scholarly journals Development of real-time polymerase chain reaction assays allowing molecular detection ofEchinococcus felidis, Echinococcus granulosussensu stricto, andEchinococcus canadensisin carnivore feces samples, animal and human hydatid cyst material from Uganda and Kenya

2017 ◽  
Author(s):  
Katharina Kopp
Keyword(s):  
Hydatid Cyst ◽  
Real Time ◽  
Real Time Pcr ◽  
Sensu Stricto ◽  
High Resolution Melt ◽  
Field Samples ◽  
Pcr Products ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Pcr Assays

AbstractFirst evaluations on field samples, including carnivore feces, animal and human hydatid cyst material from Uganda and Kenya, showed specific amplification of two target regions of the mitochondrial genome ofEchinococcusspecies according to melt and high-resolution melt curve analyses of the developed real-time PCR assays. Consecutive sequencing of PCR products revealed that, apart fromEchinococcus felidis, sequences of two other tapeworm species,Echinococcus granulosussensu stricto andEchinococcus canadensis, which are also endemic in East Africa, were detected by the developed real-time PCR assays.

Diagnosis and Causative Species of Visceral Leishmaniasis in Southwest Saudi Arabia

2021 ◽  
Author(s):  
Aymen Abdelhaleem ◽  
Nabil Dhayhi ◽  
Mohamed Salih Mahfouz ◽  
Ommer Daffalla ◽  
Mansour Mubarki ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Visceral Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Donovani ◽  
Target Genes ◽  
Sample Volume ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Robust Evidence

Visceral leishmaniasis (VL) is the most severe clinical form of the disease and has been reported in the Jazan region of southwest Saudi Arabia. This study aimed to diagnose VL by real-time polymerase chain reaction (PCR) and the direct agglutination test (DAT) and to identify the causative Leishmania species. A total of 80 participants, including 30 suspected VL patients, 30 healthy endemic control individuals, and 20 malaria disease controls, were enrolled in this study. Blood samples were collected and tested for Leishmania DNA by real-time PCR and for antibody by the DAT. Sequencing of some amplified PCR products was used to identify the causative Leishmania species. The diagnosis of VL was successfully achieved by both real-time PCR and by DAT with 100% sensitivity. Leishmania donovani and Leishmania infantum species were detected by sequencing both by the kDNA and ITS1 target genes, followed a BLASTn search. The detection of VL antibody by the DAT followed by the confirmatory detection of Leishmania DNA in patient blood by PCR could promote the adoption of the much less invasive and more sensitive methods for the routine diagnosis of VL. Further study with high sample volume to evaluate the PCR and the DAT are needed, to generate more robust evidence. Based on the sequencing results, emerging studies on VL should focus on the causative Leishmania species, reservoirs, and vectors that are important in the study area.

Real-Time Polymerase Chain Reaction Detection of Bovine DNA in Meat and Bone Meal Samples

2002 ◽  
Vol 65 (7) ◽  
pp. 1158-1165 ◽  
Author(s):  
S. LAHIFF ◽  
M. GLENNON ◽  
J. LYNG ◽  
T. SMITH ◽  
N. SHILTON ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Meat And Bone Meal ◽  
Bone Meal ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Pcr Product ◽  
Pcr Products ◽  
Polymerase Chain

We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8–ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5′ FAM reporter and a 3′ TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples.

SLCO1B1 c.521T>C Genotyping in the Austrian Population Using 2 Commercial Real-Time Polymerase Chain Reaction Assays: An Implementation Study

Pharmacology ◽  
2018 ◽  
Vol 102 (1-2) ◽  
pp. 88-90
Author(s):  
Dietmar Enko ◽  
Sophia Harringer ◽  
Christian Oberkanins ◽  
Helene Pühringer ◽  
Gabriele Halwachs-Baumann ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Performance ◽  
Chain Reaction ◽  
Increased Risk ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Austrian Population ◽  
Genotype And Allele Frequencies

Statin-induced myopathy is reported to be significantly associated with the SCLO1B1 c.521T>C polymorphism. To date, SLCO1B1 c.521T>C epidemiologic data for the Austrian population is still lacking. Therefore, this study aimed at assessing the genotype and allele frequencies of the SLCO1B1 c.521T>C variant in Austria and evaluating the clinical performance of 2 commercial real-time polymerase chain reaction (PCR) assays. Genomic DNA isolated from 181 healthy individuals was analyzed for the SLCO1B1 c.521T>C polymorphism in a comparative manner using the SLCO1B1 c.521T>C RealFastTM Assay and the BioPro SLCO1B1 Genotyping real-time PCR Kit. A total of 10 (5.5%) and 44 (24.3%) out of 181 individuals were SLCO1B1 c.521T>C C/C-homo- and ­C/T-heterozygotes, the genotypes indicative of high and increased risk of statin-induced myopathy, respectively. The SLCO1B1 c.521C allele frequency rate was 17.7%. In conclusion, the genetic predisposition of elevated statin-induced myopathy risk in the Austrian population is frequent. Both real-time PCR assays under investigation here are reliable and robust SLCO1B1 c.521T>C genotyping tools in clinical routine.

scholarly journals Improved quantitative real-time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard

2005 ◽  
Vol 3 (9) ◽  
pp. 381-391 ◽  
Author(s):  
Kathryn J. Coyne ◽  
Sara M. Handy ◽  
Elif Demir ◽  
Edward B. Whereat ◽  
David A. Hutchins ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Algal Species ◽  
Reference Standard ◽  
Quantitative Real Time Pcr ◽  
Exogenous Dna ◽  
Field Samples ◽  
Pcr Assays

scholarly journals Partial Molecular Characterization of Dahlia mosaic virus and Its Detection by PCR

Plant Disease ◽  
2003 ◽  
Vol 87 (8) ◽  
pp. 945-948 ◽  
Author(s):  
M. Nicolaisen
Keyword(s):  
Nucleotide Sequence ◽  
Mosaic Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Open Reading Frames ◽  
Dahlia Mosaic Virus ◽  
Polymerase Chain ◽  
Pcr Assays

Dahlia mosaic virus (DMV) is the causal agent of one of the most important diseases of Dahlia pinnata. The nucleotide sequence of a 1,195-bp fragment of its genome was amplified and characterized. Based on this sequence, polymerase chain reaction (PCR) assays were developed for detection of DMV. The nucleotide sequence confirmed the classification of DMV as a member of genus Caulimovirus since it was similar to a region covering partly open reading frames (ORFs) IV and V found in caulimoviruses. The two most closely related viruses on the basis of comparison of ORF V fragments were shown to be Figwort mosaic virus and Mirabilis mosaic virus with 66.6 and 68.1% identity, respectively. Two PCR assays were developed using identical primer pairs: a real-time PCR based on SYBR green chemistry and a conventional PCR. Both methods clearly discriminated DMV-infected and healthy dahlia. The real-time PCR assay detected DMV-infected material that was diluted 105-fold in healthy material.

scholarly journals Early Detection of Leifsonia xyli subsp. xyli in Sugarcane Leaves by Real-Time Polymerase Chain Reaction

Plant Disease ◽  
2007 ◽  
Vol 91 (4) ◽  
pp. 430-434 ◽  
Author(s):  
M. P. Grisham ◽  
Y.-B. Pan ◽  
E. P. Richard
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Close Agreement ◽  
Leaf Tissue ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Pcr Assays

A real-time, polymerase chain reaction (PCR) assay was developed for detecting Leifsonia xyli subsp. xyli in sugarcane leaf tissue. Real-time PCR assays were conducted on the youngest, fully expanded leaf of three cultivars collected bi-weekly from field nurseries between 11 April and 19 July 2005. L. xyli subsp. xyli infection was detected in leaves collected at all sampling dates, including those from 1-month-old plants on 11 April. Assays conducted on older, more rapidly growing plants (28 July and 21 October 2005) indicated that leaf position affects assay efficiency. Conventional PCR was less efficient than real-time PCR for detecting L. xyli subsp. xyli in leaf tissue. Real-time PCR was used to rank cultivars for susceptibility to L. xyli subsp. xyli infection based on the relative titer of L. xyli subsp. xyli in leaves of inoculated, 3- and 4-month-old greenhouse-grown plants. The ranking of cultivars by real-time PCR was in close agreement with the ranking determined by tissue-blot enzyme immunoassay performed on tissue from 7- to 9-month-old stalks.

scholarly journals Specific Detection of the Root-Lesion Nematode Pratylenchus scribneri Using Conventional and Real-Time PCR

Plant Disease ◽  
2017 ◽  
Vol 101 (2) ◽  
pp. 359-365 ◽  
Author(s):  
Danqiong Huang ◽  
Guiping Yan
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nematode Species ◽  
Economic Damage ◽  
Sequence Information ◽  
Root Lesion Nematode ◽  
Field Samples ◽  
Lesion Nematode ◽  
Pcr Assays ◽  
Pratylenchus Scribneri

Pratylenchus scribneri is a plant-parasitic root-lesion nematode causing economic damage to various crops worldwide. Identifying root-lesion nematodes to species using traditional morphological methods is an arduous task requiring extensive training on nematode taxonomy and years of experience. Thus, molecular methods for P. scribneri detection and identification were developed. Conventional and real-time polymerase chain reaction (PCR) assays with new species-specific primers were used in this study, which exclusively amplified DNA of P. scribneri but not DNA from other Pratylenchus spp. or non-Pratylenchus spp. tested. Compared with conventional PCR that was able to detect an equivalent to 1/4 of the DNA of a single nematode, real-time PCR was more sensitive and could amplify an equivalent to 1/128 of the DNA of one nematode. Both conventional and real-time PCR assays successfully identified P. scribneri and distinguished it from P. penetrans and P. neglectus isolated from field samples collected from various locations in North Dakota and Minnesota. The Blast-search based on the sequence information confirmed the reliability of the PCR assays for species identification. This is the first report of P. scribneri identification using a real-time PCR assay. The developed PCR assays are suitable for use in diagnostic laboratories and detection of field infestations with this nematode species.

scholarly journals Quantification of Diatom and Dinoflagellate Biomasses in Coastal Marine Seawater Samples by Real-Time PCR

2008 ◽  
Vol 74 (23) ◽  
pp. 7174-7182 ◽  
Author(s):  
Anna Godhe ◽  
Maria E. Asplund ◽  
Karolina Härnström ◽  
V. Saravanan ◽  
Anuj Tyagi ◽  
...  
Keyword(s):  
Linear Regression ◽  
Real Time ◽  
Real Time Pcr ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Swedish West Coast ◽  
Coastal Marine ◽  
Field Samples ◽  
Reverse Primer ◽  
Pcr Assays

ABSTRACT Two real-time PCR assays targeting the small-subunit (SSU) ribosomal DNA (rDNA) were designed to assess the proportional biomass of diatoms and dinoflagellates in marine coastal water. The reverse primer for the diatom assay was designed to be class specific, and the dinoflagellate-specific reverse primer was obtained from the literature. For both targets, we used universal eukaryotic SSU rDNA forward primers. Specificity was confirmed by using a BLAST search and by amplification of cultures of various phytoplankton taxa. Reaction conditions were optimized for each primer set with linearized plasmids from cloned SSU rDNA fragments. The number of SSU rDNA copies per cell was estimated for six species of diatoms and nine species of dinoflagellates; these were significantly correlated to the biovolumes of the cells. Nineteen field samples were collected along the Swedish west coast and subjected to the two real-time PCR assays. The linear regression of the proportion of SSU rDNA copies of dinoflagellate and diatom origin versus the proportion of dinoflagellate and diatom biovolumes or biomass per liter was significant. For diatoms, linear regression of the number of SSU rDNA copies versus biovolume or biomass per liter was significant, but no such significant correlation was detected in the field samples for dinoflagellates. The method described will be useful for estimating the proportion of dinoflagellate versus diatom biovolume or biomass and the absolute diatom biovolume or biomass in various aquatic disciplines.

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