scholarly journals Terminal Restriction Fragment Length Polymorphism for Identification of Cryptosporidium Species in Human Feces

2008 ◽  
Vol 75 (1) ◽  
pp. 108-112 ◽  
Author(s):  
L. S. Waldron ◽  
B. C. Ferrari ◽  
M. R. Gillings ◽  
M. L. Power
Keyword(s):  
Species Identification ◽  
Restriction Fragment ◽  
Rflp Analysis ◽  
Cost Effective ◽  
18S Rrna Gene ◽  
Individual Species ◽  
Rrna Gene ◽  
Species Identity ◽  
Cryptosporidium Hominis ◽  
Detection And Identification

ABSTRACT Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.

scholarly journals Detection and identification of a novel 16SrXIII subgroup phytoplasma associated with strawberry red leaf disease in Argentina

2015 ◽  
Vol 65 (Pt_8) ◽  
pp. 2741-2747 ◽  
Author(s):  
Franco D. Fernández ◽  
Natalia G. Meneguzzi ◽  
Fabiana A. Guzmán ◽  
Daniel S. Kirschbaum ◽  
Vilma C. Conci ◽  
...  
Keyword(s):  
Rflp Analysis ◽  
Rrna Gene ◽  
The Novel ◽  
Universal Primer ◽  
Mature Leaves ◽  
Leaf Disease ◽  
Detection And Identification ◽  
Rflp Profile ◽  
Young Leaves ◽  
Leaf Symptoms

Strawberry red leaf phytoplasma was found in strawberry plants from production fields in Lules (Tucumán province) and Bella Vista (Corrientes province), Argentina. Characteristic strawberry red leaf symptoms were stunting, young leaves with yellowing at the edges, mature leaves which curled and were reddish at the abaxial face, flower and fruit deformation and death. The pathogen was detected with phytoplasma-universal primer pairs P1/P7 followed by R16F2n/R16R2 as nested primers in 13 diseased plants. Based on RFLP and sequence analysis of the amplified 16S rRNA gene, the phytoplasma was related to the 16SrXIII group (Mexican periwinkle virescence). In silico the RFLP profile of all the samples analysed revealed the presence of a unique pattern, showing that the novel phytoplasma is different from all the phytoplasmas currently composing the 16SrXIII group. The phylogenetic analysis was consistent with RFLP analysis as the strawberry red leaf phytoplasma was grouped within the 16SrXIII group, but formed a particular cluster. On this basis, the Strawberry red leaf phytoplasma associated with strawberry red leaf disease was assigned to a new subgroup, 16SrXIII-F.

scholarly journals Identification of Trichomonadid Protozoa from the Bovine Preputial Cavity by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Typing

2003 ◽  
Vol 15 (4) ◽  
pp. 390-394 ◽  
Author(s):  
Dawn C. Hayes ◽  
Rebecca R. Anderson ◽  
Richard L. Walker
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Rrna Gene ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Restriction Fragment Length

Accurate identification of the bovine pathogen Tritrichomonas foetus is sometimes complicated by the presence of other trichomonadid protozoa in clinical samples. A highly specific and reproducible approach for differentiating 3 common types of bovine trichomonadid protozoa found in the bovine preputial cavity, T. foetus, Pentatrichomonas hominis, and a Tetratrichomonas species, was developed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Universal trichomonadid protozoa primers, TFR1 and TFR2, were used to amplify the 5.8S rRNA gene and internal transcribed spacer regions (ITSRs), and the products were digested with the restriction enzyme HpyCH4IV. Restriction fragment length polymorphism analysis was performed on 55 trichomonad isolates from bovine preputial washing and scraping samples. The RFLP results correlated 100% with 5.8S rRNA gene and ITSR sequence results and PCR results with primers specific for T. foetus. The results of this study demonstrate that PCR and RFLP analysis can be used in lieu of DNA sequencing to identify the specific trichomonadid protozoa isolated from the bovine preputial cavity.

scholarly journals Barcoding in trypanosomes

Parasitology ◽  
2017 ◽  
Vol 145 (5) ◽  
pp. 563-573 ◽  
Author(s):  
RACHEL HUTCHINSON ◽  
JAMIE R. STEVENS
Keyword(s):  
Genetic Markers ◽  
Molecular Diagnostics ◽  
Potential Application ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Detection And Identification ◽  
Potential Alternative ◽  
Genetic Barcoding ◽  
The Ideal

SUMMARYTrypanosomes (genus Trypanosoma) are parasites of humans, and wild and domestic mammals, in which they cause several economically and socially important diseases, including sleeping sickness in Africa and Chagas disease in the Americas. Despite the development of numerous molecular diagnostics and increasing awareness of the importance of these neglected parasites, there is currently no universal genetic barcoding marker available for trypanosomes. In this review we provide an overview of the methods used for trypanosome detection and identification, discuss the potential application of different barcoding techniques and examine the requirements of the ‘ideal’ trypanosome genetic barcode. In addition, we explore potential alternative genetic markers for barcoding Trypanosoma species, including an analysis of phylogenetically informative nucleotide changes along the length of the 18S rRNA gene.

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.

Identification of Armillaria species isolated from bigtooth aspen based on rDNA RFLP analysis

1998 ◽  
Vol 28 (1) ◽  
pp. 141-149 ◽  
Author(s):  
T M Frontz ◽  
D D Davis ◽  
B A Bunyard ◽  
D J Royse
Keyword(s):  
Restriction Fragment ◽  
Ribosomal Rna ◽  
Rflp Analysis ◽  
Rrna Gene ◽  
Polymorphism Analysis ◽  
5S Rrna Gene ◽  
Aspen Tree ◽  
Armillaria Gallica ◽  
Polymerase Chain ◽  
Armillaria Species

Restriction fragment length polymorphism analysis (RFLP) of the intergenic region (IGR-1) between the 3 ' end of the 26S ribosomal RNA gene and the 5 ' end of the 5S rRNA gene was used to identify 39 isolates of Armillaria species collected from live or recently dead bigtooth aspen (Populus grandidentata Michx.) trees and sucker sprouts in the Tioga State Forest, Pennsylvania. The unknown isolates were identified by comparing their restriction fragment patterns with 18 isolates of known Armillaria species common to the northeastern United States. Twenty of the unknown isolates (50%) were identified as either Armillaria gallica or Armillaria calvescens. Eighteen (46%) of the isolates were identified as Armillaria ostoyae. One isolate of Armillaria sinapina was obtained from a recently dead aspen tree. One isolate of Armillaria mellea, considered to be the most divergent of the Armillaria species, was obtained from basidiomes fruiting on a recently dead aspen tree near Berwick, Pennsylvania. In some instances, amplification of DNA was possible by adding mycelial scrapes directly to the polymerase chain reaction (PCR) mix, thus precluding the need for DNA extraction. Advancements in RFLP analysis may offer a method able to provide rapid and precise identification of most North American and European Armillaria isolates.

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 Real-time PCR detection and speciation of Cryptosporidium infection using Scorpion probes

2006 ◽  
Vol 55 (9) ◽  
pp. 1217-1222 ◽  
Author(s):  
Suzanne E. Stroup ◽  
Shantanu Roy ◽  
John Mchele ◽  
Venance Maro ◽  
Simon Ntabaguzi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rflp Analysis ◽  
18S Rrna Gene ◽  
Human Infection ◽  
Rrna Gene ◽  
Dna Extraction Method ◽  
Stool Samples ◽  
Stool Specimens ◽  
Human Stool

At least eight species of Cryptosporidium can cause human infection and disease. A real-time PCR (qPCR) assay based on the 18S rRNA gene and utilizing a Scorpion probe was developed to detect all human-pathogenic Cryptosporidium without the usual need for nested amplification. Sensitivity of detection in stool samples was highest using a glass bead-based DNA extraction method (under 103 oocysts per stool sample). The assay was validated against 123 human stool specimens from Bangladesh and Tanzania, exhibited a sensitivity and specificity of >91 % versus microscopy, and detected an additional eight microscopy-negative infections. Cryptosporidium parvum-specific and Cryptosporidium meleagridis-specific Scorpion qPCR assays that provided 100 % accurate speciation compared with VspI RFLP analysis and sequencing were developed subsequently. These Scorpion probe qPCR assays are simpler to perform than existing nested PCR and RFLP methods for diagnosis and epidemiological investigation of cryptosporidiosis.

scholarly journals FIGARO: An efficient and objective tool for optimizing microbiome rRNA gene trimming parameters

2019 ◽  
Author(s):  
Michael M. Weinstein ◽  
Aishani Prem ◽  
Mingda Jin ◽  
Shuiquan Tang ◽  
Jeffrey M. Bhasin
Keyword(s):  
Rapid Assessment ◽  
Cost Effective ◽  
Bioinformatic Analysis ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Sequence Information ◽  
Bioinformatic Tools ◽  
Zymo Research ◽  
High Throughput Dna Sequencing ◽  
The Cost

ABSTRACTSummaryMicrobiome studies continue to provide tremendous insight into the importance of microorganism populations to the macroscopic world. High-throughput DNA sequencing technology (i.e., Next-generation Sequencing) has enabled the cost-effective, rapid assessment of microbial populations when combined with bioinformatic tools capable of identifying microbial taxa and calculating the diversity and composition of biological and environmental samples. Ribosomal RNA gene sequencing, where 16S and 18S rRNA gene sequences are used to identify prokaryotic and eukaryotic species, respectively, is one of the most widely-used techniques currently employed in microbiome analysis. Prior to bioinformatic analysis of these sequences, trimming parameters must be set so that post-trimming sequence information is maximized while expected errors in the sequences themselves are minimized. In this application note, we present FIGARO: a Python–based application designed to maximize read retention after trimming and filtering for quality. FIGARO was designed specifically to increase reproducibility and minimize trial-and-error in trimming parameter selection for a DADA2–based pipeline and will likely be useful for optimizing trimming parameters and minimizing sequence errors in other pipelines as well where paired-end overlap is required.Availability and implementationThe FIGARO application is freely available as source code at https://github.com/Zymo-Research/figaro.

scholarly journals Development of a 16S rRNA Gene Primer and PCR-Restriction Fragment Length Polymorphism Method for Rapid Detection of Members of the Genus Megasphaera and Species-Level Identification

2011 ◽  
Vol 77 (15) ◽  
pp. 5533-5535 ◽  
Author(s):  
Akihiro Ohnishi ◽  
Shinko Abe ◽  
Shiho Nashirozawa ◽  
Sayaka Shimada ◽  
Naoshi Fujimoto ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Direct Detection ◽  
Rrna Gene ◽  
Content Type ◽  
Detection And Identification ◽  
Restriction Fragment Length

ABSTRACTThe genusMegasphaerais relevant to the environment, human health and food, and renewable energy for the future. In this study, a primer set was designed for PCR-restriction fragment length polymorphism (RFLP) analyses to detect and identify the members ofMegasphaera. Direct detection and identification were achieved for environmental samples and isolates.

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