scholarly journals ‘Ready Mixed’, improved nucleic acid amplification assays for the detection of Escherichia coli DNA and RNA

2019 ◽  
Vol 165 ◽  
pp. 105721 ◽  
Author(s):  
Jonathan S. McQuillan ◽  
Matthew W. Wilson
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Nucleic Acid Amplification ◽  
Dna And Rna

scholarly journals Pooled Nucleic Acid Amplification Test for Screening of Stool Specimens for Shiga Toxin-Producing Escherichia coli

2016 ◽  
Vol 54 (11) ◽  
pp. 2711-2715
Author(s):  
Agatha N. Jassem ◽  
Frank Y. Chou ◽  
Cathevine Yang ◽  
Matthew A. Croxen ◽  
Katarina D. M. Pintar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Shiga Toxin ◽  
Large Scale ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Accurate Estimation ◽  
Content Type ◽  
Pooling Strategy ◽  
Stool Specimens

Shiga toxin-producingEscherichia coli(STEC)-associated enteric illness is attributed to O157 and non-O157 serotypes; however, traditional culture-based methods underdetect non-O157 STEC. Labor and cost of consumables are major barriers to implementation of the CDC recommendation to test all stools for both O157 and non-O157 serotypes. We evaluated the feasibility of a pooled nucleic acid amplification test (NAAT) as an approach for screening stool specimens for STEC. For retrospective evaluation, 300 stool specimens were used to create pools of 10 samples each. The sensitivity was 83% for the preenrichment pooling strategy and 100% for the postenrichment pooling strategy compared with those for individual NAAT results. The difference in cycle threshold (CT) between individual and pooled NAAT results for specimens was significantly lower and more consistent for postenrichment pooling (stx1mean = 3.90,stx2mean = 4.28) than those for preenrichment pooling (excluding undetected specimens;stx1mean = 9.34,stx2mean = 8.96) (P≤ 0.0013). Cost of consumables and labor time savings of 48 to 81% and 6 to 66%, respectively, were estimated for the testing of 90 specimens by the postenrichment pooled NAAT strategy on the basis of an expected 1 to 2% positivity rate. A 30-day prospective head-to-head clinical trial involving 512 specimens confirmed the sensitivity and labor savings associated with the postenrichment pooled NAAT strategy. The postenrichment pooled NAAT strategy described here is suitable for efficient large-scale surveillance of all STEC serotypes. Comprehensive detection of STEC will result in accurate estimation of STEC burden and, consequently, appropriate public health interventions.

scholarly journals Clinical Evaluation and Cost Analysis of Great Basin Shiga Toxin Direct Molecular Assay for Detection of Shiga Toxin-Producing Escherichia coli in Diarrheal Stool Specimens

2016 ◽  
Vol 55 (2) ◽  
pp. 519-525 ◽  
Author(s):  
Matthew L. Faron ◽  
Nathan A. Ledeboer ◽  
Jessica Connolly ◽  
Paul A. Granato ◽  
Brenda R. Alkins ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Shiga Toxin ◽  
Nucleic Acid Amplification ◽  
Molecular Assay ◽  
Content Type ◽  
E Coli ◽  
Serotype O ◽  
Stool Specimens ◽  
O 157

ABSTRACTThe Shiga Toxin Direct molecular assay (ST Direct) relies on nucleic acid amplification and solid array-based amplicon detection to identify Shiga toxin-producingEscherichia coli(STEC) in preserved stool specimens. Genes encoding Shiga toxin (stx1andstx2), as well as theE. coliserotype O:157-specific markerrfbE, are simultaneously detected within 2 h. ST Direct was evaluated using 1,084 prospectively collected preserved stool specimens across five clinical centers. An additional 55 retrospectively collected, frozen specimens were included to increase the number of positive specimens evaluated. Results were compared to results from routine culture and an enzyme immunoassay (EIA) specific for the recovery and identification of STEC. ST Direct was found to be 93.2% sensitive and 99.3% specific for detection ofstx1andstx2and 95.7% sensitive and 99.3% specific for detection ofE. coliserotype O:157. All specimens with false-positive results were found to containstx1orstx2or were found to be positive for serotype O:157 when analyzed using alternative molecular methods. All 4 false-negativestx1orstx2results were reported for frozen, retrospectively tested specimens. In all cases, the specimens tested positive forstxby an alternative FDA-cleared nucleic acid amplification test (NAAT) but were negative forstx1andstx2following nucleic acid sequence analysis. Based on these data, culture and EIA-based methods for detection of STEC are only 33% sensitive compared to molecular tests. A retrospective cost analysis demonstrated 59% of the cost of routine stool culture to be attributable to the identification of STEC. Taken together, these data suggest that ST Direct may provide a cost-effective, rapid molecular alternative to routine culture for the identification of STEC in preserved stool specimens.

SNAPflex: a paper-and-plastic device for instrument-free RNA and DNA extraction from whole blood

2020 ◽  
Author(s):  
Nikunja Kolluri ◽  
Nikolas Albarran ◽  
Andy Fan ◽  
Alex Olson ◽  
Manish Sagar ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Whole Blood ◽  
Extraction Methods ◽  
Simulated Patient ◽  
Nucleic Acid Amplification ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Long Term Stability ◽  
Dna And Rna ◽  
Resource Limited

AbstractNucleic acid amplification tests (NAATs), which amplify and detect pathogen nucleic acids, are vital methods to diagnose diseases, particularly in cases where patients exhibit low levels of infection. For many blood-borne pathogens such as HIV or Plasmodium, it is necessary to first extract pathogen RNA or DNA from patient blood prior to analysis with NAATs. Traditional nucleic acid extraction methods are expensive, resource-intensive and are often difficult to deploy to resource-limited areas where many blood-borne infections are widespread. Here, we describe a portable, paper-and-plastic device for instrument-free nucleic acid extraction from whole blood, which we call SNAPflex, that builds upon our previous work extracting RNA in a 2D platform from nasopharyngeal swabs. We demonstrated improved extraction of HIV RNA from simulated patient samples compared to traditional extraction methods and long-term stability of extracted RNA without the need for cold storage. We further demonstrated successful extraction and recovery of Plasmodium falciparum DNA from simulated patient samples with superior recovery compared to existing extraction methods. The SNAPflex device extracts and purifies DNA and RNA from whole blood which can be amplified with traditional NAATs, and was designed to easily manufacture and integrate into existing health systems.

Rapid and simultaneous analysis of twelve virulence factor genes by a microfluidic-CFPA chip for identifying diarrheagenic Escherichia coli

The Analyst ◽  
2020 ◽  
Vol 145 (11) ◽  
pp. 3814-3821 ◽  
Author(s):  
Bin Yang ◽  
Yiling Fan ◽  
Yang Li ◽  
Jun Yan ◽  
Xueen Fang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Fluorescent Probe ◽  
Virulence Factor ◽  
Microfluidic System ◽  
Simultaneous Analysis ◽  
Nucleic Acid Amplification ◽  
Isothermal Nucleic Acid Amplification ◽  
Diarrheagenic Escherichia Coli ◽  
Virulence Factor Genes

An integrated microfluidic system based on circular fluorescent probe-mediated isothermal nucleic acid amplification for identification of five diarrheagenic Escherichia coli strains has been developed.

Infection of Escherichia coli with bacteriophages

1969 ◽  
Vol 27 ◽  
pp. 370-371
Author(s):  
Manfred E. Bayer
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Cell Surface ◽  
Virus Type ◽  
Infection Process ◽  
Adsorption Site ◽  
The Other ◽  
Specific Receptors ◽  
Bacterial Receptors

The first step in the infection of a bacterium by a virus consists of a collision between cell and bacteriophage. The presence of virus-specific receptors on the cell surface will trigger a number of events leading eventually to release of the phage nucleic acid. The execution of the various "steps" in the infection process varies from one virus-type to the other, depending on the anatomy of the virus. Small viruses like ØX 174 and MS2 adsorb directly with their capsid to the bacterial receptors, while other phages possess attachment organelles of varying complexity. In bacteriophages T3 (Fig. 1) and T7 the small conical processes of their heads point toward the adsorption site; a welldefined baseplate is attached to the head of P22; heads without baseplates are not infective.

High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level

1995 ◽  
Vol 53 ◽  
pp. 752-753
Author(s):  
B.A. Hamkalo ◽  
S. Narayanswami ◽  
A.P. Kausch
Keyword(s):  
Nucleic Acid ◽  
Interphase Nucleus ◽  
Genome Mapping ◽  
Precise Location ◽  
Electron Microscope Level ◽  
Rna Sequences ◽  
Fundamental Interest ◽  
Whole Cells ◽  
Dna And Rna

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.

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