Real-time recombinase-aided amplification assay for rapid amplification of the IS1081 gene of Mycobacterium tuberculosis

Author(s):

Yuanyuan Liu,Weicong Ren,Zhongtan Xue,Yuedong Miao,Wei Wang,Xuxia Zhang,Cong Yao,Yuanyuan Shang,Shanshan Li,Fengling Mi,Yu Pang

Journal:

European Journal of Clinical Microbiology & Infectious Diseases

Year:

2023

Abstract:

Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis (TB), is the leading cause of death due to a single infectious agent worldwide. Rapid and accurate diagnosis of MTB is critical for controlling TB especially in resource-limited countries, since any diagnosis delay increases the chances of transmission. Here, a real-time recombinase-aided amplification (RAA) assay targeting conserved positions in IS1081 gene of MTB, is successfully established to detect MTB. The intact workflow was completed within 30 min at 42 °C with no cross-reactivity observed for non-tuberculous mycobacteria and other clinical bacteria, and the detection limit for recombinant plasmid of MTB IS1081 was 163 copies/reaction at 95% probability, which was approximately 1.5-fold increase in analytical sensitivity for the detection of MTB, compared to conventional quantitative real-time PCR (qPCR; 244 copies/reaction). Furthermore, the result of clinical performance evaluation revealed an increased sensitivity of RAA assay relative to qPCR was majorly noted in the specimens with low bacteria loads. Our results demonstrate that the developed real-time RAA assay is a convenient, sensitive, and low-cost diagnostic tool for the rapid detection of MTB.

PrimerBankID	Target Pathogen	Target Gene
RPB0365	Mycobacterium tuberculosis H37Rv	IS1081 gene

Fully integrated sample-in-answer-out platform for viral detection using digital reverse transcription recombinase polymerase amplification (dRT-RPA)

Author(s):

Seder, Islam; Coronel-Tellez, Rodrigo; Helalat, Seyed Hossein; Sun, Yi;

Journal:

BIOSENS BIOELECTRON

Year:

2023

Abstract:

Recombinase polymerase amplification (RPA) is one of the most promising diagnostic methods for pathogen detection, owing to the simplified isothermal amplification technique. Using one-step digital reverse transcription RPA (dRT-RPA) to detect viral RNA provides a fast diagnosis and absolute quantification. Here, we present a chip that purifies, digitalizes, and detects viral RNA of SARS-CoV-2 in a fully automated and sensitive manner. The chip purifies the RNA using the surface charge concept of magnet bead-RNA binding, then mixes the RNA with the amplification reagents, digitalizes the amplification mixture, and performs dRT-RPA. RNA-bead complex is transported among purification buffers that are separated by an oil phase. For reagent manipulation and mixing, a magnetic valve system is integrated on the chip, where an external magnet controls the reagent direction and time of addition. Besides, a novel vacuum system is suggested to drive and regulate the reagents into two fluid systems simultaneously in ∼2 min. We also developed a cost-effective way to perform fluorescent detection for dRT-RPA on chip by using EvaGreen® dye. With integrated heating and optical detection system, the on-chip dRT-RPA presents a sample-to-answer detection platform for absolute viral RNA quantitation in 37 min and a sensitivity as low as 10 RNA copies/μL. Hence, this platform is expected to be a useful tool for accurate and automated diagnosis of infectious diseases.

PrimerBankID	Target Pathogen	Target Gene
RPB0056	SARS-CoV-2	\

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity

Author(s):

Yi Ma,Haiping Wu,Shan Chen,Chunmei Xie,Jingjing Hu,Xiemin Qi,Xueping Ma,Yanan Chu,Jingwen Shan,Yan Lu,Lunbiao Cui,Bingjie Zou,Guohua Zhou

Journal:

Biosensors and Bioelectronics

Year:

2023

Abstract:

Recombinase polymerase amplification (RPA) running at 37-42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

PrimerBankID	Target Pathogen	Target Gene
RPB0357	SARS-CoV-2	RNase P gene
RPB0358	SARS-CoV-2 (Alpha)	S gene
RPB0359	SARS-CoV-2 (Beta)	S gene
RPB0360	SARS-CoV-2 (Delta)	S gene
RPB0361	SARS-CoV-2 (Omicron)	S gene
RPB0362	SARS-CoV-2	ORF1ab gene
RPB0363	SARS-CoV-2	N gene

Rapid Detection of SARS-CoV-2 RNA Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) with Lateral Flow for N-Protein Gene and Variant-Specific Deletion-Insertion Mutation in S-Protein Gene

Author(s):

Jose L Malaga, Monica J Pajuelo, Michiko Okamoto, Emmanuel Kagning Tsinda, Kanako Otani, Pablo Tsukayama, Lucero Mascaro, Diego Cuicapuza, Masamichi Katsumi, Kazuhisa Kawamura, Hidekazu Nishimura, Akie Sakagami, Yo Ueki, Suguru Omiya, Satoshi Okamoto, Asami Nakayama, Shin-Ichi Fujimaki, Chuyao Yu, Sikandar Azam, Eiichi Kodama, Clyde Dapat, Hitoshi Oshitani, Mayuko Saito

Journal:

Viruses

Year:

2023

Abstract:

Rapid molecular testing for severe acute respiratory coronavirus 2 (SARS-CoV-2) variants may contribute to the development of public health measures, particularly in resource-limited areas. Reverse transcription recombinase polymerase amplification using a lateral flow assay (RT-RPA-LF) allows rapid RNA detection without thermal cyclers. In this study, we developed two assays to detect SARS-CoV-2 nucleocapsid (N) gene and Omicron BA.1 spike (S) gene-specific deletion-insertion mutations (del211/ins214). Both tests had a detection limit of 10 copies/µL in vitro and the detection time was approximately 35 min from incubation to detection. The sensitivities of SARS-CoV-2 (N) RT-RPA-LF by viral load categories were 100% for clinical samples with high (>9015.7 copies/µL, cycle quantification (Cq): < 25) and moderate (385.5-9015.7 copies/µL, Cq: 25-29.9) viral load, 83.3% for low (16.5-385.5 copies/µL, Cq: 30-34.9), and 14.3% for very low (<16.5 copies/µL, Cq: 35-40). The sensitivities of the Omicron BA.1 (S) RT-RPA-LF were 94.9%, 78%, 23.8%, and 0%, respectively, and the specificity against non-BA.1 SARS-CoV-2-positive samples was 96%. The assays seemed more sensitive than rapid antigen detection in moderate viral load samples. Although implementation in resource-limited settings requires additional improvements, deletion-insertion mutations were successfully detected by the RT-RPA-LF technique.

PrimerBankID	Target Pathogen	Target Gene
RPB0004	SARS-CoV-2	N
RPB0005	SARS-CoV-2	S

A CRISPR-based approach using dead Cas9-sgRNA to detect SARS-CoV-2

Author(s):

Mustapha Aouida,Maryam Saifaldeen,Dana E Al-Ansari,Sara Taleb,Ali Ait Hssain,Dindial Ramotar

Journal:

Frontiers in Molecular Biosciences

Year:

2023

Abstract:

Rapid, highly specific, and robust diagnostic kits to detect viruses and pathogens are needed to control disease spread and transmission globally. Of the many different methods proposed to diagnose COVID-19 infection, CRISPR-based detection of nucleic acids tests are among the most prominent. Here, we describe a new way of using CRISPR/Cas systems as a rapid and highly specific tool to detect the SARS-CoV-2 virus using the in vitro dCas9-sgRNA-based technique. As a proof of concept, we used a synthetic DNA of the M gene, one of the SARS-CoV-2 virus genes, and demonstrated that we can specifically inactivate unique restriction enzyme sites on this gene using CRISPR/Cas multiplexing of dCas9-sgRNA-BbsI and dCas9-sgRNA-XbaI. These complexes recognize and bind to the target sequence spanning the BbsI and XbaI restriction enzyme sites, respectively, and protect the M gene from digestion by BbsI and/or XbaI. We further demonstrated that this approach can be used to detect the M gene when expressed in human cells and from individuals infected with SARS-CoV-2. We refer to this approach as dead Cas9 Protects Restriction Enzyme Sites, and believe that it has the potential to be applied as a diagnostic tool for many DNA/RNA pathogens.

PrimerBankID	Target Pathogen	Target Gene
RPB0391	SARS-CoV-2	BbsI
RPB0392	SARS-CoV-2	XbaI
RPB0393	SARS-CoV-2	M gene

Strategies to Improve Multi-enzyme Compatibility and Coordination in One-Pot SHERLOCK

Author(s):

Hongzhao Li,Dominic M S Kielich,Guodong Liu,Greg Smith,Alexander Bello,James E Strong,Bradley S Pickering

Journal:

Analytical Chemistry

Year:

2023

Abstract:

While molecular diagnostics generally require heating elements that supply high temperatures such as 95 °C in polymerase chain reaction and 60-69 °C in loop-mediated isothermal amplification, the recently developed CRISPR-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can operate at 37 °C or a similar ambient temperature. This unique advantage may be translated into highly energy-efficient or equipment-free molecular diagnostic systems with unrestricted deployability. SHERLOCK is characterized by ultra-high sensitivity when performed in a traditional two-step format. For RNA sensing, the first step combines reverse transcription with recombinase polymerase amplification, while the second step consists of T7 transcription and CRISPR-Cas13a detection. The sensitivity drops dramatically, however, when all these components are combined into a single reaction mixture, and it largely remains an unmet need in the field to establish a high-performance one-pot SHERLOCK assay. An underlying challenge, conceivably, is the extremely complex nature of a one-pot formulation, crowding a large number of reaction types using at least eight enzymes/proteins. Although previous work has made substantial improvements by serving individual enzymes/reactions with accommodating conditions, we reason that the interactions among different enzymatic reactions could be another layer of complicating factors. In this study, we seek optimization strategies by which inter-enzymatic interference may be eliminated or reduced and cooperation created or enhanced. Several such strategies are identified for SARS-CoV-2 detection, each leading to a significantly improved reaction profile with faster and stronger signal amplification. Designed based on common molecular biology principles, these strategies are expected to be customizable and generalizable with various buffer conditions or pathogen types, thus holding broad applicability for integration into future development of one-pot diagnostics in the form of a highly coordinated multi-enzyme reaction system.

PrimerBankID	Target Pathogen	Target Gene
RPB0407	SARS-CoV-2	ORF1ab
RPB0408	SARS-CoV-2	E
RPB0409	SARS-CoV-2	S

Detecting SARS-CoV-2 BA.2, BA.4, and BA.5 Variants Utilizing a Robust RT-RPA-CRISPR/Cas12a-Based Method - China, 2023.

Author(s):

Luo, Meihui; Pan, Yang; He, Yaqing; A, Ruhan; Wu, Changcheng; Huang, Baoying; Lu, Roujian; Zhao, Li; Peng, Bo; Ye, Fei; Wang, Huijuan; Chen, Yuda; Li, Zhen; Zhang, Daitao; Wang, Wenling; Tan, Wenjie; 

Journal:

China CDC Wkly

Year:

2023

Abstract:

Introduction:Since 2019, numerous variants of concern for severe acute respiratory syndrome virus 2 (SARS-CoV-2) have emerged, leading to significant outbreaks. The development of novel, highly accurate, and rapid detection techniques for these new SARS-CoV-2 variants remains a primary focus in the ongoing efforts to control and prevent the coronavirus disease 2019 (COVID-19) pandemic.Methods:Reverse transcription-recombinase polymerase amplification combined with the clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) system was used to validate the detection of the Omicron BA.2, BA.4, and BA.5 variants of SARS-CoV-2.Results:Our results demonstrate that the CRISPR/Cas12a assay is capable of effectively detecting the SARS-CoV-2 BA.2, BA.4, and BA.5 variants with a limit of detection of 10, 1, and 10 copies/渭L, respectively. Importantly, our assay successfully differentiated the three SARS-CoV-2 Omicron strains from one another. Additionally, we evaluated 46 SARS-CoV-2 positive clinical samples consisting of BA.2 (n=20), BA.4 (n=6), and BA.5 (n=20) variants, and the sensitivity of our assay ranged from 90% to 100%, while the specificity was 100%.Discussion:This research presents a swift and reliable CRISPR-based method that may be employed to track the emergence of novel SARS-CoV-2 variants.

PrimerBankID	Target Pathogen	Target Gene
RPB0051	SARS-CoV-2 (Omicron)	ORF1ab gene C9866T
RPB0052	SARS-CoV-2 (Omicron)	S gene A23040G
RPB0053	SARS-CoV-2 (Omicron)	ORF7b gene G27788T
RPB0054	SARS-CoV-2 (Omicron)	M gene G26529A
RPB0055	SARS-CoV-2 (Omicron)	S gene C27889T

Field-deployable assay based on CRISPR-Cas13a coupled with RT-RPA in one tube for the detection of SARS-CoV-2 in wastewater

Author(s):

Yihan Yang,Fan Wang,Boyuan Xue,Xiaohong Zhou

Journal:

Journal Of Hazardous Materials

Year:

2023

Abstract:

CRISPR-based nucleic acid detection is easy to implement, field deployable, and always coupled with isothermal amplification to improve the sensitivity. However, the conventional detection requires two separate steps, which can cause long-lasting amplicon aerosol contaminants, hence leading to false-positive results. To address this problem, we proposed a one-tube assay based on CRISPR-Cas13a coupled with reverse transcription-recombinase polymerase amplification to avoid aerosol pollution. The one-tube assay could be completed within 40 min with a sensitivity of up to 180 copies of RNA per reaction, and exhibited no cross reactivity with two related coronaviruses. Our technology showed reproducibility with relative standard deviation of 4.6% responding to 1 fM nucleic acid for three times. It could be used to detect SARS-CoV-2 nucleic acids in raw wastewater with a limit of detection of 103 copies/mL. We also validated the practicability of this technique for viral detection in environmental water samples by detecting SARS-CoV-2 in wastewater, which were not detectable by RT-qPCR technology, showing resistance of this technology to wastewater matrix. It is anticipated that the robustness and high sensitivity will significantly promote the development of a point-of-care method in environmental virus monitoring.

PrimerBankID	Target Pathogen	Target Gene
RPB0439	SARS-CoV-2	WPRE
RPB0440	SARS-CoV-2	S gene

Rapid isothermal point-of-care test for screening of SARS-CoV-2 (COVID-19)

Author(s):

Jean-Marc Zingg, Yu-Ping Yang, Spencer Seely, Pratibha Joshi, Md Harun Or Roshid, Fabiola Iribarren Latasa, Gregory O'Connor, Jennifer Alfaro, Eduardo Riquelme, Sebastian Bernales, Emre Dikici, Sapna Deo, Sylvia Daunert

Journal:

Aspects of Molecular Medicine

Year:

2023

Abstract:

Rapid on-site diagnosis of emerging pathogens is key for early identification of infected individuals and for prevention of further spreading in a population. Currently available molecular diagnostic tests are instrument-based whereas rapid antibody and antigen tests are often not sufficiently sensitive for detection in pre-symptomatic subjects. There is a need for rapid point of care molecular screening tests that can be easily adapted to emerging pathogens and are selective, sensitive, reliable in different settings around the world. We have developed a simple, rapid (<30 ​min), and inexpensive test for SARS-CoV-2 that is based on combination of isothermal reverse transcription recombinase polymerase amplification (RT-RPA) using modified primers and visual detection with paper-based microfluidics. Our test (CoRapID) is specific for SARS-CoV-2 (alpha to omicron variants) and does not detect other coronaviruses and pathogens by in silico and in vitro analysis. A two-step test protocol was developed with stable lyophilized reagents that reduces handling by using portable and disposable components (droppers, microapplicators/swabs, paper-strips). After optimization of assay components and conditions, we have achieved a limit of detection (LoD) of 1 copy/reaction by adding a blocking primer to the lateral flow assay. Using a set of 138 clinical samples, a sensitivity of 88.1% (P ​< ​0.05, CI: 78.2-93.8%) and specificity of 93.9% (P ​< ​0.05, CI: 85.4-97.6%) was determined. The lack of need for instrumentation for our CoRapID makes it an ideal on-site primary screening tool for local hospitals, doctors' offices, senior homes, workplaces, and in remote settings around the world that often do not have access to clinical laboratories.

PrimerBankID	Target Pathogen	Target Gene
RPB0020	SARS-CoV-2	N

Rapid, ultrasensitive and highly specific diagnosis of Mycoplasma pneumoniae by a CRISPR-based detection platform

Author(s):

Juan Zhou,Fei Xiao,Jin Fu,Nan Jia,Xiaolan Huang,Chunrong Sun,Zheng Xu,Yu Zhang,Dong Qu,Yi Wang

Journal:

Frontiers in Cellular and Infection Microbiology

Year:

2023

Abstract:

Mycoplasma pneumoniae (MP) is an important causative agent of morbidity and mortality among all age groups, especially among patients of extreme ages. Improved and readily available tests for accurate, sensitive and rapid diagnosis of MP infection is sorely needed. Here, we developed a CRISPR-Cas12b-based detection platform on the basis of recombinase polymerase amplification (RPA) for rapid, simple, and accurate diagnosis of MP infection, named MP-RPA-CRISPR. The RPA was employed for amplifying the community-acquired respiratory distress syndrome (CARDS) toxin gene of MP strains at the optimal reaction temperature 37°C. The resulting amplicons were decoded by the CRISPR-Cas12b-based detection platform, which was interpreted by real-time PCR system and by naked eye under blue light. The MP-RPA-CRISPR can detected down to 5 fg of genomic DNA templates of MP strains and accurately distinguish MP strains from non-MP strains without any cross-reactivity. A total of 96 bronchoalveolar lavage fluid (BALF)samples collected from patients suspected of respiratory infection were used to evaluate the clinical performance of the MP-RPA-CRISPR assay. As a result, our assay accurately diagnosed 45 MP-infected samples and 51 non-MP infected sample, and the results obtained from MP-RPA-CRISPR were consistent with microfluidic chip technology. In conclusion, our MP-RPA-CRISPR assay is a simple, rapid, portable and highly sensitive method to diagnose MP infection, which can be used as a promising tool in a variety of settings including clinical, field, and resource-limited aeras.

PrimerBankID	Target Pathogen	Target Gene
RPB0390	Mycobacterium	CARDS