Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

Detection and molecular identification of Hepatozoon canis and Babesia vogeli from domestic dogs in Palestine

Parasitology ◽  
2016 ◽  
Vol 144 (5) ◽  
pp. 613-621 ◽  
Author(s):  
KIFAYA AZMI ◽  
AMER AL-JAWABREH ◽  
ABEDELMAJEED NASEREDDIN ◽  
AHMAD ABDELKADER ◽  
TAHER ZAID ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Rhipicephalus Sanguineus ◽  
18S Rrna Gene ◽  
Blood Parasites ◽  
Hepatozoon Canis ◽  
Rrna Gene ◽  
Babesia Vogeli ◽  
Wide Range ◽  
Pcr Products ◽  
Hepatozoon Felis

SUMMARYDogs serve as hosts for a great number of parasites, which may affect their health and wellbeing. This study aimed to observe tick borne pathogens in dogs from Palestine including Hepatozoon canis and Babesia species. The prevalence of both H. canis and Babesia species infections in apparently healthy dogs, from ten districts of the West Bank was surveyed. DNA was extracted from blood samples obtained from dogs (n = 362) and ticks (n = 213) collected from dogs (n = 77). A primer set that amplifies a partial sequence of the Babesia and Hepatozoon 18S rRNA gene was used for PCR and the DNA sequences of the PCR products of all samples were determined. Twenty-nine (8·0%) of the dogs were found infected including 20 with H. canis (5·5%), seven with Babesia vogeli (1·9%) and two with undefined Babesia spp. (0·6%). Twelve Rhipicephalus sanguineus s.l ticks were pathogen-positive, including ten with H. canis (4·7%), one with B. vogeli (0·5%), and one with Hepatozoon felis (0·5%). The results indicated that a wide range of tick borne pathogens is circulating in the canine population in the surveyed region. This study is the first report on the prevalence of H. canis, B. vogeli and Babesia spp. in dogs in Palestine and its results will assist in the management of diseases associated with these blood parasites.

scholarly journals Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

2004 ◽  
Vol 70 (5) ◽  
pp. 3171-3175 ◽  
Author(s):  
X. Bonjoch ◽  
E. Ballesté ◽  
A. R. Blanch
Keyword(s):  
16S Rrna ◽  
Multiplex Pcr ◽  
Fecal Pollution ◽  
Sensitivity Threshold ◽  
Rrna Gene ◽  
Specific Primers ◽  
Animal Wastewater ◽  
Wastewater Samples ◽  
Species Specific ◽  
Different Origins

ABSTRACT Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

scholarly journals Development of species-specific Cichla species eDNA primers for alien invasive species (AIS) monitoring in Malaysia

2021 ◽  
Vol 4 ◽  
Author(s):  
O. Nurul Fizatul Nabilah ◽  
A. R. Ramizah ◽  
A. B. Adibah ◽  
S. Syazwan ◽  
A.G. Intan Faraha ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Environmental Dna ◽  
Coi Gene ◽  
Alien Invasive Species ◽  
Survey Method ◽  
Specific Primers ◽  
Invasive Fishes ◽  
Specific Amplification ◽  
Species Specific ◽  
High Level

Peacock bass or the cichlids are known locally as top predator fishes which are invasive in Malaysia freshwater system. Detection probabilities for these fishes are typically low, especially using conventional capture-survey method due to the fish’s behaviour of hiding beneath the water’s surface. Hence, the environmental DNA (eDNA) monitoring is a relatively new approach that can be used to assess the distribution of these invasive fishes. Here, we report the strategy to develop small fragment (280- 400 bp) specific-specific primers for three selected invasive Cichla species namely, C. ocellaris, C. monoculus, and C. kelberi based on mitochondrial DNA (mtDNA) sequences. Current research showed that the developed species-specific primers from cytochrome oxidase I (COI) gene has high resolution at species level. Species-specific amplification tests also proved the specificity of the developed primers, securing the high- level species identification potential which may help in controlling the spread of alien invasive fish species.

scholarly journals Emergence of Babesia Conradae Infection in Coyote-hunting Greyhounds in Oklahoma, USA

2021 ◽  
Author(s):  
Erin M Stayton ◽  
Megan Lineberry ◽  
Jennifer Thomas ◽  
Tina Bass ◽  
Kelly Allen ◽  
...  
Keyword(s):  
18S Rrna ◽  
Clinical Signs ◽  
18S Rrna Gene ◽  
Post Treatment ◽  
Rrna Gene ◽  
Babesia Gibsoni ◽  
Wide Range ◽  
Pcr Products ◽  
Life Threatening ◽  
Tick Vectors

Abstract Background: Babesia species are intraerythrocytic Apicomplexan parasites that infect a wide range of vertebrate hosts. These pathogens are typically transmitted either by tick vectors or by direct blood-to-blood contact, and may cause life-threatening clinical disease such as thrombocytopenia, hemolytic anemia, and acute renal failure in canine hosts. While Babesia vogeli and Babesia gibsoni infections have both been reported in Oklahoma, reports of B. conradae infections have been limited to California. Methods: Whole blood samples were collected in EDTA tubes from all dogs in four separate kennels in Oklahoma. DNA was extracted from each blood sample and a nested PCR was performed using general Apicomplexan primers for the partial 18S rRNA gene. PCR products were electrophoresed in agarose matrix and appropriately sized amplicons were sequenced. Sequences were compared to reference 18S rRNA sequences available in GenBank, and samples with >98% homology to B. conradae (GenBank MK256976) were considered positive. B. conradae positive dogs were then treated with atovaquone (13.5 mg/kg TID) and azithromycin (10 mg/kg SID) for 10 days and retested at 30 and 60 days post treatment by PCR. Results: Fifteen of 40 dogs tested positive for B. conradae with 98–100% sequence homology to B. conradae from California. All positive cases were coyote-hunting Greyhounds. Treatment of clinically ill dogs with atovaquone and azithromycin resulted in complete clinical recovery in clinically ill dogs and all treated dogs had negative follow-up PCR at 30 and 60 days post treatment. Conclusions: Collectively, this study (i) documents the occurrence of B. conradae in Oklahoma, (ii) highlights this pathogen as a differential to be considered when clinical signs are present, and (iii) supports the use of atovaquone and azithromycin as effective treatment in these cases.

scholarly journals Molecular characterisation of Leptospira strains in Pakistan

2016 ◽  
Vol 60 (4) ◽  
pp. 417-421
Author(s):  
Muhammad Luqman Sohail ◽  
Muhammad Sarwar Khan ◽  
Muhammad Avais ◽  
Muhammad Yasir Zahoor ◽  
Irfan Khattak ◽  
...  
Keyword(s):  
16S Rrna ◽  
Animal Species ◽  
Molecular Detection ◽  
Molecular Evidence ◽  
Rrna Gene ◽  
Intermediate Species ◽  
Specific Primers ◽  
Pathogenic Leptospira ◽  
Wide Range ◽  
Serological Studies

Abstract Introduction: Leptospirosis affects a wide range of mammals, humans, and even a few poikilothermic animal species. In Pakistan, serological studies of equine leptospirosis have reported a prevalence of over 40%, but no study has ever been conducted towards molecular detection of Leptospira in horses. Material and Methods: Blood samples from 128 horses were screened using ELISA and 41 positive samples were examined for the presence of leptospiral DNA using specific primers for 16S rRNA gene. Results: Out of 41 tested samples, 20 samples were found to be PCR-positive, revealing a fragment of 306 bp after gel electrophoresis. Sequencing and phylogenetic analysis of positive samples revealed circulation of pathogenic Leptospira spp. in Pakistani horses. No evidence of circulation of intermediate species was found in this study. Conclusion: This study reports the first molecular evidence of equine leptospirosis in Pakistan and lays ground for further research in this area. It also confirms the efficiency of 16S rRNA for the diagnosis of equine leptospirosis.

Cloth-Based Hybridization Array System for Expanded Identification of the Animal Species Origin of Derived Materials in Feeds

2007 ◽  
Vol 70 (12) ◽  
pp. 2900-2905 ◽  
Author(s):  
JOHANNA MURPHY ◽  
JENNIFER ARMOUR ◽  
BURTON W. BLAIS
Keyword(s):  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Microscopic Examination ◽  
Animal Species ◽  
Specific Detection ◽  
Animal Feeds ◽  
Species Origin ◽  
Pcr Products ◽  
Species Specific ◽  
Immunoenzymatic Assay

A cloth-based hybridization array system (CHAS) previously developed for the detection of animal species for which prohibited materials have been specified (cattle, sheep, goat, elk, and deer) has been expanded to include the detection of animal species for which there are no prohibitions (pig and horse) in Canadian and American animal feeds. Animal species were identified by amplification of mitochondrial DNA sequences by PCR and subsequent hybridization of the amplicons with an array of species-specific oligonucleotide capture probes immobilized on a polyester cloth support, followed by an immunoenzymatic assay of the bound PCR products. The CHAS permitted sensitive and specific detection of meat meals from different animal species blended in a grain-based feed and should provide a useful adjunct to microscopic examination for the identification of prohibited materials in animal feeds.

scholarly journals Detection and Discrimination of Pratylenchus neglectus and P. thornei in DNA Extracts from Soil

Plant Disease ◽  
2008 ◽  
Vol 92 (11) ◽  
pp. 1480-1487 ◽  
Author(s):  
Guiping Yan ◽  
Richard W. Smiley ◽  
Patricia A. Okubara ◽  
Andrea Skantar ◽  
Sandra A. Easley ◽  
...  
Keyword(s):  
Nematode Species ◽  
Wheat Root ◽  
Fungal Species ◽  
Parasitic Nematodes ◽  
Rrna Gene ◽  
28S Rrna ◽  
Pratylenchus Neglectus ◽  
Pcr Products ◽  
Species Specific ◽  
Plant Parasitic

A species-specific polymerase chain reaction (PCR) method was developed to detect and identify the root-lesion nematodes Pratylenchus neglectus and P. thornei from soil. A primer set was designed from Pratylenchus 28S rRNA gene sequences of the D3 expansion domain. Primer specificity was confirmed with 23 isolates of 15 nematode species and other plant-parasitic and non-plant-parasitic nematodes typically present in the soil communities, and with six fungal species commonly associated with wheat root rot. DNA obtained using a commercially available kit and a method developed in our laboratory gave comparable amplification. PCR conditions were optimized and the two species were differentiated by PCR products of 144 bp for P. neglectus and 288 bp for P. thornei. With this assay, we detected a single juvenile in 1 g of sterile, inoculated soil. Examination of 30 field soil samples revealed that this method was applicable to a range of soils naturally infested with these two pathogens in Oregon. This PCR-based method is rapid, efficient, and reliable, does not require expertise in nematode taxonomy and morphology, and could be used as a rapid diagnostic tool for commercial and research applications for disease forecasting and management.

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.

Variability of Echiniscus tristis Gąsiorek & Kristensen, 2018—is morphology sufficient for taxonomic differentiation of Echiniscidae?

Zootaxa ◽  
2019 ◽  
Vol 4701 (1) ◽  
pp. 1-24 ◽  
Author(s):  
TOMASZ BARTYLAK ◽  
ADAM KULPA ◽  
DARIA GROBYS ◽  
MARTA KEPEL ◽  
ANDRZEJ KEPEL ◽  
...  
Keyword(s):  
Dna Sequences ◽  
28S Rrna ◽  
Life Stages ◽  
Significant Genetic Differentiation ◽  
Wide Range ◽  
Microscope Images ◽  
Different Types ◽  
Taxonomic Confusion ◽  
Species Specific ◽  
Range Of Variation

The majority of species in the genus Echiniscus (Heterotardigrada) have been described based on differences in the chaetotaxy or dorsal sculpture. Dorsal sculpture is, in general, considered to be species-specific and not very variable; however, many problems have arisen due to various interpretations of microscope images, which has led to taxonomic confusion in the genus Echiniscus. Conversely, chaetotaxy is generally much easier to interpret, even using low-quality microscope optics. In this study, we emended the description of Madagascan population of Echiniscus tristis Gąsiorek & Kristensen, 2018 that exhibits several different types of chaetotaxy and dorsal sculpture. The analysed specimens were characterised by two types of chaetotaxy, A-C-Dd-E and A-Dd-E, but we also found a wide range of variation in appendage number, shape and length. The observed differences are partly correlated with life stages. Additionally, we analysed DNA sequences of 28S rRNA, ITS-2 and COI of the two main morphotypes, and did not find significant genetic differentiation of the two morphotypes. This highlights the importance of analysing the morphology of both immature stages and adults, as well as of DNA markers in tardigrade species identification. 

Sign in / Sign up

Export Citation Format

Share Document