Reverse Transcription Loop-mediated Isothermal Amplification Research Articles

A microfluidic biosensor architecture for the rapid detection of COVID-19

The lack of enough diagnostic capacity to detect severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has been one of the major challenges in the control the 2019 COVID pandemic; this led to significant delay in prompt treatment of COVID-19 patients or accurately estimate disease situation. Current methods for the diagnosis of SARS-COV-2 infection on clinical specimens (e.g. nasal swabs) include polymerase chain reaction (PCR) based methods, such as real-time reverse transcription (rRT) PCR, real-time reverse transcription loop-mediated isothermal amplification (rRT-LAMP), and immunoassay based methods, such as rapid antigen test (RAT). These conventional PCR methods excel in sensitivity and specificity but require a laboratory setting and typically take up to 6 h to obtain the results whereas RAT has a low sensitivity (typically at least 3000 TCID50/ml) although with the results with 15 min. We have developed a robust micro-electro-mechanical system (MEMS) based impedance biosensor fit for rapid and accurate detection of SARS-COV-2 of clinical samples in the field with minimal training. The biosensor consisted of three regions that enabled concentrating, trapping, and sensing the virus present in low quantities with high selectivity and sensitivity in 40 min using an electrode coated with a specific SARS-COV-2 antibody cross-linker mixture. Changes in the impedance value due to the binding of the SARS-COV-2 antigen to the antibody will indicate positive or negative result. The testing results showed that the biosensor's limit of detection (LoD) for detection of inactivated SARS-COV-2 antigen in phosphate buffer saline (PBS) was as low as 50 TCID50/ml. The biosensor specificity was confirmed using the influenza virus while the selectivity was confirmed using influenza polyclonal sera. Overall, the results showed that the biosensor is able to detect SARS-COV-2 in clinical samples (swabs) in 40 min with a sensitivity of 26 TCID50/ml.

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.

One-Step Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Canna Yellow Streak Virus.

Canna yellow streak virus (CaYSV) is a potyvirus that causes severe damage to the ornamental plant canna in the United Kingdom and Brazil. Here we identified CaYSV in China by isolating total RNA from infected plant, amplifying the virus genome segments, cloning and sequencing the amplicons. After assembly, the full-length genome of the virus was obtained and uploaded to the NCBI database. Phylogenetic analysis results showed that the Guizhou isolate (OL546222) was most closley related to the KS isolate (MG545919.1). Virus detection is essential for virus disease control, but the subclinical infection of CaYSV on canna in its early development increases the difficulty of CaYSV diagnosis. The goal of this study was to develop an efficient method for detection of CaYSV. We designed the primers, optimized the reaction conditions, and finally established a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method. The product of RT-LAMP can be analyzed by both agarose gel electrophoresis and visible color change. The established one-step RT-LAMP assay showed high specificity and sensitivity in detecting CaYSV. This RT-LAMP method was also applied in analysis of 61 field samples collected from Guizhou and Jiangsu provinces. The results showed that the infection rates of CaYSV on canna samples from these two provinces were very high (63% and 96% respectively).

Paper-based colorimetric loop-mediated isothermal amplification (LAMP) assay for the identification of latent Colletotrichum in harvested fruit

Paper-based analytical devices (PADs) have gained enormous attention because of their low-cost, simple fabrication, and portability. Here, we propose a paper-based device for performing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with real-time simultaneous detection of C. gloeosporioides latent infections in tomatoes. RT-LAMP-based PAD platform comprises a paper substrate on which the DNA amplification reaction occurs. Among different types of tested papers, cellulose membrane (grade 4) enabled effective visualization of the amplification result. The assay was found highly selective for the latent stage of C. gloeosporioides with lower limit of detection (LOD) of 0.5 pg of total extracted RNA. The developed assay generated the results within 40 min and hence can be efficiently employed for identifying C. gloeosporioides in resource-limited settings.

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 1 hr 20 min. 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.

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.

Development of colloidal gold immunochromatography and reverse transcription loop-mediated isothermal amplification assays to detect lychnis mottle virus.

Lychnis mottle virus (LycMoV; genus Unassigned, family Secoviridae) infection of Angelica sinensis produces mottle and mosaic symptoms, damaging the host. Early detection of relevant pathogens is the most critical step in preventing the potential transmission of infectious disease. Polyclonal antibodies (pAbs) with high potency and high specificity were prepared using the recombinant LycMoV capsid protein as an antigen. Here, we developed and optimized a rapid colloidal gold immunochromatography assay (GICA) detection system for LycMoV using this antibody. Under optimum conditions, GICA specifically detected (up to 10,000-fold) positive LycMoV samples. A real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) system was also established by selecting the primers with high sensitivity and specificity to LycMoV. The RT-LAMP detection threshold was 1.42 fg/μl (291 copies/μl). A GICA-RT-LAMP assay system was further established and optimized. The minimum GICA detection line was calculated at 1.52x10-2 ng/μl. Although GICA did not detect positive samples after capturing 2.53x10-3 ng/μl of virus, GICA-LAMP and GICA-RT-PCR did, whose sensitivity was comparatively greater than six-fold. This is the first report showing that GICA-RT-LAMP is a cost effective approach for use in detecting LycMoV without extracting nucleic acids. These sensitive assays will help improve virus disease management in A. sinensis crops.

Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids

CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV)and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)with high specificity and accuracy. We obtain results in as little as 20min, and with the extraction process, the entire assay can be performed within an hour.

Challenges in Direct Detection of Flaviviruses: A Review.

Arthropods transmit arboviruses via mosquito and tick bites to humans and other animals. The genus flavivirus, which causes diseases, sequelae, and thousands of deaths, mainly in developing and underdeveloped countries, is among the arboviruses of interest to public health. Given the importance of early and accurate diagnosis, this review analyzes the methods of direct detection of flaviviruses, such as reverse transcription loop-mediated isothermal amplification, microfluidics, localized surface plasmon resonance, and surface-enhanced Raman scattering, and presents the advantages, disadvantages, and detection limits identified in studies reported in the literature for each methodology. Among the different methods available, it is essential to balance four fundamental indicators to determine the ideal test: good sensitivity, high specificity, low false positive rate, and rapid results. Among the methods analyzed, reverse transcription loop-mediated isothermal amplification stands out, owing to result availability within a few minutes, with good sensitivity and specificity; in addition, it is the best-characterized methodology.

A Systematic Review and Meta-Analysis Comparing the Diagnostic Accuracy Tests of COVID-19.

In this paper, we present a systematic review and meta-analysis that aims to evaluate the reliability of coronavirus disease diagnostic tests in 2019 (COVID-19). This article seeks to describe the scientific discoveries made because of diagnostic tests conducted in recent years during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Between 2020 and 2021, searches for published papers on the COVID-19 diagnostic were made in the PubMed database. Ninety-nine scientific articles that satisfied the requirements were analyzed and included in the meta-analysis, and the specificity and sensitivity of the diagnostic accuracy were assessed. When compared to serological tests such as the enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), lateral flow immunoassay (LFIA), and chemiluminescent microparticle immunoassay (CMIA), molecular tests such as reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR) performed better in terms of sensitivity and specificity. Additionally, the area under the curve restricted to the false-positive rates (AUCFPR) of 0.984 obtained by the antiviral neutralization bioassay (ANB) diagnostic test revealed significant potential for the identification of COVID-19. It has been established that the various diagnostic tests have been effectively adapted for the detection of SARS-CoV-2; nevertheless, their performance still must be enhanced to contain potential COVID-19 outbreaks, which will also help contain potential infectious agent outbreaks in the future.

Rapid and Accurate Detection of SARS-CoV-2 Using an iPad-Controlled, High-Throughput, Portable, and Multiplex Hive-Chip Platform (HiCube).

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the most effective measures to control the coronavirus disease 2019 (COVID-19) pandemic. However, there is still lack of an ideal detection platform capable of high sample throughput, portability, and multiplicity. Herein, by combining Hive-Chip (capillary microarray) and reverse transcriptional loop-mediated isothermal amplification (RT-LAMP), we developed an iPad-controlled, high-throughput (48 samples at one run), portable (smaller than a backpack), multiplex (monitoring 8 gene fragments in one reaction), and real-time detection platform for SARS-CoV-2 detection. This platform is composed of a portable Hive-Chip device (HiCube; 32.7 × 29.7 × 20 cm, 5 kg), custom-designed software, and optimized Hive-Chips. RT-LAMP primers targeting seven SARS-CoV-2 genes (S, E, M, N, ORF1ab, ORF3a, and ORF7a) and one positive control (human RNase P) were designed and prefixed in the Hive-Chip. On-chip RT-LAMP showed that the limit of detection (LOD) of SARS-CoV-2 synthetic RNAs is 1 copy/μL, and there is no cross-reaction among different target genes. The platform was validated by 100 clinical samples of SARS-CoV-2, and the results were highly consistent with those of the traditional real-time PCR assay. In addition, on-chip detection of 6 other respiratory pathogens showed no cross-reactivity. Overall, our platform has great potential for fast, accurate, and on-site detection of SARS-CoV-2.

A system model for real-time monitoring and geospatial data for the simulation of surveillance of COVID-19 in Makassar, Indonesia

The rapid spread of COVID-19 requires rapid management. Prompt treatment is needed to prevent the spread of this disease, which could be minimized or isolated in one place so that it does not spread to other places. This study was conducted to discover a model of the surveillance system in real time and to analyze the change in its distribution pattern. This study was conducted in the city of Makassar, South Sulawesi, Indonesia, involving 30 volunteers. Two devices were used, the Internet reverse transcription loop-mediated isothermal amplification (iRTLAMP) and IoT button application, to provide spatial data in the form of patient points exposed to COVID-19. Furthermore, three scenarios were applied to see the pattern of data distribution. The data recorded in the cloud database were retrieved with a created application and then analyzed using Kernel Density Estimation (KDE) and Point Pattern Analysis (PPA) to observe the distribution of patterns in real time. The analysis utilizing KDE with the Gaussian kernel function as the kernel revealed significant changes in the probability distribution, which could be seen from color changes in the map. The centrographic analysis revealed that the mean and median points of the three scenarios changed in various ways within approximately 700 m to 1.7 km. Meanwhile, the radius of minimal bounding circle behaved similarly and appeared to change depending on the scenario, from a radius of 5.57 (initial) km to 6.55 km (scenario 1), 5.57 km (scenario 2) and 6.22 km (scenario 3). The standard distance also showed a change from 4.53 km to 4.60 km (scenario 1), 4.70 km (scenario 2) and 5.40 km (scenario 3). Simulations carried out using the developed system showed that the use of internet devices could help monitor people exposed to COVID-19 by changing patterns and distribution points. Therefore, decision makers could take preventive actions earlier so that this disease does not spread quickly.

One-step Reverse Transcription-LoopMediated Isothermal Amplification (RT-LAMP) for closed-tube colorimetric detection of banana bract mosaic virus in Banana (Musa spp.).

The banana bract mosaic virus (BBrMV) is a major virus affecting bananas and plantains. Banana being propagated vegetatively, there arises a high risk of virus transmission through planting materials. Available molecular detection technique like the Reverse Transcriptase Polymerase Chain Reaction needs post-amplification sample handling, predisposing to sample cross contamination. A one-step Reverse Transcription-LoopMediated Isothermal Amplification (RT-LAMP) assay coupled with colorimetric detection was optimised for easy and quick detection of BBrMV in banana. The viral coat protein gene was amplified under isothermal conditions at 65 ºC. The RT-LAMP assay was optimised with respect to concentrations of MgSO4, dNTP, Bst polymerase enzyme and HNB dye. The total RNA purified from symptomatic samples was directly amplified under isothermal conditions by including 100 U M-MLV reverse transcriptase and 20 U RNasin® plus RNase inhibitor in the reaction. With the addition of 120µM of Hydroxy Naphthol Blue (HNB) dye in the RT-LAMP reaction, the BBrMV-positive samples had a colour change from violet to sky blue after the reaction. The RT-LAMP assay detected BBrMV in 0.1pg of total RNA isolated from symptomatic plants. Molecular characterisation of RT-LAMP products was done using restriction profiling and sequence analysis. The RT-LAMP assay was validated using field-collected banana leaf samples. The assay successfully detected the virus from symptomatic samples while the healthy samples showed no amplification. Samples sourced from banana plants with symptoms of banana bunchy top virus, banana streak virus and cucumber mosaic virus tested negative in the RT-LAMP assay, thus ensuring the specificity of the assay.

Rapid detection of apple latent spherical virus infecting Angelica sinensis by gold immunochromatography reverse transcription loop-mediated isothermal amplification assay

Apple latent spherical virus (ALSV; genus: Cheravirus, family: Secoviridae) is capable of infecting Angelica sinensis under natural conditions and causing symptoms of greenish discoloration and mottling, affecting plant growth and causing a reduction in root quality and yield. Virus detection of ALSV is an urgent need for the continued development of angelica industry. Consequently, it is necessary to develop simple, sensitive, and reliable ALSV detection methods. We describe the preparation of polyclonal antibodies with high titer and high specificity using recombinant ALSV coat protein (CP) as the antigen. Using these antibodies, we established and optimized a rapid detection system using colloidal gold immunochromatography assay (GICA) to detect ALSV. A real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) system with a minimum detection line of 2.72 × 103copys/μl was also developed to detect ALSV. This assay was further enhanced using a combination GICA-RT-LAMP assay. This GICA assay successfully detected ALSV in infected plants without cross reactivity recorded from three other plant viruses. The sensitivity of GICA was the same as that of GICA-RT-PCR, whereas the sensitivity of GICA-RT-LAMP was at least 10-fold higher than that of GICA and GICA-RT-PCR. These methods can detect and identify ALSV under field conditions and accurately measure virus concentration in the laboratory.

The use of colorimetric loop-mediated isothermal amplification assay for naked-eye detection of bean common mosaic virus

Bean common mosaic virus (BCMV), one of the most prevalent viral diseases of common beans, has been intensively studied since its initial identification. In this paper, a one-step colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method was performed by designing oligonucleotide primers to amplify the gene encoding the coat protein of BCMV. Positive LAMP reactions were observed with the naked eye using WarmStart 2 × Master Mix from NEB, changing from pink to yellowish if the test is positive. The sensitivity of this method was similar to that of conventional PCR. The method was tested on common bean and another virus, bean common mosaic necrosis virus (BCMNV), and it was shown to specifically detect BCMV and not produce false positive from other RNAs. The RT-LAMP test was quite reliable for the diagnosis of BCMV from infected samples, and the precise, sensitive. The cost analysis showed that the development of a less expensive and efficient RT-LAMP assay for BCMV detection is an important step in expanding testing capacity and improving access to accurate testing in resource-limited settings. The test can be utilized in laboratory and in the field for the diagnosis and monitoring of BCMV, and is the first reported use of RT-LAMP for BCMV detection.

A Novel RT-LAMP for the Detection of Different Genotypes of Crimean-Congo Haemorrhagic Fever Virus in Patients from Spain.

Crimean-Congo haemorrhagic fever (CCHF) is a potentially lethal tick-borne viral disease with a wide distribution. In Spain, 12 human cases of CCHF have been confirmed, with four deaths. The diagnosis of CCHF is hampered by the nonspecific symptoms, the high genetic diversity of CCHFV, and the biosafety requirements to manage the virus. RT-qPCR and serological tests are used for diagnosis with limitations. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) could be an effective alternative in the diagnosis of the disease. However, none of the few RT-LAMP assays developed to date has detected different CCHFV genotypes. Here, we designed a RT-LAMP using a degenerate primer set to compensate for the variability of the CCHFV target sequence. RT-LAMP was performed in colorimetric and real-time tests on RT-qPCR-confirmed CCHF patient samples notified in Spain in 2020 and 2021. Urine from an inpatient was analysed by RT-LAMP for the first time and compared with RT-qPCR. The amplicons obtained by RT-qPCR were sequenced and African III and European V genotypes were identified. RT-LAMP amplified both genotypes and was more sensitive than RT-qPCR in urine samples. We have developed a novel, rapid, specific, and sensitive RT-LAMP test that allows the detection of different CCHFV genotypes in clinical samples. This pan-CCHFV RT-LAMP detected viral RNA for the first time in urine samples. It can be easily performed as a single-tube isothermal colorimetric method on a portable platform in real time and without the need for expensive equipment, thus bringing molecular diagnostics closer to rural or resource-poor areas, where CCHF usually occurs.

Development of loop-mediated isothermal amplification (LAMP) assays using five primers reduces the false-positive rate in COVID-19 diagnosis

The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a cheaper and faster testing alternative for detecting SARS-CoV-2. However, a high false-positive rate due to misamplification is one of the major limitations. To overcome misamplifications, we developed colorimetric and fluorometric RT-LAMP assays using five LAMP primers, instead of six. The gold-standard RT-PCR technique verified the assays' performance. Compared to other primer sets with six primers (N, S, and RdRp), the E-ID1 primer set, including five primers, performed superbly on both colorimetric and fluorometric assays. The sensitivity of colorimetric and fluorometric assays was 89.5% and 92.2%, respectively, with a limit of detection of 20 copies/µL. The colorimetric RT-LAMP had a specificity of 97.2% and an accuracy of 94.5%, while the fluorometric RT-LAMP obtained 99% and 96.7%, respectively. No misamplification was evident even after 120 min, which is crucial for the success of this technique. These findings are important to support the use of RT-LAMP in the healthcare systems in fighting COVID-19.

An artificial positive control for routine detection of rose rosette virus and Phyllocoptes fructiphilus that fit most primers for PCR, LAMP and RPA based assays

AbstractRose (Rosa spp.) is a very important ornamental shrub cultivated worldwide and of value for the pharmaceutical industry. The plant is broadly susceptible to pathogens, including viruses. Rose rosette virus (RRV; virus species Emaravirus rosae) causes multiple symptoms typically rosettes, ultimately leading to death. The virus transmission is by grafting and a wind‐dispersed eriophyoid mite, Phyllocoptes fructiphilus, which survives in winter‐dormant plants. Due to extensive globalization RRV is a threat for the European rose, landscape, nursery and tourism industries. The most common and reliable method used for RRV detection is RT‐PCR. Positive control is indispensable for PCR reliability and can be difficult to obtain for emerging or highly contagious pathogens and are subject to BSL‐2 quarantine. A synthetic artificial positive control (APC) using custom DNA inserts of sense and anti‐sense primers was designed de novo and inserted in a circular plasmid vector to create a positive control for use with most RRV reported primers and eriophyoid mites. This study describes a functional demonstration and development of a rapid, consistent, adaptable and cost‐effective alternative to infected true‐tissue positive control for detection of RRV. The inserted RRV primers are for end point and quantitative RT‐PCR, reverse transcription loop‐mediated isothermal amplification (RT‐LAMP), recombinase‐polymerase amplification (RPA), broad detection of emaravirus and the eriophyoid mite vector Phyllocoptes fructiphilus. The APC‐RRV and RRV infected rose (leaf tissue) were tested side to side. Results demonstrated APC‐RRV is a safe, cloneable and reliable approach subjected to quality control with application in quarantine surveillance and routine diagnostics of RRV.

A triple-target reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid and accurate detection of SARS-CoV-2 virus

The spreading of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across the world has impacted people's health and lives worldwide in recent years. Rapid and accurate diagnosis is crucial for curbing the pandemic of coronavirus disease 2019 (COVID-19). Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has great potential for SARS-CoV-2 detection but fails to completely replace conventional PCR due to the high false-positive rate (FPR). We proposed a triple-target RT-LAMP method for dual-signal, sensitive, and simultaneous detection of conserved genes of SARS-CoV-2. Multiple LAMP primer sets were designed for N, E, and M genes and their amplification efficacy were screened. Then, using artificial plasmids and RNA, the optimal primer set for each gene was examined on specificity, sensitivity, and detection range. The RT-LAMP initiated by these primer sets exhibited better specificity and sensitivity than that of RT-qPCR, and the triple-target RT-LAMP could determine different variants of SARS-CoV-2. By testing 78 artificial RNA samples, the total FPR of triple-target RT-LAMP was eliminated compared with that of mono-target RT-LAMP. The triple-target RT-LAMP method precisely identified throat swab specimens through colorimetry and fluorescent signals within 60 min, and the limit of detection (LOD) was as low as 187 copies/reaction. In the future, the triple-target RT-LAMP can be applied to in-field and on-site diagnosis of symptomatic and asymptomatic virus carriers.

Scalable RT‐LAMP‐based SARS‐CoV‐2 testing for infection surveillance with applications in pandemic preparedness

Throughout the SARS‐CoV‐2 pandemic, limited diagnostic capacities prevented sentinel testing, demonstrating the need for novel testing infrastructures. Here, we describe the setup of a cost‐effective platform that can be employed in a high‐throughput manner, which allows surveillance testing as an acute pandemic control and preparedness tool, exemplified by SARS‐CoV‐2 diagnostics in an academic environment. The strategy involves self‐sampling based on gargling saline, pseudonymized sample handling, automated RNA extraction, and viral RNA detection using a semiquantitative multiplexed colorimetric reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assay with an analytical sensitivity comparable with RT‐qPCR. We provide standard operating procedures and an integrated software solution for all workflows, including sample logistics, analysis by colorimetry or sequencing, and communication of results. We evaluated factors affecting the viral load and the stability of gargling samples as well as the diagnostic sensitivity of the RT‐LAMP assay. In parallel, we estimated the economic costs of setting up and running the test station. We performed > 35,000 tests, with an average turnover time of < 6 h from sample arrival to result announcement. Altogether, our work provides a blueprint for fast, sensitive, scalable, cost‐ and labor‐efficient RT‐LAMP diagnostics, which is independent of potentially limiting clinical diagnostics supply chains.