A compact microfluidic platform for rapid multiplex detection of respiratory viruses via centrifugal polar-absorbance spectroscopy

Author(s):

Ya Su,Xiangyu Jin,Fan Yang,Xuekai Liu,Fenggang Li,Qingchen Zhao,Jialu Hou,Shuailong Zhang,Hang Li,Guoliang Huang,Rongxin Fu

Journal:

Talanta

Year:

2024

Abstract:

Nucleic acid detection technology has become a crucial tool in cutting-edge research within the life sciences and clinical diagnosis domains. Its significance is particularly highlighted during the respiratory virus pandemic, where nucleic acid testing plays a pivotal role in accurately detecting the virus. Isothermal amplification technologies have been developed and offer advantages such as rapidity, mild reaction conditions and excellent stability. Among these methods, recombinase polymerase amplification (RPA) has gained significant attention due to its simple primer design and resistance to multiple reaction inhibitors. However, the detection of RPA amplicons hinders the widespread adoption of this technology, leading to a research focus on cost-effective and convenient detection methods for RPA nucleic acid testing. In this study, we propose a novel computational absorption spectrum approach that utilizes the polar GelRed dye to efficiently detect RPA amplicons. By exploiting the asymmetry of GelRed molecules upon binding with DNA, polar electric dipoles are formed, leading to precipitate formation through centrifugal vibration and electrostatic interaction. The quantification of amplicon content is achieved by measuring the residual GelRed concentration in the supernatant. Our proposed portable and integrated microfluidic device successfully detected five respiratory virus genes simultaneously. The optimized linear detection was achieved and the sensitivity for all the targets reached 100 copies/μL. The total experiment could be finished in 27 min. The clinical experiments demonstrated the practicality and accuracy. This cost-effective and convenient detection scheme presents a promising biosensor for rapid virus detection, contributing to the advancement of RPA technology.

PrimerBankID	Target Pathogen	Target Gene
RPB0309	SARS-CoV-2	orflab gene
RPB0310	SARS-CoV-2	N gene
RPB0311	RSV A	F gene
RPB0312	Influenza A virus	M1 gene
RPB0313	Influenza B virus	NS1 gene

Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii

Author(s):

Qiming Liu,Hao Zeng,Ting Wang,HongXia Ni,Yongdong Li,Weidong Qian,Ting Fang,Guozhang Xu

Journal:

BMC Microbiology

Year:

2024

Abstract:

Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within 30-40 min, comprising a 25-30 min RPA reaction and a 5-10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33%. In conclusion, a novel, rapid, sensitive, and cost-effective RPA-Cas12a-based detection method was developed and demonstrates significant potential for on-site detection of P. jirovecii in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0248	Pneumocystis jirovecii	5.8 S ribosomal RNA

Rapid and Highly Sensitive Detection of Mycobacterium tuberculosis Utilizing the Recombinase Aided Amplification-Based CRISPR-Cas13a System

Author(s):

Qiao Li,Nenhan Wang,Mengdi Pang,Honghao Miao,Xiaowei Dai,Bo Li,Xinyu Yang,Chuanyou Li,Yi Liu

Journal:

Microorganisms

Year:

2024

Abstract:

Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (MTB) infection, remains a major threat to global public health. To facilitate early TB diagnosis, an IS6110 gene-based recombinase aided amplification (RAA) assay was coupled to a clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas13a fluorescence assay to create a rapid MTB detection assay (named RAA-CRISPR-MTB). Its diagnostic efficacy was evaluated for sensitivity and specificity through sequential testing of recombinant plasmids, mycobacterium strains, and clinical specimens. RAA-CRISPR detected IS6110 genes at levels approaching 1 copy/μL with pUC57-6110 as the template and 10 copies/μL with H37Rv as the template. There was no observed cross detection of non-tuberculosis mycobacteria (NTM) with either template. Furthermore, RAA-CRISPR testing of 151 clinical specimens yielded a diagnostic specificity rate of 100% and a diagnostic sensitivity rate of 69% that exceeded the corresponding Xpert MTB/RIF assay rate (60%). In conclusion, we established a novel RAA-CRISPR assay that achieved highly sensitive and specific MTB detection for use as a clinical TB diagnostic tool in resource-poor settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0237	Mycobacterium tuberculosis	IS6110 genes

Engineering the bacteriophage 80 alpha endolysin as a fast and ultrasensitive detection toolbox against Staphylococcus aureus

Author(s):

Feng Zhao,Yixi Yang,Wenyao Zhan,Zhiqi Li,Hui Yin,Jingjing Deng,Waner Li,Rui Li,Qi Zhao,Jian Li

Journal:

Biosensors and Bioelectronics

Year:

2024

Abstract:

The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.

PrimerBankID	Target Pathogen	Target Gene
RPB0314	Staphylococcus aureus	nuc gene

Rapid and sensitive detection of methicillin-resistant Staphylococcus aureus through the RPA- Pf Ago system

Author(s):

Weizhong Chen,Jiexiu Zhang,Huagui Wei,Jie Su,Jie Lin,Xueyan Liang,Jiangtao Chen,Rong Zhou,Lin Li,Zefang Lu,Guangyu Sun

Journal:

Front Microbiol

Year:

2024

Abstract:

Introduction: Both the incidence and mortality rates associated with methicillin-resistant Staphylococcus aureus (MRSA) have progressively increased worldwide. A nucleic acid testing system was developed in response, enabling swift and precise detection of Staphylococcus aureus (S. aureus) and its MRSA infection status. This facilitates improved prevention and control of MRSA infections. Methods: In this work, we introduce a novel assay platform developed by integrating Pyrococcus furiosus Argonaute (PfAgo) with recombinase polymerase amplification (RPA), which was designed for the simultaneous detection of the nuc and mecA genes in MRSA. Results: This innovative approach enables visual MRSA detection within 55 mins, boasting a detection limit of 102 copies/μL. Characterized by its high specificity, the platform accurately identifies MRSA infections without cross-reactivity to other clinical pathogens, highlighting its unique capability for S. aureus infection diagnostics amidst bacterial diversity. Validation of this method was performed on 40 clinical isolates, demonstrating a 95.0% accuracy rate in comparison to the established Vitek2-COMPACT system. Discussion: The RPA-PfAgo platform has emerged as a superior diagnostic tool, offering enhanced sensitivity, specificity, and identification efficacy for MRSA detection. Our findings underscore the potential of this platform to significantly improve the diagnosis and management of MRSA infection.

PrimerBankID	Target Pathogen	Target Gene
RPB0241	Staphylococcus aureus	nuc gene
RPB0242	Staphylococcus aureus	mecA gene

An extraction-free one-pot assay for rapid detection of Klebsiella pneumoniae by combining RPA and CRISPR\Cas12a

Author(s):

Jinyu Fu,Rurong Mo,Ziyao Li,Shijie Xu,Xiyu Cheng,Binghuai Lu,Shuobo Shi

Journal:

Biosensors and Bioelectronics

Year:

2025

Abstract:

Klebsiella pneumoniae poses a significant threat to global public health. Traditional clinical diagnostic methods, such as bacterial culture and microscopic identification, are not suitable for point-of-care testing. In response, based on the suboptimal protospacer adjacent motifs, this study develops an extraction-free one-pot assay, named EXORCA (EXtraction-free One-pot RPA-CRISPR/Cas12a assay), designed for the immediate, sensitive and efficient detection of K. pneumoniae. The EXORCA assay can be completed within approximately 30 min at a constant temperature and allows for the visualization of results either through a fluorescence reader or directly by the naked eye under blue light. The feasibility of the assay was evaluated using twenty unextracted clinical samples, achieving a 100% (5/5) positive predictive value and a 100% (15/15) negative predictive value in comparison to qPCR. These results suggest that the EXORCA assay holds significant potential as a point-of-care testing tool for the rapid identification of pathogens, such as K. pneumonia.

PrimerBankID	Target Pathogen	Target Gene
RPB0251	Klebsiella pneumoniae	rcsA gene

A specific and ultrasensitive Cas12a\crRNA assay with recombinase polymerase amplification and lateral flow biosensor technology for the rapid detection of Streptococcus pyogenes

Author(s):

Yu Cheng,Jiawen Lyu,Jiangfeng Han,Long Feng,Xiangmei Li,Pei Li,Shanfeng Zhang,Wenqiao Zang

Journal:

Clinical Microbiology

Year:

2024

Abstract:

The potential of CRISPR/Cas systems for nucleic acid detection in novel biosensing applications is remarkable. The current clinical diagnostic detection of Streptococcus pyogenes (S. pyogenes) is based on serological identification, culture, and PCR. We report a rapid, simple, and sensitive method for detecting and screening for S. pyogenes. This novel method is a promising supplemental test. After 10 min of the sample processing and 10 min of recombinase polymerase amplification, followed by 10 min of Cas12 reaction and 3 min of lateral flow biosensor (LFB) readout, a visible outcome can be observed without the need for magnification within 33 min. This platform is robust, inexpensive, and appropriate for on-site testing. A new technique for detection was created using CRISPR-Cas12a technology, which includes two measurements: a fluorescent-CRISPR-S. pyogenes test and a LFB-CRISPR-S. pyogenes test. An approach utilizing CRISPR Cas12a was developed, and the accuracy and precision of this technique were assessed. The LoD for the fluorescence-CRISPR- S. pyogenes assay was 1 copy/μL, and the technique effectively differentiated S. pyogenes from other microorganisms. Moreover, the detection outcomes were presented in a user-friendly manner using lateral flow biosensor strips. Conclusion: A rapid and sensitive Cas12a/crRNA assay using recombinase RPA and LFB was developed to detect S. pyogenes. The Cas12a/crRNA-based assay exhibited high specificity among different bacteria strains and extremely high sensitivity. The accuracy and rapidity of this method make it a promising tool for S. pyogenes detection and screening. Importance: Patients may experience a range of symptoms due to Streptococcus pyogenes infections, including superficial skin infections, pharyngitis, and invasive diseases in subcutaneous tissues like streptococcal toxic shock syndrome. At present, the clinical diagnostic detection of S. pyogenes is based on serological identification, culture, and PCR. These detection methods are time-consuming and require sophisticated equipment, making these methods challenging for routine laboratories. Thus, there is a need for a detection platform that is capable of quickly and accurately identifying S. pyogenes. In this study, a rapid and sensitive Cas12a/crRNA assay using recombinase RPA and LFB was developed to detect S. pyogenes. The Cas12a/crRNA-based assay exhibited high specificity among different bacteria strains and extremely high sensitivity. This method probably plays an important role for S. pyogenes detection and screening.

PrimerBankID	Target Pathogen	Target Gene
RPB0244	Streptococcus pyogenes M1 GAS	sdaB gene
RPB0511	Streptococcus pyogenes	sdaB

A Rapid Detection Method for H3 Avian Influenza Viruses Based on RT-RAA

Author(s):

Jiaqi Li,Huan Cui,Yuxin Zhang,Xuejing Wang,Huage Liu,Yingli Mu,Hongwei Wang,Xiaolong Chen,Tongchao Dong,Cheng Zhang,Ligong Chen

Journal:

Animals

Year:

2024

Abstract:

The continued evolution of H3 subtype avian influenza virus (AIV)-which crosses the interspecific barrier to infect humans-and the potential risk of genetic recombination with other subtypes pose serious threats to the poultry industry and human health. Therefore, rapid and accurate detection of H3 virus is highly important for preventing its spread. In this study, a method based on real-time reverse transcription recombinase-aided isothermal amplification (RT-RAA) was successfully developed for the rapid detection of H3 AIV. Specific primers and probes were designed to target the hemagglutinin (HA) gene of H3 AIV, ensuring highly specific detection of H3 AIV without cross-reactivity with other important avian respiratory viruses. The results showed that the detection limit of the RT-RAA fluorescence reading method was 224 copies/response within the 95% confidence interval, while the detection limit of the RT-RAA visualization method was 1527 copies/response within the same confidence interval. In addition, 68 clinical samples were examined and the results were compared with those of real-time quantitative PCR (RT-qPCR). The results showed that the real-time fluorescence RT-RAA and RT-qPCR results were completely consistent, and the kappa value reached 1, indicating excellent correlation. For visual detection, the sensitivity was 91.43%, the specificity was 100%, and the kappa value was 0.91, which also indicated good correlation. In addition, the amplified products of RT-RAA can be visualized with a portable blue light instrument, which enables rapid detection of H3 AIV even in resource-constrained environments. The H3 AIV RT-RAA rapid detection method established in this study can meet the requirements of basic laboratories and provide a valuable reference for the early diagnosis of H3 AIV.

PrimerBankID	Target Pathogen	Target Gene
RPB0353	Influenza A virus (H3N2)	HA gene

Recombinase-aided amplification assay for rapid detection of imipenem-resistant Pseudomonas aeruginosa and rifampin-resistant Pseudomonas aeruginosa

Author(s):

Yao Zhou,Ruiqing Shi,Liang Mu,Linlin Tian,Mengshan Zhou,Wenhan Lyu,Yaodong Chen

Journal:

Frontiers In Cellular And Infection Microbiology

Year:

2024

Abstract:

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.

PrimerBankID	Target Pathogen	Target Gene
RPB0219	Pseudomonas aeruginosa	oprD gene
RPB0220	Pseudomonas aeruginosa	arr gene

Isothermal recombinase polymerase amplification and silver nanoparticle assay: a sustainable approach for ultrasensitive detection of Klebsiella pneumoniae

Author(s):

Naresh Patnaik,Nidhi Orekonday,Ruchi Jain Dey

Journal:

Analytical Methods

Year:

2024

Abstract:

Our study addresses the urgent need for effective detection of Klebsiella pneumoniae, a recognized threat by the World Health Organization (WHO). Current challenges in managing K. pneumoniae infections include the lack of rapid and affordable detection tools, particularly in resource-limited point-of-care (POC) settings. To tackle this, we developed an innovative molecular detection pipeline combining three POC-compatible methods. Firstly, we employed Insta DNA™ card-based sample collection and DNA extraction for simplicity and ease of use. Next, we utilized recombinase polymerase amplification (RPA) targeting the Klebsiella hemolysin gene, khe, specific to the K. pneumoniae species complex (KpSC). Finally, we integrated a silver nanoparticle (AgNP) aggregation assay for visual detection, offering a rapid, sensitive, and specific method capable of detecting as few as ∼3 bacteria of K. pneumoniae within ∼45 minutes. This approach eliminates the need for complex equipment, making it highly suitable for field and resource-limited POC applications. Moreover, our method introduces an environmentally significant detection strategy. The method developed minimizes chemical reagent usage and reduces the carbon footprint associated with sample transportation. Furthermore, our method reduces waste compared to the traditional detection techniques, offering a safer alternative to ethidium bromide or other DNA dyes which are often genotoxic and mutagenic in nature. Silver nanoparticles, being environmentally safer, can also be recycled from the waste, contributing to sustainability in nanoparticle production and disposal. Overall, our technique presents a promising solution for detecting K. pneumoniae in various settings, including environmental, water, and food samples, as well as industrial or hospital effluents. By aligning with global efforts to improve public health and environmental sustainability, our approach holds significant potential for enhancing disease management and reducing environmental impact.

PrimerBankID	Target Pathogen	Target Gene
RPB0227	Klebsiella pneumoniae	khe gene