Determining the prevalence of Oesophagostomum bifurcum and Necator americanus infections using specific PCR amplification of DNA from faecal samples

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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Identification and Characterization of Janthinobacterium svalbardensis F19, a Novel Low-C/N-Tolerant Denitrifying Bacterium

Applied Sciences ◽

10.3390/app9091937 ◽

2019 ◽

Vol 9 (9) ◽

pp. 1937 ◽

Cited By ~ 5

Author(s):

Yinyan Chen ◽

Peng Jin ◽

Zhiwen Cui ◽

Tao Xu ◽

Ruojin Zhao ◽

...

Keyword(s):

Nitrogen Removal ◽

Removal Rate ◽

Pcr Amplification ◽

Domestic Wastewater ◽

Heterotrophic Nitrification ◽

Aerobic Denitrification ◽

Nitrite Accumulation ◽

Specific Pcr ◽

Domestic Wastewater Treatment ◽

Initial Ph

Herein, we isolated Janthinobacterium svalbardensis F19 from sludge sediment. Strain F19 can simultaneously execute heterotrophic nitrification and aerobic denitrification under aerobic conditions. The organism exhibited efficient nitrogen removal at a C/N ratio of 2:1, with an average removal rate of 0.88 mg/L/h, without nitrite accumulation. At a C/N ratio of 2, an initial pH of 10.0, a culturing temperature of 25 °C, and sodium acetate as the carbon source, the removal efficiencies of ammonium, nitrate, nitrite, and hydroxylamine were 96.44%, 92.32%, 97.46%, and 96.69%, respectively. The maximum removal rates for domestic wastewater treatment for ammonia and total nitrogen were 98.22% and 92.49%, respectively. Gene-specific PCR amplification further confirmed the presence of napA, hao, and nirS genes, which may contribute to the heterotrophic nitrification and aerobic denitrification capacity of strain F19. These results indicate that this bacterium has potential for efficient nitrogen removal at low C/N ratios from domestic wastewater.

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Detection of maternal cells in human fetal blood during the third trimester of pregnancy using allele‐specific PCR amplification

British Journal of Haematology ◽

10.1046/j.1365-2141.1997.2603076.x ◽

1997 ◽

Vol 98 (3) ◽

pp. 767-771 ◽

Cited By ~ 69

Author(s):

THIERRY PETIT ◽

MARC DOMMERGUES ◽

GÉRARD SOCIÉ ◽

YVES DUMEZ ◽

ELIANE GLUCKMAN ◽

...

Keyword(s):

Pcr Amplification ◽

Fetal Blood ◽

Third Trimester ◽

The Third ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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PCR Detection and RFLP Differentiation of Botrytis Species Associated with Neck Rot of Onion

Plant Disease ◽

10.1094/pdis.2002.86.6.682 ◽

2002 ◽

Vol 86 (6) ◽

pp. 682-686 ◽

Cited By ~ 27

Author(s):

Karsten Nielsen ◽

David S. Yohalem ◽

Dan Funck Jensen

Keyword(s):

Pcr Amplification ◽

Pcr Detection ◽

Sequence Characterized Amplified Region ◽

Specific Pcr ◽

Isolation Techniques ◽

Detection And Identification ◽

Fungal Dna ◽

Pcr Method ◽

Polymerase Chain ◽

Direct Amplification

Botrytis aclada and other Botrytis spp. can cause neck rot on onions, a storage disease that normally is very difficult to detect at harvest using traditional isolation techniques. Sequence characterized amplified region primers (BA2f/BA1r) were designed based on a previously cloned and amplified DNA fragment for direct amplification of isolates of Botrytis spp. associated with neck rot of onions. Digestion of the polymerase chain reaction (PCR) amplification product with the restriction enzyme ApoI makes it possible to distinguish the five groups: Botrytis aclada types AI and AII (B. allii); B. byssoidea; B. squamosa; and B. cinerea. The detection limit was 1 to 10 pg of pure fungal DNA. It was possible to detect B. aclada with the PCR method in artificially inoculated onion bulb tissue and in mature onion leaves showing no symptoms of the disease. The availability of a sensitive and specific PCR detection and identification method for Botrytis onion neck rot pathogens should facilitate ecological studies of this group of onion pathogens.

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Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

Applied and Environmental Microbiology ◽

10.1128/aem.68.10.5064-5081.2002 ◽

2002 ◽

Vol 68 (10) ◽

pp. 5064-5081 ◽

Cited By ~ 469

Author(s):

Alexander Loy ◽

Angelika Lehner ◽

Natuschka Lee ◽

Justyna Adamczyk ◽

Harald Meier ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Pcr Amplification ◽

Oligonucleotide Microarray ◽

16S Rrna Genes ◽

Rrna Genes ◽

Clinical Samples ◽

Rrna Gene ◽

Sulfate Reducing ◽

Specific Pcr

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

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Analysis of Immunogenetic Factors in Paroxysmal Nocturnal Hemoglobinuria Reveal Increased Frequency of IL-10 Low Secretor Genotype and Correlates with Induced IL-10 Production.

Blood ◽

10.1182/blood.v108.11.983.983 ◽

2006 ◽

Vol 108 (11) ◽

pp. 983-983

Author(s):

Giridharan Ramsingh ◽

Zachary Nearman ◽

Chris Hung ◽

Serio Bianca ◽

Antonio Risitano ◽

...

Keyword(s):

Paroxysmal Nocturnal Hemoglobinuria ◽

Direct Sequencing ◽

Pcr Amplification ◽

Hla Typing ◽

Nucleotide Polymorphisms ◽

Specific Pcr ◽

Hla Ligand ◽

Functional Consequences ◽

Immunologic Diseases ◽

Common Pathogenesis

Abstract Aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) have a common pathogenesis related to an immune attack on hematopoietic progenitor and stem cells. While the inciting events are not known, it is possible that complex immunogenetic predisposition factors exist which, in the context of exogenous influences, determine risk for these diseases. They include HLA background, KIR genotype, cytokine and immunomodulatory gene polymorphisms as well as gene variants involved in peptide processing and presentation. We have empirically selected a number of important polymorphisms that were described in the context of various immunologic diseases and studied their frequency in a large cohort (N=57) of PNH patients. As binding of KIR to the appropriate HLA ligand (KIR-L) can modulate activation of Nk-cells and cytotoxic T lymphocytes, we examined the combined impact of KIR/KIR-L genotypes on the risk of PNH and PNH/AA syndrome. PNH showed a decreased frequency of inhibitory KIR-2DL1 and KIR-2DL3 genes (79% vs. 95%, p=.0054; 67% vs. 89%, p=.0032). Analysis of the KIR genotype in correlation with the corresponding KIR-L profile, deduced from HLA typing, revealed an increased frequency of unopposed 2DS2 (2DS2/C1 type, 37% vs.10%, p=.012) and 2DL2 (2DL2/C1 mismatch 37% vs 11%, p=.031) but these mismatches have a potentially opposing functional effect. Using sequence-specific PCR amplification and/or direct sequencing, we have genotyped DNA samples derived from our cohort who presented with different subtypes of PNH and studied single nucleotide polymorphisms (SNPs) in cytokine genes such as TNF-a (−308 G/A), TGF-b 1 (C/T codon 10, C/G codon 25), IL-10 (−1082 G/A), and IFN-g (+874 A/T) and immunomodulatory receptor genes like CTLA-4 exon 6 (+49 G/A), FcγRIIIa (158 F/V) and CD45-exons 6 (+138 A/G), and 4 (+54 A/G, +77 C/G). These SNPs are responsible for intrinsic differences in cytokine production and receptor function and can thereby influence immune physiologic and pathologic responses. PNH patients showed a significantly higher frequency of A/A polymorphisms at intron −1082 of the IL-10 gene consistent with a “low secretor phenotype” (36% PNH (N=50) vs 13.8% controls (N=363), p=0.0001). In contrast to a few smaller studies, no association with any other SNP tested was found. To establish whether this finding translates into functional consequences, we examined basal and PMA-induced IL-10 secretion in PNH patients andcontrols. Upon induction, IL-10 production increased in controls (N=5, 14.11± 9.8), while in general, stimulation resulted in a much weaker IL-10 response in PNH patients (N=6, 4.51± 2.16). However, when compared genotypic IL-10 “low secretors” (−1082 A/A) among PNH patients showed significantly lower induction levels of IL-10 (N=2, 3.1± 1.58) as compared to PNH patients with “normal/high secretor” genotypes (−1082 A/G,G/G) ((N=4, 5.22 +/−3.1). The low secretor IL-10 genotype may correspond to the exaggerated TH1 response in PNH and in general supports the notion that complex inherited traits may exist that genetically determine propensity to PNH evolution.

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A simple method for discriminating Bursaphelenchus xylophilus and B. mucronatus by species-specific polymerase chain reaction primer pairs

Nematology ◽

10.1163/1568541041217960 ◽

2004 ◽

Vol 6 (2) ◽

pp. 273-277 ◽

Cited By ~ 38

Author(s):

Koji Matsunaga ◽

Katsumi Togashi

Keyword(s):

Pcr Amplification ◽

Nematode Species ◽

Bursaphelenchus Xylophilus ◽

Polymerase Chain Reaction Primer ◽

Dna Fragments ◽

Simple Method ◽

Specific Pcr ◽

Pcr Products ◽

Specific Pcr Primer ◽

Species Specific

Abstract Two species-specific PCR primer pairs were developed for identifying the two nematode species, Bursaphelenchus xylophilus and B. mucronatus. The primer pairs were developed from the sequence of ribosomal DNA (rDNA) repeats to produce DNA fragments of different lengths by PCR amplification. The DNA fragments for B. mucronatus and B. xylophilus were 210 bp and 557 bp, respectively. When mixed, neither primer pair inhibited the PCR amplification of the other. Five isolates of B. xylophilus and four isolates of B. mucronatus showed different band profiles of PCR products between the two species, but identical profiles among isolates of the same species.

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Novel Hairpin-Shaped Primer Assay To Study the Association of the −44 Single-Nucleotide Polymorphism of the DEFB1 Gene with Early-Onset Periodontal Disease

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.4.766-769.2004 ◽

2004 ◽

Vol 11 (4) ◽

pp. 766-769 ◽

Cited By ~ 24

Author(s):

Michele Boniotto ◽

Manzour Hernando Hazbón ◽

William James Jordan ◽

Greig Patrick Lennon ◽

Joyce Eskdale ◽

...

Keyword(s):

Periodontal Disease ◽

Real Time ◽

Early Onset ◽

Pcr Amplification ◽

Cost Effective ◽

Similar Distribution ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Specific Pcr ◽

Specific Allele

ABSTRACT A powerful, cost-effective new method for studying single-nucleotide polymorphisms (SNPs) is described. This method is based on the use of hairpin-shaped primers (HP), which give a sensitive and specific PCR amplification of each specific allele, without the use of costly fluorophore-labeled probes and any post-PCR manipulation. The amplification is monitored in real-time using SYBR Green I dye and takes only 2 h to yield results. The HP assay has a simple design and utilizes a conventional real-time PCR apparatus. The −44 C→G transversion in the DEFB1 gene (which encodes human β-defensin 1) has been previously associated with Candida carriage in oral epithelia. In this study, we analyzed the association between early-onset periodontal disease (EOP) and the −44 SNP. We used an HP assay to study the distribution of the −44 SNP in 264 human DNAs obtained from two cohorts of EOP patients and healthy controls from different ethnic backgrounds. The results indicate that the −44 SNP has a similar distribution between EOP and healthy patients, suggesting that it is not associated with the disease.

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