Comparative assessment of DNA extraction procedures for Ascaris spp. eggs

2019 ◽  
Vol 94 ◽  
Author(s):  
I.D. Amoah ◽  
G. Singh ◽  
K. Troell ◽  
P. Reddy ◽  
T.A. Stenström ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Molecular Detection ◽  
Dna Isolation ◽  
Quantitative Polymerase Chain Reaction ◽  
High Yield ◽  
Chain Reaction ◽  
Removal Technology ◽  
Polymerase Chain ◽  
Microbial Dna ◽  
Accurate Quantification

Abstract A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.

scholarly journals qPCR detection versus microscopy observations for assessing presence–absence of Didymosphenia geminata in Quebec rivers (Canada)

2015 ◽  
Vol 52 (2) ◽  
pp. 109-120
Author(s):  
Sandra Kim Tiam ◽  
Vincent Laderriere ◽  
Carole-Anne Gillis ◽  
Claude Fortin ◽  
Isabelle Lavoie
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Large Scale ◽  
Dna Isolation ◽  
Quantitative Polymerase Chain Reaction ◽  
Cost Effective ◽  
Chain Reaction ◽  
Didymosphenia Geminata ◽  
Large Scale Assessment ◽  
Polymerase Chain

Microscopic versus qPCR (quantitative polymerase chain reaction) analyses were compared for the detection of Didymosphenia geminata in biofilms and water filtrates from seven Gaspésie rivers (Canada). For the qPCR approach, two DNA extraction kits (QIAamp DNA Micro Kit, Qiagen and PowerSoil DNA Isolation Kit, Mo Bio Laboratories) and two pairs of primers were considered. The pair of primers D602F/D753Rext did not amplify D. geminata DNA whereas the pair of primers D602F/D753R was specific for D. geminata. Presence-absence diagnosis based on qPCR and microscopic analyses were consistent: D. geminata was detected in six of the seven rivers, both in the biofilm and filtrate samples. However, technical replications were needed at certain sites to observe the presence of D. geminata cells by microscopy. This underscores the necessity of replicate analyses, which is cost-effective to achieve when using qPCR due to the capacity to process tens of samples in a single PCR run in the context of a large scale assessment.

scholarly journals Method Comparison of DNA Isolation and Quantification for Fish and Seafood Authenticity Determination

2018 ◽  
Vol 13 (3) ◽  
pp. 115
Author(s):  
Dwiyitno Dwiyitno ◽  
Stefan Hoffman ◽  
Koen Parmentier ◽  
Chris Van Keer
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Blue Mussel ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Seafood Products ◽  
Polymerase Chain ◽  
Commercial Kit

Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen

scholarly journals Molecular detection and occurrence of equine theileriosis in Arabian horses in Al-Najaf province/Iraq

2020 ◽  
Vol 57 (3) ◽  
pp. e166996
Author(s):  
Hayder Mohammad Al-Rammahi ◽  
Abdulameer Abed Hatem ◽  
Asaad Chasib Al-Atabi
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Molecular Detection ◽  
Molecular Technique ◽  
Chain Reaction ◽  
Blood Samples ◽  
Equine Piroplasmosis ◽  
Polymerase Chain

This study was designed to detect equine piroplasmosis using the molecular technique in Al-Najaf province during the season that showed an increment in tick activities. Blood samples were collected from 110 horses with more than two signs of piroplasmosis. After DNA extraction, the product was examined by a polymerase chain reaction to amplify 18SrRNA. The results showed that the overall percentage of equine theileriosis was 38.18%. According to gender, the percentage of infection was 43.48% and 29.27% in females and males, respectively. Significant variations appeared between infected horses according to age, and the percentage of infection was 50% and 35.22% in less than 2 years and more than 2 years age, respectively. Moreover, the percentage of infection was 62.5% and 19.35% in animals with and without acariasis, respectively. Significant variations were also seen in equine theileriosis according to geographical areas, and the higher percentage was reported in Hera district (60.87%), while the lowest percentage was in the center of Al-Najaf (21.43%). This difference may be due to different distribution of vector of disease (tick), which may be the availability of the suitable weather that helped in the multiplication of the intermediate vectors. In conclusion, this study proved the variations in the occurrences of equine piroplasmosis according to gender, age, and geographical areas.

A comprehensive search for microbial DNA and RNA in sarcoidosis tissue samples by quantitative polymerase chain reaction

2017 ◽  
Vol 49 (8) ◽  
pp. 625-627
Author(s):  
Fumio Terasaki ◽  
Hitomi Fukumoto ◽  
Ryo Kawata ◽  
Yoshinobu Hirose ◽  
Shu-ichi Fujita ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Dna And Rna ◽  
Comprehensive Search ◽  
Polymerase Chain ◽  
Microbial Dna

Molecular Detection of Neospora caninum from Naturally Infected Dogs in Van Province, East Turkey

2020 ◽  
Author(s):  
Ali Bilgin Yilmaz ◽  
Yaşar Göz ◽  
Özlem Orunç Kilinç ◽  
Vural Denizhan
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Neurological Disorders ◽  
Genomic Dna ◽  
Molecular Detection ◽  
Neospora Caninum ◽  
Specific Method ◽  
Coccidian Parasite ◽  
Chain Reaction ◽  
Polymerase Chain

Neospora caninum is a coccidian parasite causing abortion in cattle and neurological problems in horses. Dogs are definitive hosts of N. caninum. Polymerase chain reaction is the most specific method used for the detection of N. caninum oocytes. In the present study, a total of 100 fecal samples were collected from naturally infected dogs. Of the 100 samples analyzed, 11 of them were detected with Hammondia/Neospora-like oocytes. Genomic DNA was isolated using a commercially available DNA extraction kit. The Nc5 gene specific to N. caninum was amplified by PCR and two of the eleven samples with Hammondia/Neospora-like oocytes formed ~337 bp repeatable band. In conclusion, N. caninum, which has been shown to cause neurological disorders in dogs and to abortion in cattle, was detected in naturally infected dogs in Van Province in Polymerase chain reaction.

Cerebral Ovine Herpesvirus 2 Infection of Cattle Is Associated With a Variable Neuropathological Phenotype

2020 ◽  
pp. 030098582097049
Author(s):  
Melanie M. Hierweger ◽  
Céline L. Boujon ◽  
Ronja V. Kauer ◽  
Mireille Meylan ◽  
Torsten Seuberlich ◽  
...  
Keyword(s):  
Molecular Detection ◽  
Viral Encephalitis ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
The Central Nervous System ◽  
Consistent Feature ◽  
Polymerase Chain ◽  
The Brain

Cross-species infection with ovine herpesvirus 2 (OvHV-2) in cattle causes malignant catarrhal fever (MCF). MCF may involve the central nervous system (CNS) with necrotizing arteritis and/or vasculitis described to be unique to MCF and discriminatory compared to other viral CNS infections. However, a systematic histopathological characterization of the neural form of MCF in cattle is lacking. We examined medulla oblongata ( n = 9) or the entire brain ( n = 9) of 18 cattle in which OvHV-2 was identified by quantitative polymerase chain reaction (qPCR), in order to pinpoint potential variations in neuropathology. In 2/18 animals (11%) no lesions were identified, while 16/18 cattle (89%) had brain lesions of varying severity. Presence and quantities of OvHV-2 nucleic acid were determined by in situ hybridization and qPCR, respectively, and were related to the severity of lesions. Fifteen of 18 animals (83%) showed vasculitis, which was mainly of the lymphohistiocytic type, while pathognomonic necrotizing arteritis was only rarely present. Neuroparenchymal lesions included gliosis and/or neuronal changes in 7/16 brains with lesions (44%). The number of CD3+ lymphocytes was highest in animals with simultaneous vascular and neuroparenchymal lesions and high viral genome load. In one animal, OvHV-2 was exclusively observed in CD3+ lymphocytes but not in neurons or microglia. In conclusion, the neuropathological phenotype of bovine MCF in the brain was variable. In some cases, lesions mimicked neurotropic viral encephalitis, while pathognomonic necrotizing arteritis was not a consistent feature of neural MCF. Therefore, molecular detection of OvHV-2 is warranted in the presence of nonsuppurative encephalitis and in the absence of necrotizing arteritis.

scholarly journals MOLECULAR DETECTION OF ASTROVIRUS USING REVERSE TRANSCRIPTASE QUANTITATIVE POLYMERASE CHAIN REACTION TECHNIQUE

2020 ◽  
Vol 51 (Special) ◽  
Author(s):  
Fadhil & et al.
Keyword(s):  
Reverse Transcriptase ◽  
Molecular Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Melting Curve ◽  
Single Infection ◽  
Chain Reaction ◽  
Qpcr Analysis ◽  
Efficient Detection ◽  
Stool Samples ◽  
Polymerase Chain

This study was aimed to highlight the importance of the melt curve-quantitative reverse transcriptase PCR (RT-qPCR) analysis in the detection of astrovirus (AstV) from both negative and positive rotavirus and enterovirus (EVs) samples, and the effectiveness of the AstV infection on the vaccine immunogenicity in the vaccinated infected children.  By RT-qPCR based Sybre green associated- melting curve assay, stool samples of 49 enterovirus suspected patients and of 39 rotavirus suspected patients were tested for AstV. Results of EVs group showed a 29 (59.2%) positive AstV contributed to 26 (89.5%) co-infection with Evs and 3 (10.3%) as a single infection in negative samples for Evs. Furthermore, AstV co-infection percentage is higher than the single infection. Moreover, the percentage of the Astrovirus among the vaccinated AFP-suspected cases was 53%, while the percentage of these viruses among the unvaccinated was 100%. Thus, MamAstrovirus- 1 MK948878 is the first local isolate recorded in the Genbank. In conclusion, the RT-qPCR based on SYBR Green showed the rapid and efficient detection of AstV with few copies number. This allow to be used for the diagnosis of AstV along with other gastroenteritis viruses in a multiplex assay to reduce processing time.

scholarly journals Real-Time PCR Quantification of Peronospora arborescens, the Opium Poppy Downy Mildew Pathogen, in Seed Stocks and Symptomless Infected Plants

Plant Disease ◽  
2011 ◽  
Vol 95 (2) ◽  
pp. 143-152 ◽  
Author(s):  
Miguel Montes-Borrego ◽  
Francisco J. Muñoz-Ledesma ◽  
Rafael M. Jiménez-Díaz ◽  
Blanca B. Landa
Keyword(s):  
Real Time ◽  
Downy Mildew ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Opium Poppy ◽  
Chain Reaction ◽  
Commercial Seed ◽  
Polymerase Chain ◽  
Pathogen Dna ◽  
Accurate Quantification

In this study, we developed a reliable, quick, and accurate quantitative polymerase chain reaction (qPCR) assay based on the MIQE (Minimum Information for publication of Quantitative Real-Time PCR Experiments) guidelines for the quantification of Peronospora arborescens in infected downy mildew–symptomless opium poppy (Papaver somniferum) tissues and commercial seed stocks. The protocol was highly reproducible and allowed accurate quantification of pathogen DNA up to 10 fg in different plant DNA backgrounds without losing specificity and efficiency. Moreover, to further overcome difficulties conferred by the strict biotrophy of this pathogen, we developed dilution series of DNA extracted from a plasmid with the target pathogen DNA as a cloned insert. This facilitated the demonstration of the robustness of the protocol in different laboratories with different qPCR equipment and reagents, which may help in its use on a broad scale. Finally, we validated the usefulness of the qPCR protocol for quarantine purposes and downy mildew resistance screening by quantifying P. arborescens in complex, naturally infested opium poppy samples. Thus, a pathogen biomass of 0.0003 to 0.007‰ or of 0.110 to 5,557 ppm was quantified in symptomless capsules in commercial seed stocks, or in stem samples from symptomless opium poppy plants systemically infected by the pathogen, respectively.

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