Group-specific differentiation of Rhizobium from clover species by PCR amplification of 23S rDNA sequences

1998 ◽  
Vol 44 (11) ◽  
pp. 1102-1105
Author(s):  
Mesfin Tesfaye ◽  
F Brian Holl
Keyword(s):  
Root Nodule ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Specific Detection ◽  
Rdna Sequences ◽  
Rhizobium Spp ◽  
Polymerase Chain ◽  
Inoculation Group ◽  
Identification Tool

Two 20-bp primers that provide group-specific detection of Rhizobium spp. by polymerase chain reaction (PCR) have been used to differentiate strains that belong to different effectiveness groups within the Rhizobium-Trifolium cross-inoculation group. The target for DNA amplification was a 370-bp fragment of the 23S rDNA region. Analysis of additional root-nodule forming, as well as root-associated bacterial species by PCR-primer assay revealed that variability within this 20-bp segment of the 23S rDNA region may be widespread and provide an effective identification tool. Our data suggest that strains of Rhizobium isolated from the perennial clover Trifolium semipilosum may be phylogenetically more closely related to Rhizobium etli.Key words: Rhizobium, Trifolium, detection, PCR, rDNA.

Detection ofTrypanosoma congolenseandTrypanosoma bruceisubspecies by DNA amplification using the polymerase chain reaction

Parasitology ◽  
1989 ◽  
Vol 99 (1) ◽  
pp. 57-66 ◽  
Author(s):  
D. R. Moser ◽  
G. A. Cook ◽  
Diane E. Ochs ◽  
Cheryl P. Bailey ◽  
Melissa R. McKane ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Large Scale ◽  
Nuclear Dna ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Detection Methods ◽  
Chain Reaction ◽  
African Trypanosomes ◽  
Polymerase Chain

SUMMARYThe nuclear DNA ofTrypanosoma congolensecontains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA ofTrypanosoma brucei brucei(Sloofet al.1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite ofT. congolenseorT. bruceispp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor inLeishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected withT. congolenseand/orT. bruceispp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

scholarly journals Molecular Bird Sexing on Fischeri Lovebird (Agapornis fischeri) by Using Polymerase Chain Reaction

2020 ◽  
Vol 20 ◽  
pp. 04003
Author(s):  
Azalea Dyah Argarini ◽  
Herjuno Ari Nugroho ◽  
Medania Purwaningrum ◽  
Aris Haryanto
Keyword(s):  
Sex Chromosome ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Total Sample ◽  
Z Chromosome ◽  
Agarose Gel Electrophoresis ◽  
Chain Reaction ◽  
Male And Female ◽  
Pcr Products ◽  
Polymerase Chain

Fischeri Lovebird (Agapornis fischeri) found originally in Africa which has spread to many countries. In Indonesia, Fischeri Lovebird is popular as a pet animal. This lovebird is a monomorphic bird, so it is difficult to differentiate morphologically between male and female birds. In general, a male lovebird has ZZ homozygotes, whereas females' lovebird has ZW heterozygous of their sex chromosome. These sex chromosomes set used as study targets for molecular bird sexing of many species of birds because this method is effective and simple to perform. This method targeted to amplify the Chromodomain Helicase DNA-binding (CHD) gene, which found into the sex chromosome of male and female birds. The objective of this study was to rapid molecular bird sexing of Fischeri Lovebird by using PCR methods. Research samples were collected from feather calamus of A. fischeri. The total sample was 11 feathers from A. fischeri. which were collected three to six feathers for each lovebird. Then the research was followed by DNA extraction from calamus feathers, DNA amplification by PCR and agarose gel electrophoresis of PCR products and visualization of PCR predicts by UV-Transilluminator in darkroom. It concluded that PCR amplification using NP, MP and P2 primers produced double DNA bands in size of 400 bp on Z chromosome and bp on W chromosome for female Fischeri Lovebird, whereas for male Fischeri Lovebird only produced a single DNA band in size of 400 bp on Z chromosome. From eleven samples of Fischeri Lovebird showed a total of five females and six male Fischeri Lovebirds.

scholarly journals Development of a Rapid PCR Assay Specific forStaphylococcus saprophyticus and Application to Direct Detection from Urine Samples

2000 ◽  
Vol 38 (9) ◽  
pp. 3280-3284 ◽  
Author(s):  
Francis Martineau ◽  
François J. Picard ◽  
Christian Ménard ◽  
Paul H. Roy ◽  
Marc Ouellette ◽  
...  
Keyword(s):  
Direct Detection ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Urine Samples ◽  
Urine Specimen ◽  
Clinical Microbiology Laboratory ◽  
Specimen Preparation ◽  
Specific Detection ◽  
Pcr Assay ◽  
Tract Infections

Staphylococcus saprophyticus is one of the most frequently encountered microorganisms associated with acute urinary tract infections (UTIs) in young, sexually active female outpatients. Conventional identification methods based on biochemical characteristics can efficiently identify S. saprophyticus, but the rapidities of these methods need to be improved. Rapid and direct identification of this bacterium from urine samples would be useful to improve time required for the diagnosis of S. saprophyticus infections in the clinical microbiology laboratory. We have developed a PCR-based assay for the specific detection ofS. saprophyticus. An arbitrarily primed PCR amplification product of 380 bp specific for S. saprophyticus was sequenced and used to design a set of S. saprophyticus-specific PCR amplification primers. The PCR assay was specific for S. saprophyticus when tested with DNA from 49 gram-positive and 31 gram-negative bacterial species. This assay was also able to amplify efficiently DNA from all 60 strains of S. saprophyticus from various origins tested. This assay was adapted for direct detection from urine samples. The sensitivity levels achieved with urine samples was 19 CFU with 30 cycles of amplification and 0.5 CFU with 40 cycles of amplification. This PCR assay for the specific detection of S. saprophyticus is simple and rapid (approximately 90 min, including the time for urine specimen preparation).

scholarly journals Deteksi Vibrio harveyi dengan Metode Amplifikasi DNA pada Gen toxR

2020 ◽  
Vol 1 (1) ◽  
pp. 29-37
Author(s):  
Calvin Wijaya Johan ◽  
Sulistyo Emantoko Dwi Putra ◽  
Ernest Suryadjaja
Keyword(s):  
False Positive ◽  
Vibrio Harveyi ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Isothermal Amplification ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Polymerase Chain ◽  
Toxr Gene ◽  
Positive Results

Abstrak -Infeksi hewan akuakultur oleh Vibrio harveyi dapat menyebabkan kematian serta kerugian ekonomi. Kemampuan untuk mendeteksi bakteri tersebut secara dini dapat mencegah penyebarannya dalam akuakultur. Penelitian ini bertujuan untuk mengembangkan metode untuk mendeteksi Vibrio harveyi melalui amplifikasi gen toxR. Amplifikasi DNA dilakukan dengan dua metode, yakni amplifikasi isotermal termediasi loop (loop-mediated isothermal amplification, LAMP) dan reaksi berantai polimerase (PCR). Amplifikasi menggunakan metode LAMP menunjukan perlu dilakukan optimasi protokol maupun desain primer untuk mencegah perolehan hasil false positive. Amplifikasi menggunakan metode PCR menghasilkan produk berukuran 229 pasang basa yang spesifik pada Vibrio harveyi dengan batas deteksi hingga 0,526 ng.µL-1 (setara 2,09 × 106 CFU.mL-1). Kata kunci: Akuakultur, LAMP, PCR, toxR, Vibrio harveyi Abstract -Vibrio harveyi infection in aquacultures may cause death and economical loss. Rapid detection of this bacteria may prevent its dispersal in aquacultures. The goal of this research was to develop method in detection of Vibrio harveyi via amplification of toxR gene. DNA amplification was carried out with two methods, loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Amplification with LAMP suggest optimization of either protocol or primer design was needed to prevents false positive results. Amplification with PCR yields 229 bp-length product specific to Vibrio harveyi with detection limit up to 0.526 ng.µL-1 (equals to 2.09 × 106 CFU.mL-1). Keywords: Aquaculture, LAMP, PCR, toxR, Vibrio harveyi

scholarly journals Using Polymerase Chain Reaction-based DNA Amplification to Fingerprint North American Potato Cultivars

HortScience ◽  
1996 ◽  
Vol 31 (1) ◽  
pp. 130-133 ◽  
Author(s):  
B. Sosinski ◽  
D.S. Douches
Keyword(s):  
Polymerase Chain Reaction ◽  
North American ◽  
Genetic Relationships ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Chain Reaction ◽  
Arbitrary Primers ◽  
Polymerase Chain ◽  
Tuber Type

DNA from 46 North American potato (Solanum tuberosum L.) cultivars was examined using the polymerase chain reaction (PCR) with 16 arbitrary primers of 10 nucleotide length (10 mers) to determine the efficiency of randomly amplified polymorphic DNA (RAPD) in delineating cultivars, both sexually derived and clonal variants. The 16 primers yielded 43 useful polymorphisms that were evaluated according to the presence or absence of fragments of equal size. All cultivars were discriminated with as few as 10 primers. The russet sport of Burbank was distinguished from a white-skinned clone by one band. More primers (29) were examined to identify a band polymorphism among six Russet Burbank clonal variants. When the cultivars were grouped by tuber type (excluding the russet clonal variants), three to four primers discriminated these commonly grown cultivars. Determination of cultivar integrity was accomplished with PCR amplification, regardless of tissue source (leaf vs. tuber) for DNA extraction. Cluster analysis based on RAPD markers was performed to examine pedigree relationships of the cultivars. Genetic relationships correlated with some pedigrees; however, many exceptions were noted.

Use of P-17 and NOX specific primer sets for assimilable organic carbon (AOC) measurements

2007 ◽  
Vol 7 (2) ◽  
pp. 157-163 ◽  
Author(s):  
T. Zhang ◽  
X.L. Qin ◽  
H.H.P. Fang
Keyword(s):  
Organic Carbon ◽  
Bacterial Species ◽  
Culture Method ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Rdna Sequences ◽  
Assimilable Organic Carbon ◽  
16S Rdna Sequences ◽  
Polymerase Chain ◽  
Primer Sets

In this study, two primer sets were at first designed specific to P-17 and NOX, the two bacterial species used for measurements of assimilable organic carbon (AOC) in water, based on their respective 16S rDNA sequences. These two primer sets were then used to measure the AOC content in 41 water samples by quantitative real time polymerase chain reaction (qRT-PCR). Results showed that P-17 and NOX cell numbers quantified by qRT-PCR using the designed primer sets were consistently higher (about 40.8% for P-17 and 54.8% for NOX measurements) than those by the traditional but time-consuming plate culture method. This proposed method not only saves the 5 days needed for plate culture, but also may detect AOC at concentrations as low as 0.3 μg-acetate-C/l and 0.6 μg-oxalate-C/l, which are lower than those measured by the plate culture and other methods. Using this new method, the water from the tap and a local reservoir were found containing 122 μg/l and 194 μg/l AOC, respectively, accounting for 5.7% and 7.8% of TOC.

scholarly journals Development of Polymerase Chain Reaction Primers Highly Specific for Xanthomonas albilineans, the Causal Bacterium of Sugarcane Leaf Scald Disease

Plant Disease ◽  
1999 ◽  
Vol 83 (3) ◽  
pp. 218-222 ◽  
Author(s):  
Y.-B. Pan ◽  
M. P. Grisham ◽  
D. M. Burner ◽  
B. L. Legendre ◽  
Q. Wei
Keyword(s):  
Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Multiple Sequence ◽  
Leaf Scald ◽  
Specific Pcr ◽  
Polymerase Chain ◽  
Xanthomonas Albilineans

New primers were developed that greatly improved the specificity of the polymerase chain reaction (PCR) protocol for Xanthomonas albilineans, the causal agent of sugarcane leaf scald disease. Length-polymorphic PCR products, amplified under the current PCR protocol from the 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) of X. albilineans and three unidentified sugarcane saprophytic bacterial species, were cloned and sequenced. Fourteen other nonredundant ITS sequences retrieved from the database were highly homologous to the sequence of X. albilineans. Two X. albilineans-specific PCR primers, namely, PGBL1 (5′ CTT TGG GTC TGT AGC TCA GG) and PGBL2 (5′ GCC TCA AGG TCA TAT TCA GC), were designed based on a multiple sequence alignment among these 18 sequences. These two primers permitted specific PCR amplification of a 288-bp DNA product from all 71 diverse X. albilineans strains tested. No amplification product was observed from any other bacterial species tested, including the three unidentified sugarcane saprophytes. The new PCR protocol has been routinely used to detect the leaf scald pathogen from infected sugarcane tissues.

scholarly journals A Disposable and Multi-Chamber Film-Based PCR Chip for Detection of Foodborne Pathogen

Sensors ◽  
2018 ◽  
Vol 18 (9) ◽  
pp. 3158 ◽  
Author(s):  
Nam Bae ◽  
Sun Lim ◽  
Younseong Song ◽  
Soon Jeong ◽  
Seol Shin ◽  
...  
Keyword(s):  
Foodborne Pathogens ◽  
Pathogen Detection ◽  
Detection System ◽  
Foodborne Pathogen ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Adhesive Film ◽  
Thermal Transfer ◽  
Polymerase Chain ◽  
Structurally Stable

Since the increment of the threat to public health caused by foodborne pathogens, researches have been widely studied on developing the miniaturized detection system for the on-site pathogen detection. In the study, we focused on the development of portable, robust, and disposable film-based polymerase chain reaction (PCR) chip containing a multiplex chamber for simultaneous gene amplification. In order to simply fabricate and operate a film-based PCR chip, different kinds of PCR chambers were designed and fabricated using polyethylene terephthalate (PET) and polyvinyl chloride (PVC) adhesive film, in comparison with commercial PCR, which employs a stereotyped system at a bench-top scale. No reagent leakage was confirmed during the PCR thermal cycling using the film PCR chip, which indicates that the film PCR chip is structurally stable for rapid heat cycling for DNA amplification. Owing to use of the thin film to fabricate the PCR chip, we are able to realize fast thermal transfer from the heat block that leads to short PCR amplification time. Moreover, using the film PCR chip, we could even amplify the target pathogen with 10 CFU mL−1. The artificially infected milk with various concentration of Bacillus cereus was successfully amplified on a single film PCR chip. On the basis of the reliable results, the developed film PCR chip could be a useful tool as a POCT device to detect foodborne pathogens via genetic analysis.

scholarly journals Quantification of Toxic Cyanobacteria in Water by Use of Competitive PCR Followed by Sequence-Specific Labeling of Oligonucleotide Probes

1998 ◽  
Vol 64 (7) ◽  
pp. 2639-2643 ◽  
Author(s):  
Knut Rudi ◽  
Olav M. Skulberg ◽  
Frank Larsen ◽  
Kjetill S. Jakobsen
Keyword(s):  
Sample Preparation ◽  
Solid Phase ◽  
Bacterial Species ◽  
Simultaneous Detection ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Oligonucleotide Probes ◽  
Competitive Pcr ◽  
Wide Range ◽  
Preparation Strategy

ABSTRACT A complete nucleic-acid-based assay which consists of sample preparation, DNA amplification, and chromogenic detection was developed for quantifying potential toxin-producing cyanobacteria of interest to the public. The sample preparation strategy involves the same solid phase for cell concentration and DNA purification. For the detection step, we used a combination of competitive PCR amplification, sequence-specific labeling of oligonucleotide probes, hybridization of the labeled oligonucleotides to immobilized complements and, finally, chromogenic detection. The complete assay was tested with water containing toxin-producing cyanobacteria belonging to the genusMicrocystis. A detection limit of 100 cells/ml and a quantitative range of more than 3 orders of magnitude were obtained. This approach can easily be adapted to a wide range of bacterial species and has the potential for simultaneous detection and quantitation of several different target organisms by a single assay.

scholarly journals Enterobacterial Repetitive Intergenic Consensus (ERIC) as a tool for genetic characterisation of bacterial isolates in Nigeria

2020 ◽  
Vol 37 (1) ◽  
pp. 122-128
Author(s):  
K. Otokunefor ◽  
C.J. Ogugbue ◽  
B.U. Fajoyomi
Keyword(s):  
16S Rrna ◽  
Bacterial Species ◽  
Pcr Analysis ◽  
Bacterial Identification ◽  
Bacterial Isolates ◽  
Enterobacterial Repetitive Intergenic Consensus ◽  
16S Rrna Sequence ◽  
Genetic Characterisation ◽  
Polymerase Chain ◽  
Identification Tool

Genetic characterisation as a tool for identification of bacterial isolates in Nigeria has been on the increase in recent years, and the 16s rRNA typing has been a preferred method. Due to cost limitations, there is a need to explore other genetic options. Enterobacterial Repetitive Intergenic Consensus (ERIC) polymerase chain reaction (PCR) analysis is a PCR- only based system which offers the advantage of reduced cost. This study set out to explore the use of ERIC-PCR in genetic characterisation of some selected bacterial isolates from Nigeria and compare it with genetic characterisation using 16s rRNA sequence typing. ERIC-PCR and 16s rRNA typing were carried out on 15 isolates following previously described protocols. Using 16s rRNA typing, thirteen different bacterial species were identified of which majority (85.7%) were Gram negative, with 57.1% belonging to the Enterobacteriaceae family. Using ERIC-PCR, only 13 of the 15 isolates (86.7%) could be typed, resulting in the identification of the 13 different types. ERIC-PCR was able to accurately differentiate between two members of the Proteus species, as well as identify the organisms as similar based on the banding pattern. The results show that ERIC-PCR may play a role as a bacterial identification tool but this role might be more suited to differentiating closely related members of a genus or typing within species rather than general bacterial identification. Keywords: Genetic characterisation, 16s rRNA, ERIC-PCR, Nigeria

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