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.

scholarly journals Detection, Distribution, and Genetic Variability of ‘Candidatus Liberibacter’ Species Associated with Zebra Complex Disease of Potato in North America

Plant Disease ◽  
2009 ◽  
Vol 93 (11) ◽  
pp. 1102-1115 ◽  
Author(s):  
A. Wen ◽  
I. Mallik ◽  
V. Y. Alvarado ◽  
J. S. Pasche ◽  
X. Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Complex Disease ◽  
Pcr Primers ◽  
23S Rrna ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Candidatus Liberibacter ◽  
Total Dna ◽  
Pcr Assays

The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for ‘Candidatus Liberibacter solanacearum’ and ‘Candidatus Liberibacter psyllaurous’ were evaluated in conventional and real-time PCR assays. All PCR primers were specific for ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. ‘Ca. Liberibacter’ species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using ‘Ca. L. solanacearum’–specific primers and primers specific for the β-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for ‘Ca. Liberibacter’ detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The ‘Ca. Liberibacter’ species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ “strains” investigated in this study. ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’ were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of ‘Ca. L. solanacearum’ and reverse primer of ‘Ca. L. psyllaurous’ in ZC-affected potato samples. This finding clarifies the current taxonomic status of ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’. The detection of ‘Ca. L. solanacearum’ from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

scholarly journals Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

2007 ◽  
Vol 73 (20) ◽  
pp. 6557-6565 ◽  
Author(s):  
Pascal E. Saikaly ◽  
Morton A. Barlaz ◽  
Francis L. de los Reyes
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Fate And Transport ◽  
Biological Warfare ◽  
Quantitative Real Time Pcr ◽  
Pcr Assays ◽  
Detection And Quantification

ABSTRACT Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R 2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R 2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

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.

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