Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP

In this paper, we asked if it is possible to identify the best primers and reaction conditions based on improvements in reaction speed when optimizing isothermal reactions. We used digital single-molecule, real-time analyses of both speed and efficiency of isothermal amplification reactions, which revealed that improvements in the speed of isothermal amplification reactions did not always correlate with improvements in digital efficiency (the fraction of molecules that amplify) or with analytical sensitivity. However, we observed that the speeds of amplification for single-molecule (in a digital device) and multi-molecule (e.g. in a PCR well plate) formats always correlated for the same conditions. Also, digital efficiency correlated with the analytical sensitivity of the same reaction performed in a multi-molecule format. Our finding was supported experimentally with examples of primer design, the use or exclusion of loop primers in different combinations, and the use of different enzyme mixtures in one-step reverse-transcription loop-mediated amplification (RT-LAMP). Our results show that measuring the digital efficiency of amplification of single-template molecules allows quick, reliable comparisons of the analytical sensitivity of reactions under any two tested conditions, independent of the speeds of the isothermal amplification reactions

A novel cassette method for probe evaluation in the designed biochips

A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip

SENSITIVE AND SPECIFIC DETECTION OF SARS-COV-2 IN SALIVA USING REVERSE TRANSCRIPTASE LOOP-MEDIATED ISOTHERMAL AMPLIFICATION

A novel coronavirus called SARS-CoV-2 has caused the emergency release of the most well-known molecular assay, the CDC N1 RT-PCR assay, causing reports of poor analytical performance resulting in false-negative results (20, 26), and inconsistent testing kits received (23, 32). An immediate and dire need for a rapid and reliable SARS-CoV-2 testing workflow specifically designed for a university setting is the purpose this project is aiming and intended to fulfill. The workflow design uses a less invasive saliva sample for rapid screening using colorimetric RT-LAMP detection of three SARS-CoV-2 gene regions for Orf1ab, envelope, and nucleocapsid. Purified SARS-CoV-2 genomic RNA is spiked into Proteinase K and heat-treated saliva from a SARS-CoV-2 negative donor to simulate a positive donor sample used to characterize and optimize this workflow, detecting 200 copies of SARS-CoV-2 genomic RNA by all three gene targets. The utility of colorimetric RT-LAMP outperformed the CDC N1 RT-PCR assay in turn-around-time and analytical performance. When applied to a small surveillance study, five out of 47 asymptomatic saliva donors had (September 2020) detectable SARS-CoV-2 genetic material, resulting in a 10% positivity rate, with the campus dashboard reportin

Sensing methods for real-time Loop-mediated Isothermal Amplification in Digital Microfluidic systems

Digital Microfluidics (DMF) is a technology capable of maneuvering picoliter to microliter droplets in an independent and individual manner, with a wide variety of uses for bioassays and biosensing. These systems are advantageous for their small volumes, higher portability and multiplex assay capabilities, proving to be very capable of lab-on-chip and point-of-care applications. One of these applications are DNA amplification assays, of which, Loop Mediated Isothermal Amplification (LAMP), that has received increased interest from the scientific community. This method is a sensitive and simple diagnostic tool for fast detection and identification of molecular biomarkers enabling real-time monitoring. Nevertheless, sensing methods coupled with DMF devices are still uncapable of measuring the progress of said reaction in real-time. This work explores two real-time LAMP measurement approaches to be coupled with a DMF system. The first approach uses an H-shaped device, where human c-Myc proto-oncogene and human 18S housekeeping gene are amplified and measured in real-time through fluorescence methods. The second approach uses interdigitated electrodes, where human c-Myc proto-oncogene is amplified and measured in real-time through Electrochemical Impedance Spectroscopy (EIS). Following development and characterization of both techniques, fluorescence measuring devices show 49% fluorescence signal difference between positive and negative controls end-points. EIS measuring devices indicate significant differences between commercial solutions with pH 4, 7 and 10, by Ciclic Voltammetry. This suggests that such devices could be used for real-time, label free, LAMP monitoring, since significant pH changes occur during a LAMP reactio

POINT OF CARE INFECTIOUS DISEASE DIAGNOSIS VIA ISOTHERMAL NUCLEIC ACID AMPLIFICATION INTEGRATED INTO A SAMPLE TO ANSWER DEVICE

Sexually transmitted infections caused by Chlamydia trachomatous (CT) and Neisseria gonorrhea (NG) are often under-diagnosed, miss-diagnosed, and not properly treated, leading to long term complications such as ectopic pregnancy and infertility, and to emergence of drug resistance. Dengue virus (DENV) infections can cause dengue fever (DF), which if not properly managed can lead to Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), causing significant morbidity and mortality. In both cases, early and accurate detection at the point of care is critical to facilitate proper patient care and disease management. Nucleic acid amplification tests (NAATs) provide suitable sensitivity and specificity, but are often complex, expensive, and difficult to implement at the point of care, especially in low resource settings. We have developed a prototype system consisting of a cartridge and compact instrument that can execute sample preparation, isothermal Loop Mediated amplification (LAMP) and lateral flow detection of these pathogens. For CT and NG, we have established singleplex and duplex LAMP assays, in liquid and paper form, with lateral flow detection. We have performed inhibition testing, demonstrated full process execution outside the cartridge, then implemented the 4 process in the cartridge and device, demonstrating detection down to 100 Elementary Bodies (EB)/mL for CT and 100 colony forming unit (CFU)/mL NG in urine. For DENV, we established a pan-serotype singleplex RT-LAMP assay and a duplex assay with MS2 as an internal amplification control, in liquid and paper form. We demonstrated that the master-mix in paper form is stable upon storage at RT and 40oC over 8 weeks. For sample preparation starting from whole blood, we performed plasma separation coupled to amplification, with inhibitor testing. The next step is a front to back experiment and implementation in a modified cartridge for the detection of all DENV serotypes. The herein described assay processes and prototype systems can be further developed to enable point of care diagnosis for these and other pathogens in low resource settings

A portable device for nucleic acid quantification powered by sunlight, a flame or electricity.

A decentralized approach to diagnostics can decrease the time to treatment of infectious diseases in resource-limited settings. Yet most modern diagnostic tools require stable electricity and are not portable. Here, we describe a portable device for isothermal nucleic-acid quantification that can operate with power from electricity, sunlight or a flame, and that can store heat from intermittent energy sources, for operation when electrical power is not available or reliable. We deployed the device in two Ugandan health clinics, where it successfully operated through multiple power outages, with equivalent performance when powered via sunlight or electricity. A direct comparison between the portable device and commercial qPCR (quantitative polymerase chain reaction) machines for samples from 71 Ugandan patients (29 of which were tested in Uganda) for the presence of Kaposi's sarcoma-associated herpesvirus DNA showed 94% agreement, with the four discordant samples having the lowest concentration of the herpesvirus DNA. The device's flexibility in power supply provides a needed solution for on-field diagnostics

Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Loop Mediated Isothermal Amplification (LAMP)

Staphylococcus aureus is one of the most common pathogens that cause a wide range of infections ranging from skin and soft tissue infections to invasive, life threatening infections. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) substantially increased healthcare burdens associated with Staphylococcal infections because of high morbidity and mortality and increasing the need for efficient and cost-effective screening methods, for high-risk patients. The objective of this study is to develop two molecular methods, real-time PCR and loop-mediated isothermal amplification (LAMP), and validate them following Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP) standards. The real time PCR assay was developed targeting mecA, mecC, nuc, and coa to detect S. aureus and methicillin-resistance. The assay had high precision, a linear range of 104-108 CFU/ml, and 95% accuracy. The assay detects MRSA, MSSA, MR-CoNS, and MS CoNS. The LAMP assay was developed targeting the same genes; however, its lower limit of detection was 106 CFU/ml, which was much higher than that of the real-time PCR assay. Additional studies are required to optimize the performance characteristics of the LAMP assay further. Nevertheless, the real-time PCR assay developed in this study will be useful for the detection of MRSA in a cost-effective manner

One-Step Detection of the 2009 Pandemic Influenza A(H1N1) Virus by the RT-SmartAmp Assay and Its Clinical Validation

<div><h3>Background</h3><p>In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society.</p> <h3>Methodology</h3><p>To address the clinical need for rapid diagnosis, we have developed a new method, the “RT-SmartAmp assay”, to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses.</p> <h3>Results and Conclusions</h3><p>We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.</p> </div

A multiplexed human papillomavirus (HPV) 16 and 18 diagnostic for cervical cancer screening

Cervical cancer is a major problem in the developing world and low- resource settings where standard screening techniques are not accessible. Cervical cancer is one of the few cancers that can be successfully treated when detected early. Therefore, there exists a high clinical need to screen for cervical cancer early. The etiological agent of cervical cancer is the human papillomavirus (HPV), with 70% of cases related to HPV genotypes 16 and 18. I sought to increase access to screening by developing a fully integrated and multiplexed molecular diagnostic assay to extract, amplify, detect, and distinguish HPV16 and HPV18 DNA on a low-cost paperfluidic platform for point- of-care (POC) applications. Isothermal (one temperature) loop-mediated amplification (LAMP) was used to amplify HPV DNA instead of the traditional polymerase chain reaction (PCR) that requires multiple temperatures. The amplified HPV16 and HPV18 DNA were differentially detected on a simple lateral flow strip – similar to that used in a pregnancy test – generating a visible colorimetric readout for each specific genotype. LAMP amplification is difficult to characterize and current methods were insufficient in providing specificity at the level needed for a multiplexed assay. Therefore, a novel characterization strategy was developed based on fluorescence to distinguish positive LAMP amplification products. This workflow used differential fluorescent tags to identify whether HPV16 DNA or HPV18 DNA was present and simplified complex nonspecific LAMP smears to a specific band pattern. After singleplex HPV16 and HPV18 LAMP assays were optimized with the new workflow, the two singleplex assays were successfully combined into one multiplex reaction with 12 primers, a nontrivial feat. Each assay step – DNA extraction, amplification, and detection – was optimized and integrated into a single chip that can control the timing of each step. Several chip configurations were tested to determine the optimal chip form factor, and a small subset of clinical samples were tested to demonstrate feasibility in low-resource settings. With this diagnostic platform, asymptomatic patients positive for HPV16 DNA and HPV18 DNA can be screened more closely, allocating precious resources to those most at risk, a beneficial use in both low-resource settings and the USA

Point-of-care diagnostics for Single Nucleotide Polymorphisms genotyping: applications to food traceability, nutrigenetics and pharmacogenetics

Single Nucleotide Polymorphisms (SNPs) are common genetic variations associated to specific phenotypes. SNPs have high relevance in different biomedical areas including both preventive/personalized medicine and non-clinical contexts, such food traceability. However, current techniques for SNP genotyping are expensive, require specialized laboratories and costly instrumentations, and are time consuming. In this framework, the development of rapid, point-of-care (POC) SNP diagnostic technologies would be of great interest, opening new possibilities for early, on-site screenings in multiple fields. In this PhD thesis, starting from state-of-art approaches, a rapid and portable diagnostic strategy based on Loop Mediated Isothermal Amplification (LAMP) has been developed for SNPs genotyping. In particular, the developed strategy was applied and validated, in a naked-eye colorimetric approach, for food varietal discrimination (i.e. durum wheat variety) in a model case of industrial relevance. Moreover, the POC technology was targeted to different salivary biomarkers, to address nutrigenetics and pharmacogenetics issues. Notably, the possibility to rapidly diagnose the genetic predisposition to lactose intolerance or the impairment of folic acid metabolism was proven. Furthermore, the developed POC-compatible protocols can work with portable, battery-powered devices, allowing for POC operation of such diagnostic tests