An expanded RT-PCR melting temperature coding assay to rapidly identify all known SARS-CoV-2 variants and sub-variants of concern.

The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs, now modified to include detection of Delta (B.1.

Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays.

Background: Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings.

Methods: We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2.

Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test.

Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2.

Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System.

, , and are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of , , and directly from patient blood samples was developed using the GeneXpert system.

The New Xpert MTB/RIF Ultra: Improving Detection of and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing.

The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance.

Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System.

Francisella tularensis is a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitive F.

A Novel Sample Processing Method for Rapid Detection of Tuberculosis in the Stool of Pediatric Patients Using the Xpert MTB/RIF Assay.

Background: Tuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available.

Methods: We developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay.

Exploring alternative biomaterials for diagnosis of pulmonary tuberculosis in HIV-negative patients by use of the GeneXpert MTB/RIF assay.

The utility of the GeneXpert MTB/RIF (Xpert) assay for detection of Mycobacterium tuberculosis in sputum samples has been extensively studied. However, the performance of the Xpert assay as applied to other readily accessible body fluids such as exhaled breath condensate (EBC), saliva, urine, and blood has not been established. We used the Xpert assay to test EBC, saliva, urine, and blood samples from HIV-negative, smear- and culture-positive pulmonary tuberculosis (TB) patients for the presence of M.

Detection of Mycobacterium tuberculosis in blood by use of the Xpert MTB/RIF assay.

We have developed a novel blood lysis-centrifugation approach for highly sensitive Mycobacterium tuberculosis detection in large volumes of blood with the Xpert MTB/RIF assay. One through 20 ml of blood was spiked with 0.25 to 10 CFU/ml of the M.

Evaluation of Xpert MTB/RIF for detection of tuberculosis from blood samples of HIV-infected adults confirms Mycobacterium tuberculosis bacteremia as an indicator of poor prognosis.

Tuberculosis (TB) remains a leading cause of death among HIV-infected adults, in part because of delayed diagnosis and therefore delayed initiation of treatment. Recently, the Gene-Xpert platform, a rapid, PCR-based diagnostic platform, has been validated for the diagnosis of TB with sputum. We have evaluated the Xpert MTB/RIF assay for the diagnosis of Mycobacterium tuberculosis bacteremia and investigated its impact on clinical outcomes.

Light-scattering sensor for real-time identification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate.

The three most common pathogenic species of Vibrio, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus, are of major concerns due to increased incidence of water- and seafood-related outbreaks and illness worldwide. Current methods are lengthy and require biochemical and molecular confirmation. A novel label-free forward light-scattering sensor was developed to detect and identify colonies of these three pathogens in real time in the presence of other vibrios in food or water samples.

Rapid, high-throughput detection of rifampin resistance and heteroresistance in Mycobacterium tuberculosis by use of sloppy molecular beacon melting temperature coding.

Rifampin resistance in Mycobacterium tuberculosis is largely determined by mutations in an 80-bp rifampin resistance determining region (RRDR) of the rpoB gene. We developed a rapid single-well PCR assay to identify RRDR mutations. The assay uses sloppy molecular beacons to probe an asymmetric PCR of the M.

Highly sensitive detection of Staphylococcus aureus directly from patient blood.

Background: Rapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing-polymerase chain reaction (PCR) platform as a model diagnostic system.

Methodology/principal Findings: We compared a short 128 bp amplicon hemi-nested PCR and a relatively shorter 79 bp amplicon nested PCR targeting the S.

Containment of bioaerosol infection risk by the Xpert MTB/RIF assay and its applicability to point-of-care settings.

The recently introduced Xpert MTB/RIF assay (Xpert) has point-of-care potential, but its capacity for biohazard containment remained to be studied. We compared the bioaerosols generated by the Xpert assay to acid-fast bacillus (AFB) microscope slide smear preparation. The Xpert assay sample treatment reagent (SR) was also studied for its sterilizing capacity, stability, and effect on assay sensitivity after prolonged treatment.

Characterization of Listeria monocytogenes isolates of food and human origins from Brazil using molecular typing procedures and in vitro cell culture assays.

The spreading of diseases through foods is a worldwide concern. Here, molecular and in vitro cell-culture assays were employed to characterize 63 Brazilian Listeria monocytogenes isolates (food, 47; clinical, 16). Serotype 4b was the most predominant (49%) followed by (1/2)b (30%), (1/2)a (10%), (1/2)c (6%), 3c (3%) and 3b (2%).

Label-free detection of multiple bacterial pathogens using light-scattering sensor.

Technologies for rapid detection and classification of bacterial pathogens are crucial for securing the food supply. This report describes a light-scattering sensor capable of real-time detection and identification of colonies of multiple pathogens without the need for a labeling reagent or biochemical processing. Bacterial colonies consisting of the progeny of a single parent cell scatter light at 635 nm to produce unique forward-scatter signatures.

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line.

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2-3 days to provide confirmatory results.

Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes.

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria-specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates.

Rapid electrical lysis of bacterial cells in a microfluidic device.

Electrical lysis of biological cells on a microfluidic platform has evoked significant interest because of its applications in rapid recovery of intracellular contents such as nucleic acids or proteins without introducing lytic agents. Applying a direct current (DC) field for cell lysis typically requires field strength of 1-10 kV/cm, which is dependent on the cell type: prokaryotes or eukaryotes. Bubble generation and Joule heating can often be induced under such high field strengths.

Analysis of time-resolved scattering from macroscale bacterial colonies.

We investigate the relationship of incubation time and forward-scattering signature for bacterial colonies grown on solid nutrient surfaces. The aim of this research is to understand the colony growth characteristics and the corresponding evolution of the scattering patterns for a variety of pathogenic bacteria relevant to food safety. In particular, we characterized time-varying macroscopic and microscopic morphological properties of the growing colonies and modeled their optical properties in terms of two-dimensional (2-D) amplitude and phase modulation distributions.

High speed classification of individual bacterial cells using a model-based light scatter system and multivariate statistics.

We describe a model-based instrument design combined with a statistical classification approach for the development and realization of high speed cell classification systems based on light scatter. In our work, angular light scatter from cells of four bacterial species of interest, Bacillus subtilis, Escherichia coli, Listeria innocua, and Enterococcus faecalis, was modeled using the discrete dipole approximation. We then optimized a scattering detector array design subject to some hardware constraints, configured the instrument, and gathered experimental data from the relevant bacterial cells.

Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier.

Biological microparticles, including bacteria, scatter light in all directions when illuminated. The complex scatter pattern is dependent on particle size, shape, refraction index, density, and morphology. Commercial flow cytometers allow measurement of scattered light intensity at forward and perpendicular (side) angles (2 degrees View Article and Find Full Text PDF

Biophysical modeling of forward scattering from bacterial colonies using scalar diffraction theory.

A model for forward scattering from bacterial colonies is presented. The colonies of interest consist of approximately 10(12) - 10(13) individual bacteria densely packed in a configuration several millimeters in diameter and approximately 0.1-0.

Optical forward-scattering for detection of Listeria monocytogenes and other Listeria species.

We demonstrate here the development of a non-invasive optical forward-scattering system, called 'scatterometer' for rapid identification of bacterial colonies. The system is based on the concept that variations in refractive indices and size, relative to the arrangement of cells in bacterial colonies growing on a semi-solid agar surface will generate different forward-scattering patterns. A 1.