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