Development of RPA-Cas12a-fluorescence assay for rapid and reliable detection of human bocavirus 1

Author(s):

Weidong Qian,Xuefei Wang,Ting Wang,Jie Huang,Qian Zhang,Yongdong Li,Si Chen

Journal:

Animal Models and Experimental Medicine

Year:

2024

Abstract:

Human bocavirus (HBoV) 1 is considered an important pathogen that mainly affects infants aged 6-24 months, but preventing viral transmission in resource-limited regions through rapid and affordable on-site diagnosis of individuals with early infection of HBoV1 remains somewhat challenging. Herein, we present a novel faster, lower cost, reliable method for the detection of HBoV1, which integrates a recombinase polymerase amplification (RPA) assay with the CRISPR/Cas12a system, designated the RPA-Cas12a-fluorescence assay. The RPA-Cas12a-fluorescence system can specifically detect target gene levels as low as 0.5 copies of HBoV1 plasmid DNA per microliter within 40 min at 37°C without the need for sophisticated instruments. The method also demonstrates excellent specificity without cross-reactivity to non-target pathogens. Furthermore, the method was appraised using 28 clinical samples, and displayed high accuracy with positive and negative predictive agreement of 90.9% and 100%, respectively. Therefore, our proposed rapid and sensitive HBoV1 detection method, the RPA-Cas12a-fluorescence assay, shows promising potential for early on-site diagnosis of HBoV1 infection in the fields of public health and health care. The established RPA-Cas12a-fluorescence assay is rapid and reliable method for human bocavirus 1 detection. The RPA-Cas12a-fluorescence assay can be completed within 40 min with robust specificity and sensitivity of 0.5 copies/μl.

PrimerBankID	Target Pathogen	Target Gene
RPB0401	Human bocavirus 1	NP1

Portable Paper-Based Nucleic Acid Enrichment for Field Testing

Author(s):

Junyang Mei, Dandan Wang, Yiheng Zhang, Dan Wu, Jinhui Cui, Mingzhe Gan, Peifeng Liu

Journal:

Advanced Science

Year:

2023

Abstract:

Point-of-care testing (POCT) can be the method of choice for detecting infectious pathogens; these pathogens are responsible for not only infectious diseases such as COVID-19, but also for certain types of cancers. For example, infections by human papillomavirus (HPV) or Helicobacter pylori (H. pylori) are the main cause of cervical and stomach cancers, respectively. COVID-19 and many cancers are treatable with early diagnoses using POCT. A variety of nucleic acid testing have been developed for use in resource-limited environments. However, questions like unintegrated nucleic acid extraction, open detection systems increase the risk of cross-contamination, and dependence on expensive equipment and alternating current (AC) power supply, significantly limit the application of POCT, especially for on-site testing. In this paper, a simple portable platform is reported capable of rapid sample-to-answer testing within 30 min based on recombinase polymerase amplification (RPA) at a lower temperature, to detect SARS-CoV-2 virus and H. pylori bacteria with a limit of detection as low as 4 × 102 copies mL-1 . The platform used a battery-powered portable reader for on-chip one-pot amplification and fluorescence detection, and can test for multiple (up to four) infectious pathogens simultaneously. This platform can provide an alternative method for fast and reliable on-site diagnostic testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0013	SARS-CoV-2	ORF1ab
RPB0396	SARS-CoV-2	ORF1ab gene

Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM

Author(s):

Kangfeng Lin,Jianguang Guo,Xiangju Guo,Qinghan Li,Xiao Li,Zhen Sun,Zehang Zhao,Jiao Weng,Jinzhun Wu,Rui Zhang,Boan Li

Journal:

Analytica Chimica Acta

Year:

2023

Abstract:

CRISPR-Cas12a (Cpf1) is widely used for pathogen detection. However, most Cas12a nucleic acid detection methods are limited by a PAM sequence requirement. Moreover, preamplification and Cas12a cleavage are separate. Here, we developed a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM sequence with high sensitivity and specificity that offers one-tube, rapid, and visually observable detection of nucleic acids. In this system, Cas12a detection and RPA amplification are performed simultaneously, without separate preamplification and product transfer steps, and 0.2 copies/μL of DNA and 0.4 copies/μL of RNA can be detected. In the ORCD system, the activity of Cas12a is the key to the nucleic acid detection; specifically, reducing Cas12a activity increases the sensitivity of ORCD assay detection of the PAM target. Furthermore, by combining this detection technique with a nucleic acid extraction-free method, our ORCD system can be used to extract, amplify and detect samples within 30 min, as verified with tests of 82 Bordetella pertussis clinical samples with a sensitivity and specificity of 97.30% and 100% compared with PCR. We also tested 13 SARS-CoV-2 samples with RT-ORCD, and the results were consistent with RT-PCR.

PrimerBankID	Target Pathogen	Target Gene
RPB0399	SARS-CoV-2	N gene
RPB0400	Bordetella pertussis CS	IS1663 gene

Centrifugal microfluidic-based multiplex recombinase polymerase amplification assay for rapid detection of SARS-CoV-2

Author(s):

Ruoxu Li,Ning Su,Xiaodong Ren,Xiange Sun,Wenman Li,Yuwei Li,Jin Li,Chen Chen,Hong Wang,Weiping Lu,Shaoli Deng,Qing Huang

Journal:

iScience

Year:

2023

Abstract:

The COVID-19 pandemic has spread worldwide, and rapid detection of the SARS-CoV-2 virus is crucial for infection surveillance and epidemic control. This study developed a centrifugal microfluidics-based multiplex reverse transcription recombinase polymerase amplification (RT-RPA) assay for endpoint fluorescence detection of the E, N, and ORF1ab genes of SARS-CoV-2. The microscope slide-shaped microfluidic chip could simultaneously accomplish three target genes and one reference human gene (i.e., ACTB) RT-RPA reactions in 30 min, and the sensitivity was 40 RNA copies/reaction for the E gene, 20 RNA copies/reaction for the N gene, and 10 RNA copies/reaction for the ORF1ab gene. The chip demonstrated high specificity, reproducibility, and repeatability. Chip performance was also evaluated using real clinical samples. Thus, this rapid, accurate, on-site, and multiplexed nucleic acid test microfluidic chip would significantly contribute to detecting patients with COVID-19 in low-resource settings and point-of-care testing (POCT) and, in the future, could be used to detect emerging new variants of SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0014	SARS-CoV-2	N
RPB0015	SARS-CoV-2	ORF1ab
RPB0016	SARS-CoV-2	E

Evaluation of three rapid low-resource molecular tests for Nipah virus

Author(s):

Nina M Pollak,Malin Olsson,Glenn A Marsh,Joanne Macdonald,David McMillan

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

Accurate and timely diagnosis of Nipah virus (NiV) requires rapid, inexpensive, and robust diagnostic tests to control spread of disease. Current state of the art technologies are slow and require laboratory infrastructure that may not be available in all endemic settings. Here we report the development and comparison of three rapid NiV molecular diagnostic tests based on reverse transcription recombinase-based isothermal amplification coupled with lateral flow detection. These tests include a simple and fast one-step sample processing step that inactivates the BSL-4 pathogen, enabling safe testing without the need for multi-step RNA purification. The rapid NiV tests targeted the Nucleocapsid protein (N) gene with analytical sensitivity down to 1,000 copies/μL for synthetic NiV RNA and did not cross-react with RNA of other flaviviruses or Chikungunya virus, which can clinically present with similar febrile symptoms. Two tests detected 50,000-100,000 TCID50/mL (100-200 RNA copies/reaction) of the two distinct strains of NiV, Bangladesh (NiVB) and Malaysia (NiVM), and took 30 min from sample to result, suggesting these tests are well suited for rapid diagnosis under resource-limited conditions due to rapidity, simplicity, and low equipment requirements. These Nipah tests represent a first step toward development of near-patient NiV diagnostics that are appropriately sensitive for first-line screening, sufficiently robust for a range of peripheral settings, with potential to be safely performed outside of biohazard containment facilities.

PrimerBankID	Target Pathogen	Target Gene
RPB0443	Nipah virus	Nucleocapsid protein (N) gene

Rapid Detection of Staphylococcus aureus in Milk and Pork via Immunomagnetic Separation and Recombinase Polymerase Amplification

Author(s):

Runan Cheng,Lei Li,Sihui Zhen,Honglei Liu,Zhouhui Wu,Yu Wang,Zhen Wang

Journal:

Microbiology spectrum

Year:

2023

Abstract:

Separation processes using immunomagnetic beads (IMBs) are advantageous for the rapid detection of Staphylococcus aureus (S. aureus). Herein, a novel method, based on immunomagnetic separation using IMBs and recombinase polymerase amplification (RPA), was employed to detect S. aureus strains in milk and pork. IMBs were formed by the carbon diimide method using rabbit anti-S. aureus polyclonal antibodies and superparamagnetic carboxyl-Fe3O4 MBs. The average capture efficiency for 2.5 to 2.5 × 105 (CFU)/mL gradient dilution of S. aureus with 6 mg of IMBs within 60 min were a range of 62.74 to 92.75%. The detection sensitivity of the IMBs-RPA method in artificially contaminated samples was 2.5 × 101 CFU/mL. The entire detection process was completed within 2.5 h, including bacteria capture, DNA extraction, amplification, and electrophoresis. Among 20 actual samples, one case of raw milk sample and two cases of pork samples were tested positive using the established IMBs-RPA method, which were verified by the standard S. aureus inspection procedure. Therefore, the novel method shows potential for food safety supervision owing to its short detection time, higher sensitivity, and high specificity. IMPORTANCE Our study established IMBs-RPA method, which simplified the steps of bacteria separation, shortened the detection time, and realized the convenient detection of S. aureus in milk and pork samples. IMBs-RPA method was also suitable for the detection of other pathogens, providing a new method for food safety monitoring and a favorable basis for rapid and early diagnosis of diseases.

PrimerBankID	Target Pathogen	Target Gene
RPB0155	Staphylococcus aureus	nuc

Recombinase-Aided Amplification Assay for Rapid Detection of Hypervirulent Klebsiella pneumoniae (hvKp) and Characterization of the hvKp Pathotype

Author(s):

Chao Yan,Yao Zhou,Shuheng Du,Bing Du,Hanqing Zhao,Yanling Feng,Guanhua Xue,Jinghua Cui,Lin Gan,Junxia Feng,Zheng Fan,Tongtong Fu,Ziying Xu,Qun Zhang,Rui Zhang,Xiaohu Cui,Ziyan Tian,Yujie Chen,Ting Zhang,Lei Huang,Jing Yuan

Journal:

Microbiology Spectrum

Year:

2023

Abstract:

Hypervirulent Klebsiella pneumoniae (hvKp) is a major human pathogen associated with liver abscess, pneumonia, meningitis, and endophthalmitis. It is challenging to differentiate hvKp from classical Klebsiella pneumoniae (cKp) using conventional methods, necessitating the development of a rapid, sensitive, and convenient assay for hvKp detection. In this study, we constructed a recombinase-aided amplification (RAA) method targeting hvKp genes peg344 and rmpA, and also analyzed the pathogenic characteristics of hvKp. We optimized the reaction temperature and system, and evaluated its sensitivity, specificity, and clinical application. The primer and probe sets peg344-set1 and rmpA-set2 delivered significant fluorescent signals at 39°C with the shortest gene amplification times (sensitivity: 20 copies/reaction). This RAA assay showed no cross-reactivity with 15 other common pathogenic bacteria. Its applicability was confirmed by the evaluation of 208 clinical specimens, of which 45 were confirmed to be hvKp. The sensitivity and specificity of the RAA assay were both 100% compared with real-time PCR as the reference standard. To verify the assay, we also assessed the diversity of molecular characteristics among the hvKp isolates and identified serotype K1 and sequence type ST23 as the dominant clone. Virulence factors iroN and iutA were highly associated with virulence level. In conclusion, our novel RAA assay is a powerful tool for early diagnosis and epidemiological surveillance of hvKp. IMPORTANCEKlebsiella pneumoniae is the most common opportunistic bacterial species and a major threat to public health. Since the 1990s, hvKp has received increasing attention from public health officials and infectious disease specialists. Hypervirulent strains differ from classical strains in terms of phenotypic features and clinical outcomes. It is hard to identify hvKp from cKp using the conventional methods including colony morphology analysis, serum killing assays, mouse lethality assays, string tests, and real-time PCR. In this study, we established a rapid, sensitive and convenient recombinase-aided amplification assay for hvKp detection targeting virulence genes peg344 and rmpA. Our RAA assay provides an important tool for the rapid diagnosis of infectious diseases caused by hvKp, particularly in primary laboratories.

PrimerBankID	Target Pathogen	Target Gene
RPB0374	Klebsiella pneumoniae	peg344 gene
RPB0375	Klebsiella pneumoniae	rmpA gene

Cas12a/Guide RNA-Based Platforms for Rapidly and Accurately Identifying Staphylococcus aureus and Methicillin-Resistant S. aureus

Author(s):

Xiaoying Cao,Yanbin Chang,Chunqing Tao,Sen Chen,Qiuxia Lin,Chao Ling,Shifeng Huang,Hengshu Zhang

Journal:

Microbiology spectrum

Year:

2023

Abstract:

In order to ensure the prevention and control of methicillin-resistant Staphylococcus aureus (MRSA) infection, rapid and accurate detection of pathogens and their resistance phenotypes is a must. Therefore, this study aimed to develop a fast and precise nucleic acid detection platform for identifying S. aureus and MRSA. We initially constructed a CRISPR-Cas12a detection system by designing single guide RNAs (sgRNAs) specifically targeting the thermonuclease (nuc) and mecA genes. To increase the sensitivity of the CRISPR-Cas12a system, we incorporated PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). Subsequently, we compared the sensitivity and specificity of the three amplification methods paired with the CRISPR-Cas12a system. Finally, the clinical performance of the methods was tested by analyzing the fluorescence readout of 111 clinical isolates. In order to visualize the results, lateral-flow test strip technology, which enables point-of-care testing, was also utilized. After comparing the sensitivity and specificity of three different methods, we determined that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a methods were the optimal detection methods. The nuc-LAMP-Cas12a platform showed a limit of detection (LOD) of 10 aM (~6 copies μL-1), while the mecA-LAMP-Cas12a platform demonstrated a LOD of 1 aM (~1 copy μL-1). The LOD of both platforms reached 4 × 103 fg/μL of genomic DNA. Critical evaluation of their efficiencies on 111 clinical bacterial isolates showed that they were 100% specific and 100% sensitive with both the fluorescence readout and the lateral-flow readout. Total detection time for the present assay was approximately 80 min (based on fluorescence readout) or 85 min (based on strip readout). These results indicated that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a platforms are promising tools for the rapid and accurate identification of S. aureus and MRSA. IMPORTANCE The spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to global health. Isothermal amplification combined with the trans-cleavage activity of Cas12a has been exploited to generate diagnostic platforms for pathogen detection. Here, we describe the design and clinical evaluation of two highly sensitive and specific platforms, nuc-LAMP-Cas12a and mecA-LAMP-Cas12a, for the detection of S. aureus and MRSA in 111 clinical bacterial isolates. With a limit of detection (LOD) of 4 × 103 fg/μL of genomic DNA and a turnaround time of 80 to 85 min, the present assay was 100% specific and 100% sensitive using either fluorescence or the lateral-flow readout. The present assay promises clinical application for rapid and accurate identification of S. aureus and MRSA in limited-resource settings or at the point of care. Beyond S. aureus and MRSA, similar CRISPR diagnostic platforms will find widespread use in the detection of various infectious diseases, malignancies, pharmacogenetics, food contamination, and gene mutations.

PrimerBankID	Target Pathogen	Target Gene
RPB0153	Staphylococcus aureus	nuc
RPB0154	Staphylococcus aureus	mecA

CESSAT: A chemical additive-enhanced single-step accurate CRISPR\Cas13 testing system for field-deployable ultrasensitive detection and genotyping of SARS-CoV-2 variants of concern

Author(s):

Yunxiang Wang,Hong Chen,Huixia Gao,Hongjuan Wei,Yuling Wang,Kai Mu,Liyan Liu,Erhei Dai,Zhen Rong,Shengqi Wang

Journal:

Biosensors and Bioelectronics

Year:

2023

Abstract:

The continued emergence of SARS-CoV-2 variants of concern (VOCs) has raised great challenges for epidemic prevention and control. A rapid, sensitive, and on-site SARS-CoV-2 genotyping technique is urgently needed for individual diagnosis and routine surveillance. Here, a field-deployable ultrasensitive CRISPR-based diagnostics system, called Chemical additive-Enhanced Single-Step Accurate CRISPR/Cas13 Testing system (CESSAT), for simultaneous screening of SARS-CoV-2 and its five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) within 40 min was reported. In this system, a single-step reverse transcription recombinase polymerase amplification-CRISPR/Cas13a assay was incorporated with optimized extraction-free viral lysis and reagent lyophilization, which could eliminate complicated sample processing steps and rigorous reagent storage conditions. Remarkably, 10% glycine as a chemical additive could improve the assay sensitivity by 10 times, making the limit of detection as low as 1 copy/μL (5 copies/reaction). A compact optic fiber-integrated smartphone-based device was developed for sample lysis, assay incubation, fluorescence imaging, and result interpretation. CESSAT could specifically differentiate the synthetic pseudovirus of SARS-CoV-2 and its five VOCs. The genotyping results for 40 clinical samples were in 100% concordance with standard method. We believe this simple but efficient enhancement strategy can be widely incorporated with existing Cas13a-based assays, thus leading a substantial progress in the development and application of rapid, ultrasensitive, and accurate nucleic acid analysis technology.

PrimerBankID	Target Pathogen	Target Gene
RPB0444	SARS-CoV-2	N gene
RPB0445	SARS-CoV-2	S gene
RPB0446	SARS-CoV-2 (Alpha)	H69V70Del
RPB0447	SARS-CoV-2 (Beta)	K417N
RPB0448	SARS-CoV-2 (Gamma)	H655Y
RPB0449	SARS-CoV-2 (Delta)	T478K
RPB0450	SARS-CoV-2 (Omicron)	49X

A Rapid Antimicrobial Resistance Diagnostic Platform for Staphylococcus aureus Using Recombinase Polymerase Amplification

Author(s):

Chuangxin Lin,Yongmei Zeng,Zhihong Zhu,Jiayu Liao,Tiandan Yang,Yaqun Liu,Huagui Wei,Jiamin Li,Jibin Ma,Xiaoqing Wu,Guangyu Lin,Liyun Lin,Liying Chen,Huiying Huang,Weizhong Chen,Junli Wang,Feiqiu Wen,Min Lin

Journal:

Microbiology spectrum

Year:

2023

Abstract:

Antimicrobial resistance (AMR) has posed a global threat to public health. The Staphylococcus aureus strains have especially developed AMR to practically all antimicrobial medications. There is an unmet need for rapid and accurate detection of the S. aureus AMR. In this study, we developed two versions of recombinase polymerase amplification (RPA), the fluorescent signal monitoring and lateral flow dipstick, for detecting the clinically relevant AMR genes retained by S. aureus isolates and simultaneously identifying such isolates at the species level. The sensitivity and specificity were validated with clinical samples. Our results showed that this RPA tool was able to detect antibiotic resistance for all the 54 collected S. aureus isolates with high sensitivity, specificity, and accuracy (all higher than 92%). Moreover, results of the RPA tool are 100% consistent with that of PCR. In sum, we successfully developed a rapid and accurate AMR diagnostic platform for S. aureus. The RPA might be used as an effective diagnostic test in clinical microbiology laboratories to improve the design and application of antibiotic therapy. IMPORTANCE Staphylococcus aureus is a species of Staphylococcus and belongs to Gram-positive. Meanwhile, S. aureus remains one of the most common nosocomial and community-acquired infections, causing blood flow, skin, soft tissue, and lower respiratory tract infections. The identification of the particular nuc gene and the other eight genes of drug-resistant S. aureus can reliably and quickly diagnose the illness, allowing doctors to prescribe treatment regimens sooner. The detection target in this work is a particular gene of S. aureus, and a POCT is built to simultaneously recognize S. aureus and analyze genes representing four common antibiotic families. We developed and assessed a rapid and on-site diagnostic platform for the specific and sensitive detection of S. aureus. This method allows the determination of S. aureus infection and 10 different AMR genes representing four different families of antibiotics within 40 min. It was easily adaptable in low-resource circumstances and professional-lacking circumstances. It should be supported in overcoming the continuous difficulty of drug-resistant S. aureus infections, which is a shortage of diagnostic tools that can swiftly detect infectious bacteria and numerous antibiotic resistance indicators.

PrimerBankID	Target Pathogen	Target Gene
RPB0132	Staphylococcus aureus	nuc gene
RPB0133	Staphylococcus aureus	blaZ
RPB0134	Staphylococcus aureus	mecA
RPB0135	Staphylococcus aureus	ermA
RPB0136	Staphylococcus aureus	ermB
RPB0137	Staphylococcus aureus	ermC
RPB0138	Staphylococcus aureus	msrA
RPB0139	Staphylococcus aureus	tetK
RPB0140	Staphylococcus aureus	tetM
RPB0141	Staphylococcus aureus	aadD
RPB0142	Staphylococcus aureus	nuc
RPB0143	Staphylococcus aureus	blaZ
RPB0144	Staphylococcus aureus	mecA
RPB0145	Staphylococcus aureus	ermA
RPB0146	Staphylococcus aureus	ermB
RPB0147	Staphylococcus aureus	ermC
RPB0148	Staphylococcus aureus	msrA
RPB0149	Staphylococcus aureus	tetK
RPB0150	Staphylococcus aureus	tetM
RPB0151	Staphylococcus aureus	aadD

A multi-country phase 2 study to evaluate the suitcase lab for rapid detection of SARS-CoV-2 in seven Sub-Saharan African countries: Lessons from the field

Author(s):

Arianna Ceruti,Ndongo Dia,Adeleye Solomon Bakarey,Judah Ssekitoleko,Soa Fy Andriamandimby,Padra Malwengo-Kasongo,Rasheeda H A Ahmed,Rea Maja Kobialka,Jean Michel Heraud,Moussa Moise Diagne,Marie Henriette Dior Dione,Idrissa Dieng,Martin Faye,Ousmane Faye,Jean Théophile Rafisandratantsoa,Jean-Pierre Ravalohery,Claudio Raharinandrasana,Tsiry H Randriambolamanantsoa,Norosoa Razanajatovo,Iony Razanatovo,Joelinotahina H Rabarison,Phillipe Dussart,Louis Kyei-Tuffuor,Abigail Agbanyo,Olubusuyi Moses Adewumi,Adeola Fowotade,Muideen Kolawole Raifu,Patient Okitale-Talunda,Gracia Kashitu-Mujinga,Christelle Mbelu-Kabongo,Steve Ahuka-Mundeke,Anguy Makaka-Mutondo,Enas M Abdalla,Sanaa M Idris,Wisal A Elmagzoub,Rahma H Ali,Eman O M Nour,Rasha S M Ebraheem,Huda H H Ahmed,Hamadelniel E Abdalla,Musab Elnegoumi,Izdihar Mukhtar,Muatsim A M Adam,Nuha Y I Mohamed,Shahinaz A Bedri,Hamdan Mustafa Hamdan,Magid Kisekka,Monica Mpumwiire,Sharley Melissa Aloyo,Joanita Nabwire Wandera,Andrew Agaba,Rogers Kamulegeya,Hosea Kiprotich,David Patrick Kateete,Paul Kadetz,Uwe Truyen,Kamal H Eltom,Anavaj Sakuntabhai,Julius Boniface Okuni,Sheila Makiala-Mandanda,Vincent Lacoste,George Olusegun Ademowo,Michael Frimpong,Amadou Alpha Sall,Manfred Weidmann,Ahmed Abd El Wahed

Journal:

Journal of Clinical Virology

Year:

2023

Abstract:

Background: The COVID-19 pandemic led to severe health systems collapse, as well as logistics and supply delivery shortages across sectors. Delivery of PCR related healthcare supplies continue to be hindered. There is the need for a rapid and accessible SARS-CoV-2 molecular detection method in low resource settings. Objectives: To validate a novel isothermal amplification method for rapid detection of SARS-CoV-2 across seven sub-Sharan African countries. Study design: In this multi-country phase 2 diagnostic study, 3,231 clinical samples in seven African sites were tested with two reverse transcription Recombinase-Aided Amplification (RT-RAA) assays (based on SARS-CoV-2 Nucleocapsid (N) gene and RNA-dependent RNA polymerase (RdRP) gene). The test was performed in a mobile suitcase laboratory within 15 min. All results were compared to a real-time RT-PCR assay. Extraction kits based on silica gel or magnetic beads were applied. Results: Four sites demonstrated good to excellent agreement, while three sites showed fair to moderate results. The RdRP gene assay exhibited an overall PPV of 0.92 and a NPV of 0.88. The N gene assay exhibited an overall PPV of 0.93 and a NPV 0.88. The sensitivity of both RT-RAA assays varied depending on the sample Ct values. When comparing sensitivity between sites, values differed considerably. For high viral load samples, the RT-RAA assay sensitivity ranges were between 60.5 and 100% (RdRP assay) and 25 and 98.6 (N assay). Conclusion: Overall, the RdRP based RT-RAA test showed the best assay accuracy. This study highlights the challenges of implementing rapid molecular assays in field conditions. Factors that are important for successful deployment across countries include the implementation of standardized operation procedures, in-person continuous training for staff, and enhanced quality control measures.

PrimerBankID	Target Pathogen	Target Gene
RPB0437	SARS-CoV-2	N
RPB0438	SARS-CoV-2	RdRP

A Novel Sample-to-Answer Visual Nucleic Acid Detection System for Adenovirus Detection

Author(s):

Kui Sun,Xiaodong Yang,Yanan Wang,Qun Guan,Wenliang Fu,Chao Zhang,Qin Liu,Wenzheng An,Yongqi Zhao,Weiwei Xing,Donggang Xu

Journal:

Microbiology Spectrum

Year:

2023

Abstract:

Human adenoviruses (HAdVs) are common viruses that can cause local outbreaks in schools, communities and military camps, posing a huge threat to public health. An ideal POCT device for adenovirus detection in resource-limited settings is critical to control the spread of the virus. In this study, we developed an integrated and electricity-independent sample-to-answer system that can complete nucleic acid extraction, amplification, and detection at room temperature. This system is suitable for field and on-site detection because of its rapidity, sensitivity, lack of contamination, and lack of requirements of high-precision instruments and skilled technicians. It consists of two separate modules, ALP FINA (alkaline lysis with the paper-based filtration isolation of nucleic acid) and SV RPA (sealed and visual recombinase polymerase amplification). The extraction efficiency of ALP FINA can reach 48 to 84%, which is close to that of the conventional centrifuge column. The detection sensitivity of SV RPA is close to 10 copies/μL of AdvB and AdvE without aerosol contamination after repeated operations. When SV RPA was applied to the detection of nasopharyngeal swab samples of 19 patients who were infected with AdvB or AdvE as well as 10 healthy volunteers, its sensitivity and specificity reached 100%, respectively. IMPORTANCE HAdV infections are readily transmittable and, in some instances, highly contagious. Early and rapid diagnosis is essential for disease control. In this work, we developed a portable, disposable, and modularized sample-to-answer detection system for AdvB and AdvE, which rendered the entire test to be completely independent of electricity and other laboratory infrastructure. Thus, this detection system can be applied in resource-limited settings, and it has the potential to be further developed as an early diagnosis method in the field.

PrimerBankID	Target Pathogen	Target Gene
RPB0419	HAdV	AdvB AdvE

CDetection.v2: One-pot assay for the detection of SARS-CoV-2

Author(s):

Xinge Wang,Yangcan Chen,Xuejia Cheng,Si-Qi Wang,Yanping Hu,Yingmei Feng,Ronghua Jin,Kangping Zhou,Ti Liu,Jianxing Wang,Kai Pan,Bing Liu,Jie Xiang,Yanping Wang,Qi Zhou,Ying Zhang,Weiye Pan,Wei Li

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important. Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b. Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples. Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.

PrimerBankID	Target Pathogen	Target Gene
RPB0405	SARS-CoV-2	RdRp

CRISPR\Cas9-based point-of-care lateral flow biosensor with improved performance for rapid and robust detection of Mycoplasma pneumonia

Author(s):

Rong Zhu,Han Jiang,Chaoyou Li,Yan Li,Min Peng,Junfeng Wang,Qian Wu,Chao Yan,Qingli Bo,Jie Wang,Chenlin Shen,Panzhu Qin

Journal:

Analytica Chimica Acta

Year:

2023

Abstract:

Screening of acute respiratory infections causes serious challenges in urgent point-of-care scenarios where conventional methods are impractical and alternative techniques suffer from low accuracy, poor robustness, and reliance on sophisticated instruments. As an improvement to this paradigm, we report a point-of-care lateral flow biosensor (LFB) based on the recognition property of clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) and apply it to the detection of Mycoplasma pneumoniae (M. pneumoniae). The designed biosensor employs CRISPR/Cas9 for secondary recognition after preamplification of target gene using specific primer set, avoiding false positives caused by nontarget factors. The high amplification efficiency and low applicable temperatures of recombinase polymerase amplification brings the detection limit of the biosensor to 3 copies even at a preamplification temperature of 25 °C. Its practical application is further demonstrated with 100% accuracy by testing with 43 M. pneumoniae-infected specimens and 80 uninfected specimens. Additionally, the entire detection, including pretreatment, preamplification, CRISPR/Cas9 recognition, and visual analysis, can be completed in 30 min. Featured with the combination of CRISPR/Cas9 and LFB, the biosensor we developed herein ensures excellent convenience, accuracy, and robustness, which endows promising point-of-care screening potential for infectious pathogens.

PrimerBankID	Target Pathogen	Target Gene
RPB0432	Mycoplasma pneumoniae	\

Droplet digital recombinase polymerase amplification for multiplexed detection of human coronavirus

Author(s):

Ji Wook Choi, Won Ho Seo, Taejoon Kang, Taewook Kang, Bong Geun Chung

Journal:

Lab On A Chip

Year:

2023

Abstract:

Since the outbreak of coronavirus 2019 (COVID-19), detection technologies have been attracting a great deal of attention in molecular diagnosis applications. In particular, the droplet digital PCR (ddPCR) has become a promising tool as it offers absolute quantification of target nucleic acids with high specificity and sensitivity. In recent years, the combination of the isothermal amplification strategies has made ddPCR a popular method for on-site testing by enabling amplification at a constant temperature. However, the current isothermal ddPCR assays are still challenging due to inherent non-specific amplification. In this paper, we present a multiplexed droplet digital recombinase polymerase amplification (MddRPA) with precise initiation of the reaction. First, the reaction temperature and dynamic range of reverse transcription (RT) and RPA were characterized by real-time monitoring of fluorescence intensities. Using a droplet-based microfluidic chip, the master mix and the initiator were fractionated and rapidly mixed within well-confined droplets. Due to the high heat transfer and mass transfer of the droplets, the precise initiation of the amplification was enabled and the entire assay could be conducted within 30 min. The concentrations of target RNA in the range from 5 copies per μL to 2500 copies per μL could be detected with high linearity (R2 > 0.999). Furthermore, the multiplexed detection of three types of human coronaviruses was successfully demonstrated with high specificity (>96%). Finally, we compared the performance of the assay with a commercial RT-qPCR system using COVID-19 clinical samples. The MddRPA assay showed a 100% concordance with the RT-qPCR results, indicating its reliability and accuracy in detecting SARS-CoV-2 nucleic acids in clinical samples. Therefore, our MddRPA assay with rapid detection, precise quantification, and multiplexing capability would be an interesting method for molecular diagnosis of viral infections.

PrimerBankID	Target Pathogen	Target Gene
RPB0006	HCoV-229E	\
RPB0007	HCoV-OC43	\
RPB0008	HCoV-NL63	\

Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation

Author(s):

Harmen J van der Veer, Eva A van Aalen , Claire M S Michielsen, Eva T L Hanckmann, Jeroen Deckers, Marcel M G J van Borren, Jacky Flipse, Anne J M Loonen, Joost P H Schoeber, Maarten Merkx

Journal:

ACS Central Science

Year:

2023

Abstract:

Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing. Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0009	SARS-CoV-2	ORF1a_L
RPB0010	SARS-CoV-2	ORF1a_S
RPB0011	SARS-CoV-2	N1_L
RPB0012	SARS-CoV-2	N1_S

A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase-aided reverse transcription PCR

Author(s):

Guohao Fan,Xiaozhou He,Ruiqing Zhang,Fengyu Tian,Xiuli Sun,Mengyi Zhang,Fengyu Li,Xinxin Shen,Xuejun Ma

Journal:

Journal of Clinical Laboratory Analysis

Year:

2023

Abstract:

Background: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. Methods: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. Results: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR. Conclusion: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.

PrimerBankID	Target Pathogen	Target Gene
RPB0422	HAdV3	\
RPB0423	HAdV7	\
RPB0424	HRSV A	\

RNA extraction-free workflow integrated with a single-tube CRISPR-Cas-based colorimetric assay for rapid SARS-CoV-2 detection in different environmental matrices

Author(s):

Yuliang Kang, Jiali Wang, Wensi Zhang, Yuhang Xu, Bohui Xu, Guangbo Qu, Yanyan Yu, Bing Yan, Gaoxing Su

Journal:

Journal Of Hazardous Materials

Year:

2023

Abstract:

On-site environmental surveillance of viruses is increasingly important for infection prevention and pandemic control. Herein, we report a facile single-tube colorimetric assay for detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from environmental compartments. Using glycerol as the phase separation additive, reverse transcription recombinase polymerase amplification (RT-RPA), CRISPR-Cas system activation, G-quadruplex (G4) cleavage, and G4-based colorimetric reaction were performed in a single tube. To further simplify the test, viral RNA genomes used for the one-tube assay were obtained via acid/base treatment without further purification. The whole assay from sampling to visual readout was completed within 30 min at a constant temperature without the need for sophisticated instruments. Coupling the RT-RPA to CRISPR-Cas improved the reliability by avoiding false positive results. Non-labeled cost-effective G4-based colorimetric systems are highly sensitive to CRISPR-Cas cleavage events, and the proposed assay reached the limit of detection of 0.84 copies/µL. Moreover, environmental samples from contaminated surfaces and wastewater were analyzed using this facile colorimetric assay. Given its simplicity, sensitivity, specificity, and cost-effectiveness, our proposed colorimetric assay is highly promising for applications in on-site environmental surveillance of viruses.

PrimerBankID	Target Pathogen	Target Gene
RPB0003	SARS-CoV-2	N

CRISPR Cas12a-enabled biosensors coupled with commercial pregnancy test strips for the visible point-of-care testing of SARS-CoV-2 Mycoplasma pneumonia

Author(s):

Peijie Shen,Zhenjun Si,Di Huang,Zhipeng Xu,Ziyi Wang,Mengjun Fang,Zhinan Xu

Journal:

Analyst

Year:

2023

Abstract:

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has promoted the development of nucleic acid diagnosis technology. Several platforms with isothermal amplification methods have achieved sensitive and specific detection of SARS-CoV-2. However, they still suffer from complicated operations, delicate instruments, and unintuitive signal output modes. Here, a system consisting of CRISPR Cas12a-based biosensors and commercial pregnancy test strips (CRISPR-PTS) was established for the point-of-care testing of SARS-CoV-2. The target viral nucleic acids were finally reflected on the test strips through four steps, namely sample pretreatment, RT-RAA amplification, CRISPR Cas12a reaction, and separation-free hCG detection. This CRISPR-PTS assay possessed an outstanding sensitivity of as low as 1 copy per μL for SARS-CoV-2 detection and showed an excellent specificity in distinguishing the SARS-CoV-2 pseudovirus as well as other SARS-like viral clinical samples. In addition, the CRISPR-PTS assay performed well in practical applications, with 96.3% agreement versus RT-qPCR in spiked samples. With the advantages of low reagent cost, simple operation procedure, and visible signal output, CRISPR-PTS assay was expected to provide a strong supplement in the prevention and early diagnosis of infectious diseases in resource-limited situations.

PrimerBankID	Target Pathogen	Target Gene
RPB0433	SARS-CoV-2	E gene
RPB0434	MERS-CoV	ORF1a
RPB0435	MERS-CoV	upE
RPB0436	MERS-CoV	ORF1b

Rapid detection of Nipah virus using the one-pot RPA-CRISPR\Cas13a assay

Author(s):

Jing Miao,Lulu Zuo,Dongmei He,Zhixin Fang,Nicolas Berthet,Chao Yu,Gary Wong

Journal:

Virus Research

Year:

2023

Abstract:

Nipah virus (NiV) is a zoonotic pathogen with airborne transmission and high case fatality rates in humans. There is currently no treatment or vaccine against NiV infection approved for humans or animals, therefore early diagnosis is the key to control any potential outbreaks. In this study, we developed an optimized one-pot assay using recombinase polymerase amplification (RPA) coupled to CRISPR/Cas13a for the molecular detection of NiV. The one-pot RPA-CRISPR/Cas13a assay for NiV detection was specific and did not cross-react against other selected (re)-emerging pathogens. The sensitivity of the one-pot RPA-CRISPR/Cas13a assay for NiV detection can detect as little as 103 cp/μL of total synthetic NiV cDNA. The assay was then validated with simulated clinical samples. The results for the one-pot RPA-CRISPR/Cas13a assay could be visualized with either fluorescence or lateral flow strips for convenient clinical or field diagnostics, providing a useful supplement to the gold-standard qRT-PCR assay for detecting NiV detections.

PrimerBankID	Target Pathogen	Target Gene
RPB0384	Nipah virus	N gene