Standardization and validation of simple PCR, duplex PCR and RAPD in comparison to blood smear examination for diagnosing bovine tropical theileriosis.

Bovine Tropical Theileriosis (BTT) is an important vector-borne protozoan disease that imposing serious constraints on the health and productivity of domestic cattle. It is matter of common fact that following recovery from primary infection, cattle become persistent carriers and act as reservoirs of infection thereby, playing a critical role in disease epidemiology. The present study describes the comparative diagnostic efficiency of simplex PCR, duplex PCR and RAPD assays for detection of Theileria annulata in cattle. An optimized simple PCR and duplex PCR assay were established using TAMS F/R as primer sets encoding for 721 bp amplicon alongside a RAPD with arbitrary primer coding for 963 bp product of T. annulata. The simple PCR and duplex PCR detected pathogen with almost same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA of another pathogen without nonspecific amplifications. RAPD failed to give comparable results and suffered from limitations of sensitivity as well as specificity. The developed assays may be seen as a good tool for epidemiological studies aiming at assessing the burden of chronic infections and improving control of the associated diseases in endemic regions.

Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA.

Lactobacillus casei strain Shirota (LcS) has been used in the production of fermented milk products for many years and is one of the most intensively studied probiotics. To evaluate the ability of LcS to proliferate in human intestines after it has been ingested, we developed a PCR-based method to identify and quantify LcS using an LcS-specific primer set (pLcS) derived from a randomly amplified polymorphic DNA (RAPD) analysis. We confirmed the high specificity of the pLcS primer set in 167 bacterial strains (57 strains of L. casei and 110 other strains of bacteria commonly isolated from human feces). The method's ability to identify LcS matched that of an ELISA using a monoclonal antibody and a RAPD analysis in a representative sample of colonies cultured from human feces. The detection limit of quantitative PCR (qPCR) using pLcS was 10(4.6) per gram of feces. The number of LcS in feces detected with qPCR was highly and significantly correlated with the number of LcS added to fecal samples within the range of 10(4.6) to 10(9.6) per gram feces (r(2)=0.999, P<0.001). After 14 healthy subjects ingested 10(11.0) CFU of LcS daily for 7 days, 10(9.1+/-0.5) LcS g(-1) (mean+/-S.D.) was detected in the fecal samples of all subjects by qPCR, and 10(8.0+/-0.9) CFU g(-1) was detected by culture; these values were significantly different (P<0.001, paired t-test). After the subjects stopped ingesting LcS, fecal LcS counts obtained with both methods decreased daily. The values produced by the 2 methods might have differed because of an overestimation in the PCR analysis due to the presence of dead LcS cells or an underestimation in the culture system due to the use of selective culture media; however, dead LcS cells can also be beneficial as immunomodulators. We confirmed that qPCR with an LcS-specific primer set was a rapid and accurate method for determining the total amount of LcS in feces including dead or less active cells which could not be detected by culture method.

Molecular epidemiology of Pseudomonas aeruginosa in adult patients with cystic fibrosis in Northern Ireland.

Isolates (n = 51) of Pseudomonas aeruginosa obtained from the sputa of 29 adult patients attending the Regional Cystic Fibrosis Centre in Northern Ireland were compared using an enterobacterial repetitive intergenic consensus sequence (ERIC2) primer in a random amplification of polymorphic DNA (RAPD) polymerase chain reaction (PCR) method. Resulting banding patterns showed a high degree of genetic heterogeneity among all isolates from the patients examined, suggesting a non-clonal relationship between isolates from these patients, when employing this genotyping technique.

[Comparison of different primers used for the genotyping of Candida albicans clinical isolates by randomly amplified polymorphic DNA method]. / Candida albicans klinik izolatlarinin "randomly amplified polymorphic DNA" yöntemiyle genotiplendirmesinde kullanilan farkli primerlerin karsilastirilmasi.

Candida albicans causes severe infections with high mortality rates especially in immunocompromised patients. The aim of this study was to evaluate the efficiency of randomly amplified polymorphic DNA (RAPD) method and compare the discriminatory powers (DP) of different primers used for genotyping Candida albicans isolates. A total of 109 C. albicans strains recovered from throat, sputum, blood, feces, urine, vagina and wound cultures of 65 hospitalized paediatric patients with haematologic malignancy were evaluated by RAPD method using 10 different primers (OPE-03, OPE-04, OPE-12, OPE-18, OPE-19, OPE-20, OPF-10, OPF-12, P1 and P2) between June 1999-April 2003. Strains were separated into groups by analyzing band patterns derived from each primer and the DP was calculated. Reproducibility of the method was determined by evaluating randomly chosen 20 isolates with the same and different PCR devices under the same PCR conditions. C. albicans isolates generated 1-16 bands and were grouped in 41-80 genotypes depending on the primers used. DP of the RAPD method was calculated as > or = 0.90 for each primer (range between 0.90-0.99), which were accepted as reliable values. However, the strains clustered in the same group when studied with a primer could be dispersed into different groups by another primer. The reproducibility of the method was poor and the comparison of band patterns was difficult especially in isolates which generated many bands. In conclusion, for obtaining reliable results by RAPD method, using more than one primer and comparative analysis of these primers are appropriate. RAPD is an adequate method for studying small outbreaks in which a few number of isolates are evaluated, but it is laborious and unreliable for many number of isolates recovered in a long time period because of its poor reproducibility and difficulties in evaluating the strains generating many bands.

Non-specificity of sequence characterised amplified region as an alternative molecular epidemiology marker for the identification of Salmonella enterica subspecies enterica serovar Typhi.

OBJECTIVE: Identification of Salmonella Typhi by conventional culture techniques is labour-intensive, time consuming, and lack sensitivity and specificity unlike high-throughput epidemiological markers that are highly specific but are not affordable for low-resource settings. SCAR, obtained from RAPD technique, is an affordable, reliable and reproducible method for developing genetic markers. Hence, this study investigated the use of SCAR as an alternative molecular epidemiological marker for easy identification of S. Typhi in low-resource settings. RESULTS: One hundred and twenty RAPD primers were screened through RAPD-PCR against a panel of common enterobacteriaceae for the best RAPD band pattern discrimination to develop SCAR primers that were used to develop a RAPD-SCAR PCR. Of this number, 10 were selected based on their calculated indices of discrimination. Four RAPD primers, SBSA02, SBSA03, SBSD08 and SBSD11 produced suitable bands ranging from 900 to 2500 bp. However, only SBSD11 was found to be specific for S. Typhi, and was cloned, sequenced and used to design new SCAR primers. The primers were used to amplify a panel of organisms to evaluate its specificity. However, the amplified regions were similar to other non-Typhi genomes denoting a lack of specificity of the primers as a marker for S. Typhi.

Genetic diversity in populations of Dalbulus maidis (DeLong and Wolcott) (Hemiptera: Cicadellidae) from distant localities in Brazil assessed by RAPD-PCR markers.

Populations of Dalbulus maidis (DeLong and Wolcott) from the northeastern and central-southern regions of Brazil differ morphologically, suggesting that they could be genetically isolated. Here we used the random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) technique to estimate genetic structuring of this leafhopper species among five geographically distant localities across those regions and to estimate gene flow between populations. Ten specimens were sampled per population and genotyped with RAPD markers generated from amplification with nine oligonucleotides. The percentage of polymorphic loci was 78% in relation to the total number of amplified loci, and genetic similarity either between or within populations was higher than 0.72. Cluster analysis grouped specimens from the northeastern population (Mossoró/RN) into a single group, whereas central-southern specimens were not grouped in relation to their places of origin. Overall, the genetic subdivision index (Fst) was low (or= 0.192 and Nm

Improvement and validation of RAPD in combination with PFGE analysis of Salmonella enterica ssp. enterica serovar Senftenberg strains isolated from feed mills.

In 1995 and 1996 a Swedish feed mill had problems due to a persistent contamination of Salmonella enterica spp. enterica serovar Senftenberg that was difficult to eliminate. Forty-eight strains isolated from the feed mill, together with unrelated strains included to evaluate the discriminatory power and reproducibility, were analysed by pulsed-field gel electrophoresis (PFGE). The source of contamination in the feed mill was identified and preventative measures were taken, that led to a resolution of the problem. A previously developed randomly amplified polymorphic DNA (RAPD) protocol was used, to evaluate a rapid and low-cost alternative to PFGE typing. The use of the alternative thermostable DNA polymerase Tth was shown to increase the reproducibility of the RAPD analysis. The reproducibility, in terms of Pearson's and Dice's similarity coefficients for duplicate runs, increased from 72.0 +/- 16.9% and 72.3 +/- 12.9% for Taq to 91.6 +/- 7.5% and 90.9 +/- 5.3% for the fingerprints obtained for the RAPD method employing Tth DNA polymerase. Simpson's index of diversity was calculated and found to be 0.580 for RAPD and 0.896 for PFGE. All of the seven RAPD types could be subdivided into one or more PFGE types, whereas none of the 22 PFGE types was divided into more than one RAPD type. RAPD provides a simple, rapid and powerful screening method that can be used to initially select isolates for further analysis by PFGE.

Identification of a rice gene (Bph 1) conferring resistance to brown planthopper (Nilaparvata lugens Stal) using STS markers.

This study was carried out to identify a high-resolution marker for a gene conferring resistance to brown planthopper (BPH) biotype 1, using japonica type resistant lines. Bulked segregant analyses were conducted using 520 RAPD primers to identify RAPD fragments linked to the BPH resistance gene. Eleven RAPDs were shown to be polymorphic amplicons between resistant and susceptible progeny. One of these primers, OPE 18, which amplified a 923 bp band tightly linked to resistance, was converted into a sequence-tagged-site (STS) marker. The STS marker, BpE18-3, was easily detectable as a dominant band with tight linkage (3.9cM) to Bph1. It promises to be useful as a marker for assisted selection of resistant progeny in backcross breeding programs to introgress the resistance gene into elite japonica cultivars.

Epidemiological study of invasive pulmonary aspergillosis in a haematology unit by molecular typing of environmental and patient isolates of Aspergillus fumigatus.

In order to determine the possible relationship between environmental contamination by Aspergillus fumigatus and occurrence of invasive aspergillosis, a one-year prospective study was carried out in the haematology ward of Hautepierre Hospital, Strasbourg, France. During the study period, 21 environmental isolates and 26 clinical isolates of A. fumigatus were collected. Each was genotyped using a random amplification of polymorphic DNA (RAPD) technique. Thirty-four distinct profiles were identified by RAPD analysis, indicating the great genetic diversity of A. fumigatus isolated from infected patients and from the environment. For two patients, RAPD analysis demonstrated concurrent infection by at least two different strains. In two cases, a genetic similarity was noted between isolates obtained from a patient and from the environment.

Optimization of random amplified polymorphic DNA techniques for use in genetic studies of Cuban Triatominae.

Random amplified polymorphic DNA (RAPD) technique is a simple and reliable method to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of assay. In this study, we analyzed the effect of changing the concentration of primer, magnesium chloride, template DNA and Taq DNA polymerase with the objective of determining their optimum concentration for the standardization of RAPD technique for genetic studies of Cuban Triatominae. Reproducible amplification patterns were obtained using 5 pmoL of primer, 2.5 mM of MgCl2, 25 ng of template DNA and 2 U of Taq DNA polymerase in 25 microL of the reaction. A panel of five random primers was used to evaluate the genetic variability of T. flavida. Three of these (OPA-1, OPA-2 and OPA-4) generated reproducible and distinguishable fingerprinting patterns of Triatominae. Numerical analysis of 52 RAPD amplified bands generated for all five primers was carried out with unweighted pair group method analysis (UPGMA). Jaccard's Similarity Coefficient data were used to construct a dendrogram. Two groups could be distinguished by RAPD data and these groups coincided with geographic origin, i.e. the populations captured in areas from east and west of Guanahacabibes, Pinar del Río. T. flavida present low interpopulation variability that could result in greater susceptibility to pesticides in control programs. The RAPD protocol and the selected primers are useful for molecular characterization of Cuban Triatominae.

Increased informativeness of RAPD analysis by detection of microsatellite motifs.

The recently developed random-amplified microsatellite polymorphism (RAMPO) technique detects second-level amplification products that are useful as molecular markers. In the first step of the procedure, genomic DNA is amplified with a single arbitrary or microsatellite-complementary primer. PCR products are then electrophoretically separated, photographed, blotted and hybridized to a 32P-labeled microsatellite probe. Autoradiography reveals highly reproducible, polymorphic, probe-dependent fingerprints, which are different from the ethidium bromide staining patterns. In this paper, we report the successful application of various mono-, tri- and tetranucleotide repeat motifs as RAMPO probes. We also compare the efficiency of arbitrary vs. microsatellite primers for the generation of RAMPO patterns. Repeated rehybridization to different probes has expanded the information contained in a single random-amplified polymorphic DNA (RAPD) gel at least fivefold. Pattern complexity varies with the length and sequence of the probe. Application of the technique to a genetic relatedness study in the genus Dioscorea (yam) yielded highly informative markers, mainly at an interspecific level.

Random amplified polymorphic DNA technique for identification and differentiation of old world Leishmania species.

The random amplified polymorphic DNA technique may be used to explore parasite DNA polymorphisms. We assessed its applicability to identification of Old World Leishmania species. A set of 6 random decamer primers (Al, A4, A5, A7, A10, and A15) was applied to a panel of DNA from 57 representatives of different Old World Leishmania species. The amplification profiles allowed discrimination among species belonging to different taxonomic complexes. Two criteria were used to analyze the profiles: the presence of consistent amplicons at the same electrophoretic position for isolates of the same species, and the presence of distinct amplicons for isolates of different species. Three primers--Al, A7 and A10--rendered such products.

Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis.

This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.

PCR typing of Corynebacterium diphtheriae by random amplification of polymorphic DNA.

The usefulness of random amplification of polymorphic DNA (RAPD) analysis with Ready-To-Go RAPD beads was investigated for the rapid differentiation of Corynebacterium diphtheriae isolates from Eastern Europe and neighbouring countries. A selection of 45 C. diphtheriae isolates of known origin, biotype, toxigenicity status and ribotype were examined by RAPD. Twenty RAPD profiles (designated Rp1-Rp20) were revealed among the 45 isolates. There was 100% correlation between RAPD profiles and ribotypes. Preliminary studies showed that the use of crude DNA preparations resulted in poor amplification and the patterns were not reproducible. Different thermal cycler models produced different RAPD profiles from the same DNA sample. Reproducibility of the technique was good when the same thermal cycler was used throughout. RAPD proved to be a simple and a rapid method for analysing C. diphtheriae and it is a method which can be used as a potential alternative to ribotyping or as a screening technique during outbreak investigations.

The possibilities and limitations of nucleic acid amplification technology in diagnostic microbiology.

Nucleic acid amplification technology is examined from the critical viewpoint of a clinical microbiologist working in a routine diagnostic bacteriology laboratory. Widely recognised limitations of amplification technology include those of false-positive and false-negative results, the difficulty of obtaining quantitative results, the problem of using this technology for susceptibility testing, and the difficulty of detecting routinely the wide range of possible pathogens contained in a clinical sample. On the positive side, amplification technology brings welcome new possibilities for rapid detection of specific pathogens in a sample, including viruses, slowly growing bacteria, fastidious or uncultivable bacteria, fungi and protozoa. Other possible applications include screening normally sterile clinical samples for non-specific bacterial contamination and the use of amplification-based DNA fingerprinting methods for identification and typing of microorganisms. Nevertheless, it is predicted that-in contrast to research and reference facilities-routine bacteriology laboratories will continue to rely on culture as the preferred 'amplification method' for most diagnostic applications.

RAPD typing in microbiology--a technical review.

Many biochemical and molecular techniques can be used for distinguishing isolates of a given bacterial species. Traditional typing techniques based on phenotypic characteristics such as serotyping are being increasingly challenged by the use of DNA-based methods. The introduction of the polymerase chain reaction (PCR) has led to typing techniques based on DNA amplification. Randomly amplified polymorphic DNA (RAPD) typing (also known as arbitrarily primed-polymerase chain reaction, APPCR) is one such technique which is being used increasingly to type micro-organisms, especially during clinical outbreaks. The application and potential problems and solutions of RAPD typing are discussed and the role of such techniques among established typing methods is addressed.

Detection of respiratory syncytial virus from clinical specimens: comparison between reverse transcription polymerase chain reaction and tissue culture.

RT-PCR was compared with tissue culture to detect RSV from nasopharyngeal aspirates. RT-PCR was more sensitive and specific than tissue culture method. RT-PCR has sensitivity and specificity of 100% and 97%, respectively. The results indicate that RT-PCR can be used for detection of RSV in clinical specimens.

Schistosoma japonicum strains: differentiation by RAPD and SSR-PCR.

Eight geographical isolates of Schistosoma japonicum from Taiwan and mainland China and one isolate of Schistosoma mansoni were studied by RAPD analysis using six arbitrary primers and SSR-PCR analysis using a (CA)8RY primer. The genetic distance was determined by the percentage of unshared bands. The RAPD and SSR-PCR results showed that the genetic distance between S. mansoni and S. japonicum was more than 0.900 and 0.850 respectively; the genetic distance between the eight geographical isolates of S. japonicum was 0.000 to 0.232 and 0.066 to 0.368 respectively. These results demonstrated the usefulness of RAPD and SSR-PCR for showing the differences of inter- and intra-species of Schistosoma. The results also suggest that there is genetic diversity among the different geographical strains of S. japonicum in China.

Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi.

Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.

[Evaluation of three methods for genotyping of nosocomial methicillin resistant Staphylococcus aureus isolates]. / Nozokomiyal metisiline dirençli Staphylococcus aureus izolatlarinin genotiplendirilmesinde üç ayri yöntemin incelenmesi.

Plasmid profile analysis (PPA), random amplification of polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE) are the three most valuable epidemiological tools for genotyping of methicillin resistant Staphylococcus aureus (MRSA) strains. Aim of this study was to evaluate these three methods in respect to their cost, reproducibility, and discriminatory power. Eighty one nosocomial MRSA isolates with unknown genetic and epidemiological relatedness from Training Hospital of Gulhane Military Medical School, were genotyped by PPA, RAPD, and PFGE methods. All isolates (100%) were typed by RAPD and PFGE, however, eight (9.9%) isolates could not be typed by PPA since they lacked plasmid DNA. Reproducibilities of all the three methods were found to be 100 percent. Discriminatory powers of PPA, RAPD and PFGE methods were calculated as 48.6%, 61.1% and 80.1%, respectively. In conclusion, out of the three methods tested, PFGE allowed the most effective discrimination of MRSA strains. However, PFGE was more time consuming and technically demanding, and required use of specialized and expensive equipment. Although PPA and RAPD were less discriminatory than PFGE, these methods were technically simple, rapid and cheaper. When PPA and RAPD were used in combination, they had equal discriminatory power to PFGE. Thus, it should be emphasized that PPA and RAPD methods could be preferred for initial screening purposes while PFGE should be used as a confirmatory test in genotyping of MRSA isolates.