Comparison of repetitive-sequence-based polymerase chain reaction with random amplified polymorphic DNA analysis for rapid genotyping of nontuberculosis mycobacteria

2012 ◽  
Vol 58 (8) ◽  
pp. 953-964 ◽  
Author(s):  
Sara Christianson ◽  
Joyce Wolfe ◽  
Hafid Soualhine ◽  
Meenu K. Sharma
Keyword(s):  
Repetitive Sequence ◽  
Dna Analysis ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Gene Sequencing ◽  
Variable Number ◽  
Outbreak Investigations ◽  
Hsp65 Gene ◽  
Polymerase Chain ◽  
Clinically Significant

Nontuberculosis mycobacteria (NTM) are an important cause of human disease and infections. Though less notorious than tuberculosis, these infections are clinically significant and have been associated with outbreaks in various settings. To accommodate outbreak investigations for the numerous species of NTM, we evaluated a DiversiLab repetitive-sequence-based PCR (rep-PCR) kit for genotyping of mycobacteria. This kit was used to genotype both rapidly and slowly growing mycobacteria and was compared with other PCR-based genotyping methods, including random amplified polymorphic DNA (RAPD) analysis, hsp65 gene sequencing, and mycobacterial interspersed repetitive unit – variable number of tandem repeat (MIRU–VNTR) analysis. Compared with RAPD analysis, rep-PCR achieved better reproducibility in testing. When compared with hsp65 gene sequencing and MIRU–VNTR for Mycobacterium avium , rep-PCR provided results that agreed with these less discriminatory genotyping methods but provided a higher level of discrimination for situations such as outbreak investigations. We also evaluated the kit for its ability to identify closely related rapidly growing NTM. While rep-PCR was informative in some cases, a much larger library of isolates would be necessary to truly evaluate it as an identification tool. Overall, rep-PCR was able to provide improved reproducibility over RAPD and a discriminatory genotyping method for the isolates evaluated in this study.

scholarly journals Genetic variation between susceptible and non-susceptible snails to Schistosoma infection using random amplified polymorphic DNA analysis (RAPDs)

1999 ◽  
Vol 41 (5) ◽  
pp. 291-295 ◽  
Author(s):  
Abdel-Hamid Zaki ABDEL-HAMID ◽  
Jeanne Blanco de MOLFETTA ◽  
Vania FERNANDEZ ◽  
Vanderlei RODRIGUES
Keyword(s):  
Dna Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Polyacrylamide Gel Electrophoresis ◽  
Genome Comparison ◽  
Gene Products ◽  
Chain Reaction ◽  
High Molecular Weight Dna ◽  
Polymerase Chain ◽  
Or Gene ◽  
Host Parasite

Susceptibility of snails to infection by certain trematodes and their suitability as hosts for continued development has been a bewildering problem in host-parasite relationships. The present work emphasizes our interest in snail genetics to determine what genes or gene products are specifically responsible for susceptibility of snails to infection. High molecular weight DNA was extracted from both susceptible and non-susceptible snails within the same species Biomphalaria tenagophila. RAPD was undertaken to distinguish between the two types of snails. Random primers (10 mers) were used to amplify the extracted DNA by the polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis (PAGE) and silver staining. The results suggest that RAPD represents an efficient means of genome comparison, since many molecular markers were detected as genetic variations between susceptible and non-susceptible snails.

Inheritance of random amplified polymorphic DNA markers in an interspecific cross in the genus Stylosanthes

Genome ◽  
1993 ◽  
Vol 36 (1) ◽  
pp. 50-56 ◽  
Author(s):  
Kemal Kazan ◽  
John M. Manners ◽  
Don F. Cameron
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Rapd Markers ◽  
Dna Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Interspecific Cross ◽  
Parental Origin ◽  
Chain Reaction ◽  
Stylosanthes Hamata ◽  
Polymerase Chain

The inheritance of random amplified polymorphic DNA (RAPD) markers generated via the polymerase chain reaction amplification of genomic DNA sequences in an F2 family of an interspecific cross between Stylosanthes hamata and S. scabra was investigated. An initial comparison between the parental species, S. hamata cv. Verano and S. scabra cv. Fitzroy, demonstrated that 34% of detected RAPD bands were polymorphic. Of 90 primers tested, 35 showed relatively simple and reliably scorable polymorphisms and were used for segregation analysis. Sixty F2 individuals were scored for the segregation of 73 RAPD markers and 55 of these markers fit a 3:1 ratio. Segregation of eight other RAPD markers deviated significantly from a 3:1 ratio. There was no bias in the inheritance of RAPD markers regarding parental origin of the segregating RAPD markers. Linkage analysis revealed 10 linkage groups containing a total of 44 RAPD loci. Another 10 RAPD markers (7 of maternal origin) that were polymorphic between the parents did not segregate in the F2 population. One of the maternally inherited RAPD bands hybridized to chloroplast DNA. Analysis of RAPD loci by DNA hybridization indicated that mainly repeated sequences were amplified. These data indicate that RAPDs are useful genetic markers in Stylosanthes spp. and they may be suitable for genetic mapping.Key words: genetic mapping, molecular markers, polymerase chain reaction, Stylosanthes hamata, Stylosanthes scabra.

Random amplified polymorphic DNA analysis in Hordeum species

Genome ◽  
1993 ◽  
Vol 36 (6) ◽  
pp. 1029-1031 ◽  
Author(s):  
Juan Manuel González ◽  
Esther Ferrer
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Polymerase ◽  
Dna Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Chain Reaction ◽  
Fragment Pattern ◽  
Polymerase Chain ◽  
Hordeum Species

Random amplified polymorphic DNA analysis was performed by applying a set of 13 arbitrary 10-mer primers to 19 Hordeum species and subspecies. High levels of variation in fragment pattern were observed both within and among species with most of the primers used. Genetic similarities between accessions and species were calculated from the fragment patterns. The resulting phenograms confirmed previous relationships among the Hordeum species.Key words: random amplified polymorphic DNA, polymerase chain reaction, polymorphism, Hordeum.

scholarly journals Characterizing the Sphaceloma Fungus, Causal Agent of Superelongation Disease in Cassava

Plant Disease ◽  
2000 ◽  
Vol 84 (4) ◽  
pp. 423-428 ◽  
Author(s):  
Elizabeth Alvarez ◽  
Martha L. Molina
Keyword(s):  
Dna Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Management Strategies ◽  
Restriction Enzymes ◽  
Colony Growth ◽  
Molecular Characteristics ◽  
Intermediate Reaction ◽  
Polymerase Chain ◽  
The Tropics ◽  
Selection Of

The fungus Sphaceloma manihoticola causes superelongation disease in cassava, a starchy root crop grown widely in the tropics. Isolates were collected from infected plants grown in six localities of Colombia. Morphological analyses of the fungus showed that colony growth and color are not stable characteristics over time. Pathogenicity studies, using the susceptible cassava variety M Col 22 and the resistant M Ven 77, showed that M Col 22 was tolerant of 29% of pathogen isolates studied and had an intermediate reaction to 71%. Variety M Ven 77 also showed tolerance of 16.2% of the isolates, had an intermediate reaction to 80.6%, and was susceptible to 3.2%. Significant cultivar × isolate interactions indicated pathogenic specialization. This study is the first to describe this pathogen's molecular characteristics. A homogeneous and reporducible band of about 545 bp was obtained with polymerase chain reaction which, when digested by restriction enzymes, showed an equal pattern of bands for all isolates. The isolates thus belonged to one species. Random amplified polymorphic DNA analysis revealed intraspecific genetic diversity. By better understanding the pathogen, we can apply more appropriate disease management strategies, such as selection of germ plasm tolerant of superelongation disease.

Isolation of Genotype- and Chromosome-specific DNA Markers in a Biotype of Colletotrichum gloeosporioides Using Random Amplified Polymorphic DNA Analysis

1998 ◽  
Vol 46 (1) ◽  
pp. 143
Author(s):  
Agnieszka M. Poplawski ◽  
John A. G. Irwin ◽  
John M. Manners
Keyword(s):  
Dna Sequences ◽  
Fungal Pathogen ◽  
Rapd Markers ◽  
Dna Analysis ◽  
Colletotrichum Gloeosporioides ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Dna Probes ◽  
Race 2 ◽  
Biotype B

Genetic markers that distinguish fungal genotypes are important tools for genetic analysis of heterokaryosis and parasexual recombination in fungi. Random amplified polymorphic DNA (RAPD) markers that distinguish two races of biotype B of Colletotrichum gloeosporioides infecting the legume Stylosanthes guianensis were sought. Eighty-five arbitrary oligonucleotide primers were used to generate 895 RAPD bands but only two bands were found to be specifically amplified from DNA of the race 3 isolate. These two RAPD bands were used as DNA probes and hybridised only to DNA of the race 3 isolate. Both RAPD bands hybridised to a dispensable 1.2 Mb chromosome of the race 3 isolate. No other genotype-specific chromosomes or DNA sequences were identified in either the race 2 or race 3 isolates. The RAPD markers hybridised to a 2 Mb chromosome in all races of the genetically distinct biotype A pathogen which infects other species of Stylosanthes as well as S. guianensis. The experiments indicate that RAPD analysis is a potentially useful tool for obtaining genotype- and chromosome-specific DNA probes in closely related isolates of one biotype of this fungal pathogen.

scholarly journals Sources of Potential Errors in the Application of Random Amplified Polymorphic DNAs in Cucumber

HortScience ◽  
1996 ◽  
Vol 31 (2) ◽  
pp. 262-266 ◽  
Author(s):  
Jack Staub ◽  
Jeffery Bacher ◽  
Karl Poetter
Keyword(s):  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Inbred Lines ◽  
Sphaerotheca Fuliginea ◽  
Chain Reaction ◽  
Banding Patterns ◽  
Taq Dna Polymerase ◽  
Polymerase Chain ◽  
Stock Solutions ◽  
Pcr Conditions

The influence of tissue age, pathogen infestation, intrapopulation contamination, and polymerase chain reaction (PCR) conditions were assessed as sources of error in random amplified polymorphic DNA (RAPD) analysis. DNA from young, uninfected tissue provided the most consistent results. Plants infected with Sphaerotheca fuliginea Schl. (ex Fr.) Poll. showed variation in RAPD banding patterns compared to those of uninfected plants. Differences in banding patterns were detectable when DNA from two inbred lines were mixed at dilution ratios of ≤20:1 but not ≥50:1. Differing lots of commercially available 10× reaction buffer, MgCl2 stock solutions, and Taq DNA polymerase affected RAPD banding patterns and overall yield. For reproducibility of RAPD assays, it may be necessary to optimize reactions for specific lots of PCR reagents from either commercial or in-house sources.

scholarly journals A DNA-Based Procedure for In Planta Detection of Fusarium oxysporum f. sp. phaseoli

2002 ◽  
Vol 92 (3) ◽  
pp. 237-244 ◽  
Author(s):  
Fernando M. Alves-Santos ◽  
Brisa Ramos ◽  
M. Asunción García-Sánchez ◽  
Arturo P. Eslava ◽  
José María Díaz-Mínguez
Keyword(s):  
Common Bean ◽  
Fusarium Oxysporum ◽  
Scar Marker ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Rapd Marker ◽  
In Planta ◽  
Sequence Characterized Amplified Region ◽  
Polymerase Chain ◽  
Highly Virulent

We have characterized strains of Fusarium oxysporum from common bean fields in Spain that were nonpathogenic on common bean, as well as F. oxysporum strains (F. oxysporum f. sp. phaseoli) pathogenic to common bean by random amplified polymorphic DNA (RAPD) analysis. We identified a RAPD marker (RAPD 4.12) specific for the highly virulent pathogenic strains of the seven races of F. oxysporum f. sp. phaseoli. Sequence analysis of RAPD 4.12 allowed the design of oligonucleotides that amplify a 609-bp sequence characterized amplified region (SCAR) marker (SCAR-B310A280). Under controlled environmental and greenhouse conditions, detection of the pathogen by polymerase chain reaction was 100% successful in root samples of infected but still symptomless plants and in stem samples of plants with disease severity of ≥4 in the Centro Internacional de Agricultura Tropical (CIAT; Cali, Colombia) scale. The diagnostic procedure can be completed in 5 h and allows the detection of all known races of the pathogen in plant samples at early stages of the disease with no visible symptoms.

scholarly journals Characterization of the Hyphal Swelling Group of Pythium: DNA Polymorphisms and Cultural and Morphological Characteristics

Plant Disease ◽  
1998 ◽  
Vol 82 (2) ◽  
pp. 218-222 ◽  
Author(s):  
K. Kageyama ◽  
H. Uchino ◽  
M. Hyakumachi
Keyword(s):  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Morphological Characteristics ◽  
Its Region ◽  
Restriction Enzymes ◽  
Dna Polymorphisms ◽  
Banding Patterns ◽  
Length Polymorphisms ◽  
Polymerase Chain

The hyphal swelling (HS) group of Pythium species and P. ultimum were studied for cultural and morphological characteristics, restriction fragment length polymorphisms of the amplified internal transcribed spacer (ITS) region in nuclear rDNA, and random amplified polymorphic DNA (RAPD) analysis of genome DNA. The shape of sporangia was spherical to subspherical or lemoniform and averaged 18.1–23.0 μm. All isolates could grow at 5 to 35°C, and the rate at the optimal temperature, 30°C, was 29–34 mm/24 h. The size of the ITS region amplified by polymerase chain reaction and the banding patterns after digestion with the restriction enzymes showed no variation between the HS group and P. ultimum. No difference in banding patterns was shown between the HS group and P. ultimum by RAPD analysis with each of three primers. Isolates examined were from Japan, and results should be confirmed from other regions.

scholarly journals Identification of the Edible Fig `Bianco del Cilento' by Random Amplified Polymorphic DNA Analysis

HortScience ◽  
1999 ◽  
Vol 34 (7) ◽  
pp. 1263-1265 ◽  
Author(s):  
U. Galderisi ◽  
M. Cipollaro ◽  
G. Di Bernardo ◽  
L. De Masi ◽  
G. Galano ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Southern Italy ◽  
Rapd Analysis ◽  
Genetic Relationships ◽  
Random Amplified Polymorphic Dna ◽  
Accurate Identification ◽  
Banding Patterns ◽  
Campania Region ◽  
Quality Of Products

Random amplified polymorphic DNA (RAPD) analysis is currently used to estimate genetic relationships in plants. We have used RAPD analysis to distinguish six different cultivars of Ficus carica, and several of their clones, that are widespread in the Campania Region of Southern Italy. Among these cultivars, `Bianco del Cilento' has unique characteristics, and is particularly useful for drying and for the manufacture of syrups. The protection of this cultivar is important to the Campania Region. We have utilized molecular markers to allow accurate identification of this cultivar, making it possible to control the quality of products and prevent fraudulent commerce. DNA was extracted from leaves and amplified by PCR using random oligonucleotide primers. The amplification patterns obtained with five decamer primers were useful for distinguishing all six cultivars analyzed. `Bianco del Cilento' was identified by two primers. The banding patterns were scored and used in similarity value calculations to estimate genetic relationships.

scholarly journals Application of Partial Internal Transcribed Spacer Sequences for the Discrimination ofArtemisia capillarisfrom OtherArtemisiaSpecies

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Eui Jeong Doh ◽  
Seung-Ho Paek ◽  
Guemsan Lee ◽  
Mi-Young Lee ◽  
Seung-Eun Oh
Keyword(s):  
Internal Transcribed Spacer ◽  
Dna Analysis ◽  
Dna Marker ◽  
Random Amplified Polymorphic Dna ◽  
Herbal Medicines ◽  
Chain Reaction ◽  
Artemisia Capillaris ◽  
Aerial Parts ◽  
Internal Transcribed Spacer Sequences ◽  
Polymerase Chain

SeveralArtemisiaspecies are used as herbal medicines including the dried aerial parts ofArtemisia capillaris, which are used as Artemisiae Capillaris Herba (known as “Injinho” in Korean medicinal terminology and “Yin Chen Hao” in Chinese). In this study, we developed tools for distinguishing betweenA. capillarisand 11 otherArtemisiaspecies that grow and/or are cultured in China, Japan, and Korea. Based on partial nucleotide sequences in the internal transcribed spacer (ITS) that differ between the species, we designed primers to amplify a DNA marker forA. capillaris. In addition, to detect otherArtemisiaspecies that are contaminants ofA. capillaris, we designed primers to amplify DNA markers ofA. japonica,A. annua,A. apiacea, andA. anomala. Moreover, based on random amplified polymorphic DNA analysis, we confirmed that primers developed in a previous study could be used to identifyArtemisiaspecies that are sources of Artemisiae Argyi Folium and Artemisiae Iwayomogii Herba. By using these primers, we found that multiplex polymerase chain reaction (PCR) was a reliable tool to distinguish betweenA. capillarisand otherArtemisiaspecies and to identify otherArtemisiaspecies as contaminants ofA. capillarisin a single PCR.

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