Cas12a\Guide RNA-Based Platform for Rapidly and Accurately Detecting blaKPC Gene in Carbapenem-Resistant Enterobacterales

Author(s):

Keke Li,Yaozhou Wu,Meng Liu,Junwen Yan,Lianhua Wei

Journal:

Infection and Drug Resistance

Year:

2024

Abstract:

Purpose: Accurate detection and identification of pathogens and their associated resistance mechanisms are essential prerequisites for implementing precision medicine in the management of Carbapenem-resistant Enterobacterales (CRE). Among the various resistance mechanisms, the production of KPC carbapenemase is the most prevalent worldwide. Consequently, this study aims to develop a convenient and precise nucleic acid detection platform specifically for the blaKPC gene. Methods: The initial phase of our research methodology involved developing a CRISPR/Cas12a detection framework, which was achieved by designing highly specific single-guide RNAs (sgRNAs) targeting the blaKPC gene. To enhance the sensitivity of this system, we incorporated three distinct amplification techniques-polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA)-into the CRISPR/Cas12a framework. Subsequently, we conducted a comparative analysis of the sensitivity and specificity of these three amplification methods when used in combination with the CRISPR/Cas12a system. Additionally, we assessed the clinical applicability of the methodologies by evaluating fluorescence readouts from 80 different clinical isolates. Furthermore, we employed lateral flow assay technology to provide a visual representation of the results, facilitating point-of-care testing. Results: Following a comparative analysis of the sensitivity and specificity of the three methods, we identified the RPA-Cas12a approach as the optimal detection technique. Our findings demonstrated that the limit of detection (LoD) of the RPA-Cas12a platform was 1 aM (~1 copy/µL) for plasmid DNA and 5 × 10³ fg/µL for genomic DNA. Furthermore, both the sensitivity and specificity of the platform achieved 100% upon validation with 80 clinical isolates. Conclusion: These findings suggest that the developed RPA-Cas12a platform represents a promising tool for the cost-effective, convenient, and accurate detection of the blaKPC gene.

PrimerBankID	Target Pathogen	Target Gene
RPB0302	Klebsiella pneumoniae	blaKPC gene

Detection and identification of SARS-CoV-2 and influenza a based on microfluidic technology

Author(s):

Yujie Liu,Guanliu Yu,Hongkun Liang,Wenbo Sun,Lulu Zhang,Michael G Mauk,Hua Li,Lei Chen

Journal:

Analytical Methods

Year:

2024

Abstract:

As of now, the global COVID-19 pandemic caused by SARS-CoV-2, which began in 2019, has been effectively controlled. However, the symptoms of influenza A virus infection were similar to those of SARS-CoV-2 infection, but they required different treatment approaches. To make the detection more accurate and the treatment more targeted. We developed a system that integrates RPA and CRISPR assays, allowing for the rapid, highly specific, and sensitive detection and differentiation of SARS-CoV-2, H1N1, and H3N2. Under isothermal amplification conditions, the RPA-CRISPR Cas12a detection system achieved a detection limit as low as 5 copies per μL, demonstrating excellent specificity. The measurement time was approximately 30 minutes. The RPA-CRISPR Cas12a detection system combined with the microfluidic chip we designed to simultaneously detect three viruses, providing a potential solution for efficient and reliable diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0231	Influenza A virus (H1N1)	\
RPB0232	Influenza A virus (H3N2)	\
RPB0233	SARS-CoV-2	\

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA

Author(s):

Qian Wang,Shiwen Wu,Jiangbing Shuai,Ye Li,Xianshu Fu,Mingzhou Zhang,Xiaoping Yu,Zihong Ye,Biao Ma

Journal:

Molecules

Year:

2024

Abstract:

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/μL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

PrimerBankID	Target Pathogen	Target Gene
RPB0294	Influenza A virus (H5N1)	HA gene
RPB0295	Influenza A virus (H5N1)	M2 gene

A rapid and ultra-sensitive dual readout platform for Klebsiella pneumoniae detection based on RPA-CRISPR\Cas12a

Author(s):

Meiying Tan,Lina Liang,Chuan Liao,Zihan Zhou,Shaoping Long,Xueli Yi,Chunfang Wang,Caiheng Wei,Jinyuan Cai,Xuebin Li,Guijiang Wei

Journal:

Frontiers in Cellular and Infection Microbiology

Year:

2024

Abstract:

The bacterium Klebsiella pneumoniae (Kp) was the primary pathogen of hospital-acquired infection, but the current detection method could not rapidly and conveniently identify Kp. Recombinase polymerase amplification (RPA) was a fast and convenient isothermal amplification technology, and the clustered regularly interspaced short palindromic repeats (CRISPR) system could rapidly amplify the signal of RPA and improve its limit of detection (LOD). In this study, we designed three pairs of RPA primers for the rcsA gene of Kp, amplified the RPA signal through single-strand DNA reporter cleavage by CRISPR/Cas12a, and finally analyzed the cleavage signal using fluorescence detection (FD) and lateral flow test strips (LFTS). Our results indicated that the RPA-CRISPR/Cas12a platform could specifically identify Kp from eleven common clinical pathogens. The LOD of FD and LFTS were 1 fg/μL and 10 fg/μL, respectively. In clinical sample testing, the RPA-CRISPR/Cas12a platform was consistent with the culture method and qPCR method, and its sensitivity and specificity were 100% (16/16) and 100% (9/9), respectively. With the advantages of detection speed, simplicity, and accuracy, the RPA-CRISPR/Cas12a platform was expected to be a convenient tool for the early clinical detection of Kp.

PrimerBankID	Target Pathogen	Target Gene
RPB0250	Klebsiella pneumoniae	rcsA gene

CRISPR-Cas12a-based ultrasensitive assay for visual detection of SARS-CoV-2 RNA

Author(s):

Shaohua Gong,Kexin Song,Wei Pan,Na Li,Bo Tang

Journal:

Analyst

Year:

2024

Abstract:

The development of ultrasensitive and visual methods is of great significance for molecular diagnosis at the point-of-care. In this study, we have integrated recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system to design an ultrasensitive strategy for visual nucleic acid testing. RPA is utilized to amplify the target nucleic acid, producing amplicons that activate the single-stranded DNase property of CRISPR-Cas12a. The activated CRISPR-Cas12a then degrades the single-stranded DNA on magnetic nanoparticles (MNPs), releasing immobilized GOx from the MNPs which catalyses the chromogenic substrate. The developed method exhibits remarkable sensitivity, successfully detecting as low as 10 aM (∼6 copies per μL) of the target nucleic acid by visual colour changes in solution. The instrumental limit of detection is calculated to be 2.86 aM (∼2 copies per μL), comparable to the sensitivity of polymerase chain reaction (PCR). Importantly, this approach only requires isothermal incubation operation and does not involve costly instruments. The method has been validated by visually detecting the SARS-CoV-2 RNA gene fragment within 50 minutes. With its ultrasensitivity, simplicity of operation, and potential for integration into a point-of-care detection kit, this strategy holds great promise for nucleic acid testing in various settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0315	SARS-CoV-2	orflab gene

Rapid and one-tube detection of human metapneumovirus using the RT-RPA and CRISPR\Cas12a

Author(s):

Yao Du,Xiaorong Liu,Hongdan Gao,Xiaoqian Liu,Meng Huang,Qiang Chai,Zhihao Xing,Tao Zhang,Dongli Ma

Journal:

Journal of Virological Methods

Year:

2024

Abstract:

Human metapneumovirus (HMPV) is a common pathogen that can cause acute respiratory tract infections and is prevalent worldwide. There is yet no effective vaccine or specific treatment for HMPV. Early, rapid, and accurate detection is essential to treat the disease and control the spread of infection. In this study, we created the One-tube assay by combining Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA) with the CRISPR/Cas12a system. By targeting the nucleoprotein (N) gene of HMPV to design specific primers and CRISPR RNAs (crRNAs), combining RT-RPA and CRISPR/Cas12a, established the One-tube assay. Meanwhile, the reaction conditions of the One-tube assay were optimized to achieve rapid and visual detection of HMPV. This assay could detect HMPV at 1 copy/μL in 30 min, without cross-reactivity with nine other respiratory pathogens. We validated the detection performance using clinical specimens and showed that the coincidence rate was 98.53 %，compared to the quantitative reverse-transcription polymerase chain reaction. The One-tube assay reduced the detection time and simplified the manual operation, while maintaining the detection performance and providing a new platform for HMPV detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0235	HMPV	nucleoprotein (N) gene

CRISPR\Cas13a-based genome editing for establishing the detection method of H9N2 subtype avian influenza virus

Author(s):

Sha-Sha Chen,Yong-Lei Yang,Hong-Yun Wang,Tian-Kui Guo,Riaz-M Azeem,Chun-Wei Shi,Gui-Lian Yang,Hai-Bin Huang,Yan-Long Jiang,Jian-Zhong Wang,Xin Cao,Nan Wang,Yan Zeng,Wen-Tao Yang,Chun-Feng Wang

Journal:

Poultry Science

Year:

2024

Abstract:

Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.

PrimerBankID	Target Pathogen	Target Gene
RPB0290	Influenza A virus (H9N2)	HA gene

RT-RPA- Pf Ago detection platform for one-tube simultaneous typing diagnosis of human respiratory syncytial virus

Author(s):

Jia-Yu Liao,Xue-Yong Feng,Jie-Xiu Zhang,Tian-Dan Yang,Min-Xuan Zhan,Yong-Mei Zeng,Wei-Yi Huang,Hao-Bin Lian,Lin Ke,Si-Si Cai,Nan-Fei Zhang,Jin-Wen Fang,Xiao-Ying Cai,Jun-Duo Chen,Guang-Yu Lin,Li-Yun Lin,Wei-Zhong Chen,Yu-Yan Liu,Fei-Fei Huang,Chuang-Xing Lin,Min Lin

Journal:

Front. Cell. Infect. Microbiol.

Year:

2024

Abstract:

Human respiratory syncytial virus (HRSV) is the most prevalent pathogen contributing to acute respiratory tract infections (ARTI) in infants and young children and can lead to significant financial and medical costs. Here, we developed a simultaneous, dual-gene and ultrasensitive detection system for typing HRSV within 60 minutes that needs only minimum laboratory support. Briefly, multiplex integrating reverse transcription-recombinase polymerase amplification (RT-RPA) was performed with viral RNA extracted from nasopharyngeal swabs as a template for the amplification of the specific regions of subtypes A (HRSVA) and B (HRSVB) of HRSV. Next, the Pyrococcus furiosus Argonaute (PfAgo) protein utilizes small 5'-phosphorylated DNA guides to cleave target sequences and produce fluorophore signals (FAM and ROX). Compared with the traditional gold standard (RT-qPCR) and direct immunofluorescence assay (DFA), this method has the additional advantages of easy operation, efficiency and sensitivity, with a limit of detection (LOD) of 1 copy/μL. In terms of clinical sample validation, the diagnostic accuracy of the method for determining the HRSVA and HRSVB infection was greater than 95%. This technique provides a reliable point-of-care (POC) testing for the diagnosis of HRSV-induced ARTI in children and for outbreak management, especially in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0347	HRSV A	\
RPB0348	HRSV B	\

Establishment of a rapid detection method for Mycoplasma pneumoniae based on RPA-CRISPR-Cas12a technology

Author(s):

Ge Li,Jing Zhou,Nana Gao,Runde Liu,Jilu Shen

Journal:

Clinica Chimica Acta

Year:

2024

Abstract:

Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 102 copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0245	Klebsiella pneumoniae	P1 gene

Fluorescent and colorimetric dual-readout platform for tuberculosis point-of-care detection based on dual signal amplification strategy and quantum dot nanoprobe

Author(s):

Ou Hu,Yingyu Gong,Yuexiang Chang,Yaoju Tan,Zuanguang Chen,Wei Bi,Zhengjin Jiang

Journal:

Biosensors and Bioelectronics

Year:

2024

Abstract:

Rapid and accurate diagnosis of tuberculosis (TB) is of great significance to control the spread of this devastating infectious disease. In this work, a sensitive and low-cost point-of-care testing (POCT) detection platform for TB was developed based on recombinase polymerase amplification (RPA)-catalytic hairpin assembly (CHA)-assisted dual signal amplification strategy. This platform could achieve homogeneous fluorescent and visual diagnosis of TB by using CdTe quantum dots (QDs) signal reporter. In the presence of target DNA (IS1081 gene fragment), RPA amplicons blocked by short oligonucleotide strands could trigger CHA signal amplification, leading to the Ag+ releasing from C-Ag+-C structure and the fluorescence quenching of CdTe QDs by the released Ag+. Furthermore, the detection performance of CdTe QDs modified by 3-mercaptopropionic acid (MPA) or thiomalic acid (TMA) (MPA-capped QDs and TMA-capped QDs) was systematically compared. Experimental results demonstrated that TMA-capped QDs exhibited better detection sensitivity due to their stronger interaction with Ag+. The limits of detection (LODs) of fluorescence and visual analysis were as low as 0.13 amol L-1 and 0.33 amol L-1. This method was successfully applied to the clinical sputum samples from 36 TB patients and 20 healthy individuals, and its quantitative results were highly consistent with those obtained by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The proposed approach has the advantages of high sensitivity and specificity, simple operation and low cost, and is expected to be applied in clinical TB screening and diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0287	Mycobacterium tuberculosis	IS10181 gene

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