Development and clinical implications of a novel CRISPR-based diagnostic test for pulmonary Aspergillus fumigatus infection

Author(s):

Zhengtu Li,Mingdie Wang,Teng Xu,Yangqing Zhan,Fengyi Chen,Ye Lin,Shaoqiang Li,Jing Cheng,Feng Ye

Journal:

Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi

Year:

2022

Abstract:

Background: Rapid and reliable diagnostic methods for Aspergillus fumigatus infection are urgently needed. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 13a (Cas13a) has high sensitivity and specificity in the diagnosis of viral infection. However, its potential use in detecting A. fumigatus remains unexplored. A highly sensitive and specific method using the CRISPR/Cas13a system was developed for the reliable and rapid detection of A. fumigatus. Methods: The conserved internal transcribed spacer (ITS) region of A. fumigatus was used to design CRISPR-derived RNA (crRNA) and the corresponding recombinase polymerase amplification (RPA) primer sequence with the T7 promoter for the CRISPR assay. Twenty-five clinical isolates and 43 bronchoalveolar lavage fluid (BALF) remaining from routine examinations of patients with confirmed pulmonary aspergillosis were collected to further validate the CRISPR assay. Results: No amplification signal was observed when genomic DNA from closely clinically related Aspergillus species, such as Aspergillus flavus, Aspergillus niger, and Aspergillus terreus, as well as Monascus purpureus Went and Escherichia coli, was tested by this assay, and the detection limit for A. fumigatus was 3 copies in a single reaction system. Validation experiments using the 25 clinical isolates demonstrated 91.7% specificity for the A. fumigatus section, and the sensitivity was 100% when first-generation sequencing was used as the standard. There was no significant difference between the PCR and CRISPR methods (P = 1.0), and the diagnosis results of the two methods were consistent (Kappa = 0.459, P = 0.003). Conclusion: The study offers a new validated CRISPR/Cas13a technique for A. fumigatus detection, providing a simple, rapid and affordable test that is ready for application in the diagnosis of A. fumigatus infection.

PrimerBankID	Target Pathogen	Target Gene
RPB0084	Aspergillus fumigatus	\

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

Author(s):

Fei Hu, Yanfei Liu, Shuhao Zhao, Zengming Zhang, Xichen Li, Niancai Peng, and Zhuangde Jiang

Journal:

Biosensors and Bioelectronics

Year:

2022

Abstract:

The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/μL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.

PrimerBankID	Target Pathogen	Target Gene
RPB0202	SARS-CoV-2	\

A Recombinase Polymerase Amplification-Coupled Cas12a Mutant-Based Module for Efficient Detection of Streptomycin-Resistant Mutations in Mycobacterium tuberculosis

Author(s):

Peng Liu,Xinjie Wang,Juan Liang,Qian Dong,Jinping Zhang,Dongxin Liu,Shuai Wang,Jing Bi,Wenqi Liu,Zhaoqin Wang,Liang Chen,Lei Liu,Xingxu Huang,Guoliang Zhang

Journal:

Frontiers in microbiology

Year:

2022

Abstract:

Drug-resistant tuberculosis (TB) is a serious public health problem and threat to global TB prevention and control. Streptomycin (STR) is the earliest and classical anti-TB drug, and it is the earliest drug that generated resistance to anti-TB treatment, which limits its use in treating TB and impedes TB control efforts. The rapid, economical, and highly sensitive detection of STR-resistant TB may help reduce disease transmission and morbimortality. CRISPR/CRISPR-associated protein (Cas) is a new-generation pathogen detection method that can detect single-nucleotide polymorphisms with high sensitivity and good specificity. In this study, a Cas12a RR detection system that can recognize more non-traditional protospacer-adjacent motif-targeting sequences was developed based on Cas12a combined with recombinase polymerase amplification technology. This system detects 0.1% of the target substance, and the entire detection process can be completed within 60 min. Its sensitivity and specificity for detecting clinical STR-resistant Mycobacterium tuberculosis were both 100%. Overall, the Cas12 RR detection system provides a novel alternative for the rapid, simple, sensitive, and specific detection of STR-resistant TB, which may contribute to the prompt treatment and prevention of disease transmission in STR-resistant TB.

PrimerBankID	Target Pathogen	Target Gene
RPB0112	Mycobacterium	rpsl gene

iSCAN-V2: A One-Pot RT-RPA-CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection.

Author(s):

Aman, Rashid; Marsic, Tin; Sivakrishna Rao, Gundra; Mahas, Ahmed; Ali, Zahir; Alsanea, Madain; Al-Qahtani, Ahmed; Alhamlan, Fatimah; Mahfouz, Magdy;

Journal:

Front Bioeng Biotechnol

Year:

2022

Abstract:

Rapid, specific, and sensitive detection platforms are prerequisites for early pathogen detection to efficiently contain and control the spread of contagious diseases. Robust and portable point-of-care (POC) methods are indispensable for mass screening of SARS-CoV-2. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based nucleic acid detection technologies coupled with isothermal amplification methods provide a straightforward and easy-to-handle platform for detecting SARS-CoV-2 at POC, low-resource settings. Recently, we developed iSCAN, a two-pot system based on coupled loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a reactions. However, in two-pot systems, the tubes must be opened to conduct both reactions; two-pot systems thus have higher inherent risks of cross-contamination and a more cumbersome workflow. In this study, we developed and optimized iSCAN-V2, a one-pot reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled CRISPR/Cas12b-based assay for SARS-CoV-2 detection, at a single temperature in less than an hour. Compared to Cas12a, Cas12b worked more efficiently in the iSCAN-V2 detection platform. We assessed and determined the critical factors, and present detailed guidelines and considerations for developing and establishing a one-pot assay. Clinical validation of our iSCAN-V2 detection module with reverse transcription-quantitative PCR (RT-qPCR) on patient samples showed 93.75% sensitivity and 100% specificity. Furthermore, we coupled our assay with a low-cost, commercially available fluorescence visualizer to enable its in-field deployment and use for SARS-CoV-2 detection. Taken together, our optimized iSCAN-V2 detection platform displays critical features of a POC molecular diagnostic device to enable mass-scale screening of SARS-CoV-2 in low-resource settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0070	SARS-CoV-2	N gene

Rapid Detection and Differentiation of Legionella pneumophila and Non -Legionella pneumophila Species by Using Recombinase Polymerase Amplification Combined With EuNPs-Based Lateral Flow Immunochromatography

Author(s):

Jungang Du,Biao Ma,Jiali Li,Yaping Wang,Tianyu Dou,Shujuan Xu,Mingzhou Zhang

Journal:

Frontiers in chemistry

Year:

2022

Abstract:

Legionella, a waterborne pathogen, is the main cause of Legionnaires' disease. Therefore, timely and accurate detection and differentiation of Legionella pneumophila and non-Legionella pneumophila species is crucial. In this study, we develop an easy and rapid recombinase polymerase amplification assay combined with EuNPs-based lateral flow immunochromatography (EuNPs-LFIC-RPA) to specifically distinguish Legionella pneumophila and non-Legionella pneumophila. We designed primers based on the mip gene of Legionella pneumophila and the 5S rRNA gene of non-Legionella pneumophila. The recombinase polymerase amplification reaction could go to completion in 10 min at 37°C, and the amplification products could be detected within 5 min with EuNPs-LFIC strips. Using a florescent test strip reader, the quantitative results were achieved by reading the colored signal intensities on the strips. The sensitivity was 1.6 × 101 CFU/ml, and a linear standard linear curve plotted from the test strip reader had a correlation coefficient for the determination of Legionella pneumophila (R 2 = 0.9516). Completed concordance for the presence or absence of Legionella pneumophila by EuNPs-LFIC-RPA and qPCR was 97.32% (κ = 0.79, 95% CI), according to an analysis of practical water samples (n = 112). In short, this work shows the feasibility of EuNPs-LFIC-RPA for efficient and rapid monitoring of Legionella pneumophila and non-Legionella pneumophila in water samples.

PrimerBankID	Target Pathogen	Target Gene
RPB0106	Legionella pneumophila	mip gene
RPB0107	Legionella pneumophila	5S rRNA gene

Enhanced Isothermal Amplification for Ultrafast Sensing of SARS-CoV-2 in Microdroplets

Author(s):

Mengyun Zhou , Chuan Fan , Lirong Wang , Tailin Xu , Xueji Zhang

Journal:

Analytical Chemistry

Year:

2022

Abstract:

Rapid and high-throughput screening is critical to control the COVID-19 pandemic. Recombinase polymerase amplification (RPA) with highly accessible and sensitive nucleic acid amplification has been widely used for point-of-care infection diagnosis. Here, we report an integrated microdroplet array platform composed of an ultrasonic unit and minipillar array to enhance the RPA for ultrafast, high-sensitivity, and high-throughput detection of SARS-CoV-2. On such a platform, the independent microvolume reactions on individual minipillars greatly decrease the consumption of reagents. The microstreaming driven by ultrasound creates on-demand contactless microagitation in the microdroplets and promotes the interaction between RPA components, thus greatly accelerating the amplification. In the presence of microstreaming, the detection time is 6-12 min, which is 38.8-59.3% shorter than that of controls without microstreaming, and the end-point fluorescence intensity also increased 1.3-1.7 times. Furthermore, the microagitation-enhanced RPA also exhibits a lower detection limit (0.42 copy/μL) for SARS-CoV-2 in comparison to the controls. This integrated microdroplet array detection platform is expected to meet the needs for high-throughput nucleic acid testing (NAT) to improve the containment of viral transmission during the epidemic, as well as provide a potential platform for the timely detection of other pathogens or viruses.

PrimerBankID	Target Pathogen	Target Gene
RPB0203	SARS-CoV-2	SARS-Cov-2 N gene

Integrated Trinity Test With RPA-CRISPR/Cas12a-Fluorescence for Real-Time Detection of Respiratory Syncytial Virus A or B

Author(s):

Ling Gong,Xiaowen Wang,Zhu Li,Guichuan Huang,Wei Zhang,Jin Nie,Chunyan Wu,Daishun Liu

Journal:

Frontiers in microbiology

Year:

2022

Abstract:

Respiratory syncytial virus (RSV) is a common virus that causes respiratory infection, especially severe respiratory infection in infants and young children, the elderly people over 65 years old, and people with weak immunity. Currently, RSV infection has no effective vaccine and antiviral treatment. The number of deaths due to RSV infection increases every year. Moreover, RSV A infection occurs in a large number and has severe clinical symptoms and complications than RSV B infection. Therefore, the development of a simple, rapid, and inexpensive detection method with high amplification efficiency, high sensitivity, and specificity is very important for the diagnosis of RSV A or RSV B infection, which can help in the early clinical medication and prevent the progress of the disease. Therefore, we developed an integrated trinity test with an RPA-CRISPR/Cas12a-fluorescence (termed IT-RAISE) assay system to detect RSV A or RSV B. The characteristic of the IT-RAISE system is that after target recognition, the reporter single-stranded DNA (ssDNA) is cleaved by Cas12a that is activated by different crRNAs to detect the generated fluorescent signal. This method is simple and helps in adding all reagents rapidly. It is a high-sensitive method that can detect 1.38 × 101 copies/μl of the target sequences, and it can distinguish RSV A or RSV B infection within 37 min. In addition, clinical specimens were detected for IT-RAISE system. It was found that the sensitivity and specificity of RSV A were 73.08 and 90%, respectively, and those of RSV B were 42.86 and 93.33%, respectively. The cost of ONE specimen for IT-RAISE system was approximately $ 2.6 (excluding rapid RNA extraction and reverse transcription costs). IT-RAISE system has good clinical application prospects for detecting RSV A or RSV B infection; it is a simple, rapid, and inexpensive method with high amplification efficiency, high sensitivity, and high specificity. The IT-RAISE system might also detect other viral or bacterial infections.

PrimerBankID	Target Pathogen	Target Gene
RPB0126	RSV A	\
RPB0127	RSV B	\

Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus

Author(s):

Yanan Li,Zhonglin Shi,Anzhong Hu,Junsheng Cui,Ke Yang,Yong Liu,Guoqing Deng,Cancan Zhu,Ling Zhu

Journal:

Diagnostics (Basel, Switzerland)

Year:

2022

Abstract:

Methicillin-resistant Staphylococcus aureus (MRSA) is a severe health threat causing high-level morbidity and mortality in health care environments and in community settings. Though existing diagnostic methods, including PCR and culture-based methods, are routinely used in clinical practice, they are not appropriate for rapid point-of-care testing (POCT). Recently, since the development of the CRISPR/Cas technology, new possibilities for rapid point-of-care detection have emerged. In this study, we developed a rapid, accurate, and contamination-free platform for MRSA detection by integrating recombinase polymerase amplification (RPA) with the Cas12 system into one tube. Using this approach, visual MRSA detection could be achieved in 20 min. Based on the one-tube RPA-CRISPR/Cas12a platform, the assay results are visualized by lateral flow test strips (LFS) and fluorescent-based methods, including real-time and end-point fluorescence. This platform allows specific MRSA detection with a sensitivity of 10 copies for the fluorescence method and a range of 10-100 copies for the LFS. The results of 23 samples from clinical MRSA isolates showed that the coincidence rate was 100% and 95.7% of the fluorescence method and LFS, respectively, compared to qPCR. In conclusion, the one-tube RPA-CRISPR/Cas12a platform is an effective method for MRSA detection with significant potential in future practical POCT applications.

PrimerBankID	Target Pathogen	Target Gene
RPB0158	Staphylococcus aureus	mecA gene

Visualized Genotyping from "Sample to Results" Within 25 Minutes by Coupling Recombinase Polymerase Amplification (RPA) With Allele-Specific Invasive Reaction Assisted Gold Nanoparticle Probes Assembling.

Author(s):

Likun Zhang , Xueping Ma , Danni Liu , Jingwen Shan , Yanan Chu , Jieyu Zhang , Xiemin Qi , Xiaohui Huang , Bingjie Zou, Guohua Zhou

Journal:

J Biomed Nanotechnol .J Biomed Nanotechnol J Biomed Nanotechnol

Year:

2022

Abstract:

A simple and rapid genotyping method with less-instrumentation is essential for realizing point-of-care detection of personalized medicine-related gene biomarkers. Herein, we developed a rapid and visualized genotyping method by coupling recombinase polymerase amplification (RPA) with allele-specific invader reaction assisted gold nanoparticle probes assembling. In the method, the DNA targets were firstly amplified by using RPA, which is a rapid isothermal amplification technology. Then an allele-specific invasion reaction was performed to recognize the single nucleotide polymorphisms (SNPs) site in the amplicons, to produce signal molecules that caused discoloration of gold nanoparticle probes. As a result, genotyping was achieved by observing the color change of the reaction by using naked eye without the requirement for any expensive instrument. In order to achieve rapid genotyping detection, the genomic DNA from oral swab lysate samples were used for the RPA templates amplification. In this way, a visualized genotyping from "samples to results" within 25 min was realized. Two clopidogrel related SNPs CYP2C19*2 and CYP2C19*3 of 56 clinical samples were correctly genotyped by using this rapid visualized genotyping assay. In addition, the feasibility for this pathogen genotyping method was also verified by detecting plasmid DNA containing three SARS-COV-2 gene mutation sites, indicating that this method has the potential for clinical sample detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0206	SARS-CoV-2	CYP2C19*2
RPB0207	SARS-CoV-2	CYP2C19*3
RPB0208	SARS-CoV-2	A570D
RPB0209	SARS-CoV-2	D80A
RPB0210	SARS-CoV-2	T473K

Establishment and Clinical Application of a RPA-LFS Assay for Detection of Capsulated and Non-Capsulated Haemophilus influenzae

Author(s):

Yan Wang,Aibo Liu,Mei Fu,Jingjing Guo,Lei Wang,Xiaohua Zuo,Fenfen Ma

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

A recombinase polymerase amplification-lateral flow strip assay was established for detection of the outer membrane protein P6 (omp6) and the capsule encoding gene bexA of Haemophilus influenzae and the detection limit, sensitivity, and specificity were determined. Specific primers and probes were designed based on the published nucleotide sequences of omp6 and bexA. The minimum detection limit was determined with standard strains and the practical applicability of the RPA-LFS assay was assessed by detection of 209 clinical samples. The results confirmed that the RPA-LFS assay was both specific and sensitive for the detection of capsulated and non-capsulated H. influenzae with a detection limit of 1 CFU/µL. The detection rate of the 209 clinical samples was 97.1%, while the detection rate of capsulated H. influenzae was 63.2%. The detection results were consistent with the traditional culture method and dual polymerase chain reaction (PCR), confirming the applicability of the RPA-LFS assay.

PrimerBankID	Target Pathogen	Target Gene
RPB0096	Haemophilus influenzae	omp6
RPB0097	Haemophilus influenzae	bexA

Clinical Validation of a Rapid Variant-Proof RT-RPA Assay for the Detection of SARS-CoV-2

Author(s):

Dounia Cherkaoui, Judith Heaney, Da Huang,Matthew Byott, Benjamin S. Miller,Eleni Nastouli, and Rachel A. McKendry

Journal:

Diagnostics (Basel).

Year:

2022

Abstract:

The COVID-19 pandemic has unveiled a pressing need to expand the diagnostic landscape to permit high-volume testing in peak demand. Rapid nucleic acid testing based on isothermal amplification is a viable alternative to real-time reverse transcription polymerase chain reaction (RT-PCR) and can help close this gap. With the emergence of SARS-CoV-2 variants of concern, clinical validation of rapid molecular tests needs to demonstrate their ability to detect known variants, an essential requirement for a robust pan-SARS-CoV-2 assay. To date, there has been no clinical validation of reverse transcription recombinase polymerase amplification (RT-RPA) assays for SARS-CoV-2 variants. We performed a clinical validation of a one-pot multi-gene RT-RPA assay with the E and RdRP genes of SARS-CoV-2 as targets. The assay was validated with 91 nasopharyngeal samples, with a full range of viral loads, collected at University College London Hospitals. Moreover, the assay was tested with previously sequenced clinical samples, including eleven lineages of SARS-CoV-2. The rapid (20 min) RT-RPA assay showed high sensitivity and specificity, equal to 96% and 97%, respectively, compared to gold standard real-time RT-PCR. The assay did not show cross-reactivity with the panel of respiratory pathogens tested. We also report on a semi-quantitative analysis of the RT-RPA results with correlation to viral load equivalents. Furthermore, the assay could detect all eleven SARS-CoV-2 lineages tested, including four variants of concern (Alpha, Beta, Delta, and Omicron). This variant-proof SARS-CoV-2 assay offers a significantly faster and simpler alternative to RT-PCR, delivering sensitive and specific results with clinical samples.

PrimerBankID	Target Pathogen	Target Gene
RPB0200	SARS-CoV-2	E gene
RPB0201	SARS-CoV-2	RdRP gene

Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay

Author(s):

Mei Lin , Huahua Yue , Tian Tian , Erhu Xiong , Debin Zhu , Yongzhong Jiang , Xiaoming Zhou

Journal:

Analytical Chemistry

Year:

2022

Abstract:

CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage. In this study, we discovered that a glycerol additive dramatically promoted the detection efficiency of the one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method. Compared with the glycerol-free version, its sensitivity was nearly 100-fold higher and was close to that of the canonical two-step method. Further investigation displayed that the enhanced detection efficiency was attributed to the phase separation of the RPA and CRISPR/Cas12a system during the initial phase of the RPA reaction caused by the glycerol viscosity. This highly efficient one-pot method has been triumphantly harnessed for the detection of African swine fever virus (ASFV) and SARS-CoV-2, achieving naked-eye readout through a smartphone-equipped device. The currently developed glycerol-enhanced one-pot RPA-CRISPR/Cas12a method can be an advantageous point-of-care nucleic acid detection platform on account of its simplicity, high sensitivity, and universality.

PrimerBankID	Target Pathogen	Target Gene
RPB0198	SARS-CoV-2	Orf1ab-gene
RPB0199	SARS-CoV-2	N-gene

Development and Clinical Application of a Recombinase Polymerase Amplification-Lateral Flow Strip Assay for Detection of Carbapenem-Resistant Acinetobacter baumannii

Author(s):

Lei Wang,Dunpo Sun,Li Chen,Ping Zhou,Kun Wang,Fang Wang,Xingqi Lei,Yan Wang,Yingzhi Lu,Guanhong Huang,Xuzhu Gao

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

Acinetobacter baumannii is a worldwide, primary cause of respiratory tract infections, septicemia, urinary apparatus infections, and secondary meningitis. It can be fatal. Rapid and accurate detection methods are needed to control the spread of carbapenem-resistant A. baumannii (CRAB). Current molecular diagnostic methods are limited and not suitable for on-site detection. In this study, an isothermal detection method using recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) was developed to target the blaOXA-51 and blaOXA-23 genes of A. baumannii. The reaction was completed in about 40 min at 37°C. This method can also effectively distinguish A. baumannii and CRAB. The limit of detection of 100-101 CFU/reaction was equal to that of other detection methods. The detection accuracy was equal to that of the qPCR method with the use of clinical samples. The RPA-LFS assay is portable, rapid, and accurate and could replace existing detection methods for on-site detection of A. baumannii and CRAB.

PrimerBankID	Target Pathogen	Target Gene
RPB0080	Acinetobacter baumannii	bla OXA-51
RPB0081	Acinetobacter baumannii	bla OXA-23

Automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) and lateral flow strip based on CRISPR/Cas13a for sensitive and visual detection of SARS-CoV-2

Author(s):

Gaihua Cao, Danqun Huo, Xiaolong Chen, Xianfeng Wang, Shiying Zhou, Shixian Zhao, Xiaogang Luo, Changjun Hou

Journal:

Talanta

Year:

2022

Abstract:

COVID-19 has erupted and quickly swept across the globe, causing huge losses to human health and wealth. It is of great value to develop a quick, accurate, visual, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we developed a biosensor based on CRISPR/Cas13a combined with recombinase polymerase amplification (RPA) to detect S and Orf1ab genes of SARS-CoV-2 within 30 min. Most important of all, we developed an automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) with a 3D-printed microfluidic chip for sensitively detecting SARS-CoV-2, which addressed aerosol contamination issue and provided a more accurate and high-throughput detection during the on-site detection process. The detection limits of S gene and Orf1ab gene were as low as 0.68 fM and 4.16 fM. Furthermore, we used the lateral flow strip to realize visualization and point of care testing (POCT) of SARS-CoV-2. Therefore, profit from the efficient amplification of RPA and the high specificity of CRISPR/Cas13a, APHF-analyzer and the lateral flow strip to simultaneous detection of S gene and Orf1ab gene would be applied as a promising tool in the field of SARS-CoV-2 detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0037	SARS-CoV-2	S
RPB0038	SARS-CoV-2	ORF1ab

Rapid Detection of Klebsiella pneumoniae Carrying Virulence Gene rmpA2 by Recombinase Polymerase Amplification Combined With Lateral Flow Strips

Author(s):

Na Li,Lei Wang,Fang Wang,Huimin Chen,Shuan Tao,Qing Zhu,Liping Liu,Wei Liang,Fang Ma

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

Highly virulent Klebsiella pneumoniae often causes invasive infections with high morbidity and mortality rates, posing an immense clinical challenge. Rapid and accurate detection of pathogenic bacteria is of great significance for treatment and preventive control. Conventional detection by polymerase chain reaction (PCR) is limited by a dependence on laboratory equipment and professional staff. Recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) can rapidly amplify and visualize target genes in a short period of time. The aim of this study was to develop an RPA-LFS technique for detection of the K. pneumoniae virulence gene rmpA2. Primers were designed against conserved sequences specific to the virulence gene, and primer probe design was optimized by introducing base substitution to obtain a specific and sensitive primer-probe combination for clinical detection. We tested 65 actual samples collected from clinics to evaluate the performance of the newly established RPA-LFS system in comparison with conventional PCR methods and qPCR methods. The RPA-LFS assay was performed at for 25 min a constant temperature of 37°C, and results could be observed without instrumentation. The system could specifically identify highly virulent K. pneumoniae carrying the virulence gene rmpA2 with a minimum detection limit of 10-1 ng/μL and 10 copies/μL. For the 65 clinical samples tested, The RPA-LFS assay results were in complete agreement with the qPCR results and PCR results. The RPA-LFS assay provides a rapid, accurate, and simple method for identification of highly virulent K. pneumoniae carrying rmpA2.

PrimerBankID	Target Pathogen	Target Gene
RPB0103	Klebsiella pneumoniae	rmpA2

Rapid, Simple, and Highly Specific Detection of Streptococcus pneumoniae With Visualized Recombinase Polymerase Amplification

Author(s):

Fang Wang,Yan Wang,Xia Liu,Lei Wang,Kun Wang,Chenglai Xu,Guanhong Huang,Xuzhu Gao

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

Streptococcus pneumoniae is a major pathogen that causes microbiological illness in humans. The introduction of polyvalent vaccines has resulted in a significant decrease in pneumococcal-related mortality. However, pneumococcal infections continue to be a leading cause of death in children under the age of 5 and adults over the age of 65 worldwide. A speedy and highly sensitive diagnostic tool is necessary for routine adoption to adequately manage patients and control the spread of infection. In this study, we investigated a new nucleic acid amplification technique, isothermal recombinase polymerase amplification (RPA), which amplifies DNA at 37°C under isothermal conditions with high specificity, efficiency, and rapidity. Using the autolysin gene lytA as the molecular diagnostic target, an RPA primer-probe combination was designed and optimized for the detection of S. pneumoniae. This RPA reaction produced amplification products labeled with specific chemical markers, to be detected with gold-nanoparticle-based lateral flow strips (LFS), reducing the reliance on equipment and trained personnel. The high specificity of the RPA-LFS technique was demonstrated with the specific detection of 22 strains of S. pneumoniae but not 25 closely related pathogenic bacteria. The assay showed good sensitivity, and detected S. pneumoniae down to 3.32 colony-forming units/μL. When used on clinical samples, the assay provided accurate and consistent results compared with PCR. The compliance with the culture-biochemistry method was 98.18% and the kappa index was 0.977. These results reveal that the RPA-LFS test significantly improved S. pneumoniae identification, particularly in resource-limited areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0165	Streptococcus pneumoniae	lytA

Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics

Author(s):

Menglu Hu, Zhiqiang Qiu, Zirong Bi, Tian Tian, Yongzhong Jiang, and Xiaoming Zhou

Journal:

Proceedings of the National Academy of Sciences of the United States of America

Year:

2022

Abstract:

CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.

PrimerBankID	Target Pathogen	Target Gene
RPB0196	SARS-CoV-2	O-gene
RPB0197	SARS-CoV-2	N-gene

Rapid detection of methicillin-resistant Staphylococcus aureus in positive blood-cultures by recombinase polymerase amplification combined with lateral flow strip

Author(s):

Arpasiri Srisrattakarn,Pimchanok Panpru,Patcharaporn Tippayawat,Aroonwadee Chanawong,Ratree Tavichakorntrakool,Jureerut Daduang,Lumyai Wonglakorn,Aroonlug Lulitanond

Journal:

PloS one

Year:

2022

Abstract:

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is an important bacterium that causes community and healthcare-related infections throughout the world. However, the current conventional detection methods are time-consuming. We therefore developed and evaluated a recombinase polymerase amplification-lateral flow strip (RPA-LF) approach for detection of MRSA in positive blood-culture samples. Sixty positive blood-cultures from a hospital were tested directly without DNA extraction and purification before the amplification reaction. RPA primers and probes were designed for nuc (encoding thermonuclease) and mecA (encoding penicillin-binding protein 2a) genes to diagnose S. aureus and its methicillin-resistance status. The RPA reaction occurred under isothermal conditions (45°C) within 20 min and a result was provided by the LF strip in a further 5 min at room temperature. The evaluation of RPA-LF using blood-culture samples showed 93.3% (14/15) sensitivity for identifying S. aureus, and no cross-amplification was seen [100% (45/45) specificity]. For detection of methicillin resistance, the RPA-LF test provided 100% (16/16) sensitivity and 97.7% (43/44) specificity. The RPA-LF is rapid, highly sensitive, robust and easy to use. It can be used for direct detection of MRSA with no requirement for special equipment.

PrimerBankID	Target Pathogen	Target Gene
RPB0156	Staphylococcus aureus	nuc
RPB0157	Staphylococcus aureus	mecA

Multiple ligation-Assisted recombinase polymerase amplification for highly sensitive and selective colorimetric detection of SARS-CoV-2

Author(s):

Tasnima Alam Asa, Pradeep Kumar, Jaehyeon Lee, Young Jun Seo

Journal:

Talanta

Year:

2023

Abstract:

In this paper we present a new method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), targeting a specific region "N gene." Under isothermal reaction conditions, we integrated ligation (Lig; high selectivity) and recombinase polymerase amplification (RPA; high sensitivity) processes, obtaining a robust method of detection. For point-of-care testing, we incorporated our laboratory-produced pyrophosphate ion (PPi)-sensing probe (PK-probe) for colorimetric analysis of the reaction. The total detection system was efficient and effective at diagnosing this RNA virus-mediated disease rapidly (30 min). In a full-genome SARS-CoV-2 study, our PK-probe/Lig-RPA system functioned with a limit of detection of 1160 copies/ml, with a single-mismatch level of selectively, and it was highly selective even in the presence of bacterial genomes commonly found in the human mouth and nose. This robust, straightforward, selective, efficient, and ultrasensitive colorimetric detection method, with potential for point-of-care analysis, should also be effective in detecting a diverse range of other RNA-based diseases.

PrimerBankID	Target Pathogen	Target Gene
RPB0019	SARS-CoV-2	N

Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection

Author(s):

Gaoxing Su,Min Zhu,Diyuan Li,Mengting Xu,Yuedong Zhu,Yan Zhang,Hongyan Zhu,Feng Li,Yanyan Yu

Journal:

Sensors and Actuators:B. Chemical

Year:

2022

Abstract:

The development of field-deployable detection platform amenable for multiplexed genes testing will significantly improve the efficiency and reliability during point-of-care testing (POCT) applications. In this regard, an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (designated as OC-MLFA) is proposed for SARS-CoV-2 genome detection. Taking the advantage of activation and cleavage preferences between Cas12a and Cas13a, orthogonal (two-independent-channel signal readout) CRISPR-Cas system is investigated. Lateral flow strips with two target lines are designed to accommodate the orthogonal CRISPR system. The interference between Cas12a and Cas13a channels can be effectively eliminated via the elaborate nucleic acids and lateral flow strips design. The high preamplification efficiency from reverse transcription recombinase polymerase amplification (RT-RPA) and Cas enzyme mediated trans-cleavage process bring the sensitivity of our OC-MLFA method to 10 copies per test (30 μL). Nasopharyngeal swab clinical samples with different cycle threshold (Ct) values according to the RT-PCR method were analyzed with the proposed OC-MLFA, during which 76 out of 76 detection accuracy was obtained. Featured with the multiplexed genes detection simultaneously in one reaction and colorimetric readout through single strip, the OC-MLFA we proposed herein ensures great accuracy and efficiency, which endows promising field-deployable POCT application feasibility.

PrimerBankID	Target Pathogen	Target Gene
RPB0033	SARS-CoV-2	ORF1ab
RPB0034	SARS-CoV-2	N