Integrating programmable DNAzymes with electrical readout for rapid and culture-free bacterial detection using a handheld platform.

The detection and identification of bacteria currently rely on enrichment steps such as bacterial culture and nucleic acid amplification to increase the concentration of target analytes. These steps increase assay time, cost and complexity, making it difficult to realize a truly rapid point-of-care test. Here we report the development of an electrical assay that uses electroactive RNA-cleaving DNAzymes (e-RCDs) to identify specific bacterial targets and subsequently release a DNA barcode for transducing a signal onto an electrical chip. Integrating e-RCDs into a two-channel electrical chip with nanostructured electrodes provides the analytical sensitivity and specificity needed for clinical analysis. The e-RCD assay is capable of detecting 10 CFU (equivalent to 1,000 CFU ml-1) of Escherichia coli selectively from a panel containing multiple non-specific bacterial species. Clinical evaluation of this assay using 41 patient urine samples demonstrated a diagnostic sensitivity of 100% and specificity of 78% at an analysis time of less than one hour compared with the several hours needed for currently used culture-based methods.

Poly(indole-5-carboxylic acid)/reduced graphene oxide/gold nanoparticles/phage-based electrochemical biosensor for highly specific detection of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis is an enteric bacterium causing yersiniosis in humans. The existing Yersinia pseudotuberculosis detection methods are time-consuming, requiring a sample pretreatment step, and are unable to discriminate live/dead cells. The current work reports a phage-based electrochemical biosensor for rapid and specific detection of Yersinia pseudotuberculosis. The conductive poly(indole-5-carboxylic acid), reduced graphene oxide, and gold nanoparticles are applied for surface modification of the electrode. They possess ultra-high redox stability and retain 97.7% of current response after performing 50 consecutive cycles of cyclic voltammetry.The specific bacteriophages vB_YepM_ZN18 we isolated from hospital sewage water were immobilized on modified electrodes by Au-NH2 bond between gold nanoparticles and phages. The biosensor fabricated with nanomaterials and phages were utilized to detect Yersinia pseudotuberculosis successfully with detection range of 5.30 × 102 to 1.05 × 107 CFU mL-1, detection limit of 3 CFU mL-1, and assay time of 35 min. Moreover, the biosensor can specifically detect live Yersinia pseudotuberculosis without responding to phage-non-host bacteria and dead Yersinia pseudotuberculosis cells. These results suggest that the proposed biosensor is a promising tool for the rapid and selective detection of Yersinia pseudotuberculosis in food, water, and clinical samples.

Highly sensitive and label-free digital detection of whole cell E. coli with Interferometric Reflectance Imaging.

Bacterial infectious diseases are a major threat to human health. Timely and sensitive pathogenic bacteria detection is crucial in bacterial contaminations identification and preventing the spread of infectious diseases. Due to limitations of conventional bacteria detection techniques there have been concerted research efforts towards developing new biosensors. Biosensors offering label-free, whole bacteria detection are highly desirable over those relying on label-based or pathogenic molecular components detection. The major advantage is eliminating the additional time and cost required for labeling or extracting the desired bacterial components. Here, we demonstrate rapid, sensitive and label-free Escherichia coli (E. coli) detection utilizing interferometric reflectance imaging enhancement allowing visualizing individual pathogens captured on the surface. Enabled by our ability to count individual bacteria on a large sensor surface, we demonstrate an extrapolated limit of detection of 2.2 CFU/ml from experimental data in buffer solution with no sample preparation. To the best of our knowledge, this level of sensitivity for whole E. coli detection is unprecedented in label-free biosensing. The specificity of our biosensor is validated by comparing the response to target bacteria E. coli and non-target bacteria S. aureus, K. pneumonia and P. aeruginosa. The biosensor's performance in tap water proves that its detection capability is unaffected by the sample complexity. Furthermore, our sensor platform provides high optical magnification imaging and thus validation of recorded detection events as the target bacteria based on morphological characterization. Therefore, our sensitive and label-free detection method offers new perspectives for direct bacterial detection in real matrices and clinical samples.

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA).

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers' safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 °C, 85 °C, or 95 °C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 µL of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of <1 log10eCFU/mL (i.e., <10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

Rapid and accurate detection of Escherichia coli O157:H7 in beef using microfluidic wax-printed paper-based ELISA.

Escherichia coli O157:H7 is a severe foodborne pathogen that causes lots of life-threatening diseases. In the search for a rapid, sensitive, portable and low-cost method to detect this pathogen, we developed a wax-printed paper-based enzyme-linked immunosorbent assay (P-ELISA) based on microfluidic paper-based analytical devices (µPADs), with the whole operation time being less than 3 h and only needing 5 µl samples for detection. The limit of detection (LOD) of E. coli O157:H7 reached 104 CFU ml-1, which is an order of magnitude higher than that of conventional ELISA (C-ELISA). The LOD in artificially contaminated beef samples is 1 CFU per 25 g after enriching the culture for 8 h. This method is superior to the molecular biology method in detection sensitivity and superior to C-ELISA and the national standard method in detection time and cost. Thus, the established P-ELISA method has good sensitivity, specificity and repeatability. It can be suitable for point-of-care testing without expensive and bulky instruments and can also provide a platform for detecting other pathogens, especially in areas that lack advanced clinical equipment.

A fully integrated bacterial pathogen detection system based on count-on-a-cartridge platform for rapid, ultrasensitive, highly accurate and culture-free assay.

Rapid, sensitive and accurate point-of-care-testing (POCT) of bacterial load from a variety of samples can help prevent human infections caused by pathogenic bacteria and mitigate their spreading. However, there is an unmet demand for a POCT device that can detect extremely low concentrations of bacteria in raw samples. Herein, we introduce the 'count-on-a-cartridge' (COC) platform for quantitation of the food-borne pathogenic bacteria Staphylococcus aureus. The system comprised of magnetic concentrator, sensing cartridge and fluorescent image reader with a built-in counting algorithm facilitated fluorescent microscopic bacterial enumeration in user-convenient manner with high sensitivity and accuracy within a couple of hours. The analytical performance of this assay is comparable to that of a standard plate count. The COC assay shows a sensitivity of 92.9% and specificity of 100% performed according to global microbiological criteria for S. aureus which is acceptable below 100 CFU/g in the food matrix. This culture-independent, rapid, ultrasensitive and highly accurate COC assay has great potential for places where prompt bacteria surveillance is in high demand.

Lab in a Pasteur pipette: low-cost, rapid and visual detection of Bacillus cereu using denaturation bubble-mediated strand exchange amplification.

Driven by a bright prospect for rapid, portable and cost-effective point-of-care testing, an assembled Pasteur pipette device to integrate nucleic acid extraction, amplification and detection was developed to detect B. cereus in a sample-to-answer format. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was chosen to perform isothermal amplification because it requires only a pair of primers and one Bst DNA polymerase. The established SEA can detect as low as 1.0 × 10-13 M genomic DNA of B. cereus, which was comparable with the previously reported method for B. cereus detection. The assembled Pasteur pipette allows sample-to-answer diagnostic in a simple, low-cost, portable, and disposable format. The inherent function of Pasteur pipette enables direct liquid handling without the need of extra pipettes, syringes or pumps. Visual readout was achieved by using a pH sensitive dye, further simplifying result judgment process. The detection limit for B. cereus is 1.0 × 104 CFU/mL in pure cultures, while the detection limit in artificially contaminated milk is 1.0 × 105 CFU/mL without enrichment and 1.0 × 100 CFU/mL following 12 h enrichment. Considering that typical cell counts in food samples associated to food poisoning are 1.0 × 105 to 1.0 × 108 CFU per gram/milliliter B. cereus, our Pasteur pipette is enough sensitive for answer-to-sample diagnosis of B. cereus even directly from foods without enrichment. The whole diagnostic procedure could be completed within 50 min, dramatically decreasing the detection time. In a word, the assembled Pasteur pipette device, combined with a homemade metal bath, possesses great potential for sample-to-answer application in resource-limited settings.

A microfluidic biosensor for online and sensitive detection of Salmonella typhimurium using fluorescence labeling and smartphone video processing.

Early screening of foodborne pathogens is a key to ensure food safety. In this study, we developed a microfluidic biosensor for online and sensitive detection of Salmonella based on immunomagnetic separation, fluorescence labeling and smartphone video processing. First, the immune magnetic nanoparticles were used to specifically separate and efficiently concentrate the target bacteria and the magnetic bacteria were formed. Then, the magnetic bacteria were labeled with the immune fluorescent microspheres and the fluorescent bacteria were formed. Finally, the fluorescent bacteria were continuously injected into the microfluidic chip on the smartphone-based fluorescent microscopic system, and the fluorescent spots were online counted using the smartphone App based on inter-frame difference algorithm to obtain the amount of the target bacteria. Under the optimal conditions, this proposed biosensor was able to quantitatively detect Salmonella typhimurium ranging from 1.4 × 102 to 1.4 × 106 CFU/mL, and its lower detection limit was 58 CFU/mL. This biosensor could be extended for detection of multiple foodborne pathogens using different fluorescent materials.

A portable microfluidic platform for rapid determination of microbial load and somatic cell count in milk.

Microfluidics systems that have been emerged in the last 20 years and used for processing the fluid in a microchannel structure at microliter levels are alternative to the conventional methods. The objective of the study is to develop a microfluidic platform for determination of the microbial load and the number of somatic cells in milk. For this purpose, a polydimethylsiloxane (PDMS) chip with a channel size of 300 µm × 60 µm was produced. Cells/bacteria labeled with fluorescent stain in milk were counted with the proposed microfluidic platform and the results were compared with the reference cell concentration/the bacterial counts by conventional method. It was found that our platform could count somatic and bacterial cells with an accuracy above 80% in 20 min run for each analysis. The portable overall platform has an overall dimension of 25x25x25 cm and weighs approximately 9 kg.

MC-Media Pad CC for Enumeration of Total Coliforms in a Variety of Foods.

BACKGROUND: Standard coliform count methods require the preparation of agar, the use of the pour-plate technique, the overlay of agar, and in some cases, the transfer of suspect colonies to broth medium for confirmation. The MC-Media Pad CC for the enumeration of coliforms is a ready-to-use dehydrated sheet medium with no agar preparation, no spreader, and no confirmation step required. OBJECTIVE: Using a paired study design, the MC-Media Pad CC was compared to standard method ISO 4832:2006 for 10 matrixes including raw ground pork, raw chicken, cream, cream cheese, ready-to-cook vegetable mix, vegetable juice, cooked prawns, crab pâté, ham sandwiches, and cooked rice. METHODS: Each matrix was tested at three levels of coliform contamination (approximately 102, 104, and 106 CFU/g). Five replicate 10 g test portions per level were tested in a paired comparison by the MC-Media Pad CC and ISO 4832:2006 methods. In addition, inclusivity/exclusivity, robustness, and product consistency and stability were evaluated. RESULTS: The candidate and reference methods demonstrated SDs ranging from 0.027 to 0.264 and 0.025 to 0.157, respectively. The difference of means ranged from -0.015 to 0.381, showing no practical difference between the methods. The MC-Media Pad CC detected 58/62 inclusivity strains and correctly excluded 26/31 exclusivity organisms, similar to the reference method. Robustness testing demonstrated no significant change in results when small changes were made to sample volume, incubation temperature, and incubation time. The product consistency study demonstrated no significant difference between lots of product and supported the 1.5 year shelf life. CONCLUSIONS: The results support the conclusions that the MC-Media Pad CC is a suitable alternative to the ISO 4832:2006 reference method for the matrixes examined and the data support AOAC Performance Tested MethodSM certification.

Label-free Bacteria Quantification in Blood Plasma by a Bioprinted Microarray Based Interferometric Point-of-Care Device.

Existing clinical methods for bacteria detection lack speed, sensitivity, and, importantly, point-of-care (PoC) applicability. Thus, finding ways to push the sensitivity of clinical PoC biosensing technologies is crucial. Here we report a portable PoC device based on lens-free interferometric microscopy (LIM). The device employs high performance nanoplasmonics and custom bioprinted microarrays and is capable of direct label-free bacteria ( E. coli) quantification. With only one-step sample handling we offer a sample-to-data turnaround time of 40 min. Our technology features detection sensitivity of a single bacterial cell both in buffer and in diluted blood plasma and is intrinsically limited by the number of cells present in the detection volume. When employed in a hospital setting, the device has enabled accurate categorization of sepsis patients (infectious SIRS) from control groups (healthy individuals and noninfectious SIRS patients) without false positives/negatives. User-friendly on-site bacterial clinical diagnosis can thus become a reality.

Cost-Effective Live Cell Density Determination of Liquid Cultured Microorganisms.

Live monitoring of microorganisms growth in liquid medium is a desired parameter for many research fields. A wildly used approach for determining microbial liquid growth quantification is based on light scattering as the result of the physical interaction of light with microbial cells. These measurements are generally achieved using costly table-top instruments; however, a live, reliable, and straight forward instrument constructed using parts that are inexpensive may provide opportunities for many researchers. Here, such an instrument has been constructed and tested. It consists of modular test tube holding chambers, each with a low power monochromatic light-emitting diode, and a monolithic photodiode. A microcontroller connects to all modular chambers to control the diodes, and send the live data to either an LCD screen, or a computer. This work demonstrate that this modular instrument can determine precise cell concentrations for the bacteria Escherichia coli and Pseudomonas syringae pv. tomato DC3000, as well as Saccharomyces cerevisiae yeast.

Mycobacterial Load Assay.

Tuberculosis is a difficult disease to treat, a process made more harder as tools to monitor treatment response only provide a result long after the patient has provided a sample. The mycobacterial load assay (MBLA) provides a simple molecular test to quantify and determine the viability of M. tuberculosis in human or other samples.

Improved detection of clinically relevant wound bacteria using autofluorescence image-guided sampling in diabetic foot ulcers.

Clinical wound assessment involves microbiological swabbing of wounds to identify and quantify bacterial species, and to determine microbial susceptibility to antibiotics. The Levine swabbing technique may be suboptimal because it samples only the wound bed, missing other diagnostically relevant areas of the wound, which may contain clinically significant bacteria. Thus, there is a clinical need to improve the reliability of microbiological wound sampling. To address this, a handheld portable autofluorescence (AF) imaging device that detects bacteria in real time, without contrast agents, was developed. Here, we report the results of a clinical study evaluating the use of real-time AF imaging to visualise bacteria in and around the wound bed and to guide swabbing during the clinical assessment of diabetic foot ulcers, compared with the Levine technique. We investigated 33 diabetic foot ulcers (n = 31 patients) and found that AF imaging more accurately identified the presence of moderate and/or heavy bacterial load compared with the Levine technique (accuracy 78% versus 52%, P = 0·048; adjusted diagnostic odds ratio 7·67, P < 0·00022 versus 3·07, P = 0·066) and maximised the effectiveness of bacterial load sampling, with no significant impact on clinical workflow. AF imaging may help clinicians better identify the wound areas with clinically significant bacteria, and maximise sampling of treatment-relevant pathogens.

Sensitive and rapid detection of pathogenic bacteria in small volumes using impedance spectroscopy technique.

We illustrate a novel impedance immunosensor which rapidly and sensitively detects typhoid-causing infectious bacteria Salmonella enterica serovar (Salmonella typhi) in 10 µL of sample volume. The bacteria are tagged with gold nanoparticles (Au NPs) via high-affinity antigen-antibody interactions for enhanced signal amplification and selectivity. The cell-particle bioconjugates are then subjected to alternating current (AC) electric fields applied through interdigitated microelectrodes. The immunosensor performance is optimized with respect to electric field frequency, cell concentration, incubation times and the type of blocking agent to achieve a low limit of detection (LOD) of 100 CFU/mL. The approach is extendable to a wide spectrum of clinical diseases and offers an efficient and cost-effective solution for point-of-care diagnosis.

Immunoliposome-based immunomagnetic concentration and separation assay for rapid detection of Cronobacter sakazakii.

This study aimed to develop an immunoliposome-based immunomagnetic concentration and separation assay for the rapid detection of Cronobacter sakazakii (C. sakazakii), an acute opportunistic foodborne pathogenic bacterium, in both pure culture and infant formula. To develop the assay, magnetic nanoparticles (diameter 30 nm) were coated with immunoglobulin G (IgG), specifically anti-C. sakazakii IgG, and applied for the sensitive and efficient detection of C. sakazakii using immunoliposomes. The binding efficiency of anti-C. sakazakii IgG to the magnetic nanoparticles was 86.23 ± 0.59%. The assay developed in this study detected as few as 3.3 × 10(3) CFUmL(-1) of C. sakazakii in pure culture within 2h 30 min; in comparison, an indirect non-competitive enzyme-linked immunosorbent assay was able to detect 6.2 × 10(5) CFUmL(-1) of C. sakazakii in pure culture after 17 h. The developed assay did not show any cross-reactivity with other Cronobacter spp. or pathogens belonging to other genera. In addition, the method was able to detect 10(3) CFUmL(-1) of C. sakazakii in infant formula without any pre-incubation. These results confirm that the immunoliposome-based immunomagnetic concentration and separation assay may facilitate highly sensitive, efficient, and rapid detection of C. sakazakii.

Construction of Au-IDE/CFP10-ESAT6 aptamer/DNA-AuNPs MSPQC for rapid detection of Mycobacterium tuberculosis.

Au-IDE/CFP10-ESAT6 aptamer/DNA-AuNPs MSPQC for rapid detection of Mycobacterium tuberculosis was constructed based on specific detection of specific fused antigen CFP10-ESAT6 which secreted only by pathogenic M. tuberculosis in its early culture time. CFP10-ESAT6 aptamer was used as sensor specific probe of CFP10-ESAT6 antigen. Au nanoparticles (NPs) was employed to increase sensor senstivity. The Au-IDE/CFP10-ESAT6 aptamer/DNA-AuNPs electrode probe was prepared by modifying of the complementary DNA-AuNPs on to interdigital array microelectrode with CFP10-ESAT6 aptamer. CFP10-ESAT6 aptamer could specifically catch CFP10-ESAT6 protein and formed a tight complex on the electrode surface and resulted in the DNA-AuNPs fragments fell away from the electrode surface. This change can be sensitively detected by IDE-MSPQC sensor. The detection time was 96.3h. Non-pathogenic Mycobacterium did not affect detection. Compared with conventional methods, this approach was specific, more sensitive, and expected to become a valuable analysis tool for the early detection of M. tuberculosis in clinical sample.

Sensitive detection of Escherichia coli O157:H7 using Pt-Au bimetal nanoparticles with peroxidase-like amplification.

Escherichia coli O157:H7 is one of the most notorious foodborne pathogens causing serious disease at low infectious dose. To protect consumers from deadly foodborne E. coli O157:H7 infection, it is vital to develop a simple, reliable, sensitive and rapid method which can detect low level E. coli O157:H7 in foods at real-time. We have successfully developed a novel immunochromatographic assay (ICA) with enhanced sensitivity for the visual and quantitative detection of E. coli O157:H7. Sandwich-type immunoreactions were performed on the ICA, and Pt-Au bimetal nanoparticles (NPs) were accumulated on the test zone. The signal amplification is based on Pt-Au bimetal NPs possessing high peroxidase activity toward 3,3',5,5'-tetramethylbenzidine, which can produce characteristic colored bands and thus, enable visual detection of E. coli O157:H7 without instrumentation. The innovative aspect of this approach lies in the visualization and quantification of target pathogen through the detection of color intensity. Due to the excellent peroxidase activity of Pt-Au NPs, they emit strong visible color intensity in less than 1 min for visual observation even in low concentration range of E. coli O157:H7. Quantification was performed using a commercial assay meter. The sensitivity was improved more than 1000-folds compared to the conventional test strip based on colored gold-colloids. Although the feasibility was demonstrated using E. coli O157:H7 as a model analyte, this approach could be easily developed to be a universal signal amplification technique and applied to detection of a wide variety of foodborne pathogens and protein biomarkers.

Comparative Evaluation of Inoculation of Urine Samples with the Copan WASP and BD Kiestra InoqulA Instruments.

This study evaluated the quantitative results from and quality of the inoculation patterns of urine specimens produced by two automated instruments, the Copan WASP and the BD InoqulA. Five hundred twenty-six urine samples submitted in 10-ml canisters containing boric acid were processed within 30 min on an InoqulA instrument plating 10 µl of specimen, and on two WASP instruments, one plating 1 µl of specimen (WASP-1), and the second plating 10 µl of WASP (WASP-10). All samples were incubated, analyzed, and digitally imaged using the BD Kiestra total lab automation system. The results were evaluated using a quantitative protocol and assessed for the presence or absence of ≥5 distinct colonies. Separate studies were conducted using quality control (QC) organisms to determine the relative accuracy of WASP-1, WASP-10, and InoqulA instruments compared to the results obtained with a calibrated pipette. The results with QC organisms were calculated as the ratios of the counts of the automated instruments divided by the counts for the calibrated pipette (the gold standard method). The ratios for the InoqulA instrument were closest to 1.0, with the smallest standard deviations indicating that compared to a calibrated pipette, the InoqulA results were more accurate than those with the WASP instrument. For clinical samples, the WASP instruments produced higher colony counts and more commensals than the InoqulA instrument, with differences most notable for WASP-1. The InoqulA instrument was significantly better at dispersing organisms with counts of ≥10(5) bacteria/ml of urine than were the WASP-1 and WASP-10 instruments (P < 0.05). Our results suggest that the InoqulA quantitative results are more accurate than the WASP results, and, moreover, the number of isolated colonies produced by the InoqulA instrument was significantly greater than that produced by the WASP instrument.

A disposable bacterial lysis cartridge (BLC) suitable for an in situ water-borne pathogen detection system.

We constructed a disposable bacterial lysis cartridge (BLC) suitable for an in situ pathogen detection system. It had an in-built micro corona discharge based ozone generator that provided ozone for cell lysis. Using a custom sample handling platform, its performance was evaluated with a Gram-positive bacterium of Bacillus subtilis. It was capable of achieving a similar degree of lysis as a commercial ultrasonic dismembrator with a P-1 microprobe in 10 min at an air pump flow rate of 29.4 ml min(-1) and an ozone generator operating voltage of 1600 V. The lysing duration could be significantly reduced to 5 min by increasing the air pump flow rate and the ozone generator operating voltage as well as by the addition of sodium dodecyl sulfate (SDS).