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