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