The pandemic coronavirus SARS-CoV-2 in the world has caused a large infected population suffering from COVID-19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. We applied a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to achieve the detection of SARS-CoV-2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS-CoV-2 in the regions of orf1ab, S gene and N gene. A colorimetric change was used to report the results, which enables the outcome of viral RNA amplification to be read by the naked eye without the need of expensive or dedicated instrument. The sensitivity can be 80 copies of viral RNA per ml in a sample. We validated the RT-LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 negatives. The testing results are consistent with the conventional RT-qPCR. In addition, we also show that one-step process without RNA extraction is feasible to achieve RNA amplification directly from a sample. This rapid, simple and sensitive RT-LAMP method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas.

https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/1751-7915.13586

RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2.

Detection of COVID-19: A review of the current literature and future perspectives.

The lateral flow assay is one of the most convenient analytical techniques for analyzing the immune response, but its applicability to precise genetic analyses is limited by the false-positive signal and tedious and inefficient hybridization steps. Here, we introduce the CRISPR (clustered regularly interspaced short palindromic repeats) /Cas system into the lateral flow assay, termed CRISPR/Cas9-mediated lateral flow nucleic acid assay (CASLFA), to address such issues. In this study, CASLFA is utilized to identify Listeria monocytogenes, genetically modified organisms (GMOs), and African swine fever virus (ASFV) at a detection limit of hundreds of copies of genome samples with high specificity within 1 h. We further evaluated the performance of CASLFA in a nonlaboratory environment and successfully confirmed 27 ASFV-infected samples from 110 suspected swine serum samples, with an accuracy of 100% when compared to real-time PCR (RT-PCR) assay. CASLFA satisfies some of the characteristics of a next-generation molecular diagnostics tool due to its rapidity and accuracy, allowing for point-of-care use without the need for technical expertise and complex ancillary equipment. This method has great potential for gene analysis in resource-poor or nonlaboratory environments.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Lateral Flow Nucleic Acid Assay.

The COVID-19 pandemic has created huge damage to society and brought panic around the world. Such panic can be ascribed to the seemingly deceptive features of COVID-19: Compared to other deadly viral outbreaks, it has medium transmission and mortality rates. As a result, the severity of the causative coronavirus, SARS-CoV-2, was deeply underestimated by society at the beginning of the COVID-19 outbreak. Based on this, in this review, we define the viruses with features similar to those of SARS-CoV-2 as the Panic Zone viruses. To contain those viruses, accurate and fast diagnosis followed by effective isolation and treatment of patients are pivotal at the early stage of virus breakouts. This is especially true when there is no cure or vaccine available for a transmissible disease, which is the case for the current COVID-19 pandemic. As of July 2020, more than 100 kits for COVID-19 diagnosis on the market have been surveyed in this review, while emerging sensing techniques for SARS-CoV-2 are also discussed. It is of critical importance to rationally use these kits for efficient management and control of the Panic Zone viruses. Therefore, we discuss guidelines to select diagnostic kits at different outbreak stages of the Panic Zone viruses, SARS-CoV-2 in particular. While it is of utmost importance to use nucleic acid based detection kits with low false negativity (high sensitivity) at the early stage of an outbreak, the low false positivity (high specificity) gains importance at later stages of the outbreak. When society is set to reopen from the lockdown stage of the COVID-19 pandemic, it becomes critical to have immunoassay based kits with high specificity to identify people who can safely return to society after their recovery from SARS-CoV-2 infections. Finally, since a massive attack from a viral pandemic requires a massive defense from the whole society, we urge both government and the private sector to research and develop affordable and reliable point-of-care testing (POCT) kits, which can be used massively by the general public (and therefore called massive POCT) to contain Panic Zone viruses in the future.

Detecting the Coronavirus (COVID-19).

Coronavirus disease 2019 (COVID-19) outbreak has become a global pandemic. The deleterious effects of coronavirus have prompted the development of diagnostic tools to manage the spread of disease. While conventional technologies such as quantitative real time polymerase chain reaction (qRT-PCR) have been broadly used to detect COVID-19, they are time-consuming, labor-intensive and are unavailable in remote settings. Point-of-care (POC) biosensors, including chip-based and paper-based biosensors are typically low-cost and user-friendly, which offer tremendous potential for rapid medical diagnosis. This mini review article discusses the recent advances in POC biosensors for COVID-19. First, the development of POC biosensors which are made of polydimethylsiloxane (PDMS), papers, and other flexible materials such as textile, film, and carbon nanosheets are reviewed. The advantages of each biosensors along with the commercially available COVID-19 biosensors are highlighted. Lastly, the existing challenges and future perspectives of developing robust POC biosensors to rapidly identify and manage the spread of COVID-19 are briefly discussed.

/pdf/development-of-point-of-care-biosensors-for-covid-19-3976zdzjx2.pdf

Development of Point-of-Care Biosensors for COVID-19

Rapid, low-cost, species-specific diagnosis, based upon DNA testing, is becoming important in the treatment of patients with infectious diseases. Here, we demonstrate an innovation that uses origami to enable multiplexed, sensitive assays that rival polymerase chain reactions (PCR) laboratory assays and provide high-quality, fast precision diagnostics for malaria. The paper-based microfluidic technology proposed here combines vertical flow sample-processing steps, including paper folding for whole-blood sample preparation, with an isothermal amplification and a lateral flow detection, incorporating a simple visualization system. Studies were performed in village schools in Uganda with individual diagnoses being completed in <50 min (faster than the standard laboratory-based PCR). The tests, which enabled the diagnosis of malaria species in patients from a finger prick of whole blood, were both highly sensitive and specific, detecting malaria in 98% of infected individuals in a double-blind first-in-human study. Our method was more sensitive than other field-based, benchmark techniques, including optical microscopy and industry standard rapid immunodiagnostic tests, both performed by experienced local healthcare teams (which detected malaria in 86% and 83% of cases, respectively). All assays were independently validated using a real-time double-blinded reference PCR assay. We not only demonstrate that advanced, low-cost DNA-based sensors can be implemented in underserved communities at the point of need but also highlight the challenges associated with developing and implementing new diagnostic technologies in the field, without access to laboratories or infrastructure.

https://www.pnas.org/content/pnas/116/11/4834.full.pdf

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities

In order to combat the growing threat of global infectious diseases, there is a need for rapid diagnostic technologies that are sensitive and that can provide species specific information (as might be needed to direct therapy as resistant strains of microbes emerge). Here, we present a convenient, enzyme-free amplification mechanism for a rational hybridization chain reaction, which is implemented in a simple format for isothermal amplification and sensing, applied to the DNA-based diagnosis of hepatitis B virus (HBV) in 54 patients. During the cycled amplification process, DNA monomers self-assemble in an organized and controllable way only when a specific target HBV sequence is present. This mechanism is confirmed using super-resolution stochastic optical reconstruction microscopy. The enabled format is designed in a manner analogous to an enzyme-linked immunosorbent assay, generating colored products with distinct tonality and with a limit of detection of ca. five copies/reaction. This routine assay al...

Cycling of Rational Hybridization Chain Reaction To Enable Enzyme-Free DNA-Based Clinical Diagnosis

Human immunodeficiency virus (HIV) continues to be a major burden on public health globally with on-going increases in the number of new infections each year. Rapid and sensitive point-of-care tests allow timely interventions and are essential to control the spread of the disease. However the highly variable nature of the virus, resulting in the evolution of many subtypes and inter-subtype recombinants, poses important challenges for its diagnosis. Here we describe a variant-tolerant reverse-transcription RT-LAMP amplification of the virus's INT gene, providing a simple to use, rapid (<30 min) in vitro point-of-care diagnostic test with a limit of detection <18 copies/reaction. The assay was first validated in clinical studies of patient samples, using both established RT-LAMP and RT-qPCR assays for reference, with results showing that this new variant-tolerant HIV-1 RT-LAMP diagnostic test is highly sensitive without compromising its high specificity for HIV-1 subtypes. The diagnostic test was subsequently configured within an easy-to-read paper microfluidic lateral flow test and was validated clinically using patient samples, demonstrating its future potential for use in timely, effective, low cost HIV diagnostics in global regions where healthcare resources may be limited.

/pdf/a-rapid-variant-tolerant-reverse-transcription-loop-mediated-5eula7k54z.pdf

Gaolian Xu

Papers

Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities

Cycling of Rational Hybridization Chain Reaction To Enable Enzyme-Free DNA-Based Clinical Diagnosis

A rapid variant-tolerant reverse transcription loop-mediated isothermal amplification assay for the point of care detection of HIV-1.