Detection of Chlamydia trachomatis and Neisseria gonorrhoeae Using Multiplex Strand Invasion Based Amplification (mSIBA).

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the most prevalent causes of sexually transmitted infections (STIs) worldwide. Timely and accurate diagnosis plays an important role in deciding appropriate treatment and preventing the spread of the infection. Strand invasion based amplification (SIBA), is an established isothermal nucleic acid amplification method for the rapid and accurate detection of infectious diseases. SIBA was applied for the simultaneous detection of CT and NG in less than 1 h. The multiplex SIBA (mSIBA) method displayed high analytical sensitivity and specificity for the detection of CT and NG. Since the method is performed at low and constant temperature, it can therefore be run on portable instruments. SIBA enables rapid screening for CT and NG within point-of-care or central laboratory settings.

Application of Loop-Mediated Isothermal Amplification Assay for the Detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

The loop-mediated isothermal amplification (LAMP) is one of the most widely used isothermal nucleic acid amplification techniques due to it its simplicity and adaptability within limited resource or point-of-care settings. Here, LAMP was utilized for the rapid amplification and detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

Simultaneous Detection of Chlamydia trachomatis and Neisseria gonorrhoeae Using Real-Time Multiplex qPCR Assay.

Real-time polymerase chain reaction (qPCR) has become a prominent technique in life science research particularly for the detection and monitoring of biomarkers, pathogens, and environmental contaminants. Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the major pathogens responsible for sexually transmitted diseases (STDs). Here, multiplex qPCR was utilized for the amplification and detection Chlamydia trachomatis and Neisseria gonorrhoeae within the same reaction tube.

Detection of human rhinoviruses by reverse transcription strand invasion based amplification method (RT-SIBA).

BACKGROUND: Rhinovirus (RV), a major cause of respiratory infection in humans, imposes an enormous economic burden due to the direct and indirect costs associated with the illness. Accurate and timely diagnosis is crucial for deciding the appropriate clinical approach and minimizing unnecessary prescription of antibiotics. Diagnosis of RV is extremely challenging due to genetic and serological variability among its numerous types and their similarity to enteroviruses. OBJECTIVE: We sought to develop a rapid nucleic acid test that can be used for the detection of Rhinovirus within both laboratory and near patient settings. STUDY DESIGN: We developed and evaluated a novel isothermal nucleic acid amplification method called Reverse Transcription Strand Invasion-Based Amplification (RT-SIBA) to rapidly detect Rhinovirus from clinical specimens. RESULT: The method, RT-SIBA, detected RV in clinical specimens with high analytical sensitivity (96%) and specificity (100%). The time to positive result was significantly shorter for the RV RT-SIBA assay than for a reference RV nucleic acid amplification method (RT-qPCR). CONCLUSION: The rapid detection time of the RV SIBA assay, as well as its compatibility with portable instruments, will facilitate prompt diagnosis of infection and thereby improve patient care.

Molecular Detection of Streptococcus pyogenes by Strand Invasion Based Amplification Assay.

INTRODUCTION: Streptococcus pyogenes (group A Streptococcus, GAS) is responsible for a variety of highly communicable infections, accounting for 5-15 and 20-30% of sore throat hospital visits in adults and children, respectively. Prompt diagnosis of GAS can improve the quality of patient care and minimize the unnecessary use of antibiotics. OBJECTIVE: Our objective was to develop an alternative nucleic acid amplification method for the diagnosis of GAS. METHOD: We developed and evaluated a strand invasion based amplification (SIBA) assay to rapidly and specifically detect GAS. The performance of the developed GAS SIBA assay was compared with an established GAS polymerase chain reaction (PCR) assay. RESULTS: The GAS SIBA assay detected small amounts (ten copies) of S. pyogenes DNA within 13 min. The rapid detection time was achieved in part by optimization of magnesium concentration and reaction temperature. The sensitivity and specificity of the GAS SIBA assay for detection of S. pyogenes from clinical specimens were both 100%, and clinical specimens were detected within ~ 8 min of starting the reaction. CONCLUSION: Because the GAS SIBA assay is performed at low and constant temperature, it can be used both in centralized laboratories and for point-of-care testing. Furthermore, given its short detection time and strong analytical performance, the GAS SIBA assay could help to improve patient care and minimize unnecessary prescription of antibiotics.

Development and evaluation of a rapid nucleic acid amplification method to detect influenza A and B viruses in human respiratory specimens.

Isothermal nucleic acid amplification methods can potentially shorten the amount of time required to diagnose influenza. We developed and evaluated a novel isothermal nucleic acid amplification method, RT-SIBA to rapidly detect and differentiate between influenza A and B viruses in a single reaction tube. The performance of the RT-SIBA Influenza assay was compared with two established RT-PCR methods. The sensitivities of the RT-SIBA, RealStar RT-PCR, and CDC RT-PCR assays for the detection of influenza A and B viruses in the clinical specimens were 98.8%, 100%, and 89.3%, respectively. All three assays demonstrated a specificity of 100%. The average time to positive result was significantly shorter with the RT-SIBA Influenza assay (<20 min) than with the two RT-PCR assays (>90 min). The method can be run using battery-operated, portable devices with a small footprint and therefore has potential applications in both laboratory and near-patient settings.

Clinical evaluation of a novel and simple-to-use molecular platform for diagnosis of respiratory syncytial virus.

Rapid molecular diagnostic testing for respiratory infections can improve patient care and minimize unnecessary prescriptions of antibiotics. We present the preliminary clinical evaluation of Orion GenRead® RSV, a novel, rapid, and easy-to-use molecular test for the diagnosis of respiratory syncytial virus (RSV) infection. The sensitivity and specificity of Orion GenRead RSV were 99% and 100%, respectively. Orion GenRead RSV detected RSV-positive specimens within 15 min. The performance of Orion GenRead RSV was similar to that of the reference method and this test could rapidly detect RSV within minutes. Orion GenRead RSV is applicable for near-patient testing.

Rapid and sensitive real-time assay for the detection of respiratory syncytial virus using RT-SIBA®.

BACKGROUND: Respiratory syncytial virus (RSV) is one of the most common causes of respiratory tract infections among young children and the elderly. Timely and accurate diagnosis of respiratory tract infections improves patient care and minimizes unnecessary prescriptions of antibiotics. We sought to develop a rapid nucleic acid tests for the detection of RSV within minutes, while retaining the high sensitivity achieved with RT-PCR. METHODS: We developed and evaluated a reverse transcription isothermal nucleic acid amplification method, reverse transcription strand invasion based amplification (RT-SIBA), for the rapid detection of RSV. RESULTS: The developed RT-SIBA assay showed good sensitivity by detecting as few as 10 copies of RSV RNA within 20 min compared with reverse transcription polymerase chain reaction, which took approximately 2 h. The performance of the RT-SIBA RSV assay was further investigated using nasopharyngeal swab specimens. The RT-SIBA assay had a sensitivity of 100% (25/25) and a specificity of 100% (15/15). CONCLUSION: RT-SIBA did not require highly purified RNA for the rapid detection of RSV and was therefore compatible with rapid specimen processing methods. This reduces the complexity of specimen preparation and further shortens the total amount of time needed to detect RSV in clinical specimens. The developed RT-SIBA assay for RSV could be a useful tool for prompt management of this infection.

Rapid molecular diagnostic test for Zika virus with low demands on sample preparation and instrumentation.

Zika virus has only recently gained attention due to recent large outbreaks worldwide. An easy to use nucleic acid amplification test could play an important role in the early detection of the infection and patient management. Here, we report a rapid and robust isothermal nucleic acid amplification assay for the detection of Zika virus. The method is cost-effective and compatible with portable instrumentation, enabling near patient testing and field use.

Multiplex Strand Invasion Based Amplification (mSIBA) assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

Nucleic acid amplification tests have become a common method for diagnosis of STIs due to their improved sensitivity over immunoassays and traditional culture-based methods. Isothermal nucleic acid amplification methods offer significant advantages over polymerase chain reaction (PCR) because they do not require sophisticated instruments needed for thermal cycling of PCR. We recently reported a novel isothermal nucleic acid amplification method, Strand Invasion-Based Amplification (SIBA), which exhibited high analytical sensitivity and specificity for amplification of DNA. However, because the reactions were detected using an intercalating dye, this method was only suitable for amplifying a single genomic target. Here, we report the development of multiplexed SIBA (mSIBA) that allows simultaneous detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and an internal control in the same reaction tube. SIBA is compatible with probes, allowing the detection of multiple DNA targets in the same reaction tube. The IC was developed to assess the quality of the isolated DNA and the integrity of the enzyme system, as well as to test oligonucleotides. The mSIBA assay retained high analytical sensitivity and specificity for the detection of CT and NG. The development of mSIBA enables rapid screening for CT and NG within point-of-care or central laboratory settings.

Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

Attenuation of Vibrio fischeri quorum sensing using rationally designed polymers.

A first attempt to attenuate the quorum sensing (QS) of a marine heterotroph microorganism, Vibrio fischeri , using signal molecule-sequestering polymers (SSPs) is presented. A set of rationally designed polymers with affinity toward a signal molecule of V. fischeri , N-(beta-ketocaproyl)-l-homoserine lactone (3-oxo-C6-AHL) was produced. It is reported that computationally designed polymers could sequester a signal molecule of V. fischeri and prevent QS-controlled phenotypes (in this case, bioluminescence) from being up-regulated. It was proven that the attenuation of bioluminescence of V. fischeri was due to sequestration of the signal molecule by specific polymers and not due to the toxicity of polymer or nonspecific depletion of nutrients. The ability to disrupt the bacterial communication using easy to synthesize and chemically inert polymers could provide a new concept for the development of pharmaceuticals and susceptible device coatings such as catheters.

The polymer physics and chemistry of microbial cell attachment and adhesion.

The attachment of microbial cells to solid substrata is a primary ecological strategy for the survival of species and the development of specific activity and function within communities. An hypothesis arising from a biological sciences perspective may be stated as follows: The attachment of microbes to interfaces is controlled by the macromolecular structure of the cell wall and the functional genes that are induced for its biological synthesis. Following logically from this is the view that diverse attached cell behaviour is mediated by the physical and chemical interactions of these macromolecules in the interfacial region and with other cells. This aspect can be reduced to its simplest form by treating physico-chemical interactions as colloidal forces acting between an isolated cell and a solid or pseudo solid substratum. These forces can be analysed by established methods rooted in DLVO (Derjaguin, Landau, Verwey and Overbeek) theory. Such a methodology provides little insight into what governs changes in the behaviour of the cell wall attached to surfaces, or indeed other cells. Nor does it shed any light on the expulsion of macromolecules that modify the interface such as formation of slime layers. These physical and chemical problems must be treated at the more fundamental level of the structure and behaviour of the individual components of the cell wall, for example biosurfactants and extracellular polysaccharides. This allows us to restate the above hypothesis in physical sciences terms: Cell attachment and related cell growth behaviour is mediated by macromolecular physics and chemistry in the interfacial environment. Ecological success depends on the genetic potential to favourably influence the interface through adaptation of the macromolecular structure, We present research that merges these two perspectives. This is achieved by quantifying attached cell growth for genetically diverse model organisms, building chemical models that capture the variations in interfacial structure and quantifying the resulting physical interactions. Experimental observations combine aqueous chemistry techniques with surface spectroscopy in order to elucidate the cell wall structure. Atomic force microscopy methods quantify the physical interactions between the solid substrata and key components of the cell wall such as macromolecular biosurfactants. Our current approach focuses on considering individually mycolic acids or longer chain polymers harvested from cells, as well as characterised whole cells. This approach allows us to use a multifactorial approach to address the relative impact of the individual components of the cell wall in contact with model surfaces. We then combine these components to increase complexity step-wise, while comparing with the behaviour of entire cells. Eventually, such an approach should allow us to estimate and understand the primary factors governing microbial cell adhesion. Although the work addresses the cell-mineral interface at a fundamental level, the research is driven by a range of technology needs. The initial rationale was improved prediction of contaminant degradation in natural environments (soils, sediments, aquifers) for environmental cleanup. However, this area of research addresses a wide range of biotechnology areas including improved understanding of pathogen survival (e.g., in surgical environments), better process intensification in biomanufacturing (biofilm technologies) and new product development.

Characterization of the extracellular polymeric substances produced by Escherichia coli using infrared spectroscopic, proteomic, and aggregation studies.

The aim of this study was twofold: first, to characterize the free extracellular polymeric substances (EPS) and bound EPS produced by Escherichia coli during different growth phases in different media, and then to investigate the role of the free EPS in promoting aggregation. EPS was extracted from a population of E. coli MG1655 cells grown in different media composition (Luria-Bertani (LB) and Luria-Bertani with the addition of 0.5 w/v% glucose at the beginning of the growth phase (LBG)) and at different growth phases (6 and 24 h). The extracted EPS was characterized using Fourier transform infrared spectroscopy and further identified using one-dimensional gel-based electrophoresis and tandem mass spectrometry. E. coli MG1655 was found to produce significantly lower amounts of bound EPS compared to free EPS under all conditions. The protein content of free EPS increased as the cells progressed from the exponential to stationary phase when grown in LB or LBG, while the carbohydrate content only increased across the growth phases for cells grown in LBG. FTIR revealed a variation in the different functional groups such as amines, carboxyl, and phosphoryl groups for free EPS extracted at the different growth conditions. Over 500 proteins were identified in the free EPS, with 40 proteins common in all growth conditions. Proteins with functionality related to amino acid and carbohydrate metabolism, as well as cell wall and membrane biogenesis were among the highest proteins identified in the free EPS extracted from E. coli MG1655 under all growth and media conditions. The role of bound and free EPS was investigated using a standardized aggregation assay. Bound EPS did not contribute to aggregation of E. coli MG1655. The readdition of free EPS to E. coli MG1655 resulted in aggregation of the cells in all growth conditions. Free EPS extracted from the 24 h E. coli MG1655 cultures grown in LB had the greatest effect on aggregation of cells grow in LBG, with a 30% increase in aggregation observed.

Investigating the surface properties of Escherichia coli under glucose controlled conditions and its effect on aggregation.

Bacteria exist as aggregates or in biofilms to help with adaptation and protection from environmental stresses. The hypothesis that is tested in this paper is that the relative presence of glucose in the media, at the beginning of the growth phase, influences the surface chemistry of the cell, which as a consequence reduces the tendency for the cells to interact and form aggregates. In this study, we used Escherichia coli (E. coli) MG1655 as a model organism and measured the change in the surface chemistry of cells harvested at different growth phases, which had been cultured in Luria-Bertani media with and without the addition of glucose, using potentiometric titration and infrared spectroscopy. Cells, cultivated with the additional supplement of glucose at the beginning of the growth phase, displayed a higher concentration of bacterial surface functional groups and a variation in outer membrane proteins. As a consequence, the tendency for cell-to-cell attachment was significantly reduced. Our findings therefore revealed that glucose limits aggregation in E. coli MG1655 by altering the concentration of functional groups from macromolecules present on the bacterial surface.

Increased dicarbonyl metabolism in endothelial cells in hyperglycemia induces anoikis and impairs angiogenesis by RGD and GFOGER motif modification.

Chronic vascular disease in diabetes is associated with disruption of extracellular matrix (ECM) interactions with adherent endothelial cells, compromising cell survival and impairing vasculature structure. Loss of functional contact with integrins activates anoikis and impairs angiogenesis. The metabolic dysfunction underlying this vascular damage and disruption is unclear. Here, we show that increased modification of vascular basement membrane type IV collagen by methylglyoxal, a dicarbonyl glycating agent with increased formation in hyperglycemia, formed arginine-derived hydroimidazolone residues at hotspot modification sites in RGD and GFOGER integrin-binding sites of collagen, causing endothelial cell detachment, anoikis, and inhibition of angiogenesis. Endothelial cells incubated in model hyperglycemia in vitro and experimental diabetes in vivo produced the same modifications of vascular collagen, inducing similar responses. Pharmacological scavenging of methylglyoxal prevented anoikis and maintained angiogenesis, and inhibition of methylglyoxal metabolism with a cell permeable glyoxalase I inhibitor provoked these responses in normoglycemia. Thus, increased formation of methylglyoxal and ECM glycation in hyperglycemia impairs endothelial cell survival and angiogenesis and likely contributes to similar vascular dysfunction in diabetes.