A field diagnostic method for rapid and sensitive detection of mpox virus.

The mpox outbreak has subdued with fewer reported cases at the present in high-income countries. It is known that mpox virus (MPXV) infection has been epidemic for more than 50 years in African countries. The ancestral MPXV strain has changed into multiple clades, indicating the ongoing evolution of MPXV, which reflects the historical neglect of mpox in Africa, especially after smallpox eradication, and bestows the danger of more severe mpox epidemics in the future. It is thus imperative to continue the development of mpox diagnostics and treatments so we can be prepared in the event of a new mpox epidemic. In this study, we have developed an MPXV detection tool that leverages the recombinase-aid amplification assay by integrating lateral flow strips (RAA-LF) and one-step sample DNA preparation, with visible readout, no need of laboratory instrument, and ready for field deployment. The detection limit reaches 10 copies per reaction. The performance of our RAA-FL assay in diagnosing mpox clinical samples is on par with that of the quantitative polymerase chain reaction (PCR) assay. Taken together, we have developed a point-of-care RAA-LF method of high accuracy and sensitivity, readily deployable for field detection of MPXV. This diagnostic tool is expected to improve and accelerate field- and self-diagnosis, allow timely isolation and treatment, reduce the spread of MPXV, thus effectively mitigate MPXV outbreak in the future.

Rapid and facile detection of largemouth bass ranavirus with CRISPR/Cas13a.

Largemouth bass ranavirus (LMBV) is an epidemic disease that seriously jeopardizes the culture of largemouth bass（Micropterus salmoides）, and it has a very high incidence in largemouth bass. Once an outbreak occurs, it may directly lead to the failure of the culture, resulting in substantial economic losses, but there is no effective vaccine or special effective drug yet. Consequently, it is important to establish an accurate, sensitive, convenient and specific detection approach for preventing LMBV infection. The recombinant enzyme-assisted amplification (RAA) technology was used in combination with clustered regularly interspaced short palindromic repeats (CRISPR), and associated protein 13a (CRISPR/Cas13a) to detect LMBV. We designed RAA primers and CRISPR RNA (crRNA) that targeted the conserved region in the LMBV main capsid protein (MCP) gene, amplified sample nucleic acids using the RAA technology, performed CRISPR/Cas13a fluorescence detection and evaluated the sensitivity and specificity of the established method with qPCR as a control method. This technique was able to determine the results by collecting fluorescence signals, visualizing fluorescence by UV excitation and combining with lateral flow strips (LFS). The sensitivity and specificity of the established method were consistent with the qPCR method. Besides, it was performed at a constant temperature of 37 °C and the sensitivity of the reaction system was 3.1 × 101 copies/µL, with no cross-reactivity with other common aquatic pathogens. Further, the positive detection rate of the proposed method in 32 clinical samples was consistent with that of qPCR. In conclusion, our established RAA-CRISPR/Cas13 method for detecting LMBV is sensitive, simple and specific, which is applicable in the rapid on-site detection and epidemiological monitoring of LMBV.

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.

Portable detection of Salmonella in food of animal origin via Cas12a-RAA combined with an LFS/PGM dual-signaling readout biosensor.

A highly specific and sensitive rapid two-signal assay was developed for the detection of Salmonella typhimurium in foods of animal origin. The invA gene of Salmonella was used as the biorecognition element and recombinase-assisted amplification (RAA) technology for signal amplification. By utilizing the specific recognition and efficient trans-cleavage activity of CRISPR/Cas12a, point-of-care testing (POCT) for S. typhimurium was achieved via lateral flow strips (LFS) and personal glucometer (PGM) biosensors as dual signal readout systems, with sensitivities of 33 CFU/mL and 20 CFU/mL, respectively. Users can select the appropriate test system on the basis of specific application requirements: LFSs are ideal for rapid onsite screening, whereas glucometer biosensors offer precise quantitative determination. This approach simplifies the use of large instruments and overcomes site constraints, demonstrating good accuracy and applicability in animal-derived samples, with significant potential for the detection of other pathogens and for use in restricted environments.

Rapid and visual detection of tomato spotted wilt virus using recombinase polymerase amplification combined with lateral flow strips.

Tomato spotted wilt virus (TSWV) is economically important in Korea as it causes significant losses to a wide range of important ornamental and vegetable crops. Therefore, a rapid detection method is imperative for TSWV diagnosis. Specific primers and probes were designed based on the conserved sequences of the TSWV coat protein gene. In this study, an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) assay, combined with lateral flow strips (LFS), was established for rapid detection of TSWV in pepper infected leaves. The RT-RPA reaction was performed at an optimal condition of 38 °C for 10 min and an LFS incubation time of approximately 5 min. There was no cross-reactivity with other viruses infecting pepper such as cucumber mosaic virus, pepper mottle virus, pepper mild mottle virus, and broad bean wilt virus 2, thus confirming the specificity of RT-RPA-LFS. The sensitivity of the RT-RPA assay was similar to that of RT-PCR, and RT-RPA-LFS was successfully applied to detect TSWV in the pepper samples collected from the field. Thus, RT-RPA-LFS assay might be a promising candidate for quick diagnosis of TSWV-infected pepper plants.

Rapid and visual detection of milk vetch dwarf virus using recombinase polymerase amplification combined with lateral flow strips.

BACKGROUND: Milk vetch dwarf virus (MDV) is an important ssDNA virus which causes yellowing, stunting and leaf rolling symptoms on legumes. In China, the virus causes great economic losses and has recently been found to infect tobacco. The expansion of its host range and its ability to spread rapidly has given rise to the urgent need for a sensitive, specific and rapid diagnostic assay that can assist in effective disease control. METHODS: Assays based on the polymerase chain reaction combined with lateral flow strip detection (PCR-LFS) and recombinase polymerase amplification combined with LFS (RPA-LFS) were developed targeting the coat protein (CP) gene of MDV. RESULTS: The PCR and RPA assays could detect respectively 103 copies or 101 copies of MDV by agarose gel electrophoresis. The PCR-LFS and RPA-LFS assays developed could both detect as few as 101 copies per reaction at 37 °C. Both methods could detect MDV in crude leaf extracts. CONCLUSIONS: The RPA-LFS assay developed is a rapid, sensitive and specific method for detecting MDV, which is convenient and has great potential for use in the field.

Development of a lateral flow recombinase polymerase amplification assay for rapid and visual detection of Cryptococcus neoformans/C. gattii in cerebral spinal fluid.

BACKGROUND: For definitive diagnosis of cryptococcal meningitis, Cryptococcus neoformans and/or C. gattii must be identified within cerebral spinal fluid from the patients. The traditional methods for detecting Cryptococcus spp. such as India ink staining and culture are not ideal. Although sensitive and specific enough, detection of cryptococcal antigen polysaccharide has a high dose hook effect. Therefore, the aim of this study was to introduce a new rapid and simple detection method of Cryptococcus neoformans and C. gattii in cerebral spinal fluid. METHODS: The lateral flow strips combined with recombinase polymerase amplification (LF-RPA) assay was constructed to detect the specific DNA sequences of C. neoformans and C. gattii. The detection limit was evaluated using serial dilutions of C. neoformans and C. gattii genomic DNA. The specificity was assessed by excessive amount of other pathogens genomic DNA. The optimal detection time and amplification temperature were also analyzed. The diagnostic parameters were first calculated using 114 clinical specimens and then compared with that of other diagnostic method. A brief analysis and comparison of different DNA extraction methods was discussed, too. RESULTS: The LF-RPA assay could detect 0.64 pg of genomic DNA of C. neoformans per reaction within 10 min and was highly specific for Cryptococcus spp.. The system could work well at a wide range of temperature from 25 to 45 °C. The overall sensitivity and specificity were 95.2 and 95.8% respectively. As amplification template for LF-RPA assay, both cell lysates and genomic DNA produce similar experimental results. CONCLUSIONS: The LF-RPA system described here is shown to be a sensitive and specific method for the visible, rapid, and accurate detection of Cryptococcus spp. in cerebral spinal fluid and might be useful for clinical preliminary screening of cryptococcal meningitis.

A SERS-based lateral flow assay for the stroke biomarker S100-ß.

A surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) is described for the quantitative analysis of the proteinic stroke biomarker S100-ß that has to be detected at very low concentration levels. The Raman reporter 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) on gold nanoparticles (GNPs) was employed as the SERS tags. They are shown to perform much better than bare GNPs in LF strips. The S100-ß protein can be detected by this method with very low detection limits by monitoring the intensity of the characteristic Raman peak of the S100-ß protein-conjugated GNPs at 1332 cm-1. Under optimized conditions, the assay works in the 1 pg·mL-1 to 40 ng·mL-1 S100-ß concentration range, and the detection limit is as low as 0.14 pg·mL-1. This is lower by a factor of 3 compared to colorimetric or fluorimetric methods. Graphical abstract Schematic illustration of the configuration (A) and the principle of the SERS-based lateral flow assay for quantification of S100-ß (B).

Rapid and visual detection of Mycobacterium tuberculosis complex using recombinase polymerase amplification combined with lateral flow strips.

To definitively diagnose active pulmonary Tuberculosis (TB), Mycobacterium tuberculosis complex (MTBC) bacilli must be identified within clinical specimens from patients. In this study, we introduced a rapid and visual detection method of MTBC using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strips. The LF-RPA assay, read results with naked eyes, could detect as few as 5 genome copies of M. tuberculosis H37Rv (ATCC 27294) per reaction and had no cross-reactions with other control bacteria even using excessive amount of template DNA. The system could work well at a broad range of temperature 25-45 °C and reach detectable level even within 5 min. When testing a total of 137 clinical specimens, the sensitivity and specificity of the LF-RPA assay were 100% (95% CI: 95.94%-100%) and 97.92% (95% CI: 88.93%-99.95%), respectively, compared to culture identification method. Therefore, the LF-RPA system we have demonstrated is a rapid, simple, robust method for MTBC detection which, subject to the availability of a suitable sample extraction method, has the potentiality to diagnose TB at the point-of-care testing.

A novel multi-walled carbon nanotube-based antibody conjugate for quantitative and semi-quantitative lateral flow assays.

In this study, the multi-walled carbon nanotubes (MWCNTs) were applied in lateral flow strips (LFS) for semi-quantitative and quantitative assays. Firstly, the solubility of MWCNTs was improved using various surfactants to enhance their biocompatibility for practical application. The dispersed MWCNTs were conjugated with the methamphetamine (MET) antibody in a non-covalent manner and then manufactured into the LFS for the quantitative detection of MET. The MWCNTs-based lateral flow assay (MWCNTs-LFA) exhibited an excellent linear relationship between the values of test line and MET when its concentration ranges from 62.5 to 1500 ng/mL. The sensitivity of the LFS was evaluated by conjugating MWCNTs with HCG antibody and the MWCNTs conjugated method is 10 times more sensitive than the one conjugated with classical colloidal gold nanoparticles. Taken together, our data demonstrate that MWCNTs-LFA is a more sensitive and reliable assay for semi-quantitative and quantitative detection which can be used in forensic analysis.

Establishment and application of a rapid visual diagnostic method for Streptococcus agalactiae based on recombinase polymerase amplification and lateral flow strips.

This study aims to establish a rapid diagnostic method for Streptococcus agalactiae (GBS) based on recombinase polymerase amplification (RPA) and lateral flow strips (LFS). The best primer pairs designed by SIP gene were screened according to the basic RPA reaction, then the probe was designed. The reaction condition was optimized based on the color development of the LFS detection line. To ascertain the reaction specificity, 10 common clinical pathogens and 10 clinical specimens of GBS were tested. Furthermore, the reaction sensitivity was assessed by utilizing a tenfold gradient dilution of GBS genomic DNA as templates. RPA-LFS method was compared to the qPCR assay and biochemical culture method for the Kappa consistency test. The RPA-LFS technique was able to complete the amplification process within 30 min and the results were observed on lateral flow strips. The method is highly sensitive, with a minimum detection limit of 1.31 ng for GBS. The RPA-LFS method showed consistent accuracy of results compared to qPCR and the culture-biochemical method. The establishment of this method is conducive to the development of on-site immediate detection, which can provide information for the timely development of a reasonable antimicrobial treatment plan, and has a greater potential for clinical application.

Development and application of a rapid visual detection technique for VanA gene in vancomycin-resistant Enterococcus faecium.

The objective of this study was to establish a rapid visual diagnosis method for vancomycin-resistant Enterococcus faecium (VREFm) based on multienzyme isothermal rapid amplification (MIRA) combined with lateral flow strips (LFSs). The MIRA primers and probes were specifically designed to maintain the sequence of the VanA gene of VREFm. We optimized the reaction time and temperature and thoroughly assessed the specificity and sensitivity of the MIRA-LFS system. We also compared the MIRA-LFS method with the polymerase chain reaction (PCR) assay and the disc diffusion method. We then evaluated the MIRA-LFS assay for consistency testing and clinical application. The MIRA-LFS technique completed the amplification process within 30 min, and the results were observed on LFS. The method demonstrated high sensitivity, with a minimum detection limit of 1.066 CFU/µL for VREFm and exhibited specificity without cross-reactivity with other pathogenic bacteria. When applied to the detection of clinical samples, the method exhibited consistency with the PCR and agar dilution methods. The combined use of MIRA and LFS in this study facilitates simplifying the workflow for detecting VREFm, which is of great significance for rapidly detecting the enterococcal infections and preventing and controlling the nosocomial infections. IMPORTANCE: One of the key approaches to treating and controlling vancomycin-resistant Enterococcus faecium (VREFm) is an accurate and rapid diagnosis. To achieve this goal, a simple and rapid method must be constructed for immediate detection in the field. Multienzyme isothermal rapid amplification (MIRA) is an isothermal rapid amplification method that allows amplification reactions to be completed under room temperature conditions. When combined with lateral flow strips (LFSs), MIRA-LFS enables the rapid detection of pathogenic microorganisms. However, the MIRA method often produces false signals. These false signals are eliminated by using base mismatches introduced in primers and probes. The MIRA-LFS system was constructed with high specificity and sensitivity for the detection of VREfm, without the limitation of sophisticated instruments. This enables the prompt formulation of diagnostic and therapeutic decisions.

Amplification-free quantitative detection of genomic DNA using lateral flow strips for milk authentication.

With the globalization and complexity of the food supply chain, the market is becoming increasingly competitive and food fraudulent activities are intensifying. The current state of food detection faced two primary challenges. Firstly, existing testing methods were predominantly laboratory-based, requiring complex procedures and precision instruments. Secondly, there was a lack of accurate and efficient quantitative detection methods. Taking cow's milk as an example, this study introduced a novel method for nucleic acid quantification in dairy products, based on lateral flow strips (LFS). The core idea of this method is to design single-stranded DNA (ssDNA) probes to hybridize with mitochondrial genes, which are abundant, stable, and species-specific in dairy products, as detection targets. Drawing inspiration from the principles of nucleic acid amplification, this research innovatively established a new DNA hybridization method, named LAMP-Like Hybridization (HybLAMP-Like). Leveraging the denaturation and DNA polymerization functions of the bst enzyme, efficient binding of the probe and template strand was achieved. This method eliminated the need for nucleic acid amplification, simplifying the procedure and mitigating aerosol contamination, thereby ensuring the accuracy of the detection results. The method exhibited exceptional sensitivity, capable of detecting extremely low to 12.5 ng in visual inspection and 3.125 ng when using a reader. In terms of practicality, it could achieve visual detection of cow's milk content as low as 1% in adulterated dairy products. When combined with a portable LFS reader, it also enabled precise quantitative analysis of milk adulteration.

Establishment of a Nucleic Acid Detection Method for Norovirus GII.2 Genotype Based on RT-RPA and CRISPR/Cas12a-LFS.

To establish a rapid detection method for norovirus GII.2 genotype, this study employed reverse transcription recombinase polymerase amplification (RT-RPA) combined with CRISPR/Cas12a and lateral flow strip (RT-RPA-Cas12a-LFS). Here, the genome of norovirus GII.2 genotype was compared to identify highly conserved sequences, facilitating the design of RT-RPA primers and crRNA specific to the conserved regions of norovirus GII.2. Subsequently, the reaction parameters of RT-RPA were optimized and evaluated using agar-gel electrophoresis and LFS. The results indicate that the conserved sequences of norovirus GII.2 were successfully amplified through RT-RPA at 37°C for 25 minutes. Additionally, CRISPR/Cas12a-mediated cleavage detection was achieved through LFS at 37°C within 10 minutes using the amplification products as templates. Including the isothermal amplification reaction time, the total time is 35 minutes. The established RT-RPA-Cas12a-LFS method demonstrated specific detection of norovirus GII.2, yielding negative results for other viral genomes, and exhibited an excellent detection limit of 10 copies/µl. The RT-RPA-Cas12a-LFS method was further compared with qRT-PCR by analyzing 60 food-contaminated samples. The positive conformity rate was 100%, the negative conformity rate was 95.45%, and the overall conformity rate reached 98.33%. This detection method for norovirus GII.2 genotype is cost-effective, highly sensitive, specific, and easy to operate, offering a promising technical solution for field-based detection of the norovirus GII.2 genotype.

Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops.

INTRODUCTION: Genetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation. OBJECTIVE: In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection. METHODS: The RPA reaction used for amplification CaMV35S and NOS targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically. RESULTS: Detection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability. CONCLUSION: The development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.

Establishment and methodological evaluation of a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes based on CRISPR-Cas12a technology.

PURPOSE: The opportunistic pathogens causing Cryptococcal meningitis are Cryptococcus neoformans and Cryptococcus gattii species complexes. At present, clinical detection methods for this condition include culture, ink staining, and cryptococcal antigen detection. In addition, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and real-time quantitative PCR (qPCR) can be applied for the detection of Cryptococcus. Nevertheless, these methods cannot achieve point-of-care detection (POCT); thus, there is a pressing need to establish a fast, sensitive, and effective detection method. METHODS: Recombinase polymerase amplification (RPA) and clustered regularly spaced short palindromic repeat (CRISPR) techniques are effective tools for achieving rapid POCT. In this study, RPA was combined with CRISPR-Cas12a to establish a fast, sensitive, and specific detection method for cryptococcal meningitis. RESULTS: This study included RPA-Cas12a fluorescence detection and RPA-Cas12a immunochromatographic detection, which can be performed within 50 min. Moreover, the detection limit was as low as 102 copies/µL. Interestingly, the developed method demonstrated satisfactory specificity and no cross-reactivity with other fungi and bacteria. 36 clinical samples were tested, and the consistency between the test results and those obtained using the commonly used clinical culture method was 100 %. CONCLUSION: In this study, a rapid detection method for Cryptococcus neoformans and Cryptococcus gattii species complexes was developed based on CRISPR-Cas12a technology, characterized by its high sensitivity and specificity, ease of use, and cost-effectiveness, making it suitable for on-site detection.

Cas12a/Guide RNA-Based Platform for Rapidly and Accurately Detecting blaKPC Gene in Carbapenem-Resistant Enterobacterales.

Purpose: Accurate detection and identification of pathogens and their associated resistance mechanisms are essential prerequisites for implementing precision medicine in the management of Carbapenem-resistant Enterobacterales (CRE). Among the various resistance mechanisms, the production of KPC carbapenemase is the most prevalent worldwide. Consequently, this study aims to develop a convenient and precise nucleic acid detection platform specifically for the blaKPC gene. Methods: The initial phase of our research methodology involved developing a CRISPR/Cas12a detection framework, which was achieved by designing highly specific single-guide RNAs (sgRNAs) targeting the blaKPC gene. To enhance the sensitivity of this system, we incorporated three distinct amplification techniques-polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA)-into the CRISPR/Cas12a framework. Subsequently, we conducted a comparative analysis of the sensitivity and specificity of these three amplification methods when used in combination with the CRISPR/Cas12a system. Additionally, we assessed the clinical applicability of the methodologies by evaluating fluorescence readouts from 80 different clinical isolates. Furthermore, we employed lateral flow assay technology to provide a visual representation of the results, facilitating point-of-care testing. Results: Following a comparative analysis of the sensitivity and specificity of the three methods, we identified the RPA-Cas12a approach as the optimal detection technique. Our findings demonstrated that the limit of detection (LoD) of the RPA-Cas12a platform was 1 aM (~1 copy/µL) for plasmid DNA and 5 × 10³ fg/µL for genomic DNA. Furthermore, both the sensitivity and specificity of the platform achieved 100% upon validation with 80 clinical isolates. Conclusion: These findings suggest that the developed RPA-Cas12a platform represents a promising tool for the cost-effective, convenient, and accurate detection of the blaKPC gene.

Meat Authenticity Made Easy: DNA Extraction-Free Rapid Onsite Detection of Duck and Pork Ingredients in Beef and Lamb Using Dual-Recombinase-Aided Amplification and Multiplex Lateral Flow Strips.

Meat adulteration is a major global concern that poses a threat to public health and consumer rights. However, current detection techniques, such as quantitative polymerase chain reaction (qPCR) and gas chromatography-mass spectrometry, are time-consuming and require sophisticated equipment. In this study, we developed a rapid onsite identification method for animal-derived ingredients by utilizing a fast nucleic acid lysis buffer to expedite the release of sample nucleic acids and combined it with dual-recombinase-aided amplification (dual-RAA) technology and visual multiplex lateral flow strips (MLFSs). Our method successfully detected duck- and bovine-derived, porcine- and bovine-derived, duck- and ovine-derived, and porcine- and ovine-derived meat in a rapid 20 min onsite detection assay, with a detection limit of 101 copies/50 µL reaction system for target genes. Moreover, our method accurately detected adulterated meat with proportions as low as 1:999. These findings have significant implications for food safety and the protection of consumer rights.

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

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.

A rapid and visual detection of Staphylococcus haemolyticus in clinical specimens with RPA-LFS.

Staphylococcus haemolyticus (S. haemolyticus), which is highly prevent in the hospital environment, is an etiological factor for nosocomial infections. Point-of-care rapid testing (POCT) of S. haemolyticus is not possible with the currently used detection methods. Recombinase polymerase amplification (RPA) is a novel isothermal amplification technology with high sensitivity and specificity. The combination of RPA and lateral flow strips (LFS) can achieve rapid pathogen detection, enabling POCT. This study developed an RPA-LFS methodology using a specific probe/primer pair to identify S. haemolyticus. A basic RPA reaction was performed to screen the specific primer from 6 primer pairs targeting mvaA gene. The optimal primer pair was selected based on agarose gel electrophoresis, and the probe was designed. To eliminate false-positive results caused by the byproducts, base mismatches were introduced in the primer/probe pair. The improved primer/probe pair could specifically identify the target sequence. To explore the optimal reaction conditions, the effects of reaction temperature and duration of the RPA-LFS method were systematically investigated. The improved system enabled optimal amplification at 37 °C for 8 min, and the results were visualized within 1 min. The S. haemolyticus detection sensitivity of the RPA-LFS method, whose performance was unaffected by contamination with other genomes, was 0.147 CFU/reaction. Furthermore, we analyzed 95 random clinical samples with RPA-LFS, quantitative polymerase chain reaction (qPCR), and traditional bacterial-culture assays and found that the RPA-LFS had 100% and 98.73% compliance rates with the qPCR and traditional culture method, respectively, which confirms its clinical applicability. In this study, we designed an improved RPA-LFS assay based on the specific probe/primer pair for the detection of S. haemolyticus via rapid POCT, free from the limitations of the precision instruments, helping to make diagnoses and treatment decisions as soon as possible.