A culture-free biphasic approach for sensitive and rapid detection of pathogens in dried whole-blood matrix.

Blood stream infections (BSIs) cause high mortality, and their rapid detection remains a significant diagnostic challenge. Timely and informed administration of antibiotics can significantly improve patient outcomes. However, blood culture, which takes up to 5 d for a negative result, followed by PCR remains the gold standard in diagnosing BSI. Here, we introduce a new approach to blood-based diagnostics where large blood volumes can be rapidly dried, resulting in inactivation of the inhibitory components in blood. Further thermal treatments then generate a physical microscale and nanoscale fluidic network inside the dried matrix to allow access to target nucleic acid. The amplification enzymes and primers initiate the reaction within the dried blood matrix through these networks, precluding any need for conventional nucleic acid purification. High heme background is confined to the solid phase, while amplicons are enriched in the clear supernatant (liquid phase), giving fluorescence change comparable to purified DNA reactions. We demonstrate single-molecule sensitivity using a loop-mediated isothermal amplification reaction in our platform and detect a broad spectrum of pathogens, including gram-positive methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria, gram-negative Escherichia coli bacteria, and Candida albicans (fungus) from whole blood with a limit of detection (LOD) of 1.2 colony-forming units (CFU)/mL from 0.8 to 1 mL of starting blood volume. We validated our assay using 63 clinical samples (100% sensitivity and specificity) and significantly reduced sample-to-result time from over 20 h to <2.5 h. The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.

Culture-free biphasic approach for sensitive detection of Escherichia coli O157:H7 from beef samples.

Foodborne illnesses are a major threat to public health also leading to significant mortality and financial and reputational damage to industry. It is very important to detect pathogen presence in food products early, rapidly, and accurately to avoid potential outbreaks and economic loss. However, "gold standard" culture methods, including enrichment of pathogens, can take up to several days. Moreover, the food matrix often interferes with nucleic acid amplification methods of detection, requiring DNA extraction from the sample for successful molecular detection of pathogens. Here, we introduce a "biphasic" amplification method that can achieve high sensitivity detection with background noise from ground beef food samples without culture or other extraction methods in 2.5 h. Homogenized ground beef is dried resulting in an increase in porosity of the dried food matrix to allowing amplification enzymes and primers to access the target DNA and initiate the reaction within the dried food matrix. Using Loop Mediated Isothermal Amplification, we demonstrate the detection of 1-3 cfu of Escherichia coli bacteria in 30 mg of dried food matrix. Our approach significantly lowers the time to result to less than a few hours and have a pronounced impact on reduction of instrumentation complexity and costs.

Portable Pathogen Diagnostics Using Microfluidic Cartridges Made from Continuous Liquid Interface Production Additive Manufacturing.

Biomedical diagnostics based on microfluidic devices have the potential to significantly benefit human health; however, the manufacturing of microfluidic devices is a key limitation to their widespread adoption. Outbreaks of infectious disease continue to demonstrate the need for simple, sensitive, and translatable tests for point-of-care use. Additive manufacturing (AM) is an attractive alternative to conventional approaches for microfluidic device manufacturing based on injection molding; however, there is a need for development and validation of new AM process capabilities and materials that are compatible with microfluidic diagnostics. In this paper, we demonstrate the development and characterization of AM cartridges using continuous liquid interface production (CLIP) and investigate process characteristics and capabilities of the AM microfluidic device manufacturing. We find that CLIP accurately produces microfluidic channels as small as 400 µm and that it is possible to routinely produce fluid channels as small as 100 µm with high repeatability. We also developed a loop-mediated isothermal amplification (LAMP) assay for detection of E. coli from whole blood directly on the CLIP-based AM microfluidic cartridges, with a 50 cfu/µL limit of detection, validating the use of CLIP processes and materials for pathogen detection. The portable diagnostic platform presented in this paper could be used to investigate and validate other AM processes for microfluidic diagnostics and could be an important component of scaling up the diagnostics for current and future infectious diseases and pandemics.

Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples.

The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. In addition, there are limited reports of saliva as the sample source, and some of these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay from saliva using a two-step RT-LAMP assay, where a short 10 min RT step is performed with only B3 and backward inner primers before the final reaction. We show that while the one-step RT-LAMP demonstrates satisfactory results, the optimized two-step approach allows detection of only few molecules per reaction and performs significantly better than the one-step RT-LAMP and conventional two-step RT-LAMP approaches with all primers included in the RT step. We show control measurements with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based assays for detection of SARS-CoV-2 from viral transport media and saliva clinical samples.

Rapid isothermal amplification and portable detection system for SARS-CoV-2.

The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per µL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.