scholarly journals Molecular Fingerprinting of Cryptosporidium Oocysts Isolated during Water Monitoring

2006 ◽  
Vol 72 (8) ◽  
pp. 5428-5435 ◽  
Author(s):  
Rosely A. B. Nichols ◽  
Brian M. Campbell ◽  
Huw V. Smith
Keyword(s):  
Environmental Water ◽  
18S Rrna Gene ◽  
Water Monitoring ◽  
Rrna Gene ◽  
Molecular Fingerprinting ◽  
Oocyst Wall ◽  
Specific Pcr ◽  
Pcr Product ◽  
Allele Specific ◽  
Microscope Slides

ABSTRACT We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.

scholarly journals Species-specific PCR assay for the detection of Babesia odocoilei

2021 ◽  
pp. 104063872110329
Author(s):  
Hilary J. Burgess ◽  
Betty P. Lockerbie ◽  
Lisanework E. Ayalew ◽  
Antonia Dibernardo ◽  
Kristýna Hrazdilová ◽  
...  
Keyword(s):  
Dna Amplification ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Species Sensitivity ◽  
Pcr Assay ◽  
Banding Patterns ◽  
Specific Pcr ◽  
Pcr Product ◽  
Specific Pcr Assay ◽  
Species Specific

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.

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.

scholarly journals Multiplex-Polymerase Chain Reaction Assay for the Authentication of the Mackerel Scomber colias in Commercial Canned Products

2006 ◽  
Vol 89 (3) ◽  
pp. 708-711 ◽  
Author(s):  
Carlos Infante ◽  
Manuel Manchado
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Nontranscribed Spacer ◽  
Specific Pcr ◽  
Scomber Colias ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Specific Fragment

Abstract A multiplex-polymerase chain reaction (PCR) system was developed for the authentication of the mackerel Scomber colias in commercial canned products. This novel method consists of an S. colias-specific fragment [159 base pairs (bp)] located in the nontranscribed spacer (NTS) sequence, and a Scomber genus-specific PCR product in the 5S rRNA gene (196201 bp) as a positive amplification control. The system was assayed using 18 different canned products labeled as S. colias. A positive identification was made in all but one sample, revealing this methodology as a potential molecular tool for direct application in the authentication of S. colias canned products.

scholarly journals Allele-Specific PCR for the Detection of Azoxystrobin Resistance in Didymella bryoniae

Plant Disease ◽  
2014 ◽  
Vol 98 (12) ◽  
pp. 1681-1684 ◽  
Author(s):  
Mavis J. Finger ◽  
Venkatesan Parkunan ◽  
Pingsheng Ji ◽  
Katherine L. Stevenson
Keyword(s):  
Dna Sequences ◽  
Cyt B ◽  
Didymella Bryoniae ◽  
Specific Pcr ◽  
Pcr Product ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Sensitive Allele ◽  
Cyt B Gene ◽  
Allele Specific Pcr

Gummy stem blight (GSB), caused by the fungus Didymella bryoniae, is considered the most widespread and destructive disease of watermelon in the southeastern United States. The quinone outside-inhibiting (QoI) fungicide azoxystrobin (AZO), which inhibits mitochondrial respiration by binding to the outer, quinone-oxidizing pocket of the cytochrome bc1 (cyt b) enzyme complex, was initially very effective in controlling GSB. However, resistance to AZO has been observed in D. bryoniae in many watermelon-producing regions. In this study, the DNA sequences of partial cyt b genes of four AZO-resistant (AZO-R) and four AZO-sensitive (AZO-S) isolates of D. bryoniae confirmed the amino acid substitution of glycine by alanine at the 143 codon (G143A) in the AZO-R isolates tested. Allele-specific primers were designed to detect the resistant or sensitive allele at codon 143 of the cyt b gene, which amplified a 165-bp polymerase chain reaction (PCR) product from genomic DNA of nine AZO-R and nine AZO-S isolates of D. bryoniae, respectively. The primer pairs did not amplify DNA from other pathogens tested in the study. The results indicated that the PCR assays developed in the study were specific in differentiating AZO-R and AZO-S isolates and could facilitate AZO resistance detection in D. bryoniae.

PCR Identification of Ruminant Tissue in Raw and Heat-Treated Meat Meals

2006 ◽  
Vol 69 (9) ◽  
pp. 2241-2247 ◽  
Author(s):  
JEONG CHUL HA ◽  
WAN TAE JUNG ◽  
YONG SUK NAM ◽  
TAE WHA MOON
Keyword(s):  
Dna Sequence ◽  
12S Rrna ◽  
Spongiform Encephalopathy ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Pcr Product ◽  
Heat Treated ◽  
Mitochondrial 12S Rrna ◽  
Animal Feedstuffs ◽  
Species Specific

To control the spread of bovine spongiform encephalopathy in cattle through contaminated animal feedstuffs, screening of feed products is essential. We designed five pairs of primers to identify specifically raw and heat-treated tissue from cattle, sheep, goat, deer, and ruminants in general. A forward common primer was designed based on a conserved DNA sequence in the mitochondrial 12S rRNA–tRNAval–16S rRNA gene, and reverse primers were designed to hybridize with a species-specific DNA sequence for each species considered. All primers were developed to create a specific PCR product small enough (less than 200 bp) to be suitable for heat-treated material. To evaluate the effect of heat treatment, a severe sterilization condition (133°C at 300 kPa for 20 min) was chosen. Species-specific amplicons were obtained from all types of heat-treated meat meals. Analysis of laboratory-contaminated vegetable meals revealed that the detection limit of the assay was 0.05% for each species analyzed. This PCR-based analysis can be used as a routine method for detecting banned animal-derived ingredients in raw and heat-treated feedstuffs.

scholarly journals Molecular Fingerprinting and Hybridity Authentication in Cowpea Using Single Nucleotide Polymorphism Based Kompetitive Allele-Specific PCR Assay

2021 ◽  
Vol 12 ◽  
Author(s):  
Patrick Obia Ongom ◽  
Christian Fatokun ◽  
Abou Togola ◽  
Stella Salvo ◽  
Oluwaseye Gideon Oyebode ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Principal Component ◽  
Snp Markers ◽  
Breeding Program ◽  
Molecular Fingerprinting ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Optimization of a breeding program for increased genetic gain requires quality assurance (QA) and quality control (QC) at key phases of the breeding process. One vital phase in a breeding program that requires QC and QA is the choice of parents and successful hybridizations to combine parental attributes and create variations. The objective of this study was to determine parental diversity and confirm hybridity of cowpea F1 progenies using KASP (Kompetitive Allele-Specific PCR)-based single nucleotide polymorphism (SNP) markers. A total of 1,436 F1 plants were derived from crossing 220 cowpea breeding lines and landraces to 2 elite sister lines IT99K-573-1-1 and IT99K-573-2-1 as male parents, constituting 225 cross combinations. The progenies and the parents were genotyped with 17 QC SNP markers via high-throughput KASP genotyping assay. The QC markers differentiated the parents with mean efficiency of 37.90% and a range of 3.4–82.8%, revealing unique fingerprints of the parents. Neighbor-Joining cladogram divided the 222 parents into 3 clusters. Genetic distances between parents ranged from 0 to 3.74 with a mean of 2.41. Principal component analysis (PCA) depicted a considerable overlap between parents and F1 progenies with more scatters among parents than the F1s. The differentiation among parents and F1s was best contributed to by 82% of the markers. As expected, parents and F1s showed a significant contrast in proportion of heterozygous individuals, with mean values of 0.02 and 0.32, respectively. KASP markers detected true hybridity with 100% success rate in 72% of the populations. Overall, 79% of the putative F1 plants were true hybrids, 14% were selfed plants, and 7% were undetermined due to missing data and lack of marker polymorphism between parents. The study demonstrated an effective application of KASP-based SNP assay in fingerprinting, confirmation of hybridity, and early detection of false F1 plants. The results further uncovered the need to deploy markers as a QC step in a breeding program.

Molecular approaches in species identification of Sarcocysts in Indian buffalo meat

2018 ◽  
Author(s):  
C. Ramakrishn ◽  
S. Vaithiyanathan ◽  
M. Muthukumar ◽  
L., P. Lavanya and V.V. Kulkarni. R. Chatlod ◽  
P. Lavanya ◽  
...  
Keyword(s):  
Restriction Enzymes ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Restriction Enzyme Digestion ◽  
Pcr Assay ◽  
Naked Eye ◽  
Molecular Approaches ◽  
Pcr Product ◽  
Pcr Products ◽  
Eye Examination

DNA was extracted from sarcocysts (visible on naked eye examination) collected from 168 buffaloes belonging to Mumbai (60), Hyderabad (54) and Kolkata (54) cities of India. They were subjected to PCR assay using 18S rRNA gene primer. All the PCR amplicons of about 900 bp were subjected to restriction enzyme digestion with four different restriction enzymes (BslI, DraI, FokI and RsaI). PCR amplicons showed two different patterns (Pattern A and Pattern B) on RFLP. Twenty one PCR products from Pattern A and one PCR product from Pattern B were subjected to DNA sequencing. S. fusiformis and S. taeniata were identified from Pattern A and S. buffalonis was identified from Pattern B on sequencing.

Helicobacter pylori 23S rRNA gene A2142G, A2143G, T2182C, and C2195T mutations associated with clarithromycin resistance detected in Sudanese patients

2020 ◽  
Author(s):  
Aalaa Mahgoub Albasha ◽  
Maram M. Alnosh ◽  
Esraa Hassan Osman ◽  
Duha M Zeinalabdin ◽  
Amira A M Fadl ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Point Mutations ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Clarithromycin Resistance ◽  
23S Rrna Gene ◽  
Allele Specific ◽  
H Pylori ◽  
Allele Specific Pcr

Abstract Background: Clarithromycin resistant Helicobacter pylori (H. pylori) strains represent a worldwide health problem. These stains are usually carrying mutations within the 23S rRNA gene associated with clarithromycin resistance. This study aimed to detect H. pylori and clarithromycin resistant associated mutations from Sudanese patients with gastritis symptoms.Materials and Methods: Two hundred and eighty-eight gastric biopsies were collected using gastrointestinal endoscopy from patients with gastritis symptoms in different hospitals in Khartoum state. H. pylori was detected by PCR using primers targeting 16S rRNA and 23S rRNA. Then allele-specific PCR and DNA sequencing were used to screen for the presence of A2142G and A2143G point mutations.Results: Out of 288 samples, H. pylori was detected in 97 (33.7%) sample. Allele-specific PCR detected the variant A2142G in 9/97 (9.3%) sample, while A2143G mutation was not found in any sample. The DNA sequencing revealed the presence of mutations associated with clarithromycin-resistance in 48% (12/25) of samples; the A2142G was present in one sample, A2143G in 5 samples, T2182C in 4 samples, and C2195T in 3 samples. There was no association of 23S rRNA gene point mutations with gender, age group and geographical distribution of patients.Conclusion: This study revealed a high frequency (48%) of mutations associated with clarithromycin resistance using DNA sequencing of the 23S rRNA gene's V domain. This information should be taken into consideration before choosing optimal therapy for H. pylori eradication.

Molecular identification and phylogenetic characterisation of Thelohanellus kitauei — short communication

2013 ◽  
Vol 61 (1) ◽  
pp. 30-35 ◽  
Author(s):  
Sang Shin ◽  
Ji Kim ◽  
Casiano Choresca ◽  
Jee Han ◽  
Jin Jun ◽  
...  
Keyword(s):  
18S Rrna ◽  
Short Communication ◽  
Genetic Relationships ◽  
18S Rrna Gene ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pcr Product ◽  
Cyprinus Carpio Haematopterus ◽  
18S Rrna Genes ◽  
Rrna Sequences

Thelohanellus kitauei was isolated from the koi Cyprinus carpio haematopterus, and the 18S rRNA gene of T. kitauei was amplified by optimised nested-PCR. The PCR product was sequenced and compared with other 18S rRNA genes of Thelohanellus species to investigate the relationships between their host specificities and infection sites. Based on the 18S rRNA sequences, T. kitauei is most closely related to T. hovorkai (which can infect the intestine). Phylogenetic analysis revealed that T. kitauei was clustered with other Thelohanellus spp. infecting Cyprininae. The present study suggests that the infection site and the host specificity (subfamily level) are reflected in the genetic relationships among Thelohanellus species.

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