Metodo LAMP como alternativa diagnostica para la deteccion del virus SARS-CoV-2

Summary Introduction: The SARS-CoV-2 coronavirus, that causes the COVID-19 disease, has become a global public health problem that requires the implementation of rapid and sensitive diagnostic tests. Aim(s): To evaluate and compare the sensitivity of LAMP assay to a standard method and use RT-LAMP for the diagnosis of SARS-CoV-2 in clinical samples from Colombian patients. Method(s): A descriptive and cross-sectional study was conducted. A total of 25 nasopharyngeal swab samples including negative and positive samples for SARS-CoV-2 were analyzed, through the RT-LAMP method compared to the RT-qPCR assay. Result(s): LAMP method detected ~18 copies of the N gene, in 30 min, evidenced a detection limit similar to the standard method, in a shorter time and a concordance in RT-LAMP of 100% with the results. Conclusion(s): RT-LAMP is a sensitive, specific, and rapid method that can be used as a diagnostic aid of COVID-19 disease.Copyright © 2021. All Rights Reserved.

Molecular Diagnostics on Smartphone

During the COVID-19 pandemic, point-of-care genetic testing (POCT) devices were used for on-time and on-site detection of the virus, which helped to prevent and control the spread of the pandemic. Smartphones, which are widely used electronic devices with many functions, have the potential to be used as a molecular diagnostic platform for universal healthcare monitoring. Several integrated diagnostics platforms for the real-time and end-point detection of COVID-19 were developed using the functions of smartphones, such as the operating system, power, sound, camera, data storage, and display. These platforms use the 5V output power of smartphones, which can be amplified to power a micro-capillary electrophoresis system or a thin-film heater, and the CMOS camera of smartphones can capture the color change during a colorimetric loop-mediated isothermal amplification test and detect fluorescence signals. Smartphones can also be used with self-written web-based apps to enable automatic and remote pathogen analysis on POCT platforms. Our lab developed a handheld micro-capillary electrophoresis device for end-point detection of SARS-CoV-2, as well as an integrated smartphone-based genetic analyzer for the qualitative and quantitative colorimetric detection of foodborne pathogens with the help of a custom mobile app. © 2023 SPIE.

Multiplexed Reverse Transcription Loop-Mediated Isothermal Amplification Coupled with a Nucleic Acid-Based Lateral Flow Dipstick as a Rapid Diagnostic Method to Detect SARS-CoV-2.

Due to the high reproduction rate of COVID-19, it is important to identify and isolate infected patients at the early stages of infection. The limitations of current diagnostic methods are speed, cost, and accuracy. Furthermore, new viral variants have emerged with higher rates of infectivity and mortality, many with mutations at various primer binding sites, which may evade detection via conventional PCR kits. Therefore, a rapid method that is sensitive, specific, and cost-effective is needed for a point-of-care molecular test. Accordingly, we developed a rapid molecular SARS-CoV-2 detection kit with high specificity and sensitivity, RT-PCR, taking advantage of the loop-mediated isothermal amplification (LAMP) technique. Four sets of six primers were designed based on conserved regions of the SARS-CoV-2 genome: two outer, two inner and two loop primers. Using the optimized protocol, SARS-CoV-2 genes were detected as quickly as 10 min but were most sensitive at 30 min, detecting as little as 100 copies of template DNA. We then coupled the RT-LAMP with a lateral flow dipstick (LFD) for multiplex detection. The LFD could detect two genic amplifications on a single strip, making it suitable for multiplexed detection. The development of a multiplexed RT-LAMP-LFD reaction on crude VTM samples would be suitable for the point-of-care diagnosis of COVID-19 in diagnostic laboratories as well as in private homes.

PATHPOD - A loop-mediated isothermal amplification (LAMP)-based point-of-care system for rapid clinical detection of SARS-CoV-2 in hospitals in Denmark.

Sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a vital goal in the ongoing COVID-19 pandemic. We present in this comprehensive work, for the first time, detailed fabrication and clinical validation of a point of care (PoC) device for rapid, onsite detection of SARS-CoV-2 using a real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) reaction on a polymer cartridge. The PoC system, namely PATHPOD, consisting of a standalone device (weight less than 1.2 kg) and a cartridge, can perform the detection of 10 different samples and two controls in less than 50 min, which is much more rapid than the golden standard real-time reverse-transcription Polymerase Chain Reaction (RT-PCR), typically taking 16-48 h. The novel total internal reflection (TIR) scheme and the reactions inside the cartridge in the PoC device allow monitoring of the diagnostic results in real-time and onsite. The analytical sensitivity and specificity of the PoC test are comparable with the current RT-PCR, with a limit of detection (LOD) down to 30-50 viral genome copies. The robustness of the PATHPOD PoC system has been confirmed by analyzing 398 clinical samples initially examined in two hospitals in Denmark. The clinical sensitivity and specificity of these tests are discussed.

Analysis of the factors that affect the detection duration of SARS-CoV-2 in Loop Mediated Isothermal Amplification among COVID-19 inpatients.

In COVID-19 patients who are severe or immunocompromised, the duration of infectious viral shedding may be longer, and a longer isolation duration is recommended. In National Sagamihara hospital, a decline in the viral load to end the isolation of COVID-19 hospitalized patients is confirmed by loop mediated isothermal amplification (LAMP). However, a subset of patients persisted in displaying LAMP positivity for more than 20 days since symptom onset. Therefore, we conducted a retrospective observational study to investigate factors impacting the persistence of LAMP positivity. The study included 102 participants. The severity of COVID-19 was mild in 25.5%, moderate in 67.6%, and severe in 6.9% of patients. The median number (interquartile range) of days until negative LAMP since symptom onset was 16 (14-19) days. Multivariate logistic regression analysis showed that age ≥55 years and the delta variant were correlated with persistently LAMP positive for more than 20 days since symptom onset. This study identified that age, the delta variant, and oxygen requirement were factors contributing to persistently positive LAMP. Therefore, it is posited that in these patients, the implementation of LAMP for de-isolation would result in a prolonged duration of isolation.

Rapid and Sensitive Diagnosis of COVID-19 Using an Electricity-Free Self-Testing System.

Rapid and sensitive detection of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for early diagnosis and effective treatment. Nucleic acid testing has been considered the gold standard method for the diagnosis of COVID-19 for its high sensitivity and specificity. However, the polymerase chain reaction (PCR)-based method in the central lab requires expensive equipment and well-trained personnel, which makes it difficult to be used in resource-limited settings. It highlights the need for a sensitive and simple assay that allows potential patients to detect SARS-CoV-2 by themselves. Here, we developed an electricity-free self-testing system based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that allows for rapid and accurate detection of SARS-CoV-2. Our system employs a heating bag as the heat source, and a 3D-printed box filled with phase change material (PCM) that successfully regulates the temperature for the RT-LAMP. The colorimetric method could be completed in 40 min and the results could be read out by the naked eye. A ratiometric measurement for exact readout was also incorporated to improve the detection accuracy of the system. This self-testing system is a promising tool for point-of-care testing (POCT) that enables rapid and sensitive diagnosis of SARS-CoV-2 in the real world and will improve the current COVID-19 screening efforts for control and mitigation of the pandemic.

Optimal Stabilization for Long-Term Storage of Nucleic Acid-Based CRISPR/Cas12a Assay for SARS-CoV-2 Detection

In this long-term storage study, we optimized the lyophilization conditions of each reaction stage of a nucleic acid-based assay for SARS-CoV-2 detection. The stability testing demonstrated that the dried reactions from all 3 steps (cDNA synthesis, isothermal amplification and detection) can be kept at ¡20 °C or 4 °C for up to 6 or 3 months, respectively, whereas, if stored at 25 °C or 37 °C, the reagents only could be stored for a few days without quality loss. This suggests that we can have the dried reactions at ¡20 °C for long-term storage until needed. Moreover, this assay is now simpler to perform as each of the 3 steps now proceeds with pre-mixed regents lyophilized in a single tube for each step. © 2023 University of Kerbala. All rights reserved.

Recent advances in RNA sample preparation techniques for the detection of SARS-CoV-2 in saliva and gargle.

Molecular detection of SARS-CoV-2 in gargle and saliva complements the standard analysis of nasopharyngeal swabs (NPS) specimens. Although gargle and saliva specimens can be readily obtained non-invasively, appropriate collection and processing of gargle and saliva specimens are critical to the accuracy and sensitivity of the overall analytical method. This review highlights challenges and recent advances in the treatment of gargle and saliva samples for subsequent analysis using reverse transcription polymerase chain reaction (RT-PCR) and isothermal amplification techniques. Important considerations include appropriate collection of gargle and saliva samples, on-site inactivation of viruses in the sample, preservation of viral RNA, extraction and concentration of viral RNA, removal of substances that inhibit nucleic acid amplification reactions, and the compatibility of sample treatment protocols with the subsequent nucleic acid amplification and detection techniques. The principles and approaches discussed in this review are applicable to molecular detection of other microbial pathogens.

A polyethylene glycol enhanced ligation-triggered self-priming isothermal amplification for the detection of SARS-CoV-2 D614G mutation.

We presented a polyethylene glycol (PEG) enhanced ligation-triggered self-priming isothermal amplification (PEG-LSPA) for the detection D614G mutation in S-glycoprotein of SARS-CoV-2. PEG was employed to improve the ligation efficiency of this assay by constructing a molecular crowding environment. Two hairpin probes (H1 and H2) were designed to contain 18 nt and 20 nt target binding site at their 3' end and 5' end, respectively. In presence of target sequence, it complemented with H1 and H2 to trigger ligation by ligase under molecular crowding condition to form ligated H1-H2 duplex. Then 3' terminus of the H2 would be extended by DNA polymerase under isothermal conditions to form a longer extended hairpin (EHP1). 5' terminus of EHP1 with phosphorothioate (PS) modification could form hairpin structure due to the lower Tm value. The resulting 3' end overhang would also fold back as a new primer to initiate the next round of polymerization, resulting in the formation of a longer extended hairpin (EHP2) containing two target sequence domains. In the circle of LSPA, long extended hairpin (EHPx) containing numerous target sequence domains was produced. The resulting DNA products can be monitored in real-time fluorescence signaling. Our proposed assay owns an excellent linear range from 10 fM to 10 nM with a detection limit down to 4 fM. Thus, this work provides a potential isothermal amplification method for monitoring mutations in SARS-CoV-2 variants.

Respiratory Syncytial Virus (RSV) infections in children: recent concepts in immunopathogenesis and diagnostic dilemma

Respiratory viral infections are important cause of morbidity and mortality in early life. The relative influence of host and viral factors possibly contribute to the disease pathogenesis. Predisposing conditions like prematurity, Low birth weight and congenital heart diseases etc. have been incriminated in the disease progression. The development of cough, wheezing, and tachypnea, usually peaking on days 4 to 5, go parallel with host cytokine responses and viral load. Various host cytokines, chemokines and molecules involved in the immune response against RSV infection might be responsible for the outcome of the disease process. Nasopharyngeal aspirates (NPAs) from children (n = 349) between 2013-2017 were subjected for IL-17A, IFN-gamma, TNF-alpha, IL-10, IL-6 levels by CBA and MMP-9 and TIMP-1 levels by ELISA. The viral load in RSV positive samples and cytokine levels were correlated with the WHO criteria for acute lower respiratory tract illness (ALRTI). RSV viral load, Pro-inflammatory cytokine (TNF-alpha) levels in severe ALRTI patients were significantly higher than the ALRTI patients [p<0.001]. Whereas Th17 cytokine (IL-17) was found to be significantly higher (p<0.05) in ALRTI patients than severe patients. MMP-9 is secreted in higher levels in severe ALRTI patients (n = 77) in comparison to Acute LRTI patients (n = 35) with an increase of thirty seven fold (p<0.001). Thus, the study highlights the role of TNF -alpha, IL-17 and Th2 cytokine biasness in the pathogenesis of RSV disease with the possible contribution of higher MMP-9/TIMP-1 ratio as a bad prognostic marker towards disease severity. To study the gene expression of autophagy and mTOR signalling pathways in RSV infected children with ALRTI. Nasopharyngeal aspirate (NPA) samples (n = 145) from children suffering from ALRTI were subjected for detection of RSV (Oct 2019 to March 2020). Semi-quantitative gene expression analysis for 5 representative genes each of mTOR signalling and autophagy pathway were performed in respiratory tract epithelial cells using 25 RSV positive cases and 10 healthy controls subjects. Autophagy gene expression analysis revealed significant upregulation in NPC1 and ATG3 autophagy genes. mTOR, AKT1 and TSC1 genes of mTOR pathway were significantly down-regulated in RSV positive patients except RICTOR gene which was significantly upregulated. Thus, survival of RSV within autophagosome might have been facilitated by upregulation of autophagy and downregulation of mTOR signalling genes. To assess the impact of SARS-CoV2 pandemic on RSV, samples were collected from children with ALRTIs admitted to emergency, PICU and indoor admissions during pre-pandemic period (October 2019 to February 2020;n = 166) and during COVID-19 Pandemic (July 2021 to July 2022;n = 189, SARS-CoV2 negative). These NP swabs were analyzed for pdm InfA H1N1, InfA H3N2, Inf B, RSV, hMPV, hBoV, hRV, PIV-2 and PIV-3 by PCR. Higher proportion of children with ALRTIs have had virus/es isolated during pre-pandemic period than during pandemic period (p<0.001). During pre-pandemic period, significantly higher proportion of children had RSV positivity (p<0.001);and significantly lower positivity for hRV (p<0.05), hMPV (p<0.05), and hBoV (p <= 0.005). The occurrence of COVID-19 pandemic has significantly impacted the frequency and pattern of detection of RSV among hospitalized children with LRTIs. RSV Fusion protein plays a critical role in the entry of the virus into the host cell by initiating the fusion of host and viral membranes. It happens to be a target of neutralizing antibodies paving the way as a vaccine candidate. Hence effort was made to introduce point mutation in hRSV fusion protein which can confer stability in its prefusion form. In-silico a stable structure of RSV fusion protein was generated making it a potential vaccine candidate. The timely diagnosis of RSV infection in this population is important for initiating therapy and instituting appropriate infection prevention measures. Serological testing is not widely used for the diagnosis of RSV. C ll Cultures including shell vial culture were used for RSV diagnosis. However, culture approaches lack sensitivity, often quite significantly, compared to nucleic acid amplification assays for the diagnosis of RSV infections. Molecular multiplex assays now offer increased sensitivity for a more accurate diagnosis. However issues with the use of these types of commercial panel assays include the requirement for substantial training, quality systems, and infrastructure to maintain and run these assays and many a times identification of viruses where the true pathogenic potential of those multiple viruses are debatable. Studies are available with laboratory- developed nucleic acid amplification test systems for the detection of RSVA and RSVB in clinical specimens either by PCRbased technologies or RT-LAMP. Gene targets of laboratory-developed molecular assays point towards M gene and the N gene in RSVA and -B with the benefits of flexibility to modify assays when targets are under evolutionary pressure to change, as well as a perceived initial low cost to carry out testing.

Validation of Commercial Kits Using Standard ICMR Protocol

Background: SARS-CoV-2 highlighted worldwide, the need of enhance testing capacity. Government of India, under Atmanirbhar Bharat provided platform to private/public companies to develop and manufacture diagnostic reagents /kits for SARS CoV 2 testing. Objective(s): * Performance evaluation of commercial kits. * Handholding of private/public companies to improve the kits quality for its diagnostic accuracy to use for Covid 19 diagnosis Material(s) and Method(s): The SOP for the validation of diagnostic kits were prepared and approved by ICMR technical committee. The ICMR NIV single tube assay was used as gold slandered. The panels of known positives and negatives were prepared. Validation of commercially developed RT-PCRs, RNA extraction kits and virus transport medium were undertaken. The sensitivity and specificity of the kit were calculated and reported as per ICMR's acceptance. Result(s): Real time RT-PCR kits evaluation: Total 165 kits were evaluated, which includes 12 LAMP assay. Among domestic kits, 31 kits were satisfactory while 83 were not satisfactory. Among the imported kits, 25 kits were satisfactory while 26 were not satisfactory. RNA extraction kits evaluation:- Total 157 kits were evaluated, Among domestic kits, 57 kits were satisfactory while 53 were not satisfactory. Among the imported kits, 31 kits were satisfactory and 17 were not satisfactory. VTM kits evaluated = Total 89 kits were evaluated among which nine kits were imported while 80 kits were of domestic origin. Performance of 10 kits was not satisfactory. Conclusion(s): Kit validation is important to access the quality of commercial kits and to enhanced the testing capacity exponentially in country.

Molecular diagnostics for clinical respiratory virus on a total integrated centrifugal microsystem using reverse transcription-loop-mediated isothermal amplification

Respiratory viruses are highly contagious agents that can cause endemic and epidemic infections in humans. Early detection of these viruses is crucial in preventing economic damage and reducing mortality rates. In this study, we present a total integrated genetic analyzer to perform a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the simultaneous detection of 7 respiratory viruses (Influenza A H1N1 and H3N2, Influenza B, Respiratory syncytial virus A and B, Adenovirus, and COVID-19). The primer sets for the RT-LAMP assay were designed and evaluated in comparison with the RT-PCR assay using clinical samples, confirming high specificity and efficiency. The entire process of viral RNA extraction, reagent mixing, gene amplification, and detection was completed on the device in 1hr 20min. The constructed portable diagnostic instrument is equipped with a rotary motor, two sets of peltier heaters, a fluorescence detector, and a touch screen for inputting experimental parameters and displaying result. The proposed point-of-care (POC) diagnostic platform correctly analyzed a total of 21 clinical samples (3 for each of the 7 viruses). The limit-of-detection (LOD) for Influenza A subtype H3N2 was 101 pfu/mL, which demonstrates the high performance of our proposed centrifugal microsystem for on-site molecular diagnostics in medical centers.

Abstracts of the papers presented in the International Conference of Indian Virological Society, VIROCON 2022 on "Emerging and Re-emerging Viral Infections Impacting Humans, Animals, Plants, Fish and Environment"

The proceedings contain 206 papers. The topics discussed include: influenza: experiences from Kashmir;outbreaks of different viral etiologies amidst COVID-19 pandemic;development of a colorimetric isothermal (LAMP) assay for rapid detection of monkeypox virus;circulation of genetically diverse non-polio enteroviruses in respiratory samples during COVID-19 pandemic period (2021-22);evolutionary analysis of all eleven genes of species C rotaviruses circulating in humans and domestic animals;molecular characterization of dengue viruses circulating in Pune district, Maharashtra from 2009-2022;isolation and genomic characterization of cell fusing agent virus from aedes aegypti mosquitoes from Assam, India;structure-based identification and evaluation of antiviral activity of potent small molecule inhibitors targeting alphavirus RNA-dependent RNA polymerase;integration of HBV receptor NTCP into hepatoma cell using grnome editing;and hepatitis B virus genome targeting using CRISPR/Cas9based gene editing tool.

Development of Duplex LAMP Technique for Detection of Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Circovirus Type 2 (PCV 2).

Porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2) are both important global pathogenic viruses which have a significant impact on the swine industry. In this study, a duplex loop-mediated isothermal amplification (duplex LAMP) method was developed in combination with lateral flow dipstick (LFD) for simultaneous detection of PEDV and PCV2 using specific sets of primers and probes designed based on the conserved regions of a spike gene (KF272920) and an ORF gene (EF493839), respectively. The limit of detection (LOD) values of the duplex LAMP-LFD for the detection of PEDV and PCV2 were 0.1 ng/µL and 0.246 ng/µL, respectively. The LOD of duplex LAMP-LFD was 10-times more sensitive than conventional PCR and RT-PCR-agarose gel-electrophoresis (PCR-AGE and RT-PCR-AGE). No cross-reaction to each other and to other pathogenic viruses that can infect pigs were observed according to analytical specificity tests. The duplex LAMP-LFD method for the simultaneous detection of PEDV and PCV2 co-infection could be completed within approximately 1.5 h, and only a simple heating block was required for isothermal amplification. The preliminary validation using 50 swine clinical samples with positive and negative PEDV and/or PCV2 revealed that the sensitivity, specificity, and accuracy of duplex LAMP-LFD were all 100% in comparison to conventional PCR and RT-PCR. Hence, this study suggests that duplex LAMP-LFD is a promising tool for the early detection and initial screening of PEDV and PCV2, which could be beneficial for prevention, planning, and epidemiological surveys of these diseases.

Strategies for the Voltammetric Detection of Loop-Mediated Isothermal Amplification.

Loop-mediated isothermal amplification (LAMP) is rapidly developing into an important tool for the point-of-use detection of pathogens for both clinical and environmental samples, largely due to its sensitivity, rapidity, and adaptability to portable devices. Many methods are used to monitor LAMP, but not all are amenable to point-of-use applications. Common methods such as fluorescence often require bulky equipment, whereas colorimetric and turbidimetric methods can lack sensitivity. Electrochemical biosensors are becoming increasingly important for these applications due to their potential for low cost, high sensitivity, and capacity for miniaturization into integrated devices. This review provides an overview of the use of voltammetric sensors for monitoring LAMP, with a specific focus on how electroactive species are used to interface between the biochemical products of the LAMP reaction and the voltammetric sensor. Various strategies for the voltammetric detection of DNA amplicons as well as pyrophosphate and protons released during LAMP are presented, ranging from direct DNA binding by electroactive species to the creative use of pyrophosphate-detecting aptamers and pH-sensitive oligonucleotide structures. Hurdles for adapting these devices to point-of-use applications are also discussed.

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection.

Nucleic acid amplification techniques have always been one of the hot spots of research, especially in the outbreak of COVID-19. From the initial polymerase chain reaction (PCR) to the current popular isothermal amplification, each new amplification techniques provides new ideas and methods for nucleic acid detection. However, limited by thermostable DNA polymerase and expensive thermal cycler, PCR is difficult to achieve point of care testing (POCT). Although isothermal amplification techniques overcome the defects of temperature control, single isothermal amplification is also limited by false positives, nucleic acid sequence compatibility, and signal amplification capability to some extent. Fortunately, efforts to integrating different enzymes or amplification techniques that enable to achieve intercatalyst communication and cascaded biotransformations may overcome the corner of single isothermal amplification. In this review, we systematically summarized the design fundamentals, signal generation, evolution, and application of cascade amplification. More importantly, the challenges and trends of cascade amplification were discussed in depth.

Accurate and ultrasensitive detection for PEDV based on photoelectrochemical sensing coupling loop-mediated isothermal amplification.

Porcine epidemic diarrhea (PED) is a serious disease requiring a simple and accurate detection method. Accordingly, this study developed a novel, ultrasensitive photoelectrochemical (PEC) sensing platform using the loop-mediated isothermal amplification (LAMP) technique (LAMP-PEC). An amino (-NH2)-modified LAMP product is obtained by amplification of the PED virus gene with specially designed primers. The generated NH2-modified LAMP product is assembled on the surface of an electrode by forming imine linkages between aldehyde and amino groups based on the Schiff base reaction. A stable photocurrent is provided by a CdIn2S4 photoactive material, which possesses high photoelectric conversion efficiency. Amplified DNA assembled on the electrode surface increases steric hindrance and hinders electrons from moving from the electrode to electron acceptors, which decreases the photocurrent. This strategy can detect PEDV with a low detection limit of 0.3 fg µL-1 and a wide linear range of 1 × 10-3-1 × 102 pg/µL. The sensing platform has excellent specificity and sensitivity and can be used for the quantitative detection of many other pathogens with the assistance of LAMP.

Multipurpose advanced split T7 promoter-based transcription amplification for ultrasensitive molecular diagnostics

An accurate, convenient, and rapid diagnostic platform, which can be applied in facility-limited or point-of-care (POC) settings, is essential to help prevent the spread of infectious diseases and enable the most effective treatment to be selected. In this study, we describe the development of a new isothermal molecular diagnostic system named multipurpose advanced split T7 promoter-based transcription amplification (MASTER) for the rapid and ultrasensitive detection of various pathogens containing single-stranded RNA and double-stranded DNA. MASTER produces a large number of RNA amplicons in the presence of target pathogens, which generate fluorescence or colorimetric signals based on light-up RNA aptamers or lateral flow assays. Implementing MASTER at 37 °C for<1 h achieved the detection of a single copy per reaction without cross-reactivity. Moreover, the testing of 40 clinical samples revealed that MASTER exhibited excellent accuracy with 100% sensitivity and specificity for SARS-CoV-2 diagnosis. Furthermore, a one-pot MASTER system capable of accelerating practical applications was demonstrated, indicating that the MASTER system is a promising platform for the effective surveillance of various pathogens. © 2023 Elsevier B.V.

Automatic Analysis of Isothermal Amplification via Impedance Time-Constant-Domain Spectroscopy: A SARS-CoV-2 Case Study

The development of sensitive and affordable testing devices for infectious diseases is essential to preserve public health, especially in pandemic scenarios. In this work, we have developed an attractive analytical method to monitor products of genetic amplification, particularly the loop-mediated isothermal amplification reaction (RT-LAMP). The method is based on electrochemical impedance measurements and the distribution of relaxation times model, to provide the so-called time-constant-domain spectroscopy (TCDS). The proposed method is tested for the SARS-CoV-2 genome, since it has been of worldwide interest due to the COVID-19 pandemic. Particularly, once the method is calibrated, its performance is demonstrated using real wastewater samples. Moreover, we propose a simple classification algorithm based on TCDS data to discriminate among positive and negative samples. Results show how a TCDS-based method provides an alternative mechanism for label-free and automated assays, exhibiting robustness and specificity for genetic detection.