Random Amplified Polymorphic DNA-polymerase Chain Reaction Technique Research Articles

Genetic Variability of the Populations of Phlebotomus papatasi, the Main Vector of Leishmania major, in Al-Madinah Al-Munawarah, Saudi Arabia

Sand flies are vectors of several human pathogens. Phlebotomus papatasi has been confirmed as the vector of Leishmania major, the causative agent of cutaneous leishmaniasis (CL) in Saudi Arabia. This species is the predominant in Al-Madinah which is in agreement with the expansion of CL. In the present study, the random amplified polymorphic DNA–polymerase chain reaction (RAPD-PCR) technique was applied with six primers to evaluate the relationships among the geographic populations of P. papatasi collected from four villages in Al-Madinah: Almondasa, Almaliliah, Abyar Al-Mashy and Agool. The results showed the mean proportion of the polymorphic loci within the populations was (84.89%), while between them it was (94.51%). The average total genetic diversity across the all studied populations was slightly higher (0.35) than the genetic diversity within the populations (0.31). The average of the gene flow between the four populations was rated to have a worth of 4.363. This higher level of gene flow corresponds to less differentiation between the populations, which could result from the limited distances between the sampling areas. The phylogenetic and genetic distance analyses generated from the RAPD-PCR profiles showed close relationships between Almondasa and Almaliliah populations, while the largest genetic distance was detected between the populations of Almondasa and Agool. In conclusion, the genetic variability among and within P. papatasi populations was identified based on the efficient RAPD-PCR technique. The identification of the vector of cutaneous leishmaniasis in Al-Madinah and the differentiation of its populations and individuals is vital to the field of medical entomology.

Identification of Lactobacillus Species Isolated From Traditional Dairy Products Using RAPD-PCR

Background: Probiotics are non-pathogenic useful microorganisms having positive effects on the host health. The aim of the present study was to discriminate Lactobacillus species extracted from traditional dairy products. Methods: This study was conducted on 26 specimens collected from traditional dairy products in Bukan. Lactobacillus species were separated and purified employing biochemical tests. Then, the intra/inter-species diversity was investigated using RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) technique. Results: Polymorphism information content (PIC) value varied between 15.9% and 34.4% with a maximum value of 34.4% associated with primer 1254. The mean of and Marker index (MI) for the 6 primers was 4.52, in which the maximum and minimum values belonged to the primers 1254 and OPA-02, respectively. The isolates were categorized into 4 main clusters according to Jaccard similarity coefficient using UPGMA (unweighted pair group method with arithmetic means) clustering method. Principle coordinates analysis (PCoA) demonstrated that the first and second components explained 30.59% and 22.48% of variances, i.e. 53.07% of variances in total. The results of RAPD marker indicated that the intra-species diversity was greater than inter-species diversity. The intra-group variance explained 94% of the all variance, while inter-group variance explained only 6% of the all variance. Moreover, the results of analysis of molecular variance (AMOVA) indicated that the highest level of discrimination occurred at the 16 groups cut-off point with a similar coefficient of 0.56. Conclusions: From the results of the present study, it can be concluded that traditional dairy products are enriched sources of probiotic bacteria which can ensure the health of general population and enhance their immunoe systems. Moreover, RAPD-PCR is an appropriate method for detection and calssification of lactobassiluses.

English

The aim of the present study was to determine the unique molecular markers among, three species of the genus Anthemis and the construction of phylogenetic tree using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique. Genetic diversity was analyzed among 15 populations of three species of Anthemis (Anthemis melampodina, Anthemis, pseudocotula and Anthemis, bornmuelleri), collected from different locations at Saudi Arabia by using RAPD primers. Pairwise genetic distance was calculated based on Nei and Li coefficient. Unweighted pair group method with an average (UPGMA) was used for construction of dendrogram, based on the similarity matrix data. Results showed wide variations among A. bornmuelleri and other two species. A wide close genetic relation was observed between A. melampodina and A. pseudocotula. RAPD-PCR technique was shown to be an accurate tool, in other to ascertain plant relationships among species of genus Anthemis. Key words: Genetic diversity, random amplified polymorphic DNA-polymerase chain reaction (RAPD- PCR), A. melampodina, A. pseudocotula, A. bornmuelleri.

RAPD-PCR as a Molecular Discriminative Technique for Human Pathogenic Bacteria - A Review

The application of random amplified polymorphic DNA- polymerase chain reaction (RAPD-PCR) was found to be a simple, cheap and rapid tool to discriminate human pathogenic bacterial isolates especially at intraspecific level. This molecular biological technique relies on the use of random oligonucleotide primers that arbitrarily amplifies specific regions of the genome which gives rise to a unique genomic fingerprint of the strains under investigations. With continued development of novel molecular-based technologies for rapid, high-throughput detection of food borne pathogenic bacteria, the future of conventional microbiological methods such as viable cell enumeration, selective isolation of bacteria on commercial media, and immunoassays seems tenuous. Approaches that enhance recovery of sub lethally injured bacteria, differentiation among species, differentiation among bacteria of interest using biochemical profiling, enumeration using impedance technology, techniques to confirm the presence of target pathogens using immunological methods, and bioluminescence applications for hygiene monitoring are of utmost need in identifying and combating the human pathogenic isolates. The aim of this study is to esti­mate the efficiency of RAPD-PCR technique in assessing the genetic diversity of diseases causing bacterial isolates. The use of RAPD-PCR in evaluating the genomic variability among the pathogenic strains belonging to different genus are also been discussed in the present report.

Differentiation by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) of Candida albicans isolated from upper respiratory tract in patients with non-small cell lung cancer.

Cancer patients are predisposed to fungal infections caused by Candida albicans, especially to oral or respiratory tract candidiasis. The aim of this study was to estimate genetic diversity by RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) of C. albicans isolated from upper respiratory tract of 100 patients with non-small cell lung cancer. Among 52 strains, 34 genotypes were defined. 10 clusters comprising 28 (53.85%) isolates with similarity coefficient ≥ 80% were formed. The remaining 24 (46.15%) isolates represented individual genotypes. The RAPD-PCR technique revealed genomic variability within C. albicans isolated from upper respiratory tract of the cancer patients.

Detection of the patulin-producing potential of some Paecilomyces variotii strains isolated from the air samples of Jeddah City, Saudi Arabia, using the RAPD-PCR technique

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis was used to detect the patulin-producing potential among seven different strains of Paecilomyces variotii isolated from air samples collected in Jeddah City, Saudi Arabia. Using five different primers, the strains showed some similarities and distinct RAPD patterns. A correlation between isolation source and clustering was noted in the constructed dendrogram. Patulin-producing strains showed identical RAPD patterns. Primer M13 produced a distinct fragment with toxin-producing strains.

RAPD-PCR in two species of Catopsilia (Pieridae, Lepidoptera)

Abstract RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) technique was used to analyze the polymorphism among the individuals of the two species of butterflies, namely Catopsilia pyranthe and Catopsilia crocale, which were captured from their natural habitat. Three decanucleotide primers viz., primer A(AAGAGCCCGT), primer B(AACGCGCAAC) and primer C(CCCGTCAGCA) were used to amplify the gDNA from the adult individuals of both the species. A series of bands ranging from below 125 bp to 1500 bp were produced by these primers. Standard curves were prepared and used to calculate the number of base pairs present in all the amplified fragments. The results obtained with these primers revealed sufficient amount of relatedness and variations in these two species. Primer A amplified some common DNA fragments of the same size in both the species, revealing the presence of conserved regions and thereby indicating their ancestral relationship. Some of the fragments were unique to the individuals of both the species which could be used to generate their diagnostic profiles. Primer B produced single bands but of different sizes in the invididuals of both the species revealing the presence of intraspecific genetic variation. Primer C did not amplify the gDNA of the individuals of C.pyranthe but produced 4 bands ranging from 480 to 1160 bp length in C.crocale. These results indicate that the RAPD-PCR technique is useful in the studies on molecular taxonomy of Lepidoptera.

Application of random amplified polymorphic DNA PCR for genomic analysis of HIV-1-infected individuals

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) is a DNA fingerprinting technique used to detect genomic polymorphisms. We employed sixteen different RAPD-PCR 10-mer primers to amplify DNA from the peripheral blood mononuclear cells (PBMC) of 80 HIV-1-infected individuals. These individuals were previously identified as either heterozygotes (+ /delta32) and homozygotes (+/+) for the CCR5 locus by PCR with gene specific primers. Four of the sixteen randomly selected RAPD primers produced distinguishable banding profiles between CCR5 (+/delta32) heterozygotes and CCR5 (+/+ ) homozygotes. Direct sequencing of some RAPD-PCR products obtained with one of the four RAPD primers that were tested yielded clearly readable, but limited sequences, which were similar to portions of the previously published sequences for (+/+ ) homozygotes (98% similarity) and (+/delta32) heterozygotes (87% similarity) of the CCR5 alleles. Thus, the RAPD-PCR technique may be useful for the identification of human molecular markers that may correlate with susceptibility to HIV-1-infection, or differences in disease progression among HIV-l-infected individuals.

Random Amplified Polymorphic Dna Used to Identify Genetic Variation in Ecotypes of the European Corn Borer (Lepidoptera: Pyralidae)

The European corn borer, Ostrinia nubilalis (Hubner), has 3 morphologically indistinguishable voltinism ecotypes. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was used to discriminate ecotypes of the European corn borer. Genomic DNA samples from the European corn borer were screened with a total of 120 random primers. Ten of these primers yielded 21 clear and reproducible RAPD markers after agarose gel electrophoresis. Dendrograms constructed using the Nei and Li distance matrix of the phylogenetic relationships, among and within ecotypes, of the European corn borer showed the multivoltine ecotype to be genetically separated from univoltine and bivoltine ecotypes. The dendrogram of individual European corn borers correctly classified each insect. These results provide evidence of genetic variation at the molecular level among European corn borer ecotypes. The RAPD-PCR technique proved to be a powerful tool for identifying intraspecific variation in the European corn borer.

The use of fecundity, lobe biometry and the RAPD-PCR technique in order to compare strains of Tetranychus sp.

In Belgium, recently two new red carmine forms of Tetranychus sp. have appeared on tomato crops in glasshouses and in maize crops. These forms maintain their red colour even during summer and on other host plants such as French bean. Our aim was to establish their status and to compare them with strains of Tetranychus urticae and Tetranychus cinnabarinus. A strain of Tetranychus kanzawai, a distant relative of the same genus, was used as a control. The five strains tested were characterized by their own demographic parameters, probably because of a long separated evolution on different host plants. The white eye strain of ir: urticae was differentiated from the four other strains by scanning electron microscopy studies of the dorsal integument folds. An investigation using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) showed T. cinnabarinus as a distinct group. However, the mixing of the individuals of the four other strains raises the question of the role of hybridization and host plant selection in the evolution of the Tetranychus complex. Exp Appl Acarol 22: 649-666 (C) 1998 Kluwer Academic Publishers.

Use of genetic polymorphisms detected by the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) for differentiation and identification of Aedes aegypti subspecies and populations.

Amplification of random regions of genomic DNA using 10-base primers in the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to differentiate and identify mosquito populations based on genetic variation. Genomic DNA was extracted from individual mosquitoes from 11 geographic populations of Aedes aegypti and amplified in PCR reactions using single primers of arbitrary nucleotide sequence. Discriminant analysis of the population frequencies of RAPD fragments produced using three different primers allowed accurate discrimination between the geographic populations in 89% of individuals and between subspecies (Ae. aegypti aegypti versus Ae. aegypti formosus) in 100% of mosquitoes tested. The genetic relatedness of the populations was estimated using three different statistical methods, and unknown populations were correctly classified in a blind test. These results indicate that the RAPD-PCR technique will be useful in studies of arthropod molecular taxonomy and in epidemiologic studies of the relatedness of geographic populations and vector movement.