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1.
Anal Chem ; 94(27): 9603-9609, 2022 07 12.
Article in English | MEDLINE | ID: covidwho-1921541

ABSTRACT

The clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection can be combined with recombinase-aided amplification (RAA) to enable rapid, accurate, and early detection of SARS-CoV-2. Current CRISPR-based approaches to detecting viral nucleic acid typically require immense manual operations to transfer RPA amplicons for CRISPR detection or suffer from compromised sensitivity by mixing the competing RPA amplification and CRISPR detection. Here, we develop dual-CRISPR/Cas12a-assisted RT-RAA assay and a ″sample-to-answer″ centrifugal microfluidic platform that can automatically detect 1 copy/µL of the SARS-CoV-2 within 30 min. This chip separates the amplification (RAA) from detection (CRISPR), such that sensitivity is maximized and the time consumption is decreased by a factor of 3. For the 26 positive and 8 negative clinical SARS-CoV-2 samples, this automated centrifugal microfluidics achieved 100% accuracy compared to the gold-standard RT-PCR technique. This point-of-care test, with the advantages of being one-step, automated, rapid, and sensitive, will have a significant potential for clinical diagnosis and disease prevention.


Subject(s)
COVID-19 , Nucleic Acids , COVID-19/diagnosis , CRISPR-Cas Systems , Humans , Microfluidics , Nucleic Acid Amplification Techniques/methods , Recombinases , SARS-CoV-2/genetics , Sensitivity and Specificity
2.
Proc Natl Acad Sci U S A ; 119(26): e2202034119, 2022 06 28.
Article in English | MEDLINE | ID: covidwho-1900769

ABSTRACT

CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.


Subject(s)
Bacterial Proteins , CRISPR-Associated Proteins , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Endodeoxyribonucleases , Reagent Kits, Diagnostic , Reverse Transcriptase Polymerase Chain Reaction , COVID-19/diagnosis , Clustered Regularly Interspaced Short Palindromic Repeats/radiation effects , Humans , RNA/radiation effects , Recombinases/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
3.
Anal Chem ; 94(23): 8277-8284, 2022 06 14.
Article in English | MEDLINE | ID: covidwho-1873390

ABSTRACT

CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage. In this study, we discovered that a glycerol additive dramatically promoted the detection efficiency of the one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method. Compared with the glycerol-free version, its sensitivity was nearly 100-fold higher and was close to that of the canonical two-step method. Further investigation displayed that the enhanced detection efficiency was attributed to the phase separation of the RPA and CRISPR/Cas12a system during the initial phase of the RPA reaction caused by the glycerol viscosity. This highly efficient one-pot method has been triumphantly harnessed for the detection of African swine fever virus (ASFV) and SARS-CoV-2, achieving naked-eye readout through a smartphone-equipped device. The currently developed glycerol-enhanced one-pot RPA-CRISPR/Cas12a method can be an advantageous point-of-care nucleic acid detection platform on account of its simplicity, high sensitivity, and universality.


Subject(s)
African Swine Fever Virus , COVID-19 , African Swine Fever Virus/genetics , Animals , CRISPR-Cas Systems/genetics , DNA/genetics , Glycerol , Nucleic Acid Amplification Techniques/methods , Recombinases , SARS-CoV-2 , Sensitivity and Specificity , Swine
4.
Talanta ; 248: 123594, 2022 Oct 01.
Article in English | MEDLINE | ID: covidwho-1867806

ABSTRACT

COVID-19 has erupted and quickly swept across the globe, causing huge losses to human health and wealth. It is of great value to develop a quick, accurate, visual, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we developed a biosensor based on CRISPR/Cas13a combined with recombinase polymerase amplification (RPA) to detect S and Orf1ab genes of SARS-CoV-2 within 30 min. Most important of all, we developed an automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) with a 3D-printed microfluidic chip for sensitively detecting SARS-CoV-2, which addressed aerosol contamination issue and provided a more accurate and high-throughput detection during the on-site detection process. The detection limits of S gene and Orf1ab gene were as low as 0.68 fM and 4.16 fM. Furthermore, we used the lateral flow strip to realize visualization and point of care testing (POCT) of SARS-CoV-2. Therefore, profit from the efficient amplification of RPA and the high specificity of CRISPR/Cas13a, APHF-analyzer and the lateral flow strip to simultaneous detection of S gene and Orf1ab gene would be applied as a promising tool in the field of SARS-CoV-2 detection.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Recombinases , SARS-CoV-2/genetics , Sensitivity and Specificity
5.
J Biomed Nanotechnol ; 18(2): 394-404, 2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1816973

ABSTRACT

A simple and rapid genotyping method with less-instrumentation is essential for realizing point-of-care detection of personalized medicine-related gene biomarkers. Herein, we developed a rapid and visualized genotyping method by coupling recombinase polymerase amplification (RPA) with allele-specific invader reaction assisted gold nanoparticle probes assembling. In the method, the DNA targets were firstly amplified by using RPA, which is a rapid isothermal amplification technology. Then an allele-specific invasion reaction was performed to recognize the single nucleotide polymorphisms (SNPs) site in the amplicons, to produce signal molecules that caused discoloration of gold nanoparticle probes. As a result, genotyping was achieved by observing the color change of the reaction by using naked eye without the requirement for any expensive instrument. In order to achieve rapid genotyping detection, the genomic DNA from oral swab lysate samples were used for the RPA templates amplification. In this way, a visualized genotyping from "samples to results" within 25 min was realized. Two clopidogrel related SNPs CYP2C19*2 and CYP2C19*3 of 56 clinical samples were correctly genotyped by using this rapid visualized genotyping assay. In addition, the feasibility for this pathogen genotyping method was also verified by detecting plasmid DNA containing three SARS-COV-2 gene mutation sites, indicating that this method has the potential for clinical sample detection.


Subject(s)
COVID-19 , Metal Nanoparticles , Alleles , COVID-19/genetics , Cytochrome P-450 CYP2C19 , DNA , Genotype , Gold , Humans , Nucleic Acid Amplification Techniques/methods , Recombinases/genetics , SARS-CoV-2
6.
BMC Vet Res ; 18(1): 140, 2022 Apr 18.
Article in English | MEDLINE | ID: covidwho-1808371

ABSTRACT

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is one of the most important enteric viruses causing diarrhea in pigs. The establishment of a rapid detection method applicable in field conditions will be conducive to early detection of pathogen and implementation of relevant treatment. A novel nucleic acid amplification method, recombinase polymerase amplification (RPA), has been widely used for infectious disease diagnosis. RESULTS: In the present study, a reverse transcription (RT)-RPA assay combined with lateral flow dipstrip (LFD) was established for the visual detection of PEDV by targeting the N gene. The RT-RPA-LFD assay detected as low as 102 copies/µL of PEDV genomic RNA standard. Moreover, the novel RT-RPA-LFD assay did not show cross-reactivity with common swine pathogens, demonstrating high specificity. The performance of the assay for detection of clinical samples was also evaluated. A total number of 86 clinical samples were tested by RT-RPA-LFD and RT-PCR. The detection results of RT-RPA-LFD were compared with those of RT-PCR, with a coincidence rate of 96.5%. CONCLUSION: The newly established RT-RPA-LFD assay in our study had high sensitivity and specificity, with a potential to use in resource-limited areas and countries.


Subject(s)
Porcine epidemic diarrhea virus , Animals , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/veterinary , Porcine epidemic diarrhea virus/genetics , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/veterinary , Recombinases/genetics , Reverse Transcription , Sensitivity and Specificity , Swine
7.
Anal Chem ; 94(10): 4135-4140, 2022 03 15.
Article in English | MEDLINE | ID: covidwho-1721379

ABSTRACT

Rapid and high-throughput screening is critical to control the COVID-19 pandemic. Recombinase polymerase amplification (RPA) with highly accessible and sensitive nucleic acid amplification has been widely used for point-of-care infection diagnosis. Here, we report an integrated microdroplet array platform composed of an ultrasonic unit and minipillar array to enhance the RPA for ultrafast, high-sensitivity, and high-throughput detection of SARS-CoV-2. On such a platform, the independent microvolume reactions on individual minipillars greatly decrease the consumption of reagents. The microstreaming driven by ultrasound creates on-demand contactless microagitation in the microdroplets and promotes the interaction between RPA components, thus greatly accelerating the amplification. In the presence of microstreaming, the detection time is 6-12 min, which is 38.8-59.3% shorter than that of controls without microstreaming, and the end-point fluorescence intensity also increased 1.3-1.7 times. Furthermore, the microagitation-enhanced RPA also exhibits a lower detection limit (0.42 copy/µL) for SARS-CoV-2 in comparison to the controls. This integrated microdroplet array detection platform is expected to meet the needs for high-throughput nucleic acid testing (NAT) to improve the containment of viral transmission during the epidemic, as well as provide a potential platform for the timely detection of other pathogens or viruses.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , Nucleic Acid Amplification Techniques , Pandemics , RNA, Viral/genetics , Recombinases , SARS-CoV-2/genetics , Sensitivity and Specificity
8.
Anal Chem ; 94(6): 2926-2933, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1721378

ABSTRACT

Recombinase polymerase amplification (RPA) is a useful pathogen identification method. Several label-free detection methods for RPA amplicons have been developed in recent years. However, these methods still lack sensitivity, specificity, efficiency, or simplicity. In this study, we propose a rapid, highly sensitive, and label-free pathogen assay system based on a solid-phase self-interference RPA chip (SiSA-chip) and hyperspectral interferometry. The SiSA-chips amplify and capture RPA amplicons on the chips, rather than irrelevant amplicons such as primer dimers, and the SiSA-chips are then analysed by hyperspectral interferometry. Optical length increases of SiSA-chips are used to demonstrate RPA detection results, with a limit of detection of 1.90 nm. This assay system can detect as few as six copies of the target 18S rRNA gene of Plasmodium falciparum within 20 min, with a good linear relationship between the detection results and the concentration of target genes (R2 = 0.9903). Single nucleotide polymorphism (SNP) genotyping of the dhfr gene of Plasmodium falciparum is also possible using the SiSA-chip, with as little as 1% of mutant gene distinguished from wild-type loci (m/wt). This system offers a high-efficiency (20 min), high-sensitivity (6 copies/reaction), high-specificity (1% m/wt), and low-cost (∼1/50 of fluorescence assays for RPA) diagnosis method for pathogen DNA identification. Therefore, this system is promising for fast identification of pathogens to help diagnose infectious diseases, including SNP genotyping.


Subject(s)
Nucleic Acid Amplification Techniques , Recombinases , Interferometry , Nucleic Acid Amplification Techniques/methods , Nucleotidyltransferases , Plasmodium falciparum/genetics , Sensitivity and Specificity
9.
Viruses ; 14(2)2022 01 18.
Article in English | MEDLINE | ID: covidwho-1715743

ABSTRACT

The African swine fever virus (ASFV) is a dsDNA virus that can cause serious, highly infectious, and fatal diseases in wild boars and domestic pigs. The ASFV has brought enormous economic loss to many countries, and no effective vaccine or treatment for the ASFV is currently available. Therefore, the on-site rapid and accurate detection of the ASFV is key to the timely implementation of control. The RNA-guided, RNA-targeting CRISPR effector CRISPR-associated 13 (Cas13a; previously known as C2c2) exhibits a "collateral effect" of promiscuous RNase activity upon the target recognition. The collateral cleavage activity of LwCas13a is activated to degrade the non-targeted RNA, when the crRNA of LwCas13a binds to the target RNA. In this study, we developed a rapid and sensitive ASFV detection method based on the collateral cleavage activity of LwCas13a, which combines recombinase-aided amplification (RAA) and a lateral flow strip (named CRISPR/Cas13a-LFD). The method was an isothermal detection at 37 °C, and the detection can be used for visual readout. The detection limit of the CRISPR/Cas13a-LFD was 101 copies/µL of p72 gene per reaction, and the detection process can be completed within an hour. The assay showed no cross-reactivity to eight other swine viruses, including classical swine fever virus (CSFV), and has a 100% coincidence rate with real-time PCR detection of the ASFV in 83 clinical samples. Overall, this method is sensitive, specific, and practicable onsite for the ASFV detection, showing a great application potential for monitoring the ASFV in the field.


Subject(s)
African Swine Fever Virus/isolation & purification , African Swine Fever/diagnosis , CRISPR-Cas Systems , African Swine Fever/virology , African Swine Fever Virus/genetics , Animals , Genotype , Reagent Strips , Recombinases/genetics , Recombinases/metabolism , Sensitivity and Specificity , Sus scrofa , Swine , Time Factors
10.
Arch Virol ; 167(2): 493-499, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1712247

ABSTRACT

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10-1 median tissue culture infection dose (TCID50) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field.


Subject(s)
Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Animals , Porcine Reproductive and Respiratory Syndrome/diagnosis , Porcine respiratory and reproductive syndrome virus/genetics , Porcine respiratory and reproductive syndrome virus/metabolism , Recombinases , Reverse Transcription , Sensitivity and Specificity , Swine
11.
Biosensors (Basel) ; 12(2)2022 Jan 27.
Article in English | MEDLINE | ID: covidwho-1700284

ABSTRACT

Pathogen detection by nucleic acid amplification proved its significance during the current coronavirus disease 2019 (COVID-19) pandemic. The emergence of recombinase polymerase amplification (RPA) has enabled nucleic acid amplification in limited-resource conditions owing to the low operating temperatures around the human body. In this study, we fabricated a wearable RPA microdevice using poly(dimethylsiloxane) (PDMS), which can form soft-but tight-contact with human skin without external support during the body-heat-based reaction process. In particular, the curing agent ratio of PDMS was tuned to improve the flexibility and adhesion of the device for better contact with human skin, as well as to temporally bond the microdevice without requiring further surface modification steps. For PDMS characterization, water contact angle measurements and tests for flexibility, stretchability, bond strength, comfortability, and bendability were conducted to confirm the surface properties of the different mixing ratios of PDMS. By using human body heat, the wearable RPA microdevices were successfully applied to amplify 210 bp from Escherichia coli O157:H7 (E. coli O157:H7) and 203 bp from the DNA plasmid SARS-CoV-2 within 23 min. The limit of detection (LOD) was approximately 500 pg/reaction for genomic DNA template (E. coli O157:H7), and 600 fg/reaction for plasmid DNA template (SARS-CoV-2), based on gel electrophoresis. The wearable RPA microdevice could have a high impact on DNA amplification in instrument-free and resource-limited settings.


Subject(s)
Body Temperature , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acids , Wearable Electronic Devices , COVID-19/diagnosis , DNA , Escherichia coli O157 , Humans , Nucleic Acid Amplification Techniques/methods , Nucleic Acids/isolation & purification , Recombinases/chemistry , Recombinases/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
12.
Talanta ; 240: 123209, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1621054

ABSTRACT

Nucleic acid testing (NAT) implemented on a portable, miniaturized, and integrated device with rapid and sensitive results readout is highly demanded for pathogen detection or genetic screening at resource-limited settings, especially after the outbreak of coronavirus disease 2019 (COVID-19). The integration of recombinase polymerase amplification (RPA) with emerging microfluidics, classified by paper-based microfluidics and chip-based microfluidics, shows great potential to perform laboratory independent NAT assays at point of care with minimal labor, time and energy consumption. This review summarizes the state-of-the-art of RPA integrated with paper-based microfluidics and chip-based microfluidics, and discusses their pros and cons. Finally, existing challenges and possible ways for optimization of microfluidics-based RPA are proposed.


Subject(s)
COVID-19 , Nucleic Acids , Humans , Microfluidics , Nucleic Acid Amplification Techniques , Point-of-Care Systems , Recombinases , SARS-CoV-2
13.
Biopolymers ; 113(4): e23485, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1620107

ABSTRACT

The early phase of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was exacerbated by a diagnostic challenge of unprecedented magnitude. In the absence of effective therapeutics or vaccines, breaking the chain of transmission through early disease detection and patient isolation was the only means to control the growing pandemic. While polymerase chain reaction (PCR)-based methods and rapid-antigen tests rose to the occasion, the analytical challenge of rapid and sequence-specific nucleic acid-sensing at a point-of-care or home setting stimulated intense developments. Herein we report a method that combines recombinase polymerase amplification and a DNA-templated reaction to achieve a dual readout with either fluorescence (microtiter plate) or naked eye (lateral flow assay: LFA) detection. The nucleic acid templated reaction is based on an SN Ar that simultaneously transfers biotin from one Peptide Nucleic Acid (PNA) strand to another PNA strand, enabling LFA detection while uncaging a coumarin for fluorescence readout. This methodology has been applied to the detection of a DNA or RNA sequence uniquely attributed to the SARS-CoV-2.


Subject(s)
COVID-19 , Nucleic Acids , COVID-19/diagnosis , DNA , Humans , Nucleic Acid Amplification Techniques/methods , Recombinases/genetics , SARS-CoV-2/genetics
14.
Int J Infect Dis ; 114: 105-111, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1587637

ABSTRACT

OBJECTIVES: The democratization of diagnostics is one of the key challenges towards containing the transmission of coronavirus disease 2019 (COVID-19) around the globe. The operational complexities of existing PCR-based methods, including sample transfer to advanced central laboratories with expensive equipment, limit their use in resource-limited settings. However, with the advent of isothermal technologies, the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possible at decentralized facilities. METHODS: In this study, two recombinase-based isothermal techniques, reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription recombinase-aided amplification (RT-RAA), were evaluated for the detection of SARS-CoV-2 in clinical samples. A total of 76 real-time reverse transcription PCR (real-time RT-PCR) confirmed COVID-19 cases and 100 negative controls were evaluated to determine the diagnostic performance of the isothermal methods. RESULTS: This investigation revealed equally promising diagnostic accuracy of the two methods, with a sensitivity of 76.32% (95% confidence interval 65.18-85.32%) when the target genes were RdRP and ORF1ab for RT-RPA and RT-RAA, respectively; the combination of N and RdRP in RT-RPA augmented the accuracy of the assay at a sensitivity of 85.53% (95% confidence interval 75.58-92.55%). Furthermore, high specificity was observed for each of the methods, ranging from 94.00% to 98.00% (95% confidence interval 87.40-9.76%). CONCLUSIONS: Considering the diagnostic accuracies, both RT-RPA and RT-RAA appear to be suitable assays for point-of-need deployment for the detection of the pathogen, understanding its epidemiology, case management, and curbing transmission.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Recombinases/metabolism , Reverse Transcription , Sensitivity and Specificity
15.
Mol Cell Probes ; 60: 101771, 2021 12.
Article in English | MEDLINE | ID: covidwho-1432043

ABSTRACT

The emergence of the influenza A(H1N1)pdm09 virus with the NA-H275Y mutation, which confers oseltamivir resistance, must be monitored, especially in patients undergoing neuraminidase inhibitor treatment. In this study, we developed a reverse transcription recombinase-aided amplification assay that has high sensitivity (detection limit: 1.0 × 101 copies/µL) and specificity for detecting the oseltamivir-resistant H275Y mutation; the assay is performed within 30 min at a constant temperature of 39° Celsius using an isothermal device. This method is suitable for the clinical application of targeted testing, thereby providing technical support for precision medicine in individual drug applications for patients with severe infection or immunosuppression.


Subject(s)
Influenza A Virus, H1N1 Subtype , Influenza, Human , Drug Resistance, Viral/genetics , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza, Human/diagnosis , Influenza, Human/drug therapy , Mutation , Mutation, Missense , Neuraminidase/genetics , Oseltamivir/pharmacology , Recombinases , Reverse Transcription
16.
Anal Chem ; 93(37): 12808-12816, 2021 09 21.
Article in English | MEDLINE | ID: covidwho-1402013

ABSTRACT

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.


Subject(s)
COVID-19 , Reverse Transcription , COVID-19 Testing , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Recombinases/genetics , SARS-CoV-2 , Sensitivity and Specificity , Technology
17.
PLoS One ; 16(7): e0254815, 2021.
Article in English | MEDLINE | ID: covidwho-1318322

ABSTRACT

African swine fever (ASF) is a serious contagious disease that causes fatal haemorrhagic fever in domestic and wild pigs, with high morbidity. It has caused devastating damage to the swine industry worldwide, necessitating the focus of attention on detection of the ASF pathogen, the African swine fever virus (ASFV). In order to overcome the disadvantages of conventional diagnostic methods (e.g. time-consuming, demanding and unintuitive), quick detection tools with higher sensitivity need to be explored. In this study, based on the conserved p72 gene sequence of ASFV, we combined the Cas12a-based assay with recombinase polymerase amplification (RPA) and a fluorophore-quencher (FQ)-labeled reporter assay for rapid and visible detection. Five crRNAs designed for Cas12a-based assay showed specificity with remarkable fluorescence intensity under visual inspection. Within 20 minutes, with an initial concentration of two copies of DNA, the assay can produce significant differences between experimental and negative groups, indicating the high sensitivity and rapidity of the method. Overall, the developed RPA-Cas12a-fluorescence assay provides a fast and visible tool for point-of-care ASFV detection with high sensitivity and specificity, which can be rapidly performed on-site under isothermal conditions, promising better control and prevention of ASF.


Subject(s)
African Swine Fever Virus/isolation & purification , African Swine Fever/diagnosis , Bacterial Proteins/genetics , CRISPR-Associated Proteins/genetics , Endodeoxyribonucleases/genetics , Swine Diseases/diagnosis , African Swine Fever/genetics , African Swine Fever/virology , African Swine Fever Virus/genetics , Animals , Bacterial Proteins/chemistry , CRISPR-Associated Proteins/chemistry , CRISPR-Cas Systems , DNA-Directed DNA Polymerase/chemistry , Endodeoxyribonucleases/chemistry , Molecular Diagnostic Techniques , Point-of-Care Systems , Recombinases/chemistry , Swine , Swine Diseases/genetics , Swine Diseases/pathology , Swine Diseases/virology
18.
J Virol Methods ; 296: 114227, 2021 10.
Article in English | MEDLINE | ID: covidwho-1294029

ABSTRACT

The rapid detection of novel pathogens including SARS-CoV-2 necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Delay in diagnosis has facilitated the rapid spread of this novel virus throughout the world resulting in global mortality that will surpass 2.5 million people. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral cDNA nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 nasopharyngeal samples using CDC's N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100 % concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/virology , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , DNA Primers , Diagnostic Tests, Routine , Humans , Point-of-Care Testing , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Recombinases/chemistry , SARS-CoV-2/genetics , Sensitivity and Specificity
19.
Anal Chem ; 93(27): 9541-9548, 2021 07 13.
Article in English | MEDLINE | ID: covidwho-1284670

ABSTRACT

Quantitative diagnostics that are rapid, inexpensive, sensitive, robust, and field-deployable are needed to contain the spread of infectious diseases and inform treatment strategies. While current gold-standard techniques are highly sensitive and quantitative, they are slow and require expensive equipment. Conversely, current rapid field-deployable assays available provide essentially binary information about the presence of the target analyte, not a quantitative measure of concentration. Here, we report the development of a molecular diagnostic test [quantitative recombinase polymerase amplification (qRPA)] that utilizes competitive amplification during a recombinase polymerase amplification (RPA) assay to provide semi-quantitative information on a target nucleic acid. We demonstrate that qRPA can quantify DNA, RNA, and viral titers in HIV and COVID-19 patient samples and that it is more robust to environmental perturbations than traditional RPA. These features make qRPA potentially useful for at-home testing to monitor the progress of viral infections or other diseases.


Subject(s)
COVID-19 , Nucleic Acid Amplification Techniques , Humans , Molecular Diagnostic Techniques , Recombinases , SARS-CoV-2 , Sensitivity and Specificity
20.
Front Cell Infect Microbiol ; 11: 680728, 2021.
Article in English | MEDLINE | ID: covidwho-1268238

ABSTRACT

The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV-2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV-2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.


Subject(s)
COVID-19 , Recombinases , China , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity
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