scholarly journals A paradigm for the molecular identification of Mycobacterium species in a routine diagnostic laboratory

2007 ◽  
Vol 56 (5) ◽  
pp. 598-602 ◽  
Author(s):  
K. J. Williams ◽  
C. L. Ling ◽  
C. Jenkins ◽  
S. H. Gillespie ◽  
T. D. McHugh
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Reference Laboratory ◽  
Mycobacterium Fortuitum ◽  
Diagnostic Laboratory ◽  
Mycobacterium Chelonae ◽  
Mycobacterium Xenopi ◽  
Pcr Assays

The aim of this study was to improve the identification of Mycobacterium species in the context of a UK teaching hospital. Real-time PCR assays were established to enable the rapid differentiation between Mycobacterium tuberculosis (MTB) complex and Mycobacterium species other than tuberculosis (MOTT), followed by 16S rRNA gene sequencing for the speciation of MOTT. Real-time PCR assays gave comparable results to those from the reference laboratory. The implementation of these PCR assays using an improved bead extraction method has enhanced the mycobacterial diagnostic service at the Royal Free Hospital by providing a rapid means of differentiating between MTB complex and MOTT, and would be simple to implement in similar laboratories. Sequence analysis successfully identified a range of Mycobacterium spp. representative of those encountered in the clinical setting of the authors, including Mycobacterium avium complex, Mycobacterium fortuitum group, Mycobacterium chelonae–Mycobacterium abscessus group, Mycobacterium xenopi and Mycobacterium gordonae. It provides a useful tool for the identification of MOTT when clinically indicated.

scholarly journals A critical assessment of two real-time PCR assays targeting the (SSU) rRNA and gdh genes for the molecular identification of Giardia intestinalis in a clinical laboratory

2014 ◽  
Vol 67 (9) ◽  
pp. 811-816 ◽  
Author(s):  
Samuel Boadi ◽  
Spencer D Polley ◽  
Sally Kilburn ◽  
Graham A Mills ◽  
Peter L Chiodini
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Laboratory ◽  
Protozoan Parasite ◽  
Molecular Techniques ◽  
Giardia Intestinalis ◽  
Diagnostic Sensitivity ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Pcr Assays

IntroductionGiardiasis is an intestinal diarrhoeal illness caused by the flagellate protozoan parasite Giardia intestinalis. Molecular techniques for the identification of G. intestinalis have generally been shown to offer a better detection rate of the parasite than the traditional faecal concentration and microscopy techniques.AimThe aim of this study was to critically assess the performance of a commercial and a published real-time PCR assay for their potential use as frontline tests for the diagnosis of giardiasis.MethodsA composite reference standard of enzyme immunoassay and rapid membrane test was used in a diagnostic accuracy study to assess the performance of Primerdesign's, and Verweij et alG. intestinalis real-time PCR assays, comparing them with the traditional ova, cysts and parasite microscopy test (OCP-M).ResultsThe Verweij real-time PCR used primers for the (SSU) rRNA gene, and produced a diagnostic sensitivity of 93.4% (95% CI 88.30% to 98.50%) and an efficiency of 100%. Primerdesign's real-time PCR used primers for the glutamate dehydrogenase gene and produced a diagnostic sensitivity of 61.5% (95% CI 51.50% to 71.50%) and an efficiency of 203%. The OCP-M sensitivity was 83.5% (95% CI 75.87% to 91.13%).ConclusionsThe Verweij real-time PCR was robust and the most sensitive assay suited for use as a first-line diagnostic test for giardiasis.

scholarly journals Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays

2004 ◽  
Vol 42 (12) ◽  
pp. 5636-5643 ◽  
Author(s):  
M. Rougemont ◽  
M. Van Saanen ◽  
R. Sahli ◽  
H. P. Hinrikson ◽  
J. Bille ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Plasmodium Species ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Pcr Assays ◽  
Species Specific

Design and testing of real-time PCR primers for the quantification ofMethanoculleus,Methanosarcina,Methanothermobacter, and a group of uncultured methanogens

2009 ◽  
Vol 55 (5) ◽  
pp. 611-616 ◽  
Author(s):  
Ingrid H. Franke-Whittle ◽  
Marta Goberna ◽  
Heribert Insam
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Biogas Production ◽  
Pcr Primers ◽  
Sybr Green ◽  
Rrna Gene ◽  
Pcr Assays ◽  
Design And Testing ◽  
Uncultured Archaea ◽  
Detection And Quantification

In this study, 16S rRNA gene primers were designed to complement the suite of already available PCR primers for the detection of different methanogens involved in biogas production through anaerobic digestion by SYBR Green real-time PCR. Primers designed for use in TaqMan real-time PCR for the organisms Methanosaeta , Methanosarcina , and Methanoculleus have been described previously; however, we found that (i) the Methanoculleus primers were not specific to members of the genus and that (ii) the Methanosarcina primers did not work specifically with SYBR Green real-time PCR. Thus, we designed new primers for these and other methanogens, and we optimized SYBR Green real-time PCR assays. Primers were tested by end-point and real-time PCR, and they were found to work specifically and sensitively. Application of these primers will allow the detection and quantification of Methanoculleus, Methanosarcina, Methanothermobacter , and a group of yet uncultured archaea from anaerobic habitats.

PCR and culture for diagnosis of Acanthamoeba keratitis

2020 ◽  
pp. bjophthalmol-2020-316730
Author(s):  
Helene Yera ◽  
Vichita Ok ◽  
Fiona Lee Koy Kuet ◽  
Naima Dahane ◽  
Frédéric Ariey ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Latent Class ◽  
Rrna Gene ◽  
Target Sequence ◽  
Small Subunit Rrna ◽  
Pcr Assay ◽  
Corneal Scraping ◽  
Pcr Assays

Background/AimsAcanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.Methods1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. Acanthamoeba PCR assays detected different regions of the Acanthamoeba nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoeba isolates were also characterised.ResultsEstimated prevalence of AK was 1.32%. The sensitivity of Acanthamoeba diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoeba genotype (84.6%).ConclusionsCulture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

scholarly journals hsp65 Sequencing for Identification of Rapidly Growing Mycobacteria

1999 ◽  
Vol 37 (3) ◽  
pp. 852-857 ◽  
Author(s):  
H. Ringuet ◽  
C. Akoua-Koffi ◽  
S. Honore ◽  
A. Varnerot ◽  
V. Vincent ◽  
...  
Keyword(s):  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Mycobacterium Abscessus ◽  
Rrna Gene ◽  
Mycobacterium Fortuitum ◽  
Mycobacterium Chelonae ◽  
Rapidly Growing Mycobacteria ◽  
Rrna Gene Sequencing ◽  
Human Infections ◽  
Type Strains

Partial sequencing of the hsp65 gene was used for the identification of rapidly growing mycobacteria (RGM). A 441-bp fragment (A. Telenti, F. Marchesi, M. Balz, F. Bally, E. Böttger, and T. Bodmer, J. Clin. Microbiol. 31:175–178, 1993) was amplified and sequenced by an automated fluorescence-based method involving capillary electrophoresis. Type strains of 10 RGM species were first studied. Each species had a unique nucleotide sequence, distinguishing it clearly from the other species. A panel of strains from the four main RGM species responsible for human infections, Mycobacterium abscessus, Mycobacterium chelonae,Mycobacterium fortuitum, and Mycobacterium peregrinum, was also studied. There were few sequence differences within each of these species (<2% of bases were different from the type strain sequence), and they had no effect on species assignment.hsp65 sequencing unambiguously differentiated M. chelonae and M. abscessus, two species difficult to identify by classical methods and 16S rRNA gene sequencing. The devised procedure is a rapid and reliable tool for the identification of RGM species.

scholarly journals Quantitative Real-Time PCR Assays for Sensitive Detection of Canada Goose-Specific Fecal Pollution in Water Sources

2010 ◽  
Vol 76 (14) ◽  
pp. 4886-4889 ◽  
Author(s):  
B. Fremaux ◽  
T. Boa ◽  
C. K. Yost
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fecal Pollution ◽  
Sensitive Detection ◽  
Rrna Gene ◽  
Canada Goose ◽  
Canada Geese ◽  
Quantitative Real Time Pcr ◽  
Gene Markers ◽  
Pcr Assays

ABSTRACT Canada geese (Branta canadensis) are prevalent in North America and may contribute to fecal pollution of water systems where they congregate. This work provides two novel real-time PCR assays (CGOF1-Bac and CGOF2-Bac) allowing for the specific and sensitive detection of Bacteroides 16S rRNA gene markers present within Canada goose feces.

scholarly journals Seasonal Change in Bacterial Flora and Biomass in Mountain Snow from the Tateyama Mountains, Japan, Analyzed by 16S rRNA Gene Sequencing and Real-Time PCR

2005 ◽  
Vol 71 (1) ◽  
pp. 123-130 ◽  
Author(s):  
Takahiro Segawa ◽  
Koji Miyamoto ◽  
Kazunari Ushida ◽  
Kiyokazu Agata ◽  
Norihiro Okada ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Flora ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
The World ◽  
Surface Snow ◽  
Rrna Gene Sequencing ◽  
Melting Season

ABSTRACT The bacterial flora and biomass in mountain snow from the Tateyama Mountains, Toyama Prefecture, Japan, one of the heaviest snowfall regions in the world, were analyzed by amplified ribosomal DNA restriction analysis followed by 16S rRNA gene sequencing and DNA quantification by real-time PCR. Samples of surface snow collected in various months during the melting season contained a psychrophilic bacterium, Cryobacterium psychrophilum, and two psychrotrophic bacteria, Variovorax paradoxus and Janthinobacterium lividum. Bacterial colonies that developed in an in situ meltwater medium at 4�C were revealed to be V. paradoxus. The biomasses of C. psychrophilum, J. lividum, and V. paradoxus, as estimated by real-time PCR, showed large increases during the melting season from March to October (2.0 � 105-fold, 1.5 � 105-fold, and 1.0 � 104-fold increases, respectively), suggesting their rapid growth in the surface snow. The biomasses of C. psychrophilum and J. lividum increased significantly from March to April, reached a maximum in August, and dropped at the end of the melting season. In contrast, the biomass of V. paradoxus did not increase as rapidly during the early melting season but continued to increase from June until October. The differences in development observed among these bacterial species suggest that their growth was promoted by different nutrients and/or environmental conditions in the snow. Since these three types of bacteria have also been reported to be present in a glacier in Antarctica and a Greenland ice core, they seem to be specialized members of the snow biota that are distributed in snow and ice environments in various parts of the world.

scholarly journals Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas

2009 ◽  
Vol 75 (9) ◽  
pp. 2945-2950 ◽  
Author(s):  
Jennifer Hodgetts ◽  
Neil Boonham ◽  
Rick Mumford ◽  
Matthew Dickinson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Multiplex Assay ◽  
The Other ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Detection ◽  
23S Rrna Gene ◽  
Pcr Assays

ABSTRACT Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays.

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