PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription.

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods use the recombinase polymerase amplification (RPA), which exhibits polymerase chain reaction (PCR)-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription) can be used to detect SARS-CoV-2 RNA with single-copy detection limits, Escherichia coli with single-cell detection limits, and 10-min detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be used for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

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.

A DNAzyme-enhanced nonlinear hybridization chain reaction for sensitive detection of microRNA.

As a typical biomarker, the expression of microRNA is closely related to the occurrence of cancer. However, in recent years, the detection methods have had some limitations in the research and application of microRNAs. In this paper, an autocatalytic platform was constructed through the combination of a nonlinear hybridization chain reaction and DNAzyme to achieve efficient detection of microRNA-21. Fluorescently labeled fuel probes can form branched nanostructures and new DNAzyme under the action of the target, and the newly formed DNAzyme can trigger a new round of reactions, resulting in enhanced fluorescence signals. This platform is a simple, efficient, fast, low-cost, and selective method for the detection of microRNA-21, which can detect microRNA-21 at concentrations as low as 0.004 nM and can distinguish sequence differences by single-base differences. In tissue samples from patients with liver cancer, the platform shows the same detection accuracy as real-time PCR but with better reproducibility. In addition, through the flexible design of the trigger chain, our method could be adapted to detect other nucleic acid biomarkers.

Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 ng of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

Biosensing Detection of the SARS-CoV-2 D614G Mutation.

The emergence of a mutant strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an amino acid change from aspartate to a glycine residue at position 614 (D614G) has been reported and this mutant appears to be now dominant in the pandemic. Efficient detection of the SARS-CoV-2 D614G mutant by biosensing technologies is therefore crucial for the control of the pandemic.

Robust Sequence Design Space for the Isothermal Exponential Amplification of Short Oligonucleotides.

Advances in isothermal amplification techniques have accelerated development in biosensing applications and the design of complex molecular devices. The exponential amplification reaction technique, or EXPAR, is uniquely positioned to process molecular information from short oligonucleotide strands (≈10 nucleotides length) typically encountered in molecular computing or microRNA detection. Despite its conceptual simplicity (requiring only a template strand and two enzymes), the issue of nonspecific background amplification has hindered broader adoption. In this work, a new system configuration is established at 37 °C to achieve significantly improved performance. Critical sequence motifs responsible for the excellent signal-to-background profile are identified and generalized as a universal adapter design framework. Orthogonal template sequences generated from the framework are implemented for a triplex reaction and successfully evaluated mixtures of multiple-target inputs in a single-step, one-pot format without the need for exogenous agents.

An Integrated Micro-Heating System for On-Chip Isothermal Amplification of African Swine Fever Virus Genes.

The loop-mediated isothermal amplification (LAMP) is widely used in the laboratory to facilitate rapid DNA or RNA detection with a streamlined operational process, whose properties are greatly dependent on the uniformity and rise rate of temperature in the reaction chambers and the design of the primers. This paper introduces a planar micro-heater equipped with an embedded micro-temperature sensor to realize temperature tunability at a low energy cost. Moreover, a control system, based on the Wheatstone bridge and proportional, integral, and derivative (PID) control, is designed to measure and adjust the temperature of the micro-heater. The maximum temperature rise rate of the designed micro-heater is ≈8 °C s-1, and it only takes ≈60 s to reach the target temperature. Furthermore, a designed plasmid, containing the B646L gene of African Swine Fever Virus (ASFV), and a set of specific primers, are used to combine with the designed micro-heating system to implement the LAMP reaction. Finally, the lateral flow assay is used to interpret the amplification results visually. This method can achieve highly sensitive and efficient detection of ASFV within 40 min. The sensitivity of this on-chip gene detection method is 8.4 copies per reaction, holding great potential for applications in DNA and RNA amplification.

Simultaneous Dual-Gene Test of Methicillin-Resistant Staphylococcus Aureus using an Argonaute-Centered Portable and Visual Biosensor.

The development of novel method for drug-resistant bacteria detection is imperative. A simultaneous dual-gene Test of methicillin-resistant Staphylococcus aureus (MRSA) is developed using an Argonaute-centered portable biosensor (STAR). This is the first report concerning Argonaute-based pathogenic bacteria detection. Simply, the species-specific mecA and nuc gene are isothermally amplified using loop-mediated isothermal amplification (LAMP) technique, followed by Argonaute-based detection enabled by its programmable, guided, sequence-specific recognition and cleavage. With the strategy, the targeted nucleic acid signals gene are dexterously converted into fluorescent signals. STAR is capable of detecting the nuc gene and mecA gene simultaneously in a single reaction. The limit of detection is 10 CFU/mL with a dynamic range from 10 to 107 CFU/mL. The sample-to-result time is <65 min. This method is successfully adapted to detect clinical samples, contaminated foods, and MRSA-infected animals. This work broadens the reach of Argonaute-based biosensing and presents a novel bacterial point-of-need (PON) detection platform.

Hybrid paper/PDMS microfluidic device integrated with RNA extraction and recombinase polymerase amplification for detection of norovirus in foods.

Human norovirus (HuNoV) is recognized as the leading causative agent of foodborne outbreaks of epidemic gastroenteritis. Consequently, there is a high demand for developing point-of-care testing for HuNoV. We developed an origami microfluidic device that facilitates rapid detection of murine norovirus 1 (MNV-1), a surrogate for HuNoV, encompassing the entire process from sample preparation to result visualization. This process includes RNA absorption via a paper strip, RNA amplification using recombinase polymerase amplification (RPA), and a lateral flow assay for signal readout. The on-chip detection of MNV-1 was completed within 35 min, demonstrating 100% specificity to MNV-1 in our settings. The detection limit of this microfluidic device for MNV-1 was 200 PFU/mL, comparable to the in-tube RPA reaction. It also successfully detected MNV-1 in lettuce and raspberries at concentrations of 170 PFU/g and 230 PFU/g, respectively, without requiring extra concentration steps. This device demonstrates high compatibility with isothermal nucleic acid amplification and holds significant potential for detecting foodborne viruses in agri-food products in remote and resource-limited settings. IMPORTANCE: HuNoV belongs to the family of Caliciviridae and is a leading cause of acute gastroenteritis that can be transmitted through contaminated foods. HuNoV causes around one out of five cases of acute gastroenteritis that lead to diarrhea and vomiting, placing a substantial burden on the healthcare system worldwide. HuNoV outbreaks can occur when food is contaminated at the source (e.g., wild mussels exposed to polluted water), on farms (e.g., during crop cultivation, harvesting, or livestock handling), during packaging, or at catered events. The research outcomes of this study expand the approaches of HuNoV testing, adding value to the framework for routine testing of food products. This microfluidic device can facilitate the monitoring of HuNoV outbreaks, reduce the economic loss of the agri-food industry, and enhance food safety.

Rapid, visual, label-based biosensor platform for identification of hepatitis C virus in clinical applications.

OBJECTIVES: In the current study, for the first time, we reported a novel HCV molecular diagnostic approach termed reverse transcription loop-mediated isothermal amplification integrated with a gold nanoparticles-based lateral flow biosensor (RT-LAMP-AuNPs-LFB), which we developed for rapid, sensitive, specific, simple, and visual identification of HCV. METHODS: A set of LAMP primer was designed according to 5'untranslated region (5'UTR) gene from the major HCV genotypes 1b, 2a, 3b, 6a, and 3a, which are prevalent in China. The HCV-RT-LAMP-AuNPs-LFB assay conditions, including HCV-RT-LAMP reaction temperature and time were optimized. The sensitivity, specificity, and selectivity of our assay were evaluated in the current study. The feasibility of HCV-RT-LAMP-AuNPs-LFB was confirmed through clinical serum samples from patients with suspected HCV infections. RESULTS: An unique set of HCV-RT-LAMP primers were successfully designed targeting on the 5'UTR gene. The optimal detection process, including crude nucleic acid extraction (approximately 5 min), RT-LAMP reaction (67℃, 30 min), and visual interpretation of AuNPs-LFB results (~ 2 min), could be performed within 40 min without specific instruments. The limit of detection was determined to be 20 copies per test. The HCV-RT-LAMP-AuNPs-LFB assay exhibited high specificity and anti-interference. CONCLUSIONS: These preliminary results confirmed that the HCV-RT-LAMP-AuNPs-LFB assay is a sensitive, specific, rapid, visual, and cost-saving assay for identification of HCV. This diagnostic approach has great potential value for point-of-care (POC) diagnostic of HCV, especially in resource-challenged regions.

Precise pathogen quantification by CRISPR-Cas: a sweet but tough nut to crack.

Pathogen detection is increasingly applied in medical diagnosis, food processing and safety, and environmental monitoring. Rapid, sensitive, and accurate pathogen quantification is the most critical prerequisite for assessing protocols and preventing risks. Among various methods evolved, those based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) have been developed as important pathogen detection strategies due to their distinct advantages of rapid target recognition, programmability, ultra-specificity, and potential for scalability of point-of-care testing (POCT). However, arguments and concerns on the quantitative capability of CRISPR-based strategies are ongoing. Herein, we systematically overview CRISPR-based pathogen quantification strategies according to the principles, properties, and application scenarios. Notably, we review future challenges and perspectives to address the of precise pathogen quantification by CRISPR-Cas. We hope the insights presented in this review will benefit development of CRISPR-based pathogen detection methods.

Rapid detection of Ebolavirus using isothermal recombinase-aided amplification.

Ebolavirus disease (EVD) is an often-lethal disease caused by the genus Ebolavirus (EBOV). Although vaccines are being developed and recently used, outbreak control still relies on a combination of various factors, including rapid identification of EVD cases. This allows rapid patient isolation and control measure implementation. Ebolavirus diagnosis is performed in treatment centers or reference laboratories, which usually takes a few hours to days to confirm the outbreak or deliver a clear result. A fast and field-deployable molecular detection method, such as the isothermal amplification recombinase-aided amplification (RAA), could significantly reduce sample-to-result time. In this study, a RT-RAA assay was evaluated for EBOV detection. Various primer and probe combinations were screened; analytical sensitivity and cross-specificity were tested. A total of 40 archived samples from the 2014 to 2016 Ebola outbreak in West Africa were tested with both the reference method real-time RT-PCR and the established RT-RAA assay. The assay could detect down to 22.6 molecular copies per microliter. No other pathogens were detected with the Ebolavirus RT-RAA assay. Testing 40 samples yield clinical sensitivity and specificity of 100% each. This rapid isothermal RT-RAA assay can replace the previous RT-RPA and continue to offer rapid EBOV diagnostics.

Single-tube, single-strip lateral flow assays utilizing loop-mediated isothermal amplification for simultaneous hepatitis B and C viral detection.

Globally, hepatitis B virus (HBV) affects over 250 million people, whereas hepatitis C virus (HCV) affects approximately 70 million people, posing major public health challenges. Despite the availability of vaccines and treatments, a lack of comprehensive diagnostic coverage has left many cases undiagnosed and untreated. To address the need for sensitive, specific, and accessible diagnostics, this study introduced a multiplex loop-mediated isothermal amplification assay with lateral flow detection for simultaneous HBV and HCV testing. This assay achieved exceptional sensitivity and was capable of detecting HBV and HCV concurrently in a single tube and on a single strip within 25 min, achieving the required clinical sensitivity (10 and 103 genomic copies/reaction for HBV and HCV, respectively). The method was validated in clinical samples of various viral genotypes, achieving an equivalent limit of detection. Additionally, a custom portable heating device was developed for field use. The assay developed here, capable of direct viral detection on the strip, shows promise in supplanting current methods that solely identify antibodies and necessitate additional qPCR for viral activity assessment. This economical and rapid assay aligns with point-of-care testing needs, offering significant advancements in enhancing viral hepatitis diagnostics in settings with limited resources.

Advanced technologies towards improved HPV diagnostics.

Persistent infection with high-risk types of human papillomaviruses (HPV) is a major cause of cervical cancer, and an important factor in other malignancies, for example, head and neck cancer. Despite recent progress in screening and vaccination, the incidence and mortality are still relatively high, especially in low-income countries. The mortality and financial burden associated with the treatment could be decreased if a simple, rapid, and inexpensive technology for HPV testing becomes available, targeting individuals for further monitoring with increased risk of developing cancer. Commercial HPV tests available in the market are often relatively expensive, time-consuming, and require sophisticated instrumentation, which limits their more widespread utilization. To address these challenges, novel technologies are being implemented also for HPV diagnostics that include for example, isothermal amplification techniques, lateral flow assays, CRISPR-Cas-based systems, as well as microfluidics, paperfluidics and lab-on-a-chip devices, ideal for point-of-care testing in decentralized settings. In this review, we first evaluate current commercial HPV tests, followed by a description of advanced technologies, explanation of their principles, critical evaluation of their strengths and weaknesses, and suggestions for their possible implementation into medical diagnostics.

Single-tube four-target lateral flow assay detects human papillomavirus types associated with majority of cervical cancers.

Isothermal nucleic acid amplification methods have many advantages for use at the point of care. However, there is a lack of multiplexed isothermal amplification tests to detect multiple targets in a single reaction, which would be valuable for many diseases, such as infection with high-risk human papillomavirus (hrHPV). In this study, we developed a multiplexed loop-mediated isothermal amplification (LAMP) reaction to detect the three most common hrHPV types that cause cervical cancer (HPV16, HPV18, and HPV45) and a cellular control for sample adequacy. First, we characterized the assay limit of detection (LOD) in a real-time reaction with fluorescence readout; after 30 min of amplification the LOD was 100, 10, and 10 copies/reaction of HPV16, HPV18, and HPV45, respectively, and 0.1 ng/reaction of human genomic DNA (gDNA). Next, we implemented the assay on lateral flow strips, and the LOD was maintained for HPV16 and HPV18, but increased to 100 copies/reaction for HPV45 and to 1 ng/reaction for gDNA. Lastly, we used the LAMP test to evaluate total nucleic acid extracted from 38 clinical samples; compared to qPCR, the LAMP test had 89% sensitivity and 95% specificity. When integrated with sample preparation, this multiplexed LAMP assay could be useful for point-of-care testing.

Endonuclease Q as a robust enhancer for nucleic acid amplification.

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.

Portable loop-mediated isothermal amplification device with spectrometric detection for rapid pathogen identification.

With the rise in extreme weather due to global warming, coupled with globalization facilitating the spread of infectious diseases, there's a pressing need for portable testing platforms offering simplicity, low cost, and remote transmission, particularly beneficial in resource-limited and non-urban areas. We have developed a portable device using loop-mediated isothermal amplification (LAMP) with spectrometric detection to identify Salmonella Typhimurium DNA. The device utilizes the LinkIt 7697 microcontroller and a microspectrometer to capture and transmit spectral signals in real-time, allowing for improved monitoring and analysis of the reaction progress. We built a hand-held box containing a microspectrometer, thermoelectric cooler, ultraviolet LED, disposable reaction tube, and homemade thermal module, all powered by rechargeable batteries. Additionally, we conducted thorough experiments to ensure temperature accuracy within 1 °C under thermal control, developed a heating module with a LinkIt 7697 IoT development board to heat the DNA mixture to the reaction temperature within 3 min, and integrated foam insulation and a 3D-printed frame to enhance the device's thermal stability. We successfully demonstrated the amplification of Salmonella Typhimurium DNA with an impressive sensitivity of 2.83 × 10-4 ng/µL. A remote webpage interface allows for monitoring the temperature and fluorescence during the LAMP process, improving usability. This portable LAMP device with real-time detection offers a cost-effective solution for detecting Salmonella Typhimurium in food products. Its unique design and capabilities make it a promising tool for ensuring food safety.

LAMPrimers iQ: New primer design software for loop-mediated isothermal amplification (LAMP).

Nucleic acids amplification is a widely used technique utilized for different manipulations with DNA and RNA. Although, polymerase chain reaction (PCR) remains the most popular amplification method, isothermal approaches are gained more attention last decades. Among these, loop-mediated isothermal amplification (LAMP) became an excellent alternative to PCR. LAMP requires an increased number of primers and, therefore, is considered a highly specific amplification reaction compared to PCR. LAMP primers design is still a non-trivial task, and all niceties should be taken into account during their selection. Here, we report on a new program called LAMPrimers iQ destined for high-quality LAMP primers design. LAMPrimers iQ is based on an original algorithm considering rigorous criteria for primers selection. Unlike alternative programs, LAMPrimers iQ can process long DNA or RNA sequences, and completely avoid primers that can form homo- and heterodimers. The quality of the primers designed was checked using SARS-CoV-2 coronavirus RNA as a model target. It was shown that primers selected with LAMPrimers iQ provide higher specificity and reliable detection of viral RNA compared to those obtained by alternative programs. The program is available at https://github.com/Restily/LAMPrimers-iQ.

Evaluation of semi-quantitative colorimetric assays based on loop-mediated isothermal amplification indicators by using image analysis.

Colorimetric assays are some of the most convenient detection methods, creating discoloration in solutions that is visible to the naked eye. However, colorimetric reactions have some limitations regarding the variability in the color perception of individuals caused by factors such as color blindness, experience, and gender. Semi-quantitative chromatic analysis has been used as an alternative method to differentiate between two colors and accurately interpret the results from a numerical value, with high confidence. Therefore, we developed and determined the optimal model between Red-Green-Blue (RGB) and Commission Internationale de l'Eclairage (CIE) Lab color spaces to establish a semi-quantitative colorimetric assay via image analysis by the ImageJ program for loop-mediated isothermal amplification (LAMP), using the dyes malachite green and phenol red. The semi-quantitative colorimetric assays using the color distance values of the CIELab color space (ΔEab) were more suitable than those using the RGB color space (ΔERGB) for chromatic differentiation between positive and negative reactions in both indicator dyes, demonstrating the feasibility of this assay to be applied in the detection of a wide range of pathogens and infectious diseases.

Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viruses infecting patchouli (Pogostemon cablin).

Patchouli (Pogostemon cablin), a highly valued medicinal plant, suffers significant economic losses following infection with Broad bean wilt virus 2 (BBWV-2) and Peanut stripe virus (PStV). In this study, a field-based isothermal technique called reverse transcription loop-mediated isothermal amplification (RT-LAMP) was established for an early and specific detection of BBWV-2 and PStV. The oligo primers were designed to target the coat protein genes of PStV and BBWV-2. The reaction conditions, such as temperature and time duration, were optimized to 65 °C for 60 min. The LAMP amplicons positive for PStV and BBWV-2 revealed characteristic ladder-type bands following agarose gel electrophoresis. Further, a colorimetric assay using a metal ion-based indicator (Hydroxy-naphthol blue, HNB) was conducted to visualize the amplified products with the naked eye, thus facilitating accessibility to field practices. The assay developed in this study was found to be virus specific, and was 100 times more sensitive than RT-PCR. Thus, the RT-LAMP assay established in this study is quick, reliable, and cost-effective for the accurate identification of BBWV-2 and PStV. It will facilitate the screening of patchouli planting materials.  Further, it may reduce the risk of virus spread and could be helpful in phytosanitary programs.