Microsporidial Keratitis in India: 16S rRNA Gene-Based PCR Assay for Diagnosis and Species Identification of Microsporidia in Clinical Samples

2006 ◽  
Vol 47 (10) ◽  
pp. 4468 ◽  
Author(s):  
Joveeta Joseph ◽  
Savitri Sharma ◽  
Somasheila I. Murthy ◽  
Pravin V. Krishna ◽  
Prashant Garg ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Species Identification ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Pcr Assay

scholarly journals Real-Time Quantitative Broad-Range PCR Assay for Detection of the 16S rRNA Gene Followed by Sequencing for Species Identification

2006 ◽  
Vol 44 (8) ◽  
pp. 2750-2759 ◽  
Author(s):  
F. Zucol ◽  
R. A. Ammann ◽  
C. Berger ◽  
C. Aebi ◽  
M. Altwegg ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Species Identification ◽  
Rrna Gene ◽  
Pcr Assay ◽  
The 16S Rrna Gene

scholarly journals 16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles

Microbiome ◽  
2014 ◽  
Vol 2 (1) ◽  
pp. 31 ◽  
Author(s):  
Jun Hang ◽  
Valmik Desai ◽  
Nela Zavaljevski ◽  
Yu Yang ◽  
Xiaoxu Lin ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Temperature Stability ◽  
Clinical Samples ◽  
Rrna Gene ◽  
16S Rrna Gene Pyrosequencing

scholarly journals Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

2002 ◽  
Vol 68 (10) ◽  
pp. 5064-5081 ◽  
Author(s):  
Alexander Loy ◽  
Angelika Lehner ◽  
Natuschka Lee ◽  
Justyna Adamczyk ◽  
Harald Meier ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Oligonucleotide Microarray ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Sulfate Reducing ◽  
Specific Pcr

ABSTRACT For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

scholarly journals Streptococcus rupicaprae sp. nov., isolated from a Pyrenean chamois (Rupicapra pyrenaica)

2011 ◽  
Vol 61 (8) ◽  
pp. 1989-1993 ◽  
Author(s):  
A. I. Vela ◽  
G. Mentaberre ◽  
I. Marco ◽  
R. Velarde ◽  
S. Lavín ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Biochemical Tests ◽  
Pyrenean Chamois

Biochemical and molecular genetic studies were performed on an unknown Gram-stain-positive, catalase-negative, coccus-shaped organism isolated from clinical samples of a Pyrenean chamois. The micro-organism was identified as a streptococcal species based on its cellular morphological and biochemical tests. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus, but the organism did not correspond to any species of this genus. The nearest phylogenetic relative of the unknown coccus from chamois was Streptococcus ovis (95.9 % 16S rRNA gene sequence similarity). The rpoB and sodA sequence analysis showed sequence similarity values of less than 85.7 % and 83.0 %, respectively, with the currently recognized species of the genus Streptococcus. The novel bacterial isolate was distinguished from S. ovis and other species of the genus Streptococcus using biochemical tests. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a novel species of the genus Streptococcus, Streptococcus rupicaprae sp. nov., with the type strain 2777-2-07T ( = CECT 7718T  = CCUG 59652T).

scholarly journals Phylogenetic tree analysis based on the 16S sequence alignment for Klebsiella spp. isolated from different sources

2019 ◽  
pp. 1957-1966
Author(s):  
Mustafa Basil Abdul Qader ◽  
Marwa Hameed AlKhafaji
Keyword(s):  
16S Rrna ◽  
Klebsiella Pneumoniae ◽  
16S Rrna Gene ◽  
Enteric Bacteria ◽  
Apium Graveolens ◽  
Clinical Samples ◽  
Identification System ◽  
Rrna Gene ◽  
Vitek 2 ◽  
Klebsiella Spp

16S ribosomal RNA (16S rRNA) gene sequences used to study bacterial phylogeny and taxonomy have been by far the most common housekeeping genetic marker utilized for identification and ancestor determination. This study aimed to investigate, for the first time, the relationship between Klebsiella spp. isolated from clinical and environmental samples in Iraq.      Fifty Klebsiella spp. isolates were isolated from clinical and environmental sources. Twenty-five isolates were collected from a fresh vegetable (Apium graveolens) and 25 from clinical samples (sputum, wound swab, urine). Enteric bacteria were isolated on selective and differential media and identified by an automatic identification system, vitek-2. The total DNA was extracted and PCR amplified for selected isolates. The 16S rRNA gene was amplified by using the universal primer 27F (5'- AGAGTTTGATCCTGGCTCAG- 3') and 1492R (5'- GGTTACCTTGTTACGACTT- 3’). The 16SrRNA gene sequence was analysed among some local isolates, and the results were compared with the standard data of similar registered strains in NCBI. The most common species of Klebsiella was Klebsiella pneumoniae pneumoniae (Kpp), followed by Klebsiella pneumoniae ozaenae (Kpo) and Klebsiella oxytoca (Ko). The results of the identification of species and sub species by using the  biochemical test (vitek-2) were more precise than those obtained by the use of the universal primer.Phylogenetic tree strategies have clearly indicated a relatively close similarity amongst all analysed Klebsiella isolates and revealed the intra-species genetic distance between the individual isolates of the Klebsiella spp. In conclusion, our results revealed the main advantage of using universal primers for the identification of Klebsiella spp. and their root from nature.

scholarly journals Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR Inhibitor Sodium Polyanetholesulfonate

1998 ◽  
Vol 36 (10) ◽  
pp. 2810-2816 ◽  
Author(s):  
David N. Fredricks ◽  
David A. Relman
Keyword(s):  
Blood Culture ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Benzyl Alcohol ◽  
Culture Media ◽  
Technical Problem ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Pcr Inhibitor ◽  
Purification Methods

Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with Escherichia coli was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted E. coli 16S rRNA gene was achieved by adding 5 μl of undiluted processed sample DNA to a 50-μl PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance.

scholarly journals First isolation and molecular phylogenetic analysis of Coxiella burnetii in lactating cows

2021 ◽  
Vol 24 (4) ◽  
pp. 508-519
Author(s):  
H. A. J. Gharban ◽  
A. A. Yousif
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Q Fever ◽  
Age Groups ◽  
Sampling Period ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Milk Samples ◽  
Lactating Cows ◽  
Specific Pathogen

Q fever is an infectious disease of animals and humans, caused by globally distributed C. burnetii. In Iraq, there are no previous studies associated with the detection of the organism in cattle. An overall of 130 lactating cows were submitted to direct collection of milk samples. Initially, the samples of milk were tested using the molecular polymerase chain reaction (PCR) assay targeting three genes (16S rRNA, IS1111a transposase, and htpB). However, positive results (18.46%; 24/130) were detected only with the 16s rRNA gene. Concerning risk factors, the highest prevalence of C. burnetii was showed in the district of Badra (42.86%), whereas the lowest - in Al-Numaniyah and Al-Suwaira districts (P=0.025). There was no significant variation in positivity between the months of sampling period (P=0.082) and between age groups (P=0.076). Crossbred cows (20.69%) showed a higher positivity than local and pure breeds (P=0.043). Milk of positive samples (n=24) was used for cultivation of C. burnetii into specific pathogen free-embryonated chicken eggs (SPF-ECEs). After three passages into SPF-ECEs, contents of yolk sac were collected, subjected for DNA extraction, and re-tested by PCR assay using the primer of 16s rRNA gene only. Of 24 cultivated milk samples, 12.5% (3/24) were positive for C. burnetii. Finally, the positive local isolates were analysed phylogenetically and reported in NCBI-Genbank under the accession numbers of MN121700.1, MN121701.1, and MN121702.1. In conclusion, this is a unique study as it detected C. burnetii in Iraqi lactating cows, and confirmed that organism was shed actively through milk, suggesting that these animals can play a role as a reservoir for organism with potential risk for transmission of infection from these animals to humans as well as to other animal species.

scholarly journals Nested PCR Assay for Detection of Granulocytic Ehrlichiae

1998 ◽  
Vol 36 (4) ◽  
pp. 1090-1095 ◽  
Author(s):  
Robert F. Massung ◽  
Kim Slater ◽  
Jessica H. Owens ◽  
William L. Nicholson ◽  
Thomas N. Mather ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Limit Of Detection ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Serum Samples ◽  
Blood Specimens ◽  
The 16S Rrna Gene ◽  
Edta Blood

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted fromIxodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.

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