Establishment of a Real-Time Fluorescence Isothermal Recombinase-Aided Amplification Method for the Detection of H9 Avian Influenza Virus

Author(s):

Yuxin Zhang,Cheng Zhang,Jiaqi Li,Yejin Yang,Ligong Chen,Heng Wang,Zitong Yang,Mingda Zhang,Huan Cui,Shishan Dong

Journal:

Diagnosis, Prevention and Control in Avian Virus Infections

Year:

2024

Abstract:

The H9 subtype of avian influenza virus (AIV) has been characterized by its rapid spread, wide range of prevalence, and continuous evolution in recent years, leading to an increasing ability for cross-species transmission. This not only severely impacts the economic benefits of the aquaculture industry, but also poses a significant threat to human health. Therefore, developing a rapid and sensitive detection method is crucial for the timely diagnosis and prevention of H9 AIVs. In this study, a real-time fluorescent reverse transcription recombinase-aided isothermal amplification (RT-RAA) technique targeting the hemagglutinin (HA) of H9 AIVs was established. This technique can be used for detection in just 30 min at a constant temperature of 42 °C, and it exhibits good specificity without cross-reactivity with other viruses. Sensitivity tests revealed that the detection limit of RT-RAA was 163 copies per reaction, and the visual detection limit was 1759 copies per reaction at a 95% confidence interval, both of which are capable of detecting low concentrations of standards. Furthermore, RT-RAA was applied to detect 155 clinical samples, and compared to real-time fluorescent quantitative PCR (RT-qPCR), RT-RAA demonstrated high accuracy, with a specificity of 100% and a kappa value of 0.96, indicating good correlation. Additionally, with the assistance of a portable blue imaging device, we can visually observe the amplification products, greatly facilitating rapid detection in resource-limited environments. The RT-RAA detection method developed in this study does not require expensive equipment or highly skilled staff, making it beneficial for the accurate and low-cost detection of H9 AIVs.

PrimerBankID	Target Pathogen	Target Gene
RPB0289	Influenza A virus (H9N2)	HA

Advanced Electrochemical Biosensing toward Staphylococcus aureus Based on the RPA-CRISPR\Cas12a System and Conductive Nanocomposite

Author(s):

Yiqing Guo,Chen Li,Wang Guo,Xinai Zhang,Li Wang,Wen Zhang,Xiaobo Zou,Zongbao Sun

Journal:

Agricultural and Food Chemistry

Year:

2024

Abstract:

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that poses significant challenges to food safety. Herein, a sensitive and specific electrochemical biosensor based on RPA-CRISPR/Cas12a is developed for evaluating S. aureus. In the presence of S. aureus, the extracted target DNA fragments are efficiently amplified by recombinase polymerase amplification (RPA). The designed crRNA, binding to Cas12a, effectively recognizes the target fragment cleaving hpDNA. The signal molecule of hpDNA is cleaved from the sensing interface, resulting in a reduction of current response. Under optimal experimental conditions, the developed electrochemical biosensor exhibits remarkable sensitivity in detecting S. aureus. The linear range for quantifying S. aureus in pure culture is 1.04 × 101-1.04 × 108 CFU/mL, with a detection limit as low as 3 CFU/mL. In addition, the biosensor enables the accurate and sensitive detection of S. aureus in milk within a linear range of 1.07 × 101-1.07 × 107 CFU/mL. The electrochemical biosensor enhances anti-interference capability owing to the specific amplification of RPA primers and the single-base recognition ability of crRNA. The RPA-CRISPR/Cas12a biosensor exhibits exceptional anti-interference capability, precision, and sensitivity, thereby establishing a robust foundation for real-time monitoring of microbial contamination.

PrimerBankID	Target Pathogen	Target Gene
RPB0320	Staphylococcus aureus	nuc gene

One-Pot Assay for Rapid Detection of Stenotrophomonas maltophilia by RPA-CRISPR\Cas12a

Author(s):

Jiangli Zhang,Ling Qin,Yingying Chang,Yulong He,Weichao Zhao,Yongyou Zhao,Yanan Ding,Jin Gao,Xiting Zhao

Journal:

ACS Synthetic Biology

Year:

2024

Abstract:

Stenotrophomonas maltophilia (S. maltophilia, SMA) is a common opportunistic pathogen that poses a serious threat to the food industry and human health. Traditional detection methods for SMA are time-consuming, have low detection rates, require complex and expensive equipment and professional technical personnel for operation, and are unsuitable for on-site detection. Therefore, establishing an efficient on-site detection method has great significance in formulating appropriate treatment strategies and ensuring food safety. In the present study, a rapid one-pot detection method was established for SMA using a combination of Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a, referred to as ORCas12a-SMA (one-pot RPA-CRISPR/Cas12a platform). In the ORCas12a-SMA detection method, all components were added into a single tube simultaneously to achieve one-pot detection and address the problems of nucleic acid cross-contamination and reduced sensitivity caused by frequent cap opening during stepwise detection. The ORCas12a-SMA method could detect at least 3 × 10° copies·μL-1 of SMA genomic DNA within 30 min at 37 °C. Additionally, this method exhibited sensitivity compared to the typical two-step RPA-CRISPR/Cas12a method. Overall, the ORCas12a-SMA detection offered the advantages of rapidity, simplicity, high sensitivity and specificity, and decreased need for complex large-scale instrumentation. This assay is the first application of the one-pot platform based on the combination of RPA and CRISPR/Cas12a in SMA detection and is highly suitable for point-of-care testing. It helps reduce losses in the food industry and provides assistance in formulating timely and appropriate antimicrobial treatment plans.

PrimerBankID	Target Pathogen	Target Gene
RPB0293	Stenotrophomonas maltophilia	N gene

Establishment of a platform based on dual RPA combined with CRISPR\Cas12a for the detection of Klebsiella pneumoniae and its KPC resistance gene

Author(s):

Meiying Tan,Xueli Yi,Chuan Liao,Zihan Zhou,Baoyan Ren,Lina Liang,Xuebin Li,Guijiang Wei

Journal:

Frontiers in Bioengineering and Biotechnology

Year:

2024

Abstract:

Carbapenem resistant Klebsiella pneumoniae (CRKP) can cause serious hospital- and community-acquired infections. Treatment for CRKP infection is limited, resulting in prolonged hospitalization and high consultation costs. The KPC genotype has the highest detection rate of CRKP, and its mortality rate is higher than the overall mortality rate of CRKP. However, traditional testing methods have disadvantages such as long time and reliance on complex and sophisticated instruments, which are not conducive to rapid screening for CRKP. Therefore, this study aimed to establish a detection platform for early screening of CRKP so that effective antimicrobial therapy could be administered promptly to prevent the widespread spread of CRKP. We integrated dual RPA with CRISPR/Cas12a to establish a dual platform for the detection of K. pneumoniae (Kp) rcsA-specific gene and KPC resistance gene. Four result reading methods were established, including fluorescence detection (FD), blue light irradiation detection (BLID), ultraviolet irradiation detection (UID), and lateral flow test strips (LFTS). For the rcsA gene, the LOD of FD was 1 × 10 pg/μL, and the other three methods could detect 1 × 101 pg/μL of bacterial DNA. As for the KPC gene, four resultant readout methods were able to detect 1 × 102 pg/μL of bacterial DNA. In 59 clinical strains tested, the dual RPA-CRISPR/Cas12a detection of the rcsA had 100% sensitivity, specificity, and accuracy compared to the culture method. Compared with the drug sensitivity test, the sensitivity of dual RPA-CRISPR/Cas12a detection for the KPC was 85.71%, the specificity was 100%, and the accuracy was 94.92%. In summary, our dual RPA-CRISPR/Cas12a platform proved to be rapid, precise, and convenient for the efficient detection of Kp with KPC in the laboratory or at the point of care.

PrimerBankID	Target Pathogen	Target Gene
RPB0283	Klebsiella pneumoniae	rcsA gene
RPB0284	Klebsiella pneumoniae	KPC gene

Duplex recombinase aided amplification-lateral flow dipstick assay for rapid distinction of Mycobacterium tuberculosis and Mycobacterium avium complex

Author(s):

Ke Chen,Junze Zhang,Simeng Wang,Zhengjun Yi,Yurong Fu

Journal:

Frontiers in Cellular and Infection Microbiology

Year:

2024

Abstract:

Objectives: This study aims to develop a novel diagnostic approach using the recombinase aided amplification-lateral flow dipstick(RAA-LFD) assay for the distinction of Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC), enabling rapid and convenient as well as accurate identification of them in clinical samples. Methods: Our study established a duplex RAA-LFD assay capable of discriminating between MTB and MAC. Based on the principles of RAA primer and probe design, specific primers and probes were developed targeting the MTB IS6110 and the MAC DT1 separately. Optimization of reaction time points and temperatures was conducted, followed by an evaluation of specificity, sensitivity, and reproducibility. The established detection method was then applied to clinical samples and compared with smear microscopy, liquid culture, LAMP, and Xpert/MTB RIF in terms of diagnostic performance. Results: The complete workflow allows for the effective amplification of the MTB IS6110 and MAC DT1 target sequences at constant 37°C within 20min, and the amplification products can be visually observed on the LFD test strip. This method exhibits high specificity, showing no cross-reactivity with nucleic acids from M. kansassi, M. abscessus, M. gordonae, M. chelonae, M. fortuitum, M. scrofulaceum, M. malmoense, M. chimaera, M. szulgai and common respiratory pathogens. It also demonstrates high sensitivity, with a detection limit as low as 102 CFU/mL. Additionally, the method's Coefficient of Variation (CV) is less than 5%, ensuring excellent repeatability and reliability. Furthermore, clinical performance evaluations, using Xpert/MTB RIF as the gold standard, demonstrated that the duplex RAA-LFD assay achieves a sensitivity of 92.86% and a specificity of 93.75%. It is also noteworthy that the assay exhibits considerable diagnostic efficacy in smear-negative patients. Conclusions: Our study introduces a rapid, specific, and sensitive duplex RAA-LFD assay for the discriminatory diagnosis of MTB and MAC. This method represents a significant advancement in the field of infectious disease diagnostics, offering a valuable tool for rapid detection and management of MTB and MAC infections. The implementation of this approach in point-of-care settings could greatly enhance TB control and prevention efforts, especially in resource-limited environments.

PrimerBankID	Target Pathogen	Target Gene
RPB0265	Mycobacterium tuberculosis	IS6110 gene

Nanoplasmonic microarray–based solid-phase amplification for highly sensitive and multiplexed molecular diagnostics: application for detecting SARS-CoV-2

Author(s):

Ji Young Lee,Hyowon Jang,Sunjoo Kim,Taejoon Kang,Sung-Gyu Park,Min-Young Lee

Journal:

Mikrochim Acta

Year:

2024

Abstract:

A novel approach is introduced using nanoplasmonic microarray-based solid-phase recombinase polymerase amplification (RPA) that offers high sensitivity and multiplexing capabilities for gene detection. Nanoplasmonic microarrays were developed through one-step immobilization of streptavidin/biotin primers and fine-tuning the amplicon size to achieve high plasmon-enhanced fluorescence (PEF) on the nanoplasmonic substrate, thereby improving sensitivity. The specificity and sensitivity of solid-phase RPA on nanoplasmonic microarrays was evaluated in detecting E, N, and RdRP genes of SARS-CoV-2. High specificity was achieved by minimizing primer-dimer formation and employing a stringent washing process and high sensitivity obtained with a limit of detection of four copies per reaction within 30 min. In clinical testing with nasopharyngeal swab samples (n = 30), the nanoplasmonic microarrays demonstrated a 100% consistency with the PCR results for detecting SARS-CoV-2, including differentiation of Omicron mutations BA.1 and BA.2. This approach overcomes the sensitivity issue of solid-phase amplification, as well as offers rapidity, high multiplexing capabilities, and simplified equipment by using isothermal reaction, making it a valuable tool for on-site molecular diagnostics.

PrimerBankID	Target Pathogen	Target Gene
RPB0303	SARS-CoV-2	E gene
RPB0304	SARS-CoV-2	N gene
RPB0305	SARS-CoV-2	RdRP gene
RPB0306	SARS-CoV-2 (Omicron)	Omicron variant BA.1
RPB0307	SARS-CoV-2 (Omicron)	Omicron variant BA.2

Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR\Cas12a detection platform

Author(s):

Xujian Mao,Jian Xu,Jingyi Jiang,Qiong Li,Ping Yao,Jinyi Jiang,Li Gong,Yin Dong,Bowen Tu,Rong Wang,Hongbing Tang,Fang Yao,Fengming Wang

Journal:

communication Biology

Year:

2024

Abstract:

CRISPR/Cas12a is a highly promising detection tool. However, detecting single nucleotide variations (SNVs) remains challenging. Here, we elucidate Cas12a specificity through crRNA engineering and profiling of single- and double-base mismatch tolerance across three targets. Our findings indicate that Cas12a specificity depends on the number, type, location, and distance of mismatches within the R-loop. We also find that introducing a wobble base pair at position 14 of the R-loop does not affect the free energy change when the spacer length is truncated to 17 bp. Therefore, we develop a new universal specificity enhancement strategy via iterative crRNA design, involving truncated spacers and a wobble base pair at position 14 of the R-loop, which tremendously increases specificity without sacrificing sensitivity. Additionally, we construct a PAM-free one-pot detection platform for SARS-CoV-2 variants, which effectively distinguishes SNV targets across various GC contents. In summary, our work reveals new insights into the specificity mechanism of Cas12a and demonstrates significant potential for in vitro diagnostics.

PrimerBankID	Target Pathogen	Target Gene
RPB0222	SARS-CoV-2	CPSIT_0429
RPB0223	SARS-CoV-2	D614G
RPB0224	SARS-CoV-2	R346T
RPB0225	SARS-CoV-2	Q183E
RPB0226	SARS-CoV-2	T1050N

A compact, palm-sized isothermal fluorescent diagnostic intelligent IoT device for personal health monitoring and beyond via one-tube\one-step LAMP-CRISPR assay

Author(s):

Fengyi Dai,Tao Zhang,Feibiao Pang,Tianjiao Jiao,Kaizheng Wang,Zhanfang Zhang,Nuo Wang,Zhiwei Xie,Yanchong Zhang,Zihao Wang,Zhiguang Chen,Mingxia Yu,Hongping Wei,Jinzhao Song

Journal:

Biosensors and Bioelectronics

Year:

2025

Abstract:

The demand for accurate, user-friendly, and sensitive at-home nucleic acid testing solutions is rising due to occasional outbreaks of various infectious diseases and a growing desire for an improved quality of life. In response, we developed the WeD-mini, a compact, palm-sized isothermal fluorescent diagnostic IoT device that weighs just 61 g. The WeD-mini features a uniquely designed, highly sensitive optical sensing system, ultra-low power consumption, a minimalist industrial design, and an intelligent operating algorithm. It integrates real-time fluorescence detection and automatic result interpretation via a smartphone, with results seamlessly uploaded to the 'EzDx Cloud' for comprehensive health management and spatio-temporal disease mapping. The device supports various assays that operate at different temperatures and with varying fluorescence emission intensities, such as RPA (39 °C, low intensity), LAMP (65 °C, high intensity), and LAMP-PfAgo (65/95 °C, high intensity), while maintaining precise temperature control and exceptional fluorescence detection sensitivity. Additionally, we engineered a more thermostable AapCRISPR-Cas12b variant that operates effectively at 63 °C, enhancing compatibility with LAMP to create a robust One-Tube/One-Step LAMP-CRISPR assay. Adaptable for at-home testing of SARS-CoV-2 and influenza viruses, the WeD-mini achieved 100% sensitivity and specificity with the newly established One-Tube/One-Step LAMP-CRISPR assay. Furthermore, the WeD-mini shows potential applications in detecting meat adulteration, monitoring respiratory diseases in pets, and conducting wastewater surveillance, making it suitable for a wide range of personal and public health use cases.

PrimerBankID	Target Pathogen	Target Gene
RPB0331	SARS-CoV-2	ORF1ab gene
RPB0332	Influenza A virus (H1N1)	H1N1

Dual recombinase polymerase amplification system combined with lateral flow immunoassay for simultaneous detection of Staphylococcus aureus and Vibrio parahaemolyticus

Author(s):

Yan Zhang,Xiaofeng Liu,Jiawei Luo,Hua Liu,You Li,Juan Liu,Lemei Zhu,Jinbin Wang,Haijuan Zeng

Journal:

Journal of Pharmaceutical and Biomedical Analysis

Year:

2025

Abstract:

Development of a highly sensitive visualization platform for multiplex genetic detection could significantly improve efficiency and reliability of on-site detection of foodborne pathogens. In this study, coupling recombinase polymerase amplification (RPA) with lateral flow immunoassay (LFIA) readout system was proposed for Staphylococcus aureus and Vibrio parahaemolyticus detection. Taking the advantage of the isothermal amplification of RPA, dual primers modified with different labeling groups were designed to realize target signal amplification. LFIA coated with anti-digoxigenin antibody and streptavidin as test line 1 and 2 were designed to detect the two RPA products. The proposed method (dual RPA-LFIA) could realize visual detection using LFIA through rapid RPA amplification within 20 min, exhibiting a lowest detection limit of 4.6 × 102 CFU/mL for Staphylococcus aureus and Vibrio parahaemolyticus. The dual RPA-LFIA is characterized by simultaneous detection of dual targets in one RPA reaction and colorimetric readout through LFIA, thus ensuring high sensitivity and efficiency, and showing great potential to address the on-site detection of foodborne pathogens in the future.

PrimerBankID	Target Pathogen	Target Gene
RPB0246	Staphylococcus aureus	nuc gene

A portable, rapid isothermal amplification kit enabling naked eye detection of SARS-CoV-2 RNAs

Author(s):

Ji-Ho Park,Kihyeun Kim,Bobin Lee,Hyungjun Jang,Min-Gon Kim

Journal:

Talanta

Year:

2025

Abstract:

Since the coronavirus disease 2019 (COVID-19) pandemic, isothermal amplification techniques have attracted attention due to their higher sensitivity and specificity, compared with immunoassays, and their potential application for point-of-care testing (POCT). A requirement of isothermal amplification-based POCT kits is the inclusion of a heating source with an electrical power supply. We developed an amplification-based rapid kit, which is a portable and naked eye-detectable reverse transcriptase (RT)-recombinase polymerase amplification (RPA) kit. The rapid RT-RPA kit consists of a flow-controllable paper chip, a nickel-chromium (NiCr)-based Joule-heating thin film, and a small-sized portable battery. We found that the Joule-heating thin film, powered by a lithium-ion battery (7.5 g, 20 mm × 35 mm size), was able to maintain the required temperature for the RPA reaction. After the RPA reaction, which takes approximately 20 min, the flow-controllable paper chip automatically enabled visualization of the amplicon by time-delayed release of gold nanoparticle-based optical probes. Using this system, we successfully detected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at levels as low as 10 copies μL-1, within 30 min.

PrimerBankID	Target Pathogen	Target Gene
RPB0292	SARS-CoV-2	N gene

Miniaturized Devices for Isothermal Amplification and Photometric Quantification of Pseudomonas Aeruginosa

Author(s):

P Ramya Priya,Satish Kumar Dubey,Sanket Goel

Journal:

IEEE Open Journal of Engineering in Medicine and Biology

Year:

2024

Abstract:

Goal: This study introduced a proof-of-concept prototype for isothermal recombinase polymerase amplification (RPA) with miniaturized photometric detection, enabling rapid P. aeruginosa detection. Methods: The researchers conducted the amplification process within a microchamber with a diameter of 10 mm, utilizing a standalone Thermostat driven thermal management setup. RPA, an amplification technique was employed, which required a lower operating temperature of 37 °C-40 °C to complete the reaction. The amplified amplicon was labeled with a fluorophore reporter, stimulated by an LED light source, and detected in real-time using a photodiode. Results: The developed prototype successfully demonstrated the rapid detection of P. aeruginosa using the RPA assay. The process only required the utilization of 0.04 ng of working concentration of DNA. The entire process, from amplification to detection, could be completed in over 15 minutes. The platform showed enhanced sensitivity and specificity, providing a cost-effective and accurate solution for on-site detection/quantification of pathogens. Conclusions: The integration of isothermal RPA with the miniaturized photometric detection platform proved successful in achieving the goal of rapid and specific pathogen detection. This study proved the benefits of Isothermal Nucleic Acid Amplification Technology (INAAT), emphasizing its potential as an accessible, user-friendly point-of-care technology for resource-constrained institutions. The RPA-based prototype demonstrated capability without requiring costly laboratory equipment or expertise. The developed platform, when combined with Internet of Things (IoT) enabled cloud platform, also allowed remote monitoring of data. Overall, the methodology presented in this study offered a cost-effective, accurate, and convenient solution for on-site testing in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0279	Pseudomonas aeruginosa	oprL gene

CRISPR-Cas12a-based detection and differentiation of Mycobacterium spp

Author(s):

Peeraphan Compiro,Nantinee Chomta,Juthamas Nimnual,Samitanan Sunantawanit,Sunchai Payungporn,Suwatchareeporn Rotcheewaphan,Pornchai Keawsapsak

Journal:

Clinica Chimica Acta

Year:

2025

Abstract:

Mycobacterium species cause several vital human diseases, including tuberculosis and non-tuberculous mycobacterial infections, which are treated with different drug regimens Therefore, accurate and rapid diagnosis is essential for effective treatment and controlling the spread of these pathogens. This study aims to develop an isothermal method combining RPA and CRISPR-Cas12a techniques, named as MyTRACK, to detect and differentiate major clinical mycobacteria at the species level. The assay has no cross-reactivity with limit of detection of 1 to 100 copies/reaction for various targeted mycobacteria. The results demonstrated 100 % specificity and 92.59 % to 100 % sensitivity in clinical isolates and were consistent with the culture technique with LPA for clinical samples. The MyTRACK assay is an effective, portable, rapid, and accurate screening method for mycobacterial detection and identification, especially in low-resource clinical settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0263	Mycobacterium	rpoB gene

A fully integrated microfluidic cartridge for rapid and ultrasensitive nucleic acid detection from oropharyngeal swabs

Author(s):

Bao Li,Baobao Lin,Wu Zeng,Yin Gu,Yulan Zhao,Peng Liu

Journal:

Lab on a Chip

Year:

2025

Abstract:

Rapid and accurate molecular diagnostics are crucial for preventing the global spread of emerging infectious diseases. However, the current gold standard for nucleic acid detection, reverse transcription polymerase chain reaction (RT-PCR), relies heavily on traditional magnetic beads or silica membranes for nucleic acid extraction, resulting in several limitations, including time-consuming processes, the need for trained personnel, and complex equipment. Therefore, there is an urgent need for fully integrated nucleic acid detection technologies that are simple to operate, rapid, and highly sensitive to meet unmet clinical needs. In this study, we developed a novel, integrated microfluidic cartridge featuring a unique needle-plug/piston microvalve, which enables stable long-term reagent storage and flexible liquid handling for on-site nucleic acid analysis. Coupled with in situ tetra-primer recombinase polymerase amplification (tp-RPA), we achieved highly sensitive nucleic acid detection with a remarkable limit of detection of 20 copies per mL (0.02 copies per μL) and a short turnaround time of less than 30 minutes. To validate this assay, we tested 48 oropharyngeal swab samples. The positive detection rate reached 64.58% (31/48), significantly exceeding the approximately 50% positive detection rate of the traditional RT-PCR method. Furthermore, our assay demonstrated a 100% concordance rate with RT-PCR in detecting positive samples. Thus, we believe our microfluidic nucleic acid analysis system represents a promising approach for enabling rapid and ultrasensitive nucleic acid detection of pathogenic microorganisms in resource-limited settings and low-income areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0323	SARS-CoV-2	N gene
RPB0324	SARS-CoV-2	ORF1ab gene

Short-Time Preamplification-Assisted One-Pot CRISPR Nucleic Acid Detection Method with Portable Self-Heating Equipment for Point-of-Care Diagnosis

Author(s):

Fei Hu,Kaihui Liu,Yunyun Zhang,Shuhao Zhao,Tianyi Zhang,Cuiping Yao,Xing Lv,Jing Wang,Xiaolong Liu,Niancai Peng

Journal:

Analytical Chemistry

Year:

2025

Abstract:

Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve "sample-in-answer-out" detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0325	SARS-CoV-2	ORF1ab gene
RPB0326	SARS-CoV-2	N gene

Visual detection of Mycobacterium tuberculosis in exhaled breath using N95 enrichment respirator, RPA, and lateral flow assay

Author(s):

Jie Cheng,Yiwei An,Qiyou Wang,Zuanguang Chen,Yanli Tong

Journal:

Talanta

Year:

2025

Abstract:

Tuberculosis (TB) is the second deadliest infectious disease worldwide. Current TB diagnostics utilize sputum samples, which are difficult to obtain, and sample processing is time-consuming and difficult. This study developed an integrated diagnostic platform for the rapid visual detection of Mycobacterium tuberculosis (Mtb) in breath samples at the point-of-care (POC), especially in resource-limited settings. The less pathogenic Mycobacterium smegmatis containing same gene fragment of Mtb served as the model bacterium. A novel respirator was designed to collect airborne mycobacteria in breath samples, with an efficiency of 38.7-61.5 % (102-109 CFU/mL). In our vision, patients only needed to wear a respirator for 1 h, and the collected pathogens were loaded into a microfluidic chip with direct-current electric field for lysis and nucleic acid extraction (20 μL, 3 s), then recombinase polymerase amplification (36 °C, 8 min) and lateral flow strip assay (5 min) were proceeded to enable visual test for the POC. Our platform completed the entire sample collection and diagnosis within 90 min, and the bacterial DNA amplification can be completed in 8 min by handheld, showing great patient compliance and eliminating the need for large equipment. Diagnostic systems involving signal detection with the naked eye are more suitable for the large-scale screening of TB. The proposed method detected low concentrations of bacterial DNA (5.0 aM, 18 copies/μL) with high reproducibility and specificity. Moreover, the system accurately detected low bacterial concentrations (102 CFU/mL). This platform provides the potential to improve the screening of TB and other airborne infectious diseases.

PrimerBankID	Target Pathogen	Target Gene
RPB0288	Mycobacterium tuberculosis H37Rv	16S rRNA M. smegmatis gene

Ultrasensitive "On-Off" Ratiometric Fluorescence Biosensor Based on RPA-CRISPR\Cas12a for Detection of Staphylococcus aureus

Author(s):

Wang Guo,Yiqing Guo,Hong Xu,Chen Li,Xinai Zhang,Xiaobo Zou,Zongbao Sun

Journal:

Agricultural and Food Chemistry

Year:

2025

Abstract:

Staphylococcus aureus (S. aureus) is a major pathogenic bacterium responsible for bacterial foodborne diseases, making its rapid, specific, and accurate detection crucial. In this study, we develop a ratiometric biosensor based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats/CRISPR associated protein 12a (RPA-CRISPR/Cas12a) system and Eu-metal-organic framework (Eu-MOF) fluorescent nanomaterials for the high-sensitivity detection of S. aureus, combining with RPA for efficient isothermal amplification, this sensor enhances specificity and sensitivity by utilizing the target activation of CRISPR/Cas12a. The Eu-MOF serves a dual function, providing stable red fluorescence as a reference signal and adsorbing FAM-labeled probes for fluorescence quenching, forming a dual-signal system that significantly reduces background interference. This ratiometric design enables accurate and quantitative detection over a wide range (7.9 × 100 to 7.9 × 108 CFU/mL) with a low detection limit of 3 CFU/mL. Overall, with these merits of simplicity, rapid response, high sensitivity, and specificity, this dual-signal biosensor offers a promising method for accurately evaluating S. aureus contamination in food under complex substrate conditions.

PrimerBankID	Target Pathogen	Target Gene
RPB0316	Staphylococcus aureus	nuc gene

A miniaturized RPA-CRISPR\Cas12a-based nucleic acid diagnostic platform for rapid and simple self-testing of SARS-CoV-2

Author(s):

Tianyi Zhang,Xiaoqin Wang,Yingtao Jiang,Yunyun Zhang,Shuhao Zhao,Jian Hu,Fei Hu,Niancai Peng

Journal:

Analytica Chimica Acta

Year:

2024

Abstract:

Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) RESULTS: In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 min with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) SIGNIFICANCE: This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52).

PrimerBankID	Target Pathogen	Target Gene
RPB0338	SARS-CoV-2	N gene

H9N2 avian influenza virus diagnostics utilizing specific high-sensitivity enzymatic molecular system termed RPA-based CRISPR-Cas13a

Author(s):

Dalin He,Saisai Zhao,Fangfang Wang,Bingrong Wu,Feng Wei,Yubo Zhao,Xinhui Wei,Hui Ren,Meijuan Zhang,Yaru Fan,Jiahao Zhang,Shumin Yu,Yi Tang,Youxiang Diao

Journal:

International Journal of Biological Macromolecules

Year:

2025

Abstract:

H9N2 avian influenza virus (AIV), a major pathogen causing respiratory infections in poultry, poses a significant threat to the poultry industry and human health. Early detection and control of H9N2 infections are essential for minimizing economic losses and preventing potential zoonotic transmission. A novel CRISPR-Cas family member called CRISPR-Cas13a comprises the CRISPR RNA (crRNA) and Cas13a nuclease. Through the crRNA-based reprogramming of Cas13a, a platform for sensing RNAs specifically is available. In this study, we developed a RPA-based CRISPR-Cas13a diagnostic method for rapid detection of the H9N2 AIV. The results demonstrated that at a limit of 10 copies/μL and 102 copies/μL could be detected within 50 min, by fluorescence detection and lateral flow strip, respectively, offering a highly sensitive method for H9N2 detection. This method exhibited excellent specificity, distinguishing H9N2 from other pathogens. Furthermore, the RPA-Cas13a-based detection system was tested on clinical samples, showing comparable performance to RT-qPCR. The detection results were visualized using either lateral flow assays or fluorescence, making it a suitable tool for on-site, field-deployable diagnostics. In a word, this RPA-Cas13a diagnostic approach offers high reliability, sensitivity, and specificity, with promising potential for rapidly detecting H9N2 and other viral pathogens in clinical and food safety applications.

PrimerBankID	Target Pathogen	Target Gene
RPB0322	Influenza A virus (H9N2)	M gene

Rapid and specific on-site H5Nx avian influenza diagnosis via RPA and PAM-independent CRISPR-Cas12a assay combined with anti-NP antibody-based viral RNA purification

Author(s):

Jin-Ha Song,Seung-Eun Son,Ho-Won Kim,Seung-Ji Kim,Se-Hee An,Chung-Young Lee,Hyuk-Joon Kwon,Kang-Seuk Choi

Journal:

Frontiers in Veterinary Science

Year:

2025

Abstract:

Rapid and accurate detection of H5Nx avian influenza viruses is critical for effective surveillance and control measures. Currently, RT-qPCR with spin column RNA extraction is the gold standard for HPAIV surveillance, but its long reaction time and need for specialized equipment limit its effectiveness for rapid response. In this study, we introduce a centrifuge-free, rapid detection method for on-site detection of H5Nx viruses that combines magnetic bead-based ribonucleoprotein (RNP) purification and concentration with a CRISPR-Cas12a system that is independent of the protospacer adjacent motif (PAM) sequence. Our approach employs anti-NP monoclonal antibodies for the targeted isolation of RNA bound to RNPs, facilitating a quick and specific RNA extraction process that negates the need for centrifugation. Additionally, by denaturing the RT-RPA amplicon using 60% DMSO, we activate the trans-ssDNA cleavage activity of the Cas12a protein without the need for a specific PAM (5'-TTTV-3') sequence. This strategy increases flexibility in CRISPR RNA design, providing a significant advantage when targeting genes with high variability. We validated the efficacy of our magnetic RNP purification and concentration method in combined with an RT-RPA/PAM-independent Cas12a assay for detecting the H5 gene. The assay achieved a sensitivity threshold of 101 EID50 with fluorescent detection and 102 EID50 using lateral flow strips. It also exhibited high specificity, yielding positive results solely for H5Nx viruses among various influenza A virus subtypes. Furthermore, in clinical samples, the assay demonstrated 80% sensitivity and 100% specificity. These results highlight the advantages of using NP-specific antibodies for RNP purification and CRISPR-Cas12a with viral gene-specific crRNA to achieve exceptional diagnostic specificity.

PrimerBankID	Target Pathogen	Target Gene
RPB0291	Influenza A virus (H5)	\

Rapid Bacterial Identification through Multiplexed Nucleic Acid Detection on a Digital Microfluidic Platform for Enhanced Clinical Intervention against Infections

Author(s):

Ruibin Xie,Jienan Shen,Lintao Zhou,Lianyu Lu,Aiping Zhi,Duo Sun,Yue Pei,Jian Yu,Lin Zeng,Guoqiang Gu,Yuye Wang,Hao Yu,Yunsheng Chen,Xiaopeng Ma,Zhongjian Xie,Hui Yang

Journal:

ACS Sensors

Year:

2025

Abstract:

Bacterial infections often lead to severe health consequences owing to their ability to infiltrate multiple anatomical sites, including the bloodstream, respiratory tract, and digestive tract, posing substantial diagnostic and therapeutic challenges. Consequently, a rapid and versatile detection method capable of identifying a broad spectrum of bacterial pathogens is urgently required to facilitate precise antibiotic prescriptions. Addressing this need, we introduce MiND-DMF (Multibacterial Infection Nucleic Acid Detection on a Digital Microfluidic Platform), a cost-effective digital microfluidic platform tailored for multiplexed bacterial detection. This system integrates DNA extraction, recombinase polymerase amplification (RPA), and CRISPR-based detection technologies, enabling the efficient identification of six common infectious bacteria. Operating at a constant temperature of 37 °C, MiND-DMF completes the entire diagnostic process in just 55 min and is compatible with human reference genes. In spiked samples, the platform demonstrated a detection limit of 100 CFU/mL, highlighting its exceptional sensitivity and quantification capability. In clinical evaluations, MiND-DMF exhibited outstanding performance, achieving 100% sensitivity and 98%-100% specificity compared to conventional PCR methods across 50 samples derived from diverse tissue sources. This robust platform demonstrates strong anti-interference capabilities, making it suitable for analyzing various tissue fluids including blood, alveolar lavage fluid, urine, nasal secretions, appendiceal pus, and ear pus. The versatility and precision of MiND-DMF support the monitoring of hospital-acquired bacterial infection origins, empowering physicians to prescribe targeted antibiotics and enhancing overall infection prevention and control strategies. By accurately detecting bacteria from multiple sources, MiND-DMF can play a pivotal role in improving patient outcomes and public health.

PrimerBankID	Target Pathogen	Target Gene
RPB0333	Staphylococcus aureus	nuc gene
RPB0334	Staphylococcus aureus	mecA gene
RPB0335	Pseudomonas aeruginosa	Ecf X
RPB0336	Klebsiella pneumoniae	groEL
RPB0337	Haemophilus influenzae	16srRNA

Advanced One-Pot RPA-CRISPR\Cas12a Reaction with Glycerol and Betaine for High-Sensitivity Diagnosis of mecA-Carrying Strains in Clinical Samples

Author(s):

Jingyuan Wang,Dan Wang,Linlin Fan,Xin Ye,Jian Hu,Xiaoqin Wang

Journal:

ACS Omega

Year:

2025

Abstract:

The mecA gene confers methicillin resistance in both MRSA and MR-CoNS by encoding the PBP2a protein and poses a significant public health threat due to its resistance to beta-lactam antibiotics. Rapid and accurate detection of mecA is critical for timely treatment, reducing morbidity, and preventing its spread in healthcare settings. In this study, we developed an advanced one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a system, enhanced with glycerol and betaine, for ultrasensitive detection of the mecA gene. Glycerol's viscosity effect prevents premature interaction between Cas12a and early amplification products, while betaine enhances nucleic acid amplification. The assay demonstrated superior sensitivity, detecting as low as 5 copies/μL of mecA DNA within 60 min. Specificity testing against a panel of bacterial species confirmed the high selectivity of the assay for mecA-carrying strains with negligible cross-reactivity. Furthermore, this method exhibited excellent performance across various clinical samples, including blood, urine, and bronchoalveolar lavage fluid. Our findings underscore the potential of this advanced RPA-CRISPR/Cas12a assay as a powerful diagnostic tool for rapid, cost-effective, and highly sensitive mecA detection, offering a promising solution for clinical diagnostics and infection control.

PrimerBankID	Target Pathogen	Target Gene
RPB0308	Staphylococcus aureus	mecA gene

Establishment and Application of Duplex Recombinase-Aided Amplification Combined with Lateral Flow Dipsticks for Rapid and Simultaneous Visual Detection of Klebsiella pneumoniae and Staphylococcus aureus in Milk

Author(s):

Ni Zhang,Laiwang Hou,Darong Li,Weiqing Lan,Yong Zhao,Xiaohong Sun

Journal:

Foods

Year:

2025

Abstract:

Staphylococcus aureus and Klebsiella pneumoniae are significant and prevalent pathogens associated with bovine mastitis on dairy farms worldwide, resulting in severe infections in both dairy cows and, subsequently, human beings. Fast and dependable pathogen diagnostics are essential to minimize the effects of cow mastitis and human infections. The aim of this research was to develop a duplex recombinase-aided amplification (RAA) combined with the lateral flow dipstick (LFD) method, which was used for rapid, simultaneous detection of S. aureus and K. pneumoniae. The SKII culture medium for S. aureus and K. pneumoniae cocultivation was developed in this study. By optimizing the duplex RAA-LFD reaction conditions in terms of primer concentration, amplification temperature, and reaction time, the duplex RAA-LFD assay could successfully detect S. aureus and K. pneumoniae when the reaction was conducted at 39 °C for 20 min. The duplex RAA-LFD method demonstrated good specificity, exhibiting no cross-reactivity with other pathogens. In addition, the detection limit of the duplex RAA-LFD for S. aureus and K. pneumoniae was 60 fg of genomic DNA and 1.78 × 103 and 2.46 × 103 CFU/mL of bacteria in pure culture. Moreover, the duplex RAA-LFD technique is capable of identifying S. aureus and K. pneumoniae in artificially spiked milk samples even at very low initial concentrations of 1.78 × 101 and 2.46 × 100 CFU/mL, respectively, after 6 h of enrichment. The result of the actual samples showed that the total concordance rate of the duplex RAA-LFD method with the biochemical identification method and PCR method could reach 92.98~98.25% with high consistency. The results of this study indicated that the duplex RAA-LFD assay, which is a precise, sensitive, and simple field testing technique, can be used to identify S. aureus and K. pneumoniae and is expected to be used for disease diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0285	Staphylococcus aureus	nuc gene
RPB0286	Klebsiella pneumoniae	rcsA gene

Space-coded microchip for multiplexed respiratory virus detection via CRISPR-Cas12a and RPA

Author(s):

Mengqiu Xiong,Yao Wang,Shuhan Lu,Nasifu Lubanga,Tao Li,Zhihao Li,Bangshun He,Ying Li

Journal:

Talanta

Year:

2025

Abstract:

Multiple infections of respiratory viruses are common in patients with clinical respiratory diseases, but current detection methods still have problems such as complex equipment and long detection time. Rapid, low-cost, and on-site detection of human respiratory viruses is crucial for both clinical diagnosis and population screening. In this research, we created a space-coded microfluidic chip (SC-Chip) for the recognition of nine respiratory viruses: influenza A virus, influenza B virus, severe acute respiratory syndrome coronavirus 2, human coronavirus OC43, human coronavirus NL63, human coronavirus HKU1, human respiratory syncytial virus, human parainfluenza virus, and human metapneumovirus. For the first time, a comprehensive sequence comparison among these viruses was performed to design the recombinase polymerase amplification (RPA) primers and Cas12a-crRNAs. The SC-Chip partitions samples amplified by RPA into spatially coded wells preloaded with CRISPR-Cas12a detection reagents, enabling the identification of all nine viral targets in a single test using a single fluorescence probe. The chip-based assay displays 9 respiratory viruses in less than 40 min with a minimum detection limit at a concentration of 10-18 M (∼1 copy/reaction). Additionally, the efficacy of the method was assessed through its application to 35 clinical patient samples identified as being at risk for respiratory virus infection, yielding a sensitivity of 90 % and a specificity of 100 %. In summary, this space-coded microfluidic CRISPR system offers several advantages, including ease of operation, cost-effectiveness, and rapid data acquisition, thereby holding great potential for multiplexed detection of nucleic acid targets in a clinical setting.

PrimerBankID	Target Pathogen	Target Gene
RPB0254	Influenza A virus	PB1
RPB0255	Influenza B virus	PB1
RPB0256	SARS-CoV-2	ORF1ab
RPB0257	HCoV-OC43	ORF1ab
RPB0258	HCoV-NL63	ORF1ab
RPB0259	HCoV-HKU1	ORF1ab
RPB0260	HRSV A	M
RPB0261	HPIV-3	M
RPB0262	HMPV	F

A Streamlined Point-of-Care CRISPR Test for Tuberculosis Detection Directly from Sputum

Author(s):

Owen R S Dunkley,Alexandra G Bell,Nisha H Modi,Yujia Huang,Soleil Tseng,Robert Reiss,Naranjargal Daivaa,J Lucian Davis,Deninson Alejandro Vargas,Padmapriya Banada,Yingda L Xie,Cameron Myhrvold

Journal:

medRxiv.

Year:

2025

Abstract:

Mycobacterium tuberculosis (Mtb) is a major threat to global health and is responsible for over one million deaths each year. To stem the tide of cases and maximize opportunities for early interventions, there is an urgent need for affordable and simple means of tuberculosis diagnosis in under-resourced areas. We sought to develop a CRISPR-based isothermal assay coupled with a compatible, straightforward sample processing technique for point-of-care use. Here, we combine Recombinase Polymerase Amplification (RPA) with Cas13a and Cas12a, to create two parallelised one-pot assays that detect two conserved elements of Mtb (IS6110 and IS1081) and an internal control targeting human DNA. These assays were shown to be compatible with lateral flow and can be readily lyophilized. Our finalized assay exhibited sensitivity over a wide range of bacterial loads (105 to 102 CFU/mL) in sputum. The limit of detection (LoD) of the assay was determined to be 69.0 (51.0 - 86.9) CFU/mL for Mtb strain H37Rv spiked in sputum and 80.5 (59.4 - 101.6) CFU/mL for M. bovis BCG. Our assay showed no cross reactivity against a wide range of bacterial/fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (6/6) sensitivity and 100% (7/7) specificity compared to culture. Our assay exhibited comparable sensitivity in clinical samples to the microbiological gold standard, TB culture, and to the nucleic acid state-of-the-art, GeneXpert MTB/RIF Ultra. This technology streamlines TB diagnosis from sample extraction to assay readout in a rapid and robust format, making it the first test to combine amplification and detection while being compatible with both lateral flow and lyophilization.

PrimerBankID	Target Pathogen	Target Gene
RPB0281	Mycobacterium tuberculosis	IS6110
RPB0282	Mycobacterium tuberculosis	IS1081

Establishment of an RPA-CRISPR\Cas12a combined diagnostic system for Pneumocystis jirovecii pneumonia

Author(s):

Yun Wu,Yuhan Shao,Wei Li,Ying Yu,Xia Rao,Jingyi Li,Nicholas R Waterfield,Guowei Yang

Journal:

PLOS Neglected Tropical Diseases

Year:

2025

Abstract:

Pneumocystis jirovecii causes severe pneumonia in immunocompromised individuals, leading to high mortality and an economic burden. There is a need for early detection methods suitable for low-resource settings and rapid point-of-care diagnostics. This study developed a detection method using Recombinase Polymerase Amplification (RPA) followed by CRISPR/Cas12a with fluorescence detection. The RPA primers and CRISPR-derived RNAs (crRNAs) were specifically designed to target the mitochondrial small subunit rRNA (mtSSU rRNA) gene of P. jirovecii. A total of 83 clinical samples were tested using this method, including 39 confirmed and 44 suspected cases of P. jirovecii infection. The combination of crRNA5 and crRNA6 demonstrated higher sensitivity compared to the current real-time PCR detection method, with a limit of detection (LOD) of 1 copy per reaction and showed no cross-reactions with other respiratory pathogens. The concordance of this method was validated with both infected and non-infected patients. In conclusion, the method developed in this study potentially provides a highly sensitive and rapid tool suitable for the early and on-site detection of P. jirovecii pneumonia. Furthermore, this method holds potential applications for the detection of other human pathogens, representing a significant advancement in diagnostic capabilities for low-resource settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0280	Pneumocystis jirovecii	mtSSU rRNA gene