Reverse Line Blot Hybridization with Species-Specific Oligonucleotide Probes: Application to Piroplasm Detection

2014 ◽  
pp. 183-192 ◽  
Author(s):  
Ana Hurtado
Keyword(s):  
Blot Hybridization ◽  
Reverse Line Blot ◽  
Oligonucleotide Probes ◽  
Species Specific

scholarly journals Simultaneous Detection of BovineTheileria and Babesia Species by Reverse Line Blot Hybridization

1999 ◽  
Vol 37 (6) ◽  
pp. 1782-1789 ◽  
Author(s):  
J. M. Gubbels ◽  
A. P. de Vos ◽  
M. van der Weide ◽  
J. Viseras ◽  
L. M. Schouls ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Bos Taurus ◽  
Blot Hybridization ◽  
Simultaneous Detection ◽  
Carrier State ◽  
Reverse Line Blot ◽  
Babesia Bovis ◽  
Rrna Gene ◽  
Blood Samples ◽  
Species Specific

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species ofTheileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to theT. sergenti-T. buffeli-T. orientalis group. TheBabesia species included were Babesia bovis,B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria andBabesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences fromBos taurus or other hemoparasites (Trypanosomaspecies, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigeminawere identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species.

scholarly journals New Method for Simultaneous Species-Specific Identification of Equine Strongyles (Nematoda, Strongylida) by Reverse Line Blot Hybridization

2007 ◽  
Vol 45 (9) ◽  
pp. 2937-2942 ◽  
Author(s):  
D. Traversa ◽  
R. Iorio ◽  
T. R. Klei ◽  
V. A. Kharchenko ◽  
J. Gawor ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Reverse Line Blot ◽  
New Method ◽  
Specific Identification ◽  
Species Specific

scholarly journals DNA-based species detection capabilities using laser transmission spectroscopy

2013 ◽  
Vol 10 (78) ◽  
pp. 20120637 ◽  
Author(s):  
A. R. Mahon ◽  
M. A. Barnes ◽  
F. Li ◽  
S. P. Egan ◽  
C. E. Tanner ◽  
...  
Keyword(s):  
Invasive Species ◽  
Dna Detection ◽  
Field Application ◽  
Economic Damage ◽  
Oligonucleotide Probes ◽  
Target Species ◽  
Dreissena Bugensis ◽  
Species Detection ◽  
Transmission Spectroscopy ◽  
Species Specific

Early detection of invasive species is critical for effective biocontrol to mitigate potential ecological and economic damage. Laser transmission spectroscopy (LTS) is a powerful solution offering real-time, DNA-based species detection in the field. LTS can measure the size, shape and number of nanoparticles in a solution and was used here to detect size shifts resulting from hybridization of the polymerase chain reaction product to nanoparticles functionalized with species-specific oligonucleotide probes or with the species-specific oligonucleotide probes alone. We carried out a series of DNA detection experiments using the invasive freshwater quagga mussel ( Dreissena bugensis ) to evaluate the capability of the LTS platform for invasive species detection. Specifically, we tested LTS sensitivity to (i) DNA concentrations of a single target species, (ii) the presence of a target species within a mixed sample of other closely related species, (iii) species-specific functionalized nanoparticles versus species-specific oligonucleotide probes alone, and (iv) amplified DNA fragments versus unamplified genomic DNA. We demonstrate that LTS is a highly sensitive technique for rapid target species detection, with detection limits in the picomolar range, capable of successful identification in multispecies samples containing target and non-target species DNA. These results indicate that the LTS DNA detection platform will be useful for field application of target species. Additionally, we find that LTS detection is effective with species-specific oligonucleotide tags alone or when they are attached to polystyrene nanobeads and with both amplified and unamplified DNA, indicating that the technique may also have versatility for broader applications.

Reverse Line Blot Hybridization Used to Identify Hemoprotozoa in Minorcan Cattle

2002 ◽  
Vol 969 (1) ◽  
pp. 78-82 ◽  
Author(s):  
SONIA ALMERIA ◽  
JOAQUIM CASTELLÀ ◽  
DAVID FERRER ◽  
JUAN FRANCISCO GUTIÉRREZ ◽  
AGUSTIN ESTRADA-PEÑA ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Reverse Line Blot

scholarly journals A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome mec (SCCmec) typing

2009 ◽  
Vol 58 (8) ◽  
pp. 1045-1057 ◽  
Author(s):  
Lin Cai ◽  
Fanrong Kong ◽  
Qinning Wang ◽  
Huiping Wang ◽  
Meng Xiao ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Clinical Laboratory ◽  
Blot Hybridization ◽  
Rapid Identification ◽  
Reverse Line Blot ◽  
Discriminatory Power ◽  
Hybridization Assay ◽  
Coagulase Negative Staphylococci ◽  
Primer Sets ◽  
Mrsa Strains

The aim of this study was to develop a new discriminatory method for MRSA SCCmec typing based on multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay to enable rapid identification and classification of MRSA SCCmec types in a clinical laboratory. Forty-five primer sets and 49 probes were designed and tested in uniplex PCR (uPCR) and mPCR/RLB. Probes were compared in silico to 14 whole-genome sequences and 18 partial SCCmec gene sequences of Staphylococcus aureus and complete genome and partial SCCmec genes of seven non-MRSA strains, including meticillin-susceptible S. aureus and meticillin-resistant coagulase-negative staphylococci. The method was tested on a set of 42 well-characterized reference MRSA strains. It identified all five epidemiologically relevant SCCmec types and 26 subtypes, including established and new subtypes of SCCmec III, IV (eight subtypes each) and V (three subtypes). The discriminatory power of mPCR/RLB SCCmec typing was similar to that of MLST and spa typing (Simpson indices of diversity of 0.916, 0.926 and 0.882, respectively; differences not statistically significant). The application of mPCR/RLB hybridization assay to MRSA SCCmec typing can improve the specificity, discriminatory power and throughput of the typing procedure. The detection of up to 43 mPCR products in a single hybridization assay transforms MRSA SCCmec typing from passive epidemiological library typing into a potential tool for near-real-time infection control surveillance and tracking of MRSA transmission in hospitals.

scholarly journals Genotyping of 27 Human Papillomavirus Types by Using L1 Consensus PCR Products by a Single-Hybridization, Reverse Line Blot Detection Method

1998 ◽  
Vol 36 (10) ◽  
pp. 3020-3027 ◽  
Author(s):  
P. E. Gravitt ◽  
C. L. Peyton ◽  
R. J. Apple ◽  
C. M. Wheeler
Keyword(s):  
Human Papillomavirus ◽  
Detection System ◽  
Sampling Error ◽  
Blot Hybridization ◽  
High Sensitivity ◽  
Oligonucleotide Probes ◽  
Dot Blot ◽  
Consensus Primer ◽  
Hpv Dna ◽  
Silver Spring

Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or GP5+/6+) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis, genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle. Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albumin-conjugated oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancer-associated, genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of β-globin probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al., p. 132–152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach, (1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics, Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92% concordance for HPV positivity (kappa = 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples resulted from weak signals and can be attributed to sampling error from specimens with low concentrations (<1 copy/μl) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of misclassification.

scholarly journals Simultaneous Detection of Nine Antibiotic Resistance-Related Genes in Streptococcus agalactiae Using Multiplex PCR and Reverse Line Blot Hybridization Assay

2006 ◽  
Vol 50 (1) ◽  
pp. 204-209 ◽  
Author(s):  
Xianyu Zeng ◽  
Fanrong Kong ◽  
Hui Wang ◽  
Archie Darbar ◽  
Gwendolyn L. Gilbert
Keyword(s):  
Antibiotic Resistance ◽  
Multiplex Pcr ◽  
Streptococcus Agalactiae ◽  
Single Gene ◽  
Blot Hybridization ◽  
Simultaneous Detection ◽  
Reverse Line Blot ◽  
Hybridization Assay ◽  
Inducible Clindamycin Resistance ◽  
Phenotypic Resistance

ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal and maternal sepsis. Penicillin is recommended for intrapartum prophylaxis, but erythromycin or clindamycin is used for penicillin-allergic carriers. Antibiotic resistance (AR) has increased recently and needs to be monitored. We have developed a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay to detect, simultaneously, seven genes encoding AR—erm(A/TR), erm(B), mef(A/E), tet(M), tet(O), aphA-3, and aad-6—and two AR-related genes, int-Tn and mreA. We tested 512 GBS isolates from Asia and Australasia and compared mPCR/RLB with antibiotic susceptibility phenotype or single-gene PCR. Phenotypic resistance to tetracycline was identified in 450 (88%) isolates, of which 442 had tet(M) (93%) and/or tet(O) (6%). Of 67 (13%) erythromycin-resistant isolates, 18 were susceptible to clindamycin, i.e., had the M phenotype, encoded by mef(A/E); 39 had constitutive (cMLSB) and 10 inducible clindamycin resistance, and of these, 34 contained erm(B) and 12 erm(A/TR). Of four additional isolates with mef(A/E), three contained erm(B) with cMLSB and one was erythromycin susceptible. Of 61 (12%) clindamycin-resistant isolates, 20 were susceptible to erythromycin and two had intermediate resistance. Based on sequencing, 21 of 22 isolates with mef had mef(E), and 8 of 353 with int-Tn had an atypical sequence. Several AR genes, erm(B), tet(O), aphA-3, aad-6, and mef(A/E), were significantly more common among Asian than Australasian isolates, and there were significant differences in distribution of AR genes between GBS serotypes. Our mPCR/RLB assay is simple, rapid, and suitable for surveillance of antibiotic resistance in GBS.

scholarly journals Species-Specific Detection of the Maize Pathogens Sporisorium reiliana and Ustilago maydis by Dot Blot Hybridization and PCR-Based Assays

Plant Disease ◽  
1999 ◽  
Vol 83 (4) ◽  
pp. 390-395 ◽  
Author(s):  
M. L. Xu ◽  
A. E. Melchinger ◽  
T. Lübberstedt
Keyword(s):  
Genomic Dna ◽  
Blot Hybridization ◽  
Pathogenic Fungi ◽  
Ustilago Maydis ◽  
Dot Blot ◽  
Cross Hybridization ◽  
Dot Blot Hybridization ◽  
Fungal Dna ◽  
Species Specific ◽  
Sporisorium Reiliana

Head smut of maize, caused by Sporisorium reiliana, may substantially reduce grain yield. The objective of the present study was to develop a highly specific and sensitive DNA-based assay for detection of S. reiliana and its differentiation from Ustilago maydis, a maize fungus inducing the symptomatically similar common smut disease. Plasmid libraries of S. reiliana and U. maydis were constructed using a shotgun cloning procedure. Clones containing strongly hybridizing species-specific DNA were selected by screening libraries with their own labeled genomic DNA, followed by cross-hybridization with genomic DNA of maize and other maize-pathogenic fungi. The selected clones were used to generate subclones with short insert fragments to facilitate PCR amplification for labeling and primer design for a PCR assay. Using Dig-dUTP labeled inserts, detection of less than 0.16 ng of fungal DNA was possible by dot blot hybridization. Sequences of insert fragments were determined to design primer pairs for a PCR-based assay. Primer pairs SR1 and SR3 are species-specific for S. reiliana, and UM11 is species-specific for U. maydis. The PCR-based assays can detect fungal DNA of less than 1.6 pg using SR1 and SR3, and 8 pg using UM11, irrespective of the presence of maize DNA. Use of SR1 and SR3 allowed detection of S. reiliana in the extracts of pith, node, and shank from S. reiliana-infected plants, but not in leaves. Thus, both the dot blot hybridization and the PCR-based assays provide a highly sensitive and reliable tool for detection and differentiation of corn smut caused either by S. reiliana or by U. maydis.

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