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PCR Protocol (pcr + protocol)
Selected AbstractsSensitive and simultaneous analysis of five transgenic maizes using multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescenceELECTROPHORESIS, Issue 14 2004Virginia García-Cañas Abstract The benefits of using multiplex polymerase chain reaction (PCR) followed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) for the simultaneous detection of five transgenic maizes (Bt11, T25, MON810, GA21, and Bt176) are demonstrated. The method uses a hexaplex PCR protocol to amplify the five mentioned transgenic amplicons plus the zein gene used as reference, followed by a CGE-LIF method to analyze the six DNA fragments. CGE-LIF was demonstrated very useful and informative for optimizing multiplex PCR parameters such as time extension, PCR buffer concentration and primers concentration. The method developed is highly sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 0.054% of Bt11, 0.057% of T25, 0.036% of MON810, 0.064% of GA21, and 0.018% of Bt176 in flour obtaining signals still far from the detection limit (namely, the signal/noise ratios for the corresponding DNA peaks were 41, 124, 98, 250, 252, and 473, respectively). These percentages are well below the minimum threshold marked by the European Regulation for transgenic food labeling (i.e., 0.5,0.9%). A study on the reproducibility of the multiplex PCR-CGE-LIF procedure was also performed. Thus, values of RSD lower than 0.67 and 6.80% were obtained for migration times and corrected peak areas, respectively, for the same sample and three different days (n = 12). On the other hand, the reproducibility of the whole procedure, including four different multiplex PCR amplifications, was determined to be better than 0.66 and 23.3% for migration times and corrected peak areas, respectively. Agarose gel electrophoresis (AGE) and CGE-LIF were compared in terms of resolution and sensitivity for detecting PCR products, demonstrating that CGE-LIF can solve false positives induced by artifacts from the multiplex PCR reaction that could not be addressed by AGE. Moreover, CGE-LIF provides better resolution and sensitivity. To our knowledge, these results demonstrate for the first time that multiplex PCR-CGE-LIF is a solid alternative to determine multiple genetically modified organisms in maize flours in a single run. [source] Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling,HUMAN MUTATION, Issue 6 2009Steven F. Dobrowolski Abstract Identifying mitochondrial DNA (mtDNA) sequence variants in human diseases is complicated. Many pathological mutations are heteroplasmic, with the mutant allele represented at highly variable percentages. High-resolution melt (HRM or HRMA) profiling was applied to comprehensive assessment of the mitochondrial genome and targeted assessment of recognized pathological mutations. The assay panel providing comprehensive coverage of the mitochondrial genome utilizes 36 overlapping fragments (301,658,bp) that employ a common PCR protocol. The comprehensive assay identified heteroplasmic mutation in 33 out of 33 patient specimens tested. Allele fraction among the specimens ranged from 1 to 100%. The comprehensive assay panel was also used to assess 125 mtDNA specimens from healthy donors, which identified 431 unique sequence variants. Utilizing the comprehensive mtDNA panel, the mitochondrial genome of a patient specimen may be assessed in less than 1 day using a single 384-well plate or two 96-well plates. Specific assays were used to identify the myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) mutation m.3243A>G, myoclonus epilepsy, ragged red fibers (MERRF) mutation m.8344A>G, and m.1555A>G associated with aminoglycoside hearing loss. These assays employ a calibrated, amplicon-based strategy that is exceedingly simple in design, utilization, and interpretation, yet provides sensitivity to detect variants at and below 10% heteroplasmy. Turnaround time for the genotyping tests is about 1,hr. Hum Mutat 30,1,8, 2009. © 2009 Wiley-Liss, Inc. [source] Detection and identification of aquatic mycobacteria in formalin-fixed, paraffin-embedded fish tissuesJOURNAL OF FISH DISEASES, Issue 5 2009F Pourahmad Abstract The isolation of mycobacteria from field samples is problematic, and isolation of the bacterium is sometimes not even attempted. The detection of mycobacteria through traditional histology using formalin-fixed, paraffin-embedded (FFPE) tissues is neither sensitive nor specific. However, detection of mycobacterial DNA from FFPE specimens, suspected of being infected with mammalian mycobacteriosis, is a routine clinical procedure. In the present study, a polymerase chain reaction (PCR)-based method was used to detect and identify mycobacteria in FFPE specimens sampled from fish suspected of being infected with fish mycobacteriosis. A total of 45 fish tissue samples, comprising of 12 tissue samples obtained from experimentally infected fish and the remainder from fish naturally infected with mycobacteria, were analysed using a PCR protocol which amplifies a fragment of the mycobacterial 65 kDa heat-shock protein (hsp65) gene. PCR-restriction enzyme analysis and/or sequencing were employed to further analyse the PCR amplicons. The PCR results were compared with those obtained by histology and culture. Mycobacterial DNA was detected in 34 of the 45 samples examined, of which 16 samples (47%) showed granulomatous reactions on histological examination. Using histology as the gold standard, no false-negative PCR results were obtained. Also, considering the presence or absence of granulomas as a diagnostic criterion, the sensitivity and specificity of PCR in 42 of the FFPE tissues were 16/16 (100%) and 8/26 (,30.8%), respectively. Corresponding microbiological cultures were available for 15 cases, of which 13 were pure Mycobacterium cultures. Of these, 13 were PCR positive (100% sensitivity and 50% specificity). The PCR-based methods used here proved sensitive, specific and rapid for the detection of mycobacteria in routinely processed paraffin wax-embedded and formalin-fixed histological samples, and the results of the study suggest that this method has potential use in retrospective epidemiological studies. [source] Detection of Ralstonia solanacearum in Potato Tubers by Polymerase Chain ReactionJOURNAL OF PHYTOPATHOLOGY, Issue 11-12 2000K.-H. Pastrik Abstract A new polymerase chain reaction (PCR) assay was developed for the detection of Ralstonia solanacearum in potato tubers. The designed primers PS-1/PS-2 based on the sequence data of the 16S rRNA gene. Using the optimized PCR protocol, it was possible to detect R. solanacearum cells artificially added to concentrated potato extracts in the range of 1,10 colony-forming units (CFU) per PCR reaction mixture (10,100 CFU/ml potato homogenate). No amplification products were obtained, when bacteria belonging to other species or genera were submitted to PCR under the same conditions. A total of 10 different DNA extraction methods were adapted for the isolation of R. solanacearum DNA from potato homogenates and were compared for their suitability as pre-PCR procedures. Zusammenfassung Es wurde ein neuer PCR-Test entwickelt für die Detektion von Ralstonia solanacearum in Kartoffel-Knollen. Die entwickelten Primer PS-1/PS-2 basierten auf Sequenzdaten des 16S rRNA Gens. Mit dem optimierten PCR Protokoll war es möglich künstlich zugegebene R. solanacearum Zellen in konzentrierten Kartoffel-Homogenaten zu detektieren, bei einer Nachweis-Empfindlichkeit von 1,10 CFU pro PCR-Mix (10,100 CFU pro ml Kartoffel-Homogenat). Mit dem optimierten PCR Protokoll wurden keine Amplifikationsprodukte bei Bakterien anderer Arten oder Gattungen erhalten. Außerdem wurden 10 unterschiedliche DNA-Extraktionsmethoden getestet zur Isolierung von Ralstonia solanacearum DNA aus Kartoffel-Homogenat und ihre Eignung für die PCR verglichen. [source] Identification of Microsporum canis from dermatophytic pseudomycetoma in paraffin-embedded veterinary specimens using a common PCR protocolMYCOSES, Issue 3 2007Simona Nardoni Summary The effectiveness of a simple PCR protocol performed on paraffin-embedded tissues, obtained from histopathologically and culturally diagnosed cases of dermatophytic pseudomycetoma DPM was tested. The specimens were investigated using previously described primers (DH1L and DH1R) targeting the 18S rDNA gene and amplifying a 183-bp fragment. Microsporum canis was identified from all samples. The PCR protocol described in the present work demonstrated a 100% concordant result comparing the molecular characterisation with phenotypic characterisation of dermatophytes. Molecular biology could represent a valid identification tool in dermatophytic deep infections, when diagnosis cannot be achieved by cultural methods. [source] PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic bandPLANT PATHOLOGY, Issue 2 2003H. W. Kang A 24-mer primer pair was generated by sequencing a URP-PCR fingerprinting-derived polymorphic band that is uniquely shared in Pectobacterium carotovorum ssp. carotovorum strains (Pcc). The primer set (EXPCCF/EXPCCR) amplified a single band of expected size (0·55 kb) from genomic DNA obtained from 29 Pcc strains and three Pectobacterium carotovorum ssp. wasabiae (Pcw) strains, but not from other P. carotovorum subspecies atrosepticum, betavasculorum or odoriferum, or from other Erwinia spp. or bacterial genera. The RsaI digestion profile of the amplified bands divided Pcc strains into five groups with a unique profile from Pcw strains. First-round PCR detected between 5 × 102 and 1 × 103 colony forming units (CFU) mL,1 and detection sensitivity was increased to as few as 2,4 CFU mL,1 after second-round (nested) PCR. This PCR protocol was used directly to detect Pcc strains in infected plant tissues. [source] Additional reverse transcription,polymerase chain reaction of peripheral slices is not superior to analysis of the central slice in sentinel lymph nodes from melanoma patientsBRITISH JOURNAL OF DERMATOLOGY, Issue 3 2004H-J. Blaheta Summary Background The status of the sentinel lymph node (SLN) is an important prognostic factor in patients with cutaneous melanoma. Reverse transcription,polymerase chain reaction (RT,PCR) has been used as a sensitive means of detecting tumour cells in SLNs. Objectives To determine whether RT,PCR analysis of the SLN using both the central and the peripheral slices is more sensitive than molecular analysis of the central slice only. Methods Eighty-three SLNs from 59 patients with primary cutaneous melanoma were identified by SLN mapping. All SLNs were bisected along their longitudinal axis to produce two equal halves. One half was used for histology and immunohistochemistry, and the other was analysed by RT,PCR for tyrosinase and MelanA. Parallel to the longitudinal axis, one central slice (approximately 2 mm in thickness) was cut manually. This central slice was used for our standard RT,PCR protocol. In the current study, up to eight additional peripheral slices (each approximately 2 mm in thickness) were cut parallel to the existing cut surface. These peripheral slices were analysed by additional RT,PCR. Results Standard RT,PCR of the central slice yielded positive results in 34 of 59 patients (57%). Additional RT,PCR of peripheral slices demonstrated positive findings in six additional patients (10%) who were initially negative by standard RT,PCR of the central slice. In detail, seven of those 34 patients positive by standard RT,PCR of the central slice had positive histological findings. In each of these seven patients, RT,PCR was positive both in the central slice as well as in the peripheral slices. The remaining 27 patients with positive RT,PCR results of the central slice showed negative histological findings. Only nine (33%) of these 27 patients had a positive RT,PCR also in the peripheral slices. Finally, all 25 patients with negative RT,PCR results in the central slice showed negative histological findings. Six of these patients (24%) revealed positive RT,PCR results on the analysis of peripheral slices. However, three of these patients expressed only MelanA but not tyrosinase. Thirty lymph nodes from 24 nonmelanoma patients served as negative controls for RT,PCR. In three of these 24 patients (13%) expression of MelanA but not tyrosinase was detected by RT,PCR. Conclusions Molecular analysis of peripheral slices yielded six additional patients (10%) positive by RT,PCR who were initially negative by standard RT,PCR of the central slice. However, three of these six patients were found to express only MelanA but not tyrosinase. As MelanA expression was also found in 13% of control lymph nodes, positive MelanA expression alone in SLNs of melanoma patients requires cautious interpretation in order to avoid false-positive findings. Thus, additional molecular processing of peripheral slices did not significantly increase the number of patients with RT,PCR-positive SLNs. [source] Direct amplification of rhizobial nodC sequences from soil total DNA and comparison to nodC diversity of root nodule isolatesFEMS MICROBIOLOGY ECOLOGY, Issue 1 2005Sarita Sarita Abstract A group-specific primer set was developed using nodC as a target gene for the amplification of rhizobial sequence diversity from nodule isolates and total soil DNA preparations. The primer set was tested on 209 nodule isolates, recovered from six different trap plant species which were grown in two soil samples collected from a chickpea and a wheat field site in India. We also amplified and cloned PCR products from total DNA isolated from the same soil samples. The total diversity within the resulting clone libraries (, 218 clones) was higher than that recovered from trap plants, but differed depending on the PCR protocols and primers used. However, some plant-selected genotypes could not be obtained using the community approach, probably due to variable detection limits and limited clone library sizes. [source] A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNAJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007A. Lorusso Abstract Aims:, The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. Methods and Results:, A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 101 DNA copies per 10 ,l of plasmid template and 6·5 × 100 colour changing units of reference strain Ba/2. Conclusions:, The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2,3 h compared with at least 1 week). Significance and Impact of the Study:, The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies. [source] A STUDY ON SUITABILITY OF FOUR ENRICHMENT BROTHS FOR PCR-BASED DETECTION OF LISTERIA MONOCYTOGENES FROM RAW MEATJOURNAL OF FOOD SAFETY, Issue 1 2006J. BALAMURUGAN ABSTRACT Four enrichment broths were evaluated for their compatibility with the polymerase chain reaction (PCR) for detection of Listeria monocytogenes from raw meat after single-step enrichment. Standardized PCR protocols for listeriolysin O (hlyA) gene were used for the species-specific identification of L. monocytogenes. Four broths, namely, modified University of Vermont broth (MUVM), Listeria enrichment broth (LEB), Fraser broth (FB) and polymyxin, acriflavin, lithium chloride, ceftazidime, aesculin, mannitol, egg yolk broth (PALCAM) , were inoculated with L. monocytogenes. The enriched cultures were subjected for PCR. Similarly, meat samples were artificially spiked with various concentrations of L. monocytogenes, these spiked samples were enriched in the above-mentioned four broths and subjected to PCR to determine the medium that was most compatible for PCR-based detection of L. monocytogenes. The aliquots taken during different incubation periods were subjected to three different procedures for the concentration of the target organism for use in PCR. Results revealed that MUVM was better than other broths for the detection of L. monocytogenes by both PCR and cultural method; moreover, it was able to support the growth of as low as 10 cfu/g of meat. Concentration of the target organisms by centrifugation and washing with PCR buffer was the most suitable method for improving PCR performance for detection of L. monocytogenes. Goat (n = 67) and buffalo (n = 45) meat samples from local markets were also screened by both PCR and cultural method to validate the results obtained from the spiking studies. Both results were in agreement in spiking studies as well as screening of market meat samples. [source] Development and demonstration of RNA isolation and RT,PCR procedures to detect Escherichia coli O157:H7 gene expression on beef carcass surfacesLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2000E.D. Berry Preventing the development of pathogen resistance to processing and preservation techniques will require an understanding of the genetic mechanisms that pathogens use in situ to adapt and develop tolerance to stresses they encounter in the food environment. RNA isolation and reverse-transcription (RT),PCR protocols were developed as tools to detect gene expression in bacteria on beef carcass surfaces. The utility of these procedures was demonstrated by detecting the expression of a selectively-inducible green fluorescent protein (GFP) gene in a plasmid-transformed strain of Escherichia coli O157:H7 inoculated onto beef carcass surface tissue. These procedures should serve as useful tools for studying the genetic responses of bacteria when exposed to antimicrobial interventions applied to food animal carcasses. [source] Diagnostic approaches for immunocompromised paediatric patients with pulmonary infiltratesCLINICAL MICROBIOLOGY AND INFECTION, Issue 3 2006K. Bochennek Abstract Pulmonary infiltrates in immunocompromised children often pose problems in terms of deciding on further diagnostic and therapeutic procedures, but few studies have evaluated the value of non-invasive and invasive diagnostic methods in paediatric populations. Both galactomannan ELISA and PCR protocols appear to be less useful in children than in adults. Invasive procedures, such as bronchoalveolar lavage or lung biopsy, can yield a pathohistological diagnosis and/or the isolation of a pathogen. Prospective studies in paediatric patients are needed urgently to assess the value of different diagnostic procedures and to define an effective and safe diagnostic strategy for the individual child. [source] | |||||||||||||