Simple, Visual, Point-of-Care SARS-CoV-2 Detection Incorporating Recombinase Polymerase Amplification and Target DNA–Protein Crosslinking Enhanced Chemiluminescence

Author(s):

Hui Chen,Zhiyuan Zhuang,Naihan Xu,Ying Feng,Kaixin Fang,Chunyan Tan,Ying Tan

Journal:

Biosensors

Year:

2024

Abstract:

The ongoing COVID-19 pandemic, driven by persistent SARS-CoV-2 transmission, threatens human health worldwide, underscoring the urgent need for an efficient, low-cost, rapid SARS-CoV-2 detection method. Herein, we developed a point-of-care SARS-CoV-2 detection method incorporating recombinase polymerase amplification (RPA) and DNA-protein crosslinking chemiluminescence (DPCL) (RPADPCL). RPADPCL involves the crosslinking of biotinylated double-stranded RPA DNA products with horseradish peroxidase (HRP)-labeled streptavidin (SA-HRP). Modified products are captured using SA-labeled magnetic beads, and then analyzed using a chemiluminescence detector and smartphone after the addition of a chemiluminescent substrate. Under optimal conditions, the RPADPCL limit of detection (LOD) was observed to be 6 copies (within the linear detection range of 1-300 copies) for a plasmid containing the SARS-CoV-2 N gene and 15 copies (within the linear range of 10-500 copies) for in vitro transcribed (IVT) SARS-CoV-2 RNA. The proposed method is convenient, specific, visually intuitive, easy to use, and does not require external excitation. The effective RPADPCL detection of SARS-CoV-2 in complex matrix systems was verified by testing simulated clinical samples containing 10% human saliva or a virus transfer medium (VTM) spiked with a plasmid containing a SARS-CoV-2 N gene sequence or SARS-CoV-2 IVT RNA. Consequently, this method has great potential for detecting targets in clinical samples.

PrimerBankID	Target Pathogen	Target Gene
RPB0240	SARS-CoV-2	SARS-CoV-2 N gene

A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC-2, and blaNDM-1 Genes in Klebsiella pneumoniae

Author(s):

Shao-Wei Hua,Jie Wang,Zi-Jin Zhao,Feng-Yu Tian,Meng Zhao,Yu-Xin Wang,Rui-Qing Zhang,Zhi-Qiang Han,Shi-Jue Gao,Xiao-Na Lv,Hong-Yi Li,Xin-Xin Shen,Xue-Jun Ma,Zhi-Shan Feng

Journal:

Clinical Laboratory Analysis

Year:

2024

Abstract:

Objective: This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. Methods: mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. Results: mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). Conclusion: mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.

PrimerBankID	Target Pathogen	Target Gene
RPB0317	Klebsiella pneumoniae	khe gene
RPB0318	Klebsiella pneumoniae	blaKPC-2 gene
RPB0319	Klebsiella pneumoniae	blaNDM-1gene

Automatic Microfluidic Harmonized RAA-CRISPR Diagnostic System for Rapid and Accurate Identification of Bacterial Respiratory Tract Infections

Author(s):

Xinran Xiang,Xiaoqing Ren,Qianyu Wen,Gaowa Xing,Yuting Liu,Xiaowei Xu,Yuhuan Wei,Yuhan Ji,Tingting Liu,Huwei Song,Shenghang Zhang,Yuting Shang,Minghui Song

Journal:

Analytical Chemistry

Year:

2024

Abstract:

Respiratory tract infections (RTIs) pose a grave threat to human health, with bacterial pathogens being the primary culprits behind severe illness and mortality. In response to the pressing issue, we developed a centrifugal microfluidic chip integrated with a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR) system to achieve rapid detection of respiratory pathogens. The limitations of conventional two-step CRISPR-mediated systems were effectively addressed by employing the all-in-one RAA-CRISPR detection method, thereby enhancing the accuracy and sensitivity of bacterial detection. Moreover, the integration of a centrifugal microfluidic chip led to reduced sample consumption and significantly improved the detection throughput, enabling the simultaneous detection of multiple respiratory pathogens. Furthermore, the incorporation of Chelex-100 in the sample pretreatment enabled a sample-to-answer capability. This pivotal addition facilitated the deployment of the system in real clinical sample testing, enabling the accurate detection of 12 common respiratory bacteria within a set of 60 clinical samples. The system offers rapid and reliable results that are crucial for clinical diagnosis, enabling healthcare professionals to administer timely and accurate treatment interventions to patients.

PrimerBankID	Target Pathogen	Target Gene
RPB0269	Acinetobacter baumannii	\
RPB0270	Klebsiella pneumoniae	\
RPB0271	Pseudomonas aeruginosa	\
RPB0272	Stenotrophomonas maltophilia	\
RPB0273	Staphylococcus aureus	\
RPB0274	Haemophilus influenzae	\
RPB0275	Streptococcus pneumoniae	\

SARS-CoV-2 and Its Omicron Variants Detection with RT-RPA -CRISPR\Cas13a-Based Method at Room Temperature

Author(s):

Jia Li,Xiaojun Wang,Liujie Chen,Lili Duan,Fenghua Tan,Kai Li,Zheng Hu

Journal:

Reports of Biochemistry & Molecular Biology logo

Year:

2023

Abstract:

Background: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, with genetic mutations and evolution further creating uncertainty about epidemic risk. It is imperative to rapidly determine the nucleic acid sequence of SARS-CoV-2 and its variants to combat the coronavirus pandemic. Our goal was to develop a rapid, room-temperature, point-of-care (POC) detection system to determine the nucleic acid sequences of SARS-CoV-2 isolates, especially omicron variants. Methods: Based on the conserved nucleotide sequence of SARS-CoV-2, bioinformatics software was used to analyze, design, and screen optimal enzymatic isothermal amplification primers and efficient CRISPR RNAs (crRNAs) of CRISPR/Cas13a to the target sequences. Reverse transcription-recombinase polymerase amplification (RT-RPA) was used to amplify the virus, and CRISPR/Cas13a-crRNA was used to cleave the SARS-CoV-2 target sequence. The sensitivity of nucleic acid detection was assessed by serial dilution of plasmid templates. All reactions were performed at room temperature. Results: RT-RPA, combined with CRISPR/Cas13a, can detect the SARS-CoV-2 with a minimum content of 102 copies/μL, and can effectively distinguish between the original strain and the Omicron variant with a minimum limit of detection (LOD) of 103 copies/μL. Conclusions: The method developed in this study has potential application in clinical detection of SARS-CoV-2 and its omicron variants.

PrimerBankID	Target Pathogen	Target Gene
RPB0370	SARS-CoV-2	S gene
RPB0371	SARS-CoV-2	ORF1ab gene
RPB0372	SARS-CoV-2	N gene
RPB0373	SARS-CoV-2 (Omicron)	Spike gene

PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification–in vitro transcription

Author(s):

Pavana Khan,Lauren M Aufdembrink,Katarzyna P Adamala,Aaron E Engelhart

Journal:

RNA

Year:

2024

Abstract:

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods use the recombinase polymerase amplification (RPA), which exhibits polymerase chain reaction (PCR)-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription) can be used to detect SARS-CoV-2 RNA with single-copy detection limits, Escherichia coli with single-cell detection limits, and 10-min detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be used for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0234	SARS-CoV-2	Nucleocapsid N3 gene

Rapid, sensitive, and user-friendly detection of Pseudomonas aeruginosa using the RPA\CRISPR\Cas12a system

Author(s):

Wenjing Zhang,Hai Qu,Xin Wu,Jingjing Shi,Xinling Wang

Journal:

BMC microbiology

Year:

2024

Abstract:

Background: Pseudomonas aeruginosa (P. aeruginosa) is a life-threatening bacterium known for its rapid development of antibiotic resistance, posing significant challenges in clinical treatment, biosecurity, food safety, and environmental monitoring. Early and accurate identification of P. aeruginosa is crucial for effective intervention. Methods: The lasB gene of P. aeruginosa was selected as the target for the detection. RPA primers for recombinase polymerase amplification (RPA) and crRNA for CRISPR/Cas12a detection were meticulously designed to target specific regions within the lasB gene. The specificity of the RPA/CRISPR/Cas12a detection platform was assessed using 15 strains. The detection limit of RPA/CRISPR/Cas12a detection platform was determined by utilizing a pseudo-dilution series of the P. aeruginosa DNA. The practical applicability of the RPA/CRISPR/Cas12a detection platform was validated by comparing it with qPCR on 150 samples (35 processed meat product samples, 55 cold seasoned vegetable dishes, 60 bottled water samples). Results: The RPA/CRISPR/Cas12a detection platform demonstrates high specificity, with no cross-reactivity with non-P. aeruginosa strains. This assay exhibits remarkable sensitivity, with a limit of detection (LOD) of 100 copies/µL for fluorescence assay and 101 copies/µL for the LFTS method. Furthermore, the performance of the RPA/CRISPR/Cas12a detection platform is comparable to that of the well-established qPCR method, while offering advantages such as shorter reaction time, simplified operation, and reduced equipment requirements. Conclusions: The RPA/CRISPR/Cas12a detection platform presents a straightforward, accurate, and sensitive approach for early P. aeruginosa detection and holds great promise for diverse applications requiring rapid and reliable identification.

PrimerBankID	Target Pathogen	Target Gene
RPB0228	Pseudomonas aeruginosa	lasB gene

A Method for Detecting Five Carbapenemases in Bacteria Based on CRISPR-Cas12a Multiple RPA Rapid Detection Technology

Author(s):

Huaming Xu,Chunhui Lin,Hao Tang,Rongrong Li,Zhaoxin Xia,Yi Zhu,Zhen Liu,Jilu Shen

Journal:

Infection and Drug Resistance

Year:

2024

Abstract:

Introduction: As the last line of defense for clinical treatment, Carbapenem antibiotics are increasingly challenged by multi-drug resistant bacteria containing carbapenemases. The rapid spread of these multidrug-resistant bacteria is the greatest threat to severe global health problems. Methods: To solve the problem of rapid transmission of this multidrug-resistant bacteria, we have developed a rapid detection technology using CRPSPR-Cas12a gene editing based on multiple Recombinase polymerase amplification. This technical method can directly isolate the genes of carbapenemase-containing bacteria from samples, with a relatively short detection time of 30 minutes. The instrument used for the detection is relatively inexpensive. Only a water bath can complete the entire experiment of Recombinase polymerase amplification and trans cleavage. This reaction requires no lid during the entire process while reducing a large amount of aerosol pollution. Results: The detection sensitivity of this method is 1.5 CFU/mL, and the specificity is 100%. Discussion: This multi-scene detection method is suitable for screening populations in wild low-resource environments and large-scale indoor crowds. It can be widely used in hospital infection control and prevention and to provide theoretical insights for clinical diagnosis and treatment.

PrimerBankID	Target Pathogen	Target Gene
RPB0297	Klebsiella pneumoniae	KPC gene
RPB0298	Klebsiella pneumoniae	NDM gene
RPB0299	Klebsiella pneumoniae	OXA gene
RPB0300	Klebsiella pneumoniae	IMP gene
RPB0301	Klebsiella pneumoniae	VIM gene

Detection method for reverse transcription recombinase-aided amplification of avian influenza virus subtypes H5, H7, and H9

Author(s):

Zongshu Zhang,Zichuang Zhang,Chunguang Wang,Xianghe Zhai,Wenjing Wang,Xi Chen,Tie Zhang

Journal:

BMC Veterinary Research

Year:

2024

Abstract:

Background: Avian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA. Results: The results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%. Conclusions: RT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.

PrimerBankID	Target Pathogen	Target Gene
RPB0341	Influenza A virus (H5)	HA gene
RPB0342	Influenza A virus (H7)	HA gene
RPB0343	Influenza A virus (H9)	HA gene
RPB0344	Influenza A virus (H5)	HA gene
RPB0345	Influenza A virus (H7)	HA gene
RPB0346	Influenza A virus (H9)	HA gene

FARPA-based tube array coupled with quick DNA extraction enables ultra-fast bedside detection of antibiotic-resistant pathogens

Author(s):

Jinling Huang,Huijie Yue,Wei Wei,Jingwen Shan,Yue Zhu,Liying Feng,Yi Ma,Bingjie Zou,Haiping Wu,Guohua Zhou

Journal:

Analyst

Year:

2024

Abstract:

Rapid and accurate detection of pathogens and antimicrobial-resistant (AMR) genes of the pathogens are crucial for the clinical diagnosis and effective treatment of infectious diseases. However, the time-consuming steps of conventional culture-based methods inhibit the precise and early application of anti-infection therapy. For the prompt treatment of pathogen-infected patients, we have proposed a novel tube array strategy based on our previously reported FARPA (FEN1-aided recombinase polymerase amplification) principle for the ultra-fast detection of antibiotic-resistant pathogens on site. The entire process from "sample to result" can be completed in 25 min by combining quick DNA extraction from a urine sample with FARPA to avoid the usually complicated DNA extraction step. Furthermore, a 36-tube array made from commercial 384-well titre plates was efficiently introduced to perform FARPA in a portable analyser, achieving an increase in the loading sample throughput (from several to several tens), which is quite suitable for the point-of-care testing (POCT) of multiple pathogens and multiple samples. Finally, we tested 92 urine samples to verify the performance of our proposed method. The sensitivities for the detection of E. coli, K. pneumoniae, E. faecium, and E. faecalis were 92.7%, 93.8%, 100% and 88.9%, respectively. The specificities for the detection of the four pathogens were 100%. Consequently, our rapid, low-cost and user-friendly POCT method holds great potential for guiding the rational use of antibiotics and reducing bacterial resistance.

PrimerBankID	Target Pathogen	Target Gene
RPB0327	Klebsiella pneumoniae	KPC
RPB0328	Klebsiella pneumoniae	NDM
RPB0329	Klebsiella pneumoniae	K.pneumoniae

Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA

Author(s):

Qinlong Zeng,Miaojin Zhou,Weiheng Deng,Qian Gao,Zhuo Li,Lingqian Wu,Desheng Liang

Journal:

Analytica Chimica Acta

Year:

2024

Abstract:

Background: CRISPR-Cas12a based one-step assays are widely used for nucleic acid detection, particularly for pathogen detection. However, the detection capability of the one-step assay is reduced because the Cas12a protein competes with the isothermal amplification enzymes for the target DNA and cleaves it. Therefore, the key to improving the sensitivity of the one-step assay is to address the imbalance between isothermal amplification and CRISPR detection. In previous study, we developed a Cas12a one-step assay using single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA) and applied this method for the detection of pathogenic DNA. Results: Here, we utilized mD-crRNA to establish a sensitive one-step assay that enables the visual detection of SARS-CoV-2 under ultraviolet light, achieving a detection limit of 5 aM without cross-reactivity. The sensitivity of mD-crRNA in the one-step assay was 100-fold higher than that of wild-type crRNA. Mechanistic studies revealed that the addition of ssDNA at the 3' end of mD-crRNA attenuates the binding affinity between the Cas12a-mD-crRNA complex and the target DNA. Consequently, this reduction in binding affinity decreases the cis-cleavage activity of Cas12a, mitigating its cleavage of the target DNA in the one-step assay. As a result, there is an augmentation in the amplification and accumulation of target DNA, thereby enhancing detection sensitivity. In the clinical testing of 40 SARS-CoV-2 RNA samples, the concordance between the results of the one-step assay and known qPCR results was 97.5 %. Significance: The one-step assay using mD-crRNA proves to be highly sensitive and specificity and visually effective for the detection of SARS-CoV-2. Our study delves into the application of the mD-crRNA-mediated one-step assay in nucleic acid detection and its associated reaction mechanism. This holds great significance in addressing the inherent incompatibility issues between isothermal amplification and CRISPR detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0339	SARS-CoV-2	N gene