One-step colorimetric isothermal detection of COVID-19 with AI-assisted automated result analysis: A platform model for future emerging point-of-care RNA/DNA disease diagnosis.

Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the diagnosis of COVID-19 owing to their unmatched feasibility in low-resource settings. However, the vast majority of them are restricted to proprietary pH-sensitive dyes that limit downstream assay optimization or hinder efficient result interpretation. To address this problem, we developed a novel dual colorimetric RT-LAMP assay using in-house pH-dependent indicators to maximize the visual detection and assay simplicity, and further integrated it with the artificial intelligence (AI) operated tool (RT-LAMP-DETR) to enable a more precise and rapid result analysis in large scale testing. The dual assay leverages xylenol orange (XO) and a newly formulated lavender green (LG) dye for distinctive colorimetric readouts, which enhance the test accuracy when performed and analyzed simultaneously. Our RT-LAMP assay has a detection limit of 50 viral copies/reaction with the cycle threshold (Ct) value ≤ 39.7 ± 0.4 determined by the WHO-approved RT-qPCR assay. RT-LAMP-DETR exhibited a complete concordance with the results from naked-eye observation and RT-qPCR, achieving 100% sensitivity, specificity, and accuracy that altogether render it suitable for ultrasensitive point-of-care COVID-19 screening efforts. From the perspective of pandemic preparedness, our method offers a simpler, faster, and cheaper (∼$8/test) approach for COVID-19 testing and other emerging pathogens with respect to RT-qPCR.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with colorimetric gold nanoparticle (AuNP) probe assay for visual detection of tilapia lake virus (TiLV) in Nile and red hybrid tilapia.

Tilapia is one of the major aquaculture species with a global economic significance. Despite a high scale of production worldwide, mortality in many tilapia cultures has recently become a problem concerned with not only intensive farming but also the prevalence of infectious pathogens. Tilapia lake virus (TiLV) has emerged as a serious single-stranded RNA disease agent that thus far has continued to cause a number of incidences across the continents. Conventional PCR-based molecular detection techniques, despite having high sensitivity for TiLV, are not best suited for the onsite identification of infected fish mainly due to their requirement of laboratory resources and extended assay turnaround time. To address this practical limitation, we have developed a novel colorimetric assay based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and gold nanoparticle (AuNP)-labelled oligonucleotide reporter probe targeting the viral genomic segment 9 that enables the assay to be completed within an hour. This technique has been shown to be compatible with a rapid nucleic extraction method that does not demand centrifugation steps or any benchtop laboratory equipment. When validated with field-acquired tilapia samples, our RT-LAMP-AuNP assay exhibited a near-perfect agreement with the semi-nested RT-PCR assay recommended by OIE with Cohen's κ coefficient of .869, yet requiring significantly less time to perform.

Sensitivity Validation of EWOD Devices for Diagnosis of Early Mortality Syndrome (EMS) in Shrimp Using Colorimetric LAMP-XO Technique.

Electrowetting-on-dielectric (EWOD) is a microfluidic technology used for manipulating liquid droplets at microliter to nanoliter scale. EWOD has the ability to facilitate the accurate manipulation of liquid droplets, i.e., transporting, dispensing, splitting, and mixing. In this work, EWOD fabrication with suitable and affordable materials is proposed for creating EWOD lab-on-a-chip platforms. The EWOD platforms are applied for the diagnosis of early mortality syndrome (EMS) in shrimp by utilizing the colorimetric loop-mediated isothermal amplification method with pH-sensitive xylenol orange (LAMP-XO) diagnosis technique. The qualitative sensitivity is observed by comparing the limit of detection (LOD) while performing the LAMP-XO diagnosis test on the proposed lab-on-a-chip EWOD platform, alongside standard LAMP laboratory tests. The comparison results confirm the reliability of EMS diagnosis on the EWOD platform with qualitative sensitivity for detecting the EMS DNA plasmid concentration at 102 copies in a similar manner to the common LAMP diagnosis tests.

Ultrasensitive detection of Mycobacterium tuberculosis by a rapid and specific probe-triggered one-step, simultaneous DNA hybridization and isothermal amplification combined with a lateral flow dipstick.

Mycobacterium tuberculosis (Mtb) is an insidious scourge that has afflicted millions of people worldwide. Although there are many rapid methods to detect it based on loop-mediated isothermal amplification (LAMP) and a lateral flow dipstick (LFD), this study made further improvements using a new set of primers to enhance LAMP performance and a novel DNA probe system to simplify detection and increase specificity. The new probe system eliminates the post-LAMP hybridization step typically required for LFD assays by allowing co-hybridization and amplification of target DNA in one reaction while preventing self-polymerization that could lead to false-positive results. The improved assay was named Probe-Triggered, One-Step, Simultaneous DNA Hybridization and LAMP Integrated with LFD (SH-LAMP-LFD). SH-LAMP-LFD was simpler to perform and more sensitive than previously reported LAMP-LFD and PCR methods by 100 and 1000 times, respectively. It could detect a single cell of Mtb. The absence of cross-reactivity with 23 non-TB bacteria, and accurate test results with all 104 blind clinical samples have highlighted its accuracy. Its robustness and portability make SH-LAMP-LFD suitable for users in both low and high resource settings.

Graphene-based electrochemical genosensor incorporated loop-mediated isothermal amplification for rapid on-site detection of Mycobacterium tuberculosis.

Tuberculosis (TB) is one of the most contagious and lethal infectious diseases that affects more than 10 million individuals worldwide. A lack of rapid TB diagnosis is partly responsible for its alarming spread and prevalence in many regions. To address this problem, we report a novel integrated point-of-care platform to detect a TB-causative bacterium, Mycobacterium tuberculosis (Mtb). This leverages loop-mediated isothermal amplification (LAMP) for Mtb-DNA amplification and the screen-printed graphene electrode (SPGE) for label-free electrochemical analysis of DNA amplicons. When implemented on a portable potentiostat device developed in-house, the system (LAMP-EC) offers a rapid end-point qualitative analysis of specific DNA amplicons that will be displayed as a discrete positive/negative readout on the LCD screen. Under optimized conditions, LAMP-EC showed a comparable detection limit to the previously developed LAMP assay with a lateral flow readout at 1 pg total DNA or 40 Mtb genome equivalents. This highly specific technique detected the presence of TB in all 104 blinded sputum samples with a 100% accuracy. Our technique can also easily be clinically adopted due to its affordability (∼USD2.5/test), rapidity (<65 min turnaround time) and feasibility (lack of advanced instrumental requirement). This serves as a practical incentive, appealing to users in both high- and low-resource settings across the TB endemic regions and economic backgrounds.

Point-of-care rapid detection of Vibrio parahaemolyticus in seafood using loop-mediated isothermal amplification and graphene-based screen-printed electrochemical sensor.

Vibrio parahaemolyticus is one of the most important foodborne pathogens that cause various life-threatening diseases in human and animals. Here, we present a rapid detection platform for V. parahaemolyticus by combining loop-mediated isothermal amplification (LAMP) and disposable electrochemical sensors based on screen-printed graphene electrodes (SPGEs). The LAMP reactions using primers targeting V. parahaemolyticus toxR gene were optimized at an isothermal temperature of 65 °C, providing specific detection of V. parahaemolyticus within 45 min at the detection limit of 0.3 CFU per 25 g of raw seafood. The LAMP amplicons can be effectively detected using unmodified SPGEs, redox active molecules namely Hoechst-33258 and a portable potentiostat. Therefore, the proposed system is particularly suitable as a point-of-care device for on-site detection of foodborne pathogens.

Simple detection of single nucleotide polymorphism in Plasmodium falciparum by SNP-LAMP assay combined with lateral flow dipstick.

The significant strides made in reducing global malaria burden over the past decades are being threatened by the emergence of multi-drug resistant malaria. Mechanisms of resistance to several classes of antimalarial drugs have been linked to key mutations in the Plasmodium falciparum genes. Pyrimethamine targets the dihydrofolate reductase of the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS), and specific point mutations in the dhfr-ts gene have been assigned to resistant phenotypes. Several molecular methods are available to detect the mutant genotypes including DNA sequencing and PCR-based methods. In this study, we report the development of PfSNP-LAMP to detect nucleotide polymorphism in the dhfr gene associated with N51I mutation and antifolate resistance. The PfSNP-LAMP method was validated with genomic DNA samples and parasite lysates prepared from sensitive and pyrimethamine resistant strains of P. falciparum.

Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes.

Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non-AHPND bacteria commonly found in shrimp ponds (including other Vibrio species). The new method significantly reduced the time, difficulty and cost for molecular detection of VPAHPND in shrimp hatchery and farm settings.

Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method.

Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria.

Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms.

Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP-calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10(2) F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP-calcein method had 97% homology with the DNA sequence of F. columnare.

Rapid and sensitive detection of shrimp infectious myonecrosis virus using a reverse transcription loop-mediated isothermal amplification and visual colorogenic nanogold hybridization probe assay.

This study reports a novel strategy for the detection of reverse transcription loop-mediated isothermal amplification (RT-LAMP) products derived from infectious myonecrosis virus (IMNV), causes a serious myonecrosis in Penaeus (Litopenaeus) vannamei, by using a ssDNA-labeled with gold nanoparticle (AuNP) probe. This technique relies on a self-aggregation method, when the AuNP aggregation is induced by an increasing of salt concentrations with visual detection. The presence of IMNV-LAMP target prevented an AuNP aggregation and a solution remained as pink color of AuNP, while non-complementary targets cannot prevent AuNP aggregation, resulting in a visible color change to purple color after addition of salt. By using the combination of LAMP and AuNP probe system, the total assay interval required approximately 50 min (exclude RNA preparation). Detection limit was 10 copies of IMNV RNA in vitro transcript that comparable to that of LAMP followed by LFD and nested RT-PCR, but it was 100-times more sensitive than RT-PCR methods. This assay can be adapted easily for rapid detection of other shrimp infectious diseases agents at low-cost with robust reagents and using a simple colorimetric detection method.