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