scholarly journals Identification of 8 Foodborne Pathogens by Multicolor Combinational Probe Coding Technology in a Single Real-Time PCR

2007 ◽  
Vol 53 (10) ◽  
pp. 1741-1748 ◽  
Author(s):  
Qiuying Huang ◽  
Qinghua Hu ◽  
Qingge Li
Keyword(s):  
Real Time ◽  
Species Identification ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Target Genes ◽  
Molecular Beacon ◽  
Universal Primers ◽  
Pcr Analysis ◽  
Bacterial Strains ◽  
Blind Test

Abstract Background: Real-time PCR assays have been widely used for detecting foodborne pathogens but have been much less frequently applied in species identification, mainly because of the low number of species they can distinguish in 1 reaction. The present study used a new probe coding/labeling strategy, termed multicolor combinational probe coding (MCPC), to increase the number of targets that can be distinguished in a single real-time PCR for rapid and reliable species identification. Methods: With MCPC, 8 pairs of species-specific tagged primers, 1 pair of universal primers, and 8 unilabeled or mix-labeled molecular beacon probes were included in a single reaction tube. Real-time PCR was performed, and the identity of each of the 8 pathogens was determined by amplification profile comparison. The method was validated via blind assessment of 118 bacterial strains, including clinical isolates and isolates from food products. Results: The blind test with 118 samples gave no false-positive or -negative results for the target genes. The template DNA suitable for MCPC analysis was simply prepared by heating lysis, and the total PCR analysis was finished within 2.5 h, excluding template preparation. Conclusions: MCPC is suitable for rapid and reliable identification of foodborne pathogens at the species level.

scholarly journals Real-Time PCR Quantitation of Clostridia in Feces of Autistic Children

2004 ◽  
Vol 70 (11) ◽  
pp. 6459-6465 ◽  
Author(s):  
Yuli Song ◽  
Chengxu Liu ◽  
Sydney M. Finegold
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Late Onset ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Pcr Analysis ◽  
Bacterial Cells ◽  
Autistic Children ◽  
Bacterial Strains ◽  
And Control

ABSTRACT Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT ) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 � 108 CFU/g in autistic children and 4.8 � 108 CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.

MicroSEQ® Salmonella spp. Detection Kit Using the Pathatrix® 10-Pooling Salmonella spp. Kit Linked Protocol Method Modification

2014 ◽  
Vol 97 (2) ◽  
pp. 484-491 ◽  
Author(s):  
Jason Wall ◽  
Rick Conrad ◽  
Kathy Latham ◽  
Eric Liu
Keyword(s):  
Real Time ◽  
Study Design ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Reference Method ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Salmonella Spp ◽  
Culture Methods ◽  
Design For All

Abstract Real-time PCR methods for detecting foodborne pathogens offer the advantages of simplicity and quick time to results compared to traditional culture methods. The addition of a recirculating pooled immunomagnetic separation method prior to real-time PCR analysis increases processing output while reducing bothcost and labor. This AOAC Research Institute method modification study validates the MicroSEQ®Salmonella spp. Detection Kit [AOAC Performance Tested Method(PTM) 031001] linked with the Pathatrix® 10-Pooling Salmonella spp. Kit (AOAC PTM 090203C) in diced tomatoes, chocolate, and deli ham. The Pathatrix 10-Pooling protocol represents a method modification of the enrichment portion of the MicroSEQ Salmonella spp. protocol. The results of the method modification were compared to standard cultural reference methods for diced tomatoes, chocolate, and deli ham. All threematrixes were analyzed in a paired study design. An additional set of chocolate test portions was analyzed using an alternative enrichment medium in an unpaired study design. For all matrixes tested, there were no statistically significant differences in the number of positive test portions detected by the modified candidate method compared to the appropriate reference method. The MicroSEQ Salmonella spp. protocol linked with the Pathatrix individual or 10-Pooling procedure demonstrated reliabilityas a rapid, simplified, method for the preparation of samples and subsequent detection of Salmonella in diced tomatoes, chocolate, and deliham.

scholarly journals Simultaneous Screening of 24 Target Genes of Foodborne Pathogens in 35 Foodborne Outbreaks Using Multiplex Real-Time SYBR Green PCR Analysis

2010 ◽  
Vol 2010 ◽  
pp. 1-18 ◽  
Author(s):  
Hiroshi Fukushima ◽  
Jun Kawase ◽  
Yoshiki Etoh ◽  
Kumiko Sugama ◽  
Shunshuke Yashiro ◽  
...  
Keyword(s):  
Real Time ◽  
Foodborne Pathogens ◽  
Target Genes ◽  
Simultaneous Detection ◽  
Sybr Green ◽  
Pcr Analysis ◽  
Foodborne Outbreak ◽  
Pcr Assay ◽  
Foodborne Outbreaks ◽  
Stool Specimens

A set of 8 multiplex real-time SYBR Green PCR (SG-PCR) assays including 3 target primers and an internal amplification control (IAC) primer was simultaneously evaluated in 3 h or less with regard to detection of 24 target genes of 23 foodborne pathogens in 7 stool specimens of foodborne outbreak using a 96-well reaction plate. This assay, combined with DNA extraction (QIAamp DNA Stool Mini kit), offered detection of greater than103-104foodborne pathogens per g in stool specimens. The products formed were identified using melting point temperature (Tm) curve analysis. This assay was evaluated for the detection of foodborne pathogens in 33 out of 35 cases of foodborne outbreak, using 4 different PCR instruments in 5 different laboratories. No interference from the multiplex real-time SG-PCR assay, including IAC, was observed in stool specimens in any analysis. We found multiplex real-time SG-PCR assay for simultaneous detection of 24 target genes of foodborne pathogens to be comprehensive, rapid, inexpensive, accurate, of high selectivity, and good for screening probability.

scholarly journals Detection of Genes Involved in Biodegradation and Biotransformation in Microbial Communities by Using 50-Mer Oligonucleotide Microarrays

2004 ◽  
Vol 70 (7) ◽  
pp. 4303-4317 ◽  
Author(s):  
Sung-Keun Rhee ◽  
Xueduan Liu ◽  
Liyou Wu ◽  
Song C. Chong ◽  
Xiufeng Wan ◽  
...  
Keyword(s):  
Microbial Community ◽  
Real Time ◽  
Real Time Pcr ◽  
Genomic Dna ◽  
Target Genes ◽  
Metal Resistance ◽  
Detection Sensitivity ◽  
Pcr Analysis ◽  
Polyaromatic Hydrocarbon ◽  
Soil Microcosms

ABSTRACT To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50°C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was ∼5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 × 107 cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r2 = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r2 = 0.74). In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.

scholarly journals Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI) – ITI target genes in mouse ovary identified by microarray analysis

2004 ◽  
Vol 183 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Mika Suzuki ◽  
Hiroshi Kobayashi ◽  
Yoshiko Tanaka ◽  
Naohiro Kanayama ◽  
Toshihiko Terao
Keyword(s):  
Real Time ◽  
Trypsin Inhibitor ◽  
Target Genes ◽  
Reproductive Function ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Reproductive Process ◽  
Female Mice ◽  
Retinoid Metabolism ◽  
Regulatory Effects

Bikunin, a Kunitz-type protease inhibitor, is found in blood and urine. It has been established by two laboratories independently that the bikunin knockout female mice display a severe reduction in fertility: the cumulus oophorus has a defect in forming the extracellular hyaluronan-rich matrix during expansion. Proteins of the inter-alpha-trypsin inhibitor (ITI) family are eliminated in mice in which the bikunin gene has been inactivated, since bikunin is essential for their biosynthesis. Proteins of the ITI family may contribute to the microenvironment in which ovulation takes place. It is not clear, however, whether a single mechanism affects the reproductive function including ovulation. For identifying the full repertoire of the ITI deficiency-related genes, a cDNA microarray hybridization screening was conducted using mRNA from ovaries of wild-type or bik−/− female mice. A number of genes were identified and their regulation was confirmed by real-time RT-PCR analysis. Our screen identified that 29 (0.7%) and 5 genes (0.1%) of the genes assayed were, respectively, up- and down-regulated twofold or more. The identified genes can be classified into distinct subsets. These include stress-related, apoptosis-related, proteases, signaling molecules, aging-related, cytokines, hyaluronan metabolism and signaling, reactive oxygen species-related, and retinoid metabolism, which have previously been implicated in enhancing follicle development and/or ovulation. Real-time RT-PCR analysis confirmed that these genes were up- and down-regulated two- to tenfold by bikunin knockout. These studies demonstrate that proteins of the ITI family may exert potent regulatory effects on a major physiological reproductive process, ovulation.

ID: 316 Real-time PCR analysis of BCL2L12, a novel member of BCL2 family of the apoptosis-related genes, in human leukemia.

2006 ◽  
Vol 4 (s1) ◽  
pp. 82-82
Author(s):  
K. Floros ◽  
H. Thomadaki ◽  
S. Pavlovic ◽  
M. Talieri ◽  
M. Colovic ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Human Leukemia ◽  
Bcl2 Family

Diagnosis and Causative Species of Visceral Leishmaniasis in Southwest Saudi Arabia

2021 ◽  
Author(s):  
Aymen Abdelhaleem ◽  
Nabil Dhayhi ◽  
Mohamed Salih Mahfouz ◽  
Ommer Daffalla ◽  
Mansour Mubarki ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Visceral Leishmaniasis ◽  
Real Time ◽  
Real Time Pcr ◽  
Leishmania Donovani ◽  
Target Genes ◽  
Sample Volume ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Robust Evidence

Visceral leishmaniasis (VL) is the most severe clinical form of the disease and has been reported in the Jazan region of southwest Saudi Arabia. This study aimed to diagnose VL by real-time polymerase chain reaction (PCR) and the direct agglutination test (DAT) and to identify the causative Leishmania species. A total of 80 participants, including 30 suspected VL patients, 30 healthy endemic control individuals, and 20 malaria disease controls, were enrolled in this study. Blood samples were collected and tested for Leishmania DNA by real-time PCR and for antibody by the DAT. Sequencing of some amplified PCR products was used to identify the causative Leishmania species. The diagnosis of VL was successfully achieved by both real-time PCR and by DAT with 100% sensitivity. Leishmania donovani and Leishmania infantum species were detected by sequencing both by the kDNA and ITS1 target genes, followed a BLASTn search. The detection of VL antibody by the DAT followed by the confirmatory detection of Leishmania DNA in patient blood by PCR could promote the adoption of the much less invasive and more sensitive methods for the routine diagnosis of VL. Further study with high sample volume to evaluate the PCR and the DAT are needed, to generate more robust evidence. Based on the sequencing results, emerging studies on VL should focus on the causative Leishmania species, reservoirs, and vectors that are important in the study area.

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