scholarly journals Detection of fish allergen by droplet digital PCR

2019 ◽  
Vol 7 (4) ◽  
Author(s):  
Cinzia Daga ◽  
Simona Cau ◽  
Maria Giovanna Tilocca ◽  
Barbara Soro ◽  
Aldo Marongiu ◽  
...  
Keyword(s):  
18S Rrna ◽  
Annealing Temperature ◽  
Pcr Primers ◽  
Digital Pcr ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Chain Reaction ◽  
European Regulation ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Fish is one of fourteen allergens that must be highlighted on the label within the ingredients list. It should be noted that the European regulation, is very restrictive to allergens with zero tolerance. Therefore it is important to establish sensitive and specific methods for detecting fish allergen. Applicability to detect and quantify fish allergen by droplet digital polymerase chain reaction (ddPCR) has been evaluated in this work. Genomic DNA of three fish species belonging to the most common fish families were analyzed. PCR primers were designed to amplify a 166 bp region of the 18S rRNA gene. Comparative studies were performed to establish the optimal primer and probe concentrations.  Annealing temperature was determined by using thermal gradient. The results have shown good applicability of the optimized 18S rRNA gene-method to detect and quantify small amounts of the target in all samples analyzed. However, validation studies are needed in order to apply ddPCR technology for routine allergens analysis.  

scholarly journals AMPLIFIKASI FRAGMEN GEN 18S rRNA PADA DNA METAGENOMIK MADU DENGAN TEKNIK PCR (POLYMERASE CHAIN REACTION)

2017 ◽  
Vol 7 ◽  
pp. 1
Author(s):  
Satriya Putra Prakoso ◽  
I Nengah Wirajana ◽  
I Wayan Suarsa
Keyword(s):  
Polymerase Chain Reaction ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Honey Sample ◽  
Rrna Gene ◽  
In Vitro Test ◽  
Chain Reaction ◽  
Metagenomic Dna ◽  
Polymerase Chain

The aim of this research was to amplificate 18S rRNA gene fragment from honey’s metagenomic DNA using Polymerase Chain Reaction (PCR). The honey sample was collected from Seraya Tengah village, Karangasem regency. The best result of primer design from in silico test was continued to in vitro test using PCR method. The optimum conditions for amplification was obtained as follows: pre-denaturation at 95oC for 3 minutes and continued with 30 of amplification cycle (denaturation at 95°C for 1 minutes, annealing at 55°C for 1 minutes and elongation at 72°C for 1 minutes) and the last step continued with extension process at 72°C for 2 minutes. The size of DNA fragment band of amplified product was about 100 bp which obtained from the honey’s metagenomic DNA.

scholarly journals Fatal Hymenolepis nana cestodiasis in a ring-tailed lemur (Lemur catta)

2021 ◽  
pp. 030098582110425
Author(s):  
Esther E. V. Crouch ◽  
Charlotte Hollinger ◽  
Stephanie Zec ◽  
Denise McAloose
Keyword(s):  
Polymerase Chain Reaction ◽  
18S Rrna ◽  
Lemur Catta ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Hymenolepis Nana ◽  
Chain Reaction ◽  
Fecal Shedding ◽  
Polymerase Chain ◽  
Common Parasite

The cestode Hymenolepis nana is a common parasite of humans and mice. Fecal shedding in the absence of clinical disease has previously been reported in ring-tailed lemurs ( Lemur catta). This report describes fatal, disseminated H. nana cestodiasis infection in an aged ring-tailed lemur in a zoological collection. The parasites were associated with severe multifocal to coalescing and regionally extensive pyogranulomatous hepatitis and moderate multifocal pneumonia. The morphology of the parasites was highly unusual. Profiles were variably sized, ellipsoid to irregularly serpiginous, lined by a thin tegument, and filled with lightly eosinophilic fibrillar stroma and numerous, round basophilic cells. Polymerase chain reaction targeting a portion of the 18S rRNA gene and DNA sequencing of the amplicon showed 100% homology with H. nana.

scholarly journals Droplet Digital PCR (ddPCR) Analysis for the Detection and Quantification of Cow DNA in Buffalo Mozzarella Cheese

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1270
Author(s):  
Anna Cutarelli ◽  
Andrea Fulgione ◽  
Pasquale Fraulo ◽  
Francesco Paolo Serpe ◽  
Pasquale Gallo ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Buffalo Milk ◽  
Cow Milk ◽  
Mozzarella Cheese ◽  
Chain Reaction ◽  
New Methods ◽  
Protected Designation Of Origin ◽  
Commission Regulation ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Buffalo mozzarella cheese is one of the most appreciated traditional Italian products and it is certified as a Protected Designation of Origin (PDO) product under the European Commission Regulation No. 1151/2012. It is obtained exclusively from buffalo milk. If made from cow milk, or a mixture of buffalo and cow milk, buffalo mozzarella cheese does not qualify as a PDO product. In order to maximize their profits, some producers market buffalo mozzarella that also contains cow milk as a PDO product, thus defrauding consumers. New methods for revealing this fraud are therefore needed. One such method is the droplet digital Polymerase Chain Reaction (ddPCR). Thanks to its high precision and sensitivity, the ddPCR could prove an efficacious means for detecting the presence of cow milk in buffalo mozzarella cheese that is marketed as a PDO product. ddPCR has proved able to detect the DNA of cow and/or buffalo milk in 33 buffalo mozzarella cheeses labelled as PDO products, and experimental evidence could support its application in routine analyses.

scholarly journals Molecular Characterization of Ciliate Diversity in Stream Biofilms

2008 ◽  
Vol 74 (6) ◽  
pp. 1740-1747 ◽  
Author(s):  
Andrew Dopheide ◽  
Gavin Lear ◽  
Rebecca Stott ◽  
Gillian Lewis
Keyword(s):  
18S Rrna ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Sequence Diversity ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Specific Pcr ◽  
Stream Biofilms

ABSTRACT Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

First report on optimization of loop-mediated isothermal amplification (LAMP) for the diagnosis of Babesia felis

2017 ◽  
Author(s):  
Muhammad Awais Salim ◽  
Raheela Akhtar ◽  
Muhammad Lateef ◽  
Imran Rashid ◽  
Harron Akbar ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Cost Effective ◽  
Conventional Polymerase Chain Reaction ◽  
18S Rrna Gene ◽  
Isothermal Amplification ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain ◽  
Effective Diagnosis

The objective of present study was to optimize loop mediated isothermal amplification (LAMP) assay for the diagnosis of Babesia felis in cats. LAMP primers were designed recognizing four sections of 18SribosomalRNA (18S rRNA) gene of B. felis. The blood samples of cats microscopically positive for Babesia felis were further used to extract deoxyribo neuclic acid (DNA) and the reaction mixture of 25 µL was standardized at 63°C temperature for 1 hour. LAMP assay provided more positive samples than conventional polymerase chain reaction (PCR). The prevalence of B. felis was also determined in cats using this optimized LAMP assay and it was found that the prevalence was more in younger cats as compare to adults. The application of LAMP can be helpful in rapid, reliable and cost effective diagnosis of B. felis in field.

Development of a Polymerase Chain Reaction-Based Assay for the Detection of Alternaria Fungal Contamination in Food Products

1999 ◽  
Vol 62 (10) ◽  
pp. 1191-1197 ◽  
Author(s):  
GIDEON ZUR ◽  
ERIC M. HALLERMAN ◽  
RACHEL SHARF ◽  
YECHEZKEL KASHI
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Tomato Sauce ◽  
5.8S Rrna Gene ◽  
Polymerase Chain ◽  
Tomato Products ◽  
Fungal Contaminants ◽  
Tomato Powder

Alternaria sp. are important fungal contaminants of vegetable, fruit, and grain products, including Alternaria alternata, a contaminant of tomato products. To date, the Howard method, based on microscopic observation of fungal filaments, has been the standard examination for inspection of tomato products. We report development of a polymerase chain reaction (PCR)-based method for detection of Alternaria DNA. PCR primers were designed to anneal to the internal transcribed regions ITS1 and ITS2 of the 5.8S rRNA gene of Alternaria but not to other microbial or tomato DNA. We demonstrate use of the PCR assay to detect Alternaria DNA in experimentally infested and commercially obtained tomato sauce and tomato powder. Use of the PCR method offers a rapid and sensitive assay for the presence of Alternaria DNA in tomato products. The apparent breakdown of DNA in tomato sauce may limit the utility of the assay to freshly prepared products. The assay for tomato powder is not affected by storage time.

scholarly journals Morphological and Molecular Identification of Fusarium oxysporum Sch. Isolated From Guava Wilt in Bangladesh

2012 ◽  
Vol 41 (1) ◽  
pp. 49-54 ◽  
Author(s):  
M Zakir Hussain ◽  
MA Rahman ◽  
Mohammad Nurul Islam ◽  
MA Latif ◽  
MA Bashar
Keyword(s):  
Fusarium Oxysporum ◽  
Psidium Guajava ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Species Identity ◽  
Specific Primers ◽  
Polymerase Chain ◽  
Serious Disease ◽  
Species Specific

Wilt of guava plants (Psidium guajava L.) is a serious disease in Bangladesh. Sixteen isolates of Fusarium oxysporum Sch. were collected from the root and stem fragments of guava plants growing in six districts of Bangladesh. Species identity was based on the colony character, nature of conidiogenous cell, morphology of microconidia, macroconidia and chlamydospores. Eleven isolates were confirmed as F. oxysporum through polymerase chain reaction (PCR) using species specific primers designed from the conserved regions of 18S rRNA gene. DOI: http://dx.doi.org/10.3329/bjb.v41i1.11082 Bangladesh J. Bot. 41(1): 49-54, 2012 (June)

scholarly journals Digital PCR: A Reliable Tool for Analyzing and Monitoring Hematologic Malignancies

2020 ◽  
Vol 21 (9) ◽  
pp. 3141 ◽  
Author(s):  
Nicoletta Coccaro ◽  
Giuseppina Tota ◽  
Luisa Anelli ◽  
Antonella Zagaria ◽  
Giorgina Specchia ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Residual Disease ◽  
Hematologic Malignancies ◽  
Digital Pcr ◽  
Future Perspectives ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction ◽  
Generation Sequencing ◽  
Minimal Residual

The digital polymerase chain reaction (dPCR) is considered to be the third-generation polymerase chain reaction (PCR), as it yields direct, absolute and precise measures of target sequences. dPCR has proven particularly useful for the accurate detection and quantification of low-abundance nucleic acids, highlighting its advantages in cancer diagnosis and in predicting recurrence and monitoring minimal residual disease, mostly coupled with next generation sequencing. In the last few years, a series of studies have employed dPCR for the analysis of hematologic malignancies. In this review, we will summarize these findings, attempting to focus on the potential future perspectives of the application of this promising technology.

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