Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities

Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.

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Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

Virology Journal ◽

10.1186/s12985-019-1264-z ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 4

Author(s):

Xin-na Li ◽

Xin-xin Shen ◽

Ming-hui Li ◽

Ju-ju Qi ◽

Rui-huan Wang ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Limit Of Detection ◽

Enterovirus 71 ◽

Cross Reactivity ◽

Lateral Flow ◽

Coxsackievirus A16 ◽

Fluorescence Detector ◽

Lateral Flow Strip ◽

Clinical Diagnostic

Abstract Background Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. Methods Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. Results The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. Conclusions The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

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Identification of Mycoses in Developing Countries

Journal of Fungi ◽

10.3390/jof5040090 ◽

2019 ◽

Vol 5 (4) ◽

pp. 90 ◽

Cited By ~ 15

Author(s):

Amir Arastehfar ◽

Brian L. Wickes ◽

Macit Ilkit ◽

David H. Pincus ◽

Farnaz Daneshnia ◽

...

Keyword(s):

Developing Countries ◽

Real Time ◽

Real Time Pcr ◽

Fungal Infections ◽

Point Of Care ◽

Fungal Species ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Diagnostic Methods ◽

Diagnostic Tools

Extensive advances in technology offer a vast variety of diagnostic methods that save time and costs, but identification of fungal species causing human infections remains challenging in developing countries. Since the echinocandins, antifungals widely used to treat invasive mycoses, are still unavailable in developing countries where a considerable number of problematic fungal species are present, rapid and reliable identification is of paramount importance. Unaffordability, large footprints, lack of skilled personnel, and high costs associated with maintenance and infrastructure are the main factors precluding the establishment of high-precision technologies that can replace inexpensive yet time-consuming and inaccurate phenotypic methods. In addition, point-of-care lateral flow assay tests are available for the diagnosis of Aspergillus and Cryptococcus and are highly relevant for developing countries. An Aspergillus galactomannan lateral flow assay is also now available. Real-time PCR remains difficult to standardize and is not widespread in countries with limited resources. Isothermal and conventional PCR-based amplification assays may be alternative solutions. The combination of real-time PCR and serological assays can significantly increase diagnostic efficiency. However, this approach is too expensive for medical institutions in developing countries. Further advances in next-generation sequencing and other innovative technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tools may lead to efficient, alternate methods that can be used in point-of-care assays, which may supplement or replace some of the current technologies and improve the diagnostics of fungal infections in developing countries.

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Use of biochemical miniaturized galleries, rRNA based lateral flow assay and Real Time PCR for Cronobacter spp. confirmation

Food Microbiology ◽

10.1016/j.fm.2018.05.008 ◽

2018 ◽

Vol 76 ◽

pp. 189-195

Author(s):

David Tomas ◽

Mingzhen Fan ◽

Sha Zhu ◽

Adrianne Klijn

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Lateral Flow ◽

Lateral Flow Assay

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Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae

10.21203/rs.3.rs-17865/v2 ◽

2020 ◽

Author(s):

Jinfeng Wang ◽

Ruiwen Li ◽

Xiaoxia Sun ◽

Libing Liu ◽

Xuepiao Hao ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Lateral Flow ◽

Recombinase Polymerase Amplification ◽

Metal Bath ◽

Lateral Flow Strip ◽

The Real ◽

Mycoplasmal Pneumonia ◽

Mycoplasma Ovipneumoniae

Abstract Background: Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. Results: The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16S rRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae , as there were no cross-reactions with other microorganisms tested, especially the pathogens involved in respiratory complex and other mycoplasmas frequently identified in ruminant s . The limit of detection of LFS RPA assay was 1.0×10 1 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times lower than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 111 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 29 samples in the real-time RPA, 31 samples in the LFS RPA and 32 samples in the real-time PCR assay. Compared to real-time PCR, the real-time RPA and LFS RPA showed diagnostic specificity of 100% and 98.73%, diagnostic sensitivity of 90.63% and 93.75%, and a kappa coefficient of 0.932 and 0.934, respectively. Conclusions: The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae , especially in resource-limited settings.

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Real-Time Connection Monitoring of Ubiquitous Networks for Intrusion Prediction: A SequentialKNN Voting Approach

International Journal of Distributed Sensor Networks ◽

10.1155/2015/387462 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10

Author(s):

Bokyoung Kang ◽

Dongsoo Kim ◽

Minsoo Kim

Keyword(s):

Real Time ◽

Partial Information ◽

Early Stage ◽

Threshold Value ◽

Network Intrusion ◽

Preventive Actions ◽

Novel Approach ◽

Predicted Probability ◽

Wide Range ◽

Ongoing Behavior

In the ubiquitous network environment where numerous devices are connecting each other, it is believed that security will play an important role in overall network management. And the wireless sensor network (WSN) is commonly considered to be one of such networks prone to a wide range of attacks due to its inherent characteristics. For the sound operation of WSN, it is important to block malicious connections from the network as early as possible. This paper proposes a novel approach to real-time monitoring of network by using the sequentialKNN voting. When connection data is sequentially recorded on the log, the final result of ongoing behavior is predicted probabilistically with only partial data, which iterates consecutively as additional connection data are accumulated to the log. Once this predicted probability reaches certain preset threshold value for possible network intrusion, then we can do some preventive actions for this ongoing connection. The value of this research lies in that the eventualities are predicted at the early stage of connection with partial information available. Since the prediction uses sequentialKNN voting, the accuracy of our approach can be even more enhanced as with the volume of log grows.

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Comparative Study of Gold and Carbon Nanoparticles in Nucleic Acid Lateral Flow Assay

Nanomaterials ◽

10.3390/nano11030741 ◽

2021 ◽

Vol 11 (3) ◽

pp. 741

Author(s):

Juan Carlos Porras ◽

Mireia Bernuz ◽

Jennifer Marfa ◽

Arnau Pallares-Rusiñol ◽

Mercè Martí ◽

...

Keyword(s):

Nucleic Acid ◽

Comparative Study ◽

Carbon Nanoparticles ◽

Limit Of Detection ◽

Early Stage ◽

High Sensitivity ◽

Lateral Flow ◽

Lateral Flow Assay ◽

Complex Samples ◽

Increased Sensitivity

A lateral flow assay (LFA) is a paper-based, point-of-need test designed to detect a specific analyte in complex samples in low-resource settings. Although LFA has been successfully used in different applications, its use is still limited when high sensitivity is required, especially in the diagnosis of an early-stage condition. The limit of detection (LOD) is clearly related to the signal-generating system used to achieve the visual readout, in many cases involving nanoparticles coupled to a biomolecule, which, when combined, provides sensitivity and specificity, respectively. While colloidal gold is currently the most-used label, other detection systems are being developed. Carbon nanoparticles (CNPs) demonstrate outstanding features to improve the sensitivity of this technology by producing an increased contrast in the paper background. Based on the necessity of sensitivity improvement, the aim of this work is a comparative study, in terms of analytical performance, between commercial streptavidin gold nanoparticles (streptAv-AuNPs) and avidin carbon nanoparticles (Av-CNPs) in a nucleic acid lateral flow assay. The visual LOD of the method was calculated by serial dilution of the DNA template, ranging from 0.0 to 7 pg μL−1/1.5 × 104 CFU mL−1). The LFA achieved visual detection of as low as 2.2 × 10−2 pg μL−1 using Av-CNPs and 8.4 × 10−2 pg μL−1 using streptAv-AuNPs. These LODs could be obtained without the assistance of any instrumentation. The results demonstrate that CNPs showed an increased sensitivity, achieving the nanomolar range even by visual inspection. Furthermore, CNPs are the cheapest labels, and the suspensions are very stable and easy to modify.

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Direct and Rapid Detection of Mycoplasma bovis in Bovine Milk Samples by Recombinase Polymerase Amplification Assays

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.639083 ◽

2021 ◽

Vol 11 ◽

Author(s):

Ruiwen Li ◽

Jinfeng Wang ◽

Xiaoxia Sun ◽

Libing Liu ◽

Jianchang Wang ◽

...

Keyword(s):

Real Time ◽

Bovine Milk ◽

Bovine Mastitis ◽

High Specificity ◽

Lateral Flow ◽

Recombinase Polymerase Amplification ◽

Mycoplasma Bovis ◽

Lateral Flow Strip ◽

The Real ◽

Milk Samples

This study aimed to detetct Mycoplasma bovis (M. bovis) in bovine milk quickly and directly by developing and validating isothermal recombinase polymerase amplification (RPA) assays. Targeting the uvrC gene of M. bovis, an RPA assay based on the fluorescence monitoring (real-time RPA) and an RPA assay combined with a lateral flow strip (LFS RPA) were conducted. It took 20 min for the real-time RPA to finish in a Genie III at 39°C, and 15 min were required to perform the LFS RPA in an incubator block at 39°C, followed by the visualization of the products on the lateral flow strip within 5 min. Both of the two assays showed high specificity for M. bovis without any cross-reaction with the other tested pathogens. With the standard recombinant plasmid pMbovis-uvrC serving as a template, both RPA assays had a limit of detcion of 1.0 × 101 copies per reaction, equivalent to that of a real-time PCR assay. In the 65 milk samples collected from cattle with mastitis, the M. bovis genomic DNA was detected in 24 samples by both the real-time RPA and the LFS RPA assays. The developed RPA assays could detect M. bovis in bovine milk in an efficient, convenient, and credible manner as attractive and promising tools, and the assays would be helpful in the rapid response to M. bovis infection causing bovine mastitis.

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Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae

10.21203/rs.3.rs-17865/v1 ◽

2020 ◽

Author(s):

Jinfeng Wang ◽

Ruiwen Li ◽

Xiaoxia Sun ◽

Libing Liu ◽

Xuepiao Hao ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Lateral Flow ◽

Recombinase Polymerase Amplification ◽

Metal Bath ◽

Lateral Flow Strip ◽

The Real ◽

Mycoplasmal Pneumonia ◽

Mycoplasma Ovipneumoniae

Abstract Background Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. It is an urgent need to develop a rapid and accurate method to detect M. ovipneumoniae . Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. Results The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16SrRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae , as there were no cross-reactions with other pathogens tested, especially the M. capricolum subsp. capripneumoniae . The limit of detection of LFS RPA assay was 1.0×10 1 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times higher than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 46 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 17 samples in the RPA assays and 19 samples in the real-time PCR assay. The real-time RPA and LFS RPA showed diagnostic specificity of 100%, diagnostic sensitivity of 89.47%, and a kappa coefficient of 0.909. Conclusions The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae , especially in resource-limited settings.

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