Development and evaluation of rapid and accurate one-tube RPA-CRISPR-Cas12b-based detection of mcr-1 and tet(X4).

The emergence and quick spread of the plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 have posed a great threat to public health and raised global concerns. It is imperative to develop rapid and accurate detection systems for the onsite surveillance of mcr-1 and tet(X4). In this study, we developed one-tube recombinase polymerase amplification (RPA) and CRISPR-Cas12b integrated mcr-1 and tet(X4) detection systems. We identified mcr-1- and tet(X4)-conserved and -specific protospacers through a comprehensive BLAST search based on the NCBI nt database and used them for assembling the detection systems. Our developed one-tube RPA-CRISPR-Cas12b-based detection systems enabled the specific detection of mcr-1 and tet(X4) with a sensitivity of 6.25 and 9 copies within a detection time of ~ 55 and ~ 40 min, respectively. The detection results using pork and associated environmental samples collected from retail markets demonstrated that our developed mcr-1 and tet(X4) detection systems could successfully monitor mcr-1 and tet(X4), respectively. Notably, mcr-1- and tet(X4)-positive strains were isolated from the positive samples, as revealed using the developed detection systems. Whole-genome sequencing of representative strains identified an mcr-1-carrying IncI2 plasmid and a tet(X4)-carrying IncFII plasmid, which are known as important vectors for mcr-1 and tet(X4) transmission, respectively. Taken together, our developed one-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems show promising potential for the onsite detection of mcr-1 and tet(X4). KEY POINTS: • One-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems were developed based on identified novel protospacers. • Both detection systems exhibited high sensitivity and specification with a sample-to-answer time of less than 1 h. • The detection systems show promising potential for onsite detection of mcr-1 and tet(X4).

Rapid visual detection of Giardia duodenalis in faecal samples using an RPA-CRISPR/Cas12a system.

Giardiasis is a common intestinal infection caused by Giardia duodenalis, which is a major economic and health burden for humans and livestock. Currently, a convenient and effective detection method is urgently needed. CRISPR/Cas12a-based diagnostic methods have been widely used for nucleic acid-based detection of pathogens due to their high efficiency and sensitivity. In this study, a technique combining CRISPR/Cas12a and RPA was established that allows the detection of G. duodenalis in faecal samples by the naked eye with high sensitivity (10-1 copies/µL) and specificity (no cross-reactivity with nine common pathogens). In clinical evaluations, the RPA-CRISPR/Cas12a-based detection assay detected Giardia positivity in 2% (1/50) of human faecal samples and 47% (33/70) of cattle faecal samples, respectively, which was consistent with the results of nested PCR. Our study demonstrated that the RPA-CRISPR/Cas12a technique for G. duodenalis is stable, efficient, sensitive, specific and has low equipment requirements. This technique offers new opportunities for on-site detection in remote and poor areas.

Cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of Shigella flexneri.

Pathogen infections including Shigella flexneri have posed a significant threat to human health for numerous years. Although culturing and qPCR were the gold standards for pathogen detection, time-consuming and instrument-dependent restrict their application in rapid diagnosis and economically less-developed regions. Thus, it is urgently needed to develop rapid, simple, sensitive, accurate, and low-cost detection methods for pathogen detection. In this study, an immunomagnetic beads-recombinase polymerase amplification-CRISPR/Cas12a (IMB-RPA-CRISPR/Cas12a) method was built based on a cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of S. flexneri in the laboratory. Firstly, S. flexneri was specifically captured and enriched by IMB (Shigella antibody-coated magnetic beads), and the genomic DNA was released and used as the template in the RPA reaction. Then, the RPA products were mixed with the pre-loaded CRISPR/Cas12a for fluorescence visualization. The results were observed by naked eyes under LED blue light, with a sensitivity of 5 CFU/mL in a time of 70 min. With no specialized equipment or complicated technical requirements, the IMB-RPA-CRISPR/Cas12a diagnostic method can be used for visual, rapid, and simple detection of S. flexneri and can be easily adapted to monitoring other pathogens.

Identification of Viruses Infecting Phalaenopsis Orchids Using Nanopore Sequencing and Development of an RT-RPA-CRISPR/Cas12a for Rapid Visual Detection of Nerine Latent Virus.

Phalaenopsis orchids are one of the most popular ornamental plants. More than thirty orchid viruses have been reported, and virus-infected Phalaenopsis orchids significantly lose their commercial value. Therefore, the development of improved viral disease detection methods could be useful for quality control in orchid cultivation. In this study, we first utilized the MinION, a portable sequencing device based on Oxford Nanopore Technologies (ONT) to rapidly detect plant viruses in Phalaenopsis orchids. Nanopore sequencing revealed the presence of three plant viruses in Phalaenopsis orchids: odontoglossum ringspot virus, cymbidium mosaic virus, and nerine latent virus (NeLV). Furthermore, for the first time, we detected NeLV infection in Phalaenopsis orchids using nanopore sequencing and developed the reverse transcription-recombinase polymerase amplification (RT-RPA)-CRISPR/Cas12a method for rapid, instrument-flexible, and accurate diagnosis. The developed RT-RPA-CRISPR/Cas12a technique can confirm NeLV infection in less than 20 min and exhibits no cross-reactivity with other viruses. To determine the sensitivity of RT-RPA-CRISPR/Cas12a for NeLV, we compared it with RT-PCR using serially diluted transcripts and found a detection limit of 10 zg/µL, which is approximately 1000-fold more sensitive. Taken together, the ONT platform offers an efficient strategy for monitoring plant viral pathogens, and the RT-RPA-CRISPR/Cas12a method has great potential as a useful tool for the rapid and sensitive diagnosis of NeLV.

Rapid and multi-target genotyping of Helicobacter pylori with digital microfluidics.

Helicobacter pylori (H. pylori) infection correlates closely with gastric diseases such as gastritis, ulcers, and cancer, influencing more than half of the world's population. Establishing a rapid, precise, and automated platform for H. pylori diagnosis is an urgent clinical need and would significantly benefit therapeutic intervention. Recombinase polymerase amplification (RPA)-CRISPR recently emerged as a promising molecular diagnostic assay due to its rapid detection capability, high specificity, and mild reaction conditions. In this work, we adapted the RPA-CRISPR assay on a digital microfluidics (DMF) system for automated H. pylori detection and genotyping. The system can achieve multi-target parallel detection of H. pylori nucleotide conservative genes (ureB) and virulence genes (cagA and vacA) across different samples within 30 min, exhibiting a detection limit of 10 copies/rxn and no false positives. We further conducted tests on 80 clinical saliva samples and compared the results with those derived from real-time quantitative polymerase chain reaction, demonstrating 100% diagnostic sensitivity and specificity for the RPA-CRISPR/DMF method. By automating the assay process on a single chip, the DMF system can significantly reduce the usage of reagents and samples, minimize the cross-contamination effect, and shorten the reaction time, with the additional benefit of losing the chance of experiment failure/inconsistency due to manual operations. The DMF system together with the RPA-CRISPR assay can be used for early detection and genotyping of H. pylori with high sensitivity and specificity, and has the potential to become a universal molecular diagnostic platform.

Sensitive and specific CRISPR-Cas12a assisted nanopore with RPA for Monkeypox detection.

Monkeypox virus (MPXV) poses a global health emergency, necessitating rapid, simple, and accurate detection to manage its spread effectively. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique has emerged as a promising next-generation molecular diagnostic approach. Here, we developed a highly sensitive and specific CRISPR-Cas12a assisted nanopore (SCAN) with isothermal recombinase polymerase amplification (RPA) for MPXV detection. The RPA-SCAN method offers a sensitivity unachievable with unamplified SCAN while also addressing the obstacles of PCR-SCAN for point-of-care applications. We demonstrated that size-counting of single molecules enables analysis of reaction-time dependent distribution of the cleaved reporter. Our MPXV-specific RPA assay achieved a limit of detection (LoD) of 19 copies in a 50 µL reaction system. By integrating 2 µL of RPA amplifications into a 20 µL CRISPR reaction, we attained an overall LoD of 16 copies/µL (26.56 aM) of MPXV at a 95% confidence level using the SCAN sensor. We also verified the specificity of RPA-SCAN in distinguishing MPXV from cowpox virus with 100% accuracy. These findings suggest that the isothermal RPA-SCAN device is well-suited for highly sensitive and specific Monkeypox detection. Given its electronic nature and miniaturization potential, the RPA-SCAN system paves the way for diagnosing a wide array of other infectious pathogens at the point of care.

Ultrasensitive one-pot detection of monkeypox virus with RPA and CRISPR in a sucrose-aided multiphase aqueous system.

IMPORTANCE: The monkeypox virus was declared as a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO) and continues to cause infection cases worldwide. Given the risk of virus evolution, it is essential to identify monkeypox virus infection in a timely manner to prevent outbreaks. This study establishes a novel one-pot recombinase polymerase amplification-Clustered Regularly Interspaced Short Palindromic Repeats (RPA-CRISPR) assay for monkeypox virus with an ultra-high sensitivity. The assay shows good specificity, accuracy, and the rapidness and convenience important for point-of-care testing. It provides an effective tool for the early diagnosis of monkeypox, which is useful for the prevention of an epidemic.

A rapid simultaneous detection of duck hepatitis A virus 3 and novel duck reovirus based on RPA CRISPR Cas12a/Cas13a.

The mixed infection of duck hepatitis A virus 3 (DHAV-3) and novel duck reovirus (NDRV) has caused significant losses to the global duck farming industry. On-site point-of-care testing of viruses plays a crucial role in the early diagnosis, prevention, and disease control. Here, we proposed an RPA-CRISPR Cas12a/Cas13a one-pot strategy (DRCFS) for rapid and simultaneous detection of DHAV-3 and NDRV. This method integrated the reaction of RPA and CRISPR Cas12a/Cas13a in a single tube, eliminating the need to open the lid during the intermediate processes and thereby avoiding aerosol contamination. On this basis, we proposed a dual RPA-CRISPR strategy coupled with a lateral flow analysis platform (DRC-LFA). This circumvented the necessity for complex instruments, enabling direct visual interpretation of results, making the test more accessible and user-friendly. Our findings demonstrated that the DRCFS method could detect DHAV-3 and NDRV at concentrations as low as 100 copy/µL, while DRC-LFA achieved limit of 101 copies/µL within 35 min. Furthermore, when DRCFS, DRC-LFA, and qPCR were employed collectively for clinical samples analysis, all three methods yielded consistent results. The specificity, sensitivity, and user-friendly of these methods rendered them invaluable for on-site virus detection.

Establishment of portable Pseudomonas aeruginosa detection platform based on one-tube CRISPR/Cas12a combined with recombinase polymerase amplification technology.

Pseudomonas aeruginosa, a common Gram-negative bacterium, is associated with diverse diseases. Its increasing resistance to antibiotics presents challenges in clinical treatment. The predominant diagnostic approach involves conventional biochemical cultures, known for their time and labor intensiveness. Despite progress in isothermal amplification studies, limitations persist, including reliance on specialized equipment, intricate primer design, and aerosol contamination. Therefore, there is a demand for enhanced clinical assays. This study successfully combined RPA and CRISPR/Cas12a techniques. Through a series of experiments involving the design and screening of lasB crRNA, the creation of lasB RPA primers, and the establishment of a streamlined RPA-CRISPR/Cas12a assay, the study developed a one-tube detection method targeting P. aeruginosa's lasB gene. The assay demonstrated inclusive behavior across standard and 21 isolates, while specifically discerning P. aeruginosa from diverse strains. Sensitivity reached 15.9 CFU/reaction. Clinical validation revealed a 97.62% concordance with traditional methods. The one-tube assay's protocol mitigated aerosol contamination. Offering precision, specificity, and sensitivity, this method shows promise for field applications in resource-scarce regions, enabling early detection and improved management of P. aeruginosa infections.

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China.

Toxoplasma gondii, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii. The lowest limit of detection of the assay was 31 copies/µL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii, showing promise for on-site surveillance. IMPORTANCE: Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii. Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.

Advances in plant pathogen detection: integrating recombinase polymerase amplification with CRISPR/Cas systems.

Plant pathogens are causing substantial economic losses and thus became a significant threat to global agriculture. Effective and timely detection methods are prerequisite for combating the damages caused by the plant pathogens. In the realm of plant pathogen detection, the isothermal amplification techniques, e.g., recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), have emerged as a fast, precise, and most sensitive alternative to conventional PCR but they often comprise high rates of non-specific amplification and operational complexity. In recent advancements, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems, particularly Cas12, have emerged as powerful tools for highly sensitive, specific, and rapid pathogen detection. Exploiting the collateral activities of Cas12, which selectively cleaves single-stranded DNA (ssDNA), novel detection platforms have been developed. The mechanism employs the formation of a triple complex molecule comprising guide RNA, Cas12 enzyme, and the substrate target nucleotide sequence. Upon recognition of the target, Cas12 indiscriminately cleaves the DNA strand, leading to the release of fluorescence from the cleaved ssDNA reporter. Integration of isothermal amplification methods with CRISPR/Cas12 enables one-step detection assays, facilitating rapid pathogen identification within 30 min at a single temperature. This integrated RPA-CRISPR/Cas12a approach eliminates the need for RNA extraction and cDNA conversion, allowing direct use of crude plant sap as a template. With an affordable fluorescence visualization system, this portable method achieves 100-fold greater sensitivity than conventional techniques. This review summarizes recent advances in RPA-CRISPR/Cas12a for detecting plant pathogens, covering primer design, field-level portability, and enhanced sensitivity.

Efficient magnetic enrichment cascade single-step RPA-CRISPR/Cas12a assay for rapid and ultrasensitive detection of Staphylococcus aureus in food samples.

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that could cause severe food poisoning. Thus, rapid, efficient, and ultrasensitive detection of S. aureus in food samples is urgently needed. Here, we report an efficient magnetic enrichment cascade single-step recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay for the ultrasensitive detection of S. aureus. Magnetic beads (MBs) functionalized with S. aureus-specific antibodies were initially used for S. aureus enrichment from the complex matrix, with 98% capture efficiency in 5 min and 100-fold sensitivity improvement compared with unenriched S. aureus. Next, a single-step RPA-CRISPR/Cas12a-based diagnostic system with optimized extraction-free bacteria lysis was constructed. This assay could detect as low as 1 copy/µL (five copies/reaction) of extracted DNA template and 10 CFU/mL of S. aureus within 40 min. Furthermore, the assay could effectively detect S. aureus in real food samples such as lake water, orange juice, pork, and lettuce, with concordant results to qPCR assays. The proposed cascade signal-amplification assay eliminates the need for lengthy bacterial culture and complex sample preparation steps. Hence, the proposed assay shows great application potential for rapid, efficient, and ultrasensitive detection of pathogens in real food samples.

A 3D-Printed Integrated Handheld Biosensor for the Detection of Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the important seafood-borne pathogens that cause a serious gastrointestinal disorder in humans. Recently, biosensors have attracted serious attention for precisely detecting and tracking risk factors in foods. However, a major consideration when fabricating biosensors is to match the low cost of portable devices to broaden its application. In this study, a 3D-printed integrated handheld biosensor (IHB) that combines RPA-CRISPR/Cas12a, a lateral flow strip (LFS), and a handheld device was developed for the ultrasensitive detection of V. parahaemolyticus. Using the preamplification of RPA on tlh gene of V. parahaemolyticus, a specific duplex DNA product was obtained to activate the trans-cleavage activity of CRISPR/Cas12a, which was then utilized to cleave the ssDNA probe. The ssDNA probe was then detected by the LFS, which was negatively correlated with the content of amplified RPA products of the tlh gene. The IHB showed high selectivity and excellent sensitivity for V. parahaemolyticus detection, and the limit of detection was 4.9 CFU/mL. The IHB also demonstrated great promise for the screening of V. parahaemolyticus in samples and had the potential to be applied to the rapid screening of other pathogen risks for seafood and marine environmental safety.

Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for naked-eye detection of Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions. RESULTS: In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min. SIGNIFICANCE: We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis.

One-Pot Molecular Diagnosis of Acute Hepatopancreatic Necrosis Disease by Recombinase Polymerase Amplification and CRISPR/Cas12a with Specially Designed crRNA.

Acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating diseases in aquaculture, causing significant economic losses in seafood supplies worldwide. Early detection is critical for its prevention, which requires reliable and fast-responding diagnosis tools with point-of-care testing (POCT) capacity. Recombinase polymerase amplification (RPA) has been combined with CRISPR/Cas12a for AHPND diagnosis with a two-step procedure, but the operation is inconvenient and has the risk of carryover contamination. Here, we develop an RPA-CRISPR one-pot assay that integrates RPA and CRISPR/Cas12a cleavage into simultaneous reactions. Using the special design of crRNA, which is based on suboptimal protospacer adjacent motifs (PAM), RPA and Cas12a are made compatible in one pot. The assay is highly specific with a good sensitivity of 102 copies/reaction. This study provides a new choice for AHPND diagnosis with a POCT facility and sets a good example for developing RPA-CRISPR one-pot molecular diagnosis assays.

Rapid and Ultrasensitive Detection of Plasmodium spp. Parasites via the RPA-CRISPR/Cas12a Platform.

Microscopic examination of thick and thin blood smears stained with Giemsa dye is considered the primary diagnostic tool for the confirmation and management of suspected clinical malaria. However, detecting gametocytes is relatively insensitive, particularly in asymptomatic individuals with low-density Plasmodium infections. To complement existing diagnostic methods, a rapid and ultrasensitive point-of-care testing (POCT) platform for malaria detection is urgently needed and necessary. A platform based on recombinase polymerase amplification (RPA) followed by CRISPR/Cas12a (referred to as RPA-CRISPR/Cas12a) was developed and optimized for the determination of Plasmodium spp. parasites, particularly Plasmodium falciparum, using a fluorescence-based assay (FBDA), lateral flow test strips (LFTS), or naked eye observation (NEO). Then, the established platform was assessed with clinical malaria isolates. Under optimal conditions, the detection threshold was 1 copy/µL for the plasmid, and the limit of detection was 3.11-7.27 parasites/µL for dried blood spots. There was no cross-reactivity against blood-borne pathogens. For the accuracies of RPA-CRISPR/Cas12a, Plasmodium spp. and P. falciparum testing were 98.68 and 94.74%, respectively. The method was consistent with nested PCR results and superior to the qPCR results. RPA-CRISPR/Cas12a is a rapid, ultrasensitive, and reliable platform for malaria diagnosis. The platform requires no or minimal instrumentation for nucleic acid amplification reactions and can be read with the naked eye. Compared with similar diagnostic methods, this platform improves the reaction speed while reducing detection requirements. Therefore, this platform has the potential to become a true POCT for malaria parasites.

Pneumatic Nano-Sieve for CRISPR-based Detection of Drug-resistant Bacteria.

The increasing prevalence of antibiotic-resistant bacterial infections, particularly methicillin-resistant Staphylococcus aureus (MRSA), presents a significant public health concern. Timely detection of MRSA is crucial to enable prompt medical intervention, limit its spread, and reduce antimicrobial resistance. Here, we introduce a miniaturized nano-sieve device featuring a pneumatically-regulated chamber for highly efficient MRSA purification from human plasma samples. By using packed magnetic beads as a filter and leveraging the deformability of the nano-sieve channel, we achieve an on-chip concentration factor of 15 for MRSA. We integrated this device with recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas detection system, resulting in an on-chip limit of detection (LOD) of approximately 100 CFU/mL. This developed approach provides a rapid, precise, and centrifuge-free solution suitable for point-of-care diagnostics, with the potential to significantly improve patient outcomes in resource-limited medical conditions.

A rapid and high-throughput Helicobacter pylori RPA-CRISPR/Cas12a-based nucleic acid detection system.

BACKGROUND: Helicobacter pylori lives in the human stomach and causes gastric cancer and other gastric diseases. The development of molecular technology has facilitated low-cost, rapid, and high-throughput detection of H. pylori. MATERIALS AND METHODS: The combination of isothermal recombinase polymerase amplification (RPA) and CRISPR-Cas12a was used for early diagnosis and monitoring of H. pylori in clinical settings. The UreB genes from 242 H. pylori strains were subjected to cluster analysis, and we designed corresponding RPA primers and screened 2 sets of CRISPR-derived RNAs (crRNAs) for accurate H. pylori recognition. We then performed specificity and sensitivity validation of seven strains using this RPA-CRISPR/Cas12a method. In addition, the cut-off values of this RPA-CRISPR/Cas12a method based on fluorescence values (i.e., RPA-CRISPR/Cas12a-FT) were determined by comparison with quantitative PCR (qPCR), and further experiments comparing different methods were performed using clinical samples. RESULTS: We developed a rapid detection system based on the combination of RPA and CRISPR-Cas12a, which was applied to the early diagnosis and monitoring of H. pylori in clinical settings. The RPA-CRISPR/Cas12a system was used to detect the UreB gene. We found that the limit of detection (LOD) for the CRISPR/Cas12a method based on the lateral flow dipstick result (i.e., CRISPR/Cas12a-LFD) was 100 copies, the cut-off value was 1.4; and for CRISPR/Cas12a-FT the LOD was 50 copies. This system was used to assess clinical samples and showed high reproducibility with proof-of-concept sensitivity, and the whole detection process was completed within 40 min. CONCLUSION: As a diagnostic method that can detect the UreB gene of H. pylori in gastric tissue samples rapidly, sensitively, visually, and in a high throughput manner, our method provides a new diagnostic option for clinicians. This system is ideal for hospitals or testing sites with limited medical resources.

Generation and application of a novel high-throughput detection based on RPA-CRISPR technique to sensitively monitor pathogenic microorganisms in the environment.

Staphylococcus aureus (S. aureus) is an important opportunistic human and animal pathogen that can cause a wide diversity of infections. Due to its environmental health risks, it is crucial to establish a time-saving, high-throughput, and highly sensitive technique for water quality surveillance. In this study, we developed a novel method to detect S. aureus in the water environment based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a. This method utilizes isothermal amplification of nucleic acids and the trans-cleavage activity of the CRISPR/Cas12a system to generate fluorescence signals with a single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporter and a naked-eye detected lateral flow assay (LFA). Our RPA-CRISPR/Cas12a detection system can reduce the detection time to 35 min and enhance the high-throughput detection threshold to ≥5 copies of pathogen DNA, which is more sensitive than that of reported. Moreover, in the lower reaches of the Jialing River in Chongqing, China, 10 water samples from the mainstream and 7 ones from tributaries were successfully monitored S. aureus for less than 35 min using RPA-CRISPR/Cas12a detection system. Taken together, a novel high-throughput RPA-CRISPR detection was established and firstly applied for sensitively monitoring S. aureus in the natural water environment.

RPA-Cas12aDS: A visual and fast molecular diagnostics platform based on RPA-CRISPR-Cas12a method for infectious bursal disease virus detection.

Infectious bursal disease (IBD), a major disease of birds, is caused by infectious bursal disease virus (IBDV). The disease can lead to immunosuppression, resulting in huge economic losses in the poultry industry. A specific, rapid, and simple detection method is important for the early diagnosis and prevention and control of IBDV. In this study, we established a naked-eye visual IBDV detection method, named "RPA-Cas12aDS", by combining recombinase polymerase amplification (RPA) with CRISPR-Cas12a-based nucleic acid detection. The detection process can be accomplished in 50 min, and uncapping contamination can be avoided. The detection results can be observed under blue or UV light. We used the RPA-Cas12aDS method to detect IBDV in bursa of Fabricius tissue samples of chickens, and the results were consistent with those obtained using commercial RT-PCR kits. This method presents great potential for visual, rapid, and point-of-care molecular diagnostics of IBDV in poultry.