 105 CFU/g/mL, which is consistent with probiotic and powdered milk products with a declared quality of more than 106 CFU/g/mL.

scholarly journals A TaqMan real-time PCR method based on alternative oxidase genes for detection of plant species in animal feed samples

PLoS ONE ◽  
2018 ◽  
Vol 13 (1) ◽  
pp. e0190668 ◽  
Author(s):  
Maria Doroteia Campos ◽  
Vera Valadas ◽  
Catarina Campos ◽  
Laura Morello ◽  
Luca Braglia ◽  
...  
Keyword(s):  
Real Time ◽  
Plant Species ◽  
Real Time Pcr ◽  
Alternative Oxidase ◽  
Animal Feed ◽  
Pcr Method ◽  
Feed Samples

Assessment of Microbiological Safety and Quality of Marinades Used To Treat Beef and That Were Collected over a 12-Month Period from Specialty Retailers Near Raleigh, North Carolina

2018 ◽  
Vol 81 (3) ◽  
pp. 490-496 ◽  
Author(s):  
Yangjin Jung ◽  
Christopher L. Rupert ◽  
Benjamin Chapman ◽  
Anna C. S. Porto Fett ◽  
John B. Luchansky
Keyword(s):  
Escherichia Coli ◽  
North Carolina ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Plate Count ◽  
Pcr Assay ◽  
E Coli ◽  
Aerobic Plate Count

ABSTRACT In total, 115 marinade samples (58 fresh marinades and 57 spent marinades) were collected over 12 months from specialty retailers (four individual stores) near Raleigh, NC. These marinades were screened for total mesophilic aerobic plate count (M-APC), total psychrotrophic aerobic plate count (P-APC), and Enterobacteriaceae. These marinades were also screened for the seven regulated serogroups of Shiga toxin–producing Escherichia coli. Stores A and B used immersion to marinade raw beef cuts, whereas stores C-1 and C-2 used vacuum tumbling. In general, marinade temperatures at the stores ranged from 1.8 to 6.6°C, and beef cuts were marinated from a few minutes to up to 3 days. Regardless of the process used to marinade meat, levels of M-APC and P-APC in fresh marinades ranged from 3.4 to 4.7 and 1.4 to 1.8 log CFU/mL, respectively, whereas Enterobacteriaceae were not detected in any fresh marinades, even after enrichment. However, levels of M-APC, P-APC, and Enterobacteriaceae in spent marinades collected from stores C-1 and C-2 (ca. 3.6 to 7.1 log CFU/mL) were significantly higher (P < 0.05) compared with levels of these same types of bacteria enumerated from spent marinades collected at stores A and B (ca. ≤0.7 to 4.9 log CFU/mL). None of the 115 marinade samples tested positive for Shiga toxin–producing E. coli by using a BAX system real-time PCR assay. No significant (P > 0.05) association was observed between microbial levels (i.e., M-APC, P-APC, and Enterobacteriaceae) and the temperature or duration of the marination process. Levels of M-APC, P-APC, and Enterobacteriaceae in spent marinades were significantly affected by the marination method (P < 0.05), with levels, in general, being higher in marinades used for tumbling. Thus, retailers must continue to keep marinade solutions and meat at a safe temperature (i.e., ≤4°C) and to properly and frequently sanitize the equipment and environment in both the processing area and deli case.

scholarly journals Comparison of laboratory methods for diagnosis of Trichomonas vaginalis

2014 ◽  
Vol 63 (1) ◽  
pp. 5-9
Author(s):  
Zinaida Mikhaylovna Martikaynen ◽  
Aleksey Nikolayevich Grigoryev ◽  
Olga Sergeyevna Ryzhkova ◽  
Yelena Vasilyevna Shipitsyna ◽  
Alevtina Mikhaylovna Savicheva ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Trichomonas Vaginalis ◽  
Culture Media ◽  
High Sensitivity ◽  
Laboratory Methods ◽  
Pcr Method

The paper presents data on comparison of microscopy, culture and molecular (PCR) methods for diagnosis of trichomoniasis, as well as on evaluation of the quality of culture media for isolation of trichomonads produced by Russian and international manufacturers. The highest sensitivity was shown for the molecular (real-time PCR) method. Microscopy of stained and wet smears demonstrated relatively high sensitivity. The culture media that are widely used in our country for Trichomonas vaginalis isolation need optimization.

A real-time PCR assay to differentiate the B and Q biotypes of the Bemisia tabaci complex in Cyprus

2009 ◽  
Vol 99 (6) ◽  
pp. 573-582 ◽  
Author(s):  
L.C. Papayiannis ◽  
J.K. Brown ◽  
N.A. Seraphides ◽  
M. Hadjistylli ◽  
N. Ioannou ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Real Time ◽  
Plant Species ◽  
Bemisia Tabaci ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Middle Eastern ◽  
Polymorphism Analysis ◽  
Pcr Assay ◽  
The Real

AbstractA real-time PCR assay based on TaqMan® technology was developed and evaluated for the rapid detection of the B and Q biotypes of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). A survey was conducted during 2005–2007 in order to identify the distribution and prevalence of B. tabaci biotypes in Cyprus using the real-time PCR assay. More than 700 adult whiteflies collected from 35 cultivated and weed plant species were individually haplotyped using TaqMan® PCR, and the results of the assay were validated by restriction fragment length polymorphism analysis and DNA sequencing of the mitochondrial cytochrome oxidase I (mtCOI) gene. Two biotypes, B and Q, were identified in the collected plant species on the island. The real-time PCR and RFLP assay consistently yielded the same results, although the real-time assay was more sensitive and less time consuming. Phylogenetic analysis of the mtCOI DNA sequences corroborated the identity of the B and Q biotypes 100% of the time and by phylogenetic analysis the haplotypes grouped, as expected, in the major North African-Mediterranean-Middle Eastern clade of the B. tabaci complex.

scholarly journals The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

2009 ◽  
Vol 55 (4) ◽  
pp. 611-622 ◽  
Author(s):  
Stephen A Bustin ◽  
Vladimir Benes ◽  
Jeremy A Garson ◽  
Jan Hellemans ◽  
Jim Huggett ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Scientific Literature ◽  
Quantitative Real Time Pcr ◽  
Reliability Of Results ◽  
Experimental Conditions ◽  
Full Disclosure ◽  
Minimum Information ◽  
Online Supplement

Abstract Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments. Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

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