Clinical Validation of a Rapid Variant-Proof RT-RPA Assay for the Detection of SARS-CoV-2

Author(s):

Dounia Cherkaoui, Judith Heaney, Da Huang,Matthew Byott, Benjamin S. Miller,Eleni Nastouli, and Rachel A. McKendry

Journal:

Diagnostics (Basel).

Year:

2022

Abstract:

The COVID-19 pandemic has unveiled a pressing need to expand the diagnostic landscape to permit high-volume testing in peak demand. Rapid nucleic acid testing based on isothermal amplification is a viable alternative to real-time reverse transcription polymerase chain reaction (RT-PCR) and can help close this gap. With the emergence of SARS-CoV-2 variants of concern, clinical validation of rapid molecular tests needs to demonstrate their ability to detect known variants, an essential requirement for a robust pan-SARS-CoV-2 assay. To date, there has been no clinical validation of reverse transcription recombinase polymerase amplification (RT-RPA) assays for SARS-CoV-2 variants. We performed a clinical validation of a one-pot multi-gene RT-RPA assay with the E and RdRP genes of SARS-CoV-2 as targets. The assay was validated with 91 nasopharyngeal samples, with a full range of viral loads, collected at University College London Hospitals. Moreover, the assay was tested with previously sequenced clinical samples, including eleven lineages of SARS-CoV-2. The rapid (20 min) RT-RPA assay showed high sensitivity and specificity, equal to 96% and 97%, respectively, compared to gold standard real-time RT-PCR. The assay did not show cross-reactivity with the panel of respiratory pathogens tested. We also report on a semi-quantitative analysis of the RT-RPA results with correlation to viral load equivalents. Furthermore, the assay could detect all eleven SARS-CoV-2 lineages tested, including four variants of concern (Alpha, Beta, Delta, and Omicron). This variant-proof SARS-CoV-2 assay offers a significantly faster and simpler alternative to RT-PCR, delivering sensitive and specific results with clinical samples.

PrimerBankID	Target Pathogen	Target Gene
RPB0200	SARS-CoV-2	E gene
RPB0201	SARS-CoV-2	RdRP gene

Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay

Author(s):

Mei Lin , Huahua Yue , Tian Tian , Erhu Xiong , Debin Zhu , Yongzhong Jiang , Xiaoming Zhou

Journal:

Analytical Chemistry

Year:

2022

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.

PrimerBankID	Target Pathogen	Target Gene
RPB0198	SARS-CoV-2	Orf1ab-gene
RPB0199	SARS-CoV-2	N-gene

Development and Clinical Application of a Recombinase Polymerase Amplification-Lateral Flow Strip Assay for Detection of Carbapenem-Resistant Acinetobacter baumannii

Author(s):

Lei Wang,Dunpo Sun,Li Chen,Ping Zhou,Kun Wang,Fang Wang,Xingqi Lei,Yan Wang,Yingzhi Lu,Guanhong Huang,Xuzhu Gao

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

Acinetobacter baumannii is a worldwide, primary cause of respiratory tract infections, septicemia, urinary apparatus infections, and secondary meningitis. It can be fatal. Rapid and accurate detection methods are needed to control the spread of carbapenem-resistant A. baumannii (CRAB). Current molecular diagnostic methods are limited and not suitable for on-site detection. In this study, an isothermal detection method using recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) was developed to target the blaOXA-51 and blaOXA-23 genes of A. baumannii. The reaction was completed in about 40 min at 37°C. This method can also effectively distinguish A. baumannii and CRAB. The limit of detection of 100-101 CFU/reaction was equal to that of other detection methods. The detection accuracy was equal to that of the qPCR method with the use of clinical samples. The RPA-LFS assay is portable, rapid, and accurate and could replace existing detection methods for on-site detection of A. baumannii and CRAB.

PrimerBankID	Target Pathogen	Target Gene
RPB0080	Acinetobacter baumannii	bla OXA-51
RPB0081	Acinetobacter baumannii	bla OXA-23

Automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) and lateral flow strip based on CRISPR/Cas13a for sensitive and visual detection of SARS-CoV-2

Author(s):

Gaihua Cao, Danqun Huo, Xiaolong Chen, Xianfeng Wang, Shiying Zhou, Shixian Zhao, Xiaogang Luo, Changjun Hou

Journal:

Talanta

Year:

2022

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.

PrimerBankID	Target Pathogen	Target Gene
RPB0037	SARS-CoV-2	S
RPB0038	SARS-CoV-2	ORF1ab

Rapid Detection of Klebsiella pneumoniae Carrying Virulence Gene rmpA2 by Recombinase Polymerase Amplification Combined With Lateral Flow Strips

Author(s):

Na Li,Lei Wang,Fang Wang,Huimin Chen,Shuan Tao,Qing Zhu,Liping Liu,Wei Liang,Fang Ma

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

Highly virulent Klebsiella pneumoniae often causes invasive infections with high morbidity and mortality rates, posing an immense clinical challenge. Rapid and accurate detection of pathogenic bacteria is of great significance for treatment and preventive control. Conventional detection by polymerase chain reaction (PCR) is limited by a dependence on laboratory equipment and professional staff. Recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) can rapidly amplify and visualize target genes in a short period of time. The aim of this study was to develop an RPA-LFS technique for detection of the K. pneumoniae virulence gene rmpA2. Primers were designed against conserved sequences specific to the virulence gene, and primer probe design was optimized by introducing base substitution to obtain a specific and sensitive primer-probe combination for clinical detection. We tested 65 actual samples collected from clinics to evaluate the performance of the newly established RPA-LFS system in comparison with conventional PCR methods and qPCR methods. The RPA-LFS assay was performed at for 25 min a constant temperature of 37°C, and results could be observed without instrumentation. The system could specifically identify highly virulent K. pneumoniae carrying the virulence gene rmpA2 with a minimum detection limit of 10-1 ng/μL and 10 copies/μL. For the 65 clinical samples tested, The RPA-LFS assay results were in complete agreement with the qPCR results and PCR results. The RPA-LFS assay provides a rapid, accurate, and simple method for identification of highly virulent K. pneumoniae carrying rmpA2.

PrimerBankID	Target Pathogen	Target Gene
RPB0103	Klebsiella pneumoniae	rmpA2

Rapid, Simple, and Highly Specific Detection of Streptococcus pneumoniae With Visualized Recombinase Polymerase Amplification

Author(s):

Fang Wang,Yan Wang,Xia Liu,Lei Wang,Kun Wang,Chenglai Xu,Guanhong Huang,Xuzhu Gao

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

Streptococcus pneumoniae is a major pathogen that causes microbiological illness in humans. The introduction of polyvalent vaccines has resulted in a significant decrease in pneumococcal-related mortality. However, pneumococcal infections continue to be a leading cause of death in children under the age of 5 and adults over the age of 65 worldwide. A speedy and highly sensitive diagnostic tool is necessary for routine adoption to adequately manage patients and control the spread of infection. In this study, we investigated a new nucleic acid amplification technique, isothermal recombinase polymerase amplification (RPA), which amplifies DNA at 37°C under isothermal conditions with high specificity, efficiency, and rapidity. Using the autolysin gene lytA as the molecular diagnostic target, an RPA primer-probe combination was designed and optimized for the detection of S. pneumoniae. This RPA reaction produced amplification products labeled with specific chemical markers, to be detected with gold-nanoparticle-based lateral flow strips (LFS), reducing the reliance on equipment and trained personnel. The high specificity of the RPA-LFS technique was demonstrated with the specific detection of 22 strains of S. pneumoniae but not 25 closely related pathogenic bacteria. The assay showed good sensitivity, and detected S. pneumoniae down to 3.32 colony-forming units/μL. When used on clinical samples, the assay provided accurate and consistent results compared with PCR. The compliance with the culture-biochemistry method was 98.18% and the kappa index was 0.977. These results reveal that the RPA-LFS test significantly improved S. pneumoniae identification, particularly in resource-limited areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0165	Streptococcus pneumoniae	lytA

Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics

Author(s):

Menglu Hu, Zhiqiang Qiu, Zirong Bi, Tian Tian, Yongzhong Jiang, and Xiaoming Zhou

Journal:

Proceedings of the National Academy of Sciences of the United States of America

Year:

2022

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.

PrimerBankID	Target Pathogen	Target Gene
RPB0196	SARS-CoV-2	O-gene
RPB0197	SARS-CoV-2	N-gene

Rapid detection of methicillin-resistant Staphylococcus aureus in positive blood-cultures by recombinase polymerase amplification combined with lateral flow strip

Author(s):

Arpasiri Srisrattakarn,Pimchanok Panpru,Patcharaporn Tippayawat,Aroonwadee Chanawong,Ratree Tavichakorntrakool,Jureerut Daduang,Lumyai Wonglakorn,Aroonlug Lulitanond

Journal:

PloS one

Year:

2022

Abstract:

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is an important bacterium that causes community and healthcare-related infections throughout the world. However, the current conventional detection methods are time-consuming. We therefore developed and evaluated a recombinase polymerase amplification-lateral flow strip (RPA-LF) approach for detection of MRSA in positive blood-culture samples. Sixty positive blood-cultures from a hospital were tested directly without DNA extraction and purification before the amplification reaction. RPA primers and probes were designed for nuc (encoding thermonuclease) and mecA (encoding penicillin-binding protein 2a) genes to diagnose S. aureus and its methicillin-resistance status. The RPA reaction occurred under isothermal conditions (45°C) within 20 min and a result was provided by the LF strip in a further 5 min at room temperature. The evaluation of RPA-LF using blood-culture samples showed 93.3% (14/15) sensitivity for identifying S. aureus, and no cross-amplification was seen [100% (45/45) specificity]. For detection of methicillin resistance, the RPA-LF test provided 100% (16/16) sensitivity and 97.7% (43/44) specificity. The RPA-LF is rapid, highly sensitive, robust and easy to use. It can be used for direct detection of MRSA with no requirement for special equipment.

PrimerBankID	Target Pathogen	Target Gene
RPB0156	Staphylococcus aureus	nuc
RPB0157	Staphylococcus aureus	mecA

Multiple ligation-Assisted recombinase polymerase amplification for highly sensitive and selective colorimetric detection of SARS-CoV-2

Author(s):

Tasnima Alam Asa, Pradeep Kumar, Jaehyeon Lee, Young Jun Seo

Journal:

Talanta

Year:

2023

Abstract:

In this paper we present a new method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), targeting a specific region "N gene." Under isothermal reaction conditions, we integrated ligation (Lig; high selectivity) and recombinase polymerase amplification (RPA; high sensitivity) processes, obtaining a robust method of detection. For point-of-care testing, we incorporated our laboratory-produced pyrophosphate ion (PPi)-sensing probe (PK-probe) for colorimetric analysis of the reaction. The total detection system was efficient and effective at diagnosing this RNA virus-mediated disease rapidly (30 min). In a full-genome SARS-CoV-2 study, our PK-probe/Lig-RPA system functioned with a limit of detection of 1160 copies/ml, with a single-mismatch level of selectively, and it was highly selective even in the presence of bacterial genomes commonly found in the human mouth and nose. This robust, straightforward, selective, efficient, and ultrasensitive colorimetric detection method, with potential for point-of-care analysis, should also be effective in detecting a diverse range of other RNA-based diseases.

PrimerBankID	Target Pathogen	Target Gene
RPB0019	SARS-CoV-2	N

Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection

Author(s):

Gaoxing Su,Min Zhu,Diyuan Li,Mengting Xu,Yuedong Zhu,Yan Zhang,Hongyan Zhu,Feng Li,Yanyan Yu

Journal:

Sensors and Actuators:B. Chemical

Year:

2022

Abstract:

The development of field-deployable detection platform amenable for multiplexed genes testing will significantly improve the efficiency and reliability during point-of-care testing (POCT) applications. In this regard, an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (designated as OC-MLFA) is proposed for SARS-CoV-2 genome detection. Taking the advantage of activation and cleavage preferences between Cas12a and Cas13a, orthogonal (two-independent-channel signal readout) CRISPR-Cas system is investigated. Lateral flow strips with two target lines are designed to accommodate the orthogonal CRISPR system. The interference between Cas12a and Cas13a channels can be effectively eliminated via the elaborate nucleic acids and lateral flow strips design. The high preamplification efficiency from reverse transcription recombinase polymerase amplification (RT-RPA) and Cas enzyme mediated trans-cleavage process bring the sensitivity of our OC-MLFA method to 10 copies per test (30 μL). Nasopharyngeal swab clinical samples with different cycle threshold (Ct) values according to the RT-PCR method were analyzed with the proposed OC-MLFA, during which 76 out of 76 detection accuracy was obtained. Featured with the multiplexed genes detection simultaneously in one reaction and colorimetric readout through single strip, the OC-MLFA we proposed herein ensures great accuracy and efficiency, which endows promising field-deployable POCT application feasibility.

PrimerBankID	Target Pathogen	Target Gene
RPB0033	SARS-CoV-2	ORF1ab
RPB0034	SARS-CoV-2	N