Recombinase-aided Amplification Research Articles

Simultaneous Detection of Helicobacter pylori and Clarithromycin Resistance Mutations Using RAA-CRISPR/Cas13a Assay.

Infection caused by Helicobacter pylori (H. pylori) affects approximately 50% of the global population. It is a major pathogenic factor for chronic gastritis and gastric cancer. Besides, the resistance to antibiotics such as clarithromycin could reduce the eradication rate. Currently, there is an urgent need for a swift, easy to perform, and highly sensitive detection method for H. pylori and clarithromycin resistance. We used FAM/Digoxin labeled primers to amplify specific H. pylori 23S rRNA fragments by Recombinase Aided Amplification (RAA), and resistance mutations were distinguished using CRISPR/Cas13a system combined with lateral flow strip. Twenty-eight saliva samples were analyzed using qPCR, gene sequencing and this method to evaluate the detection efficiency. We developed a simultaneous detection method for H. pylori and clarithromycin resistance mutations named sensitive H. pylori easy-read dual detection (SHIELD). The results showed both A2142G and A2143G mutant DNAs causing clarithromycin resistance could be distinguished from the wild type with a concentration of 50 copies/μL, and no cross-reaction with other 5 common gastrointestinal bacteria was observed. For the detection of H. pylori in 28 saliva samples, the positive predictive value of this method was 100% (19/19) in comparison with qPCR. For detecting clarithromycin resistance, the positive predictive value of this method was 84.6% (11/13) compared with gene sequencing. SHIELD assay showed high sensitivity and specificity in detecting H. pylori and clarithromycin resistance mutations. It could be a potential measure in the rapid detection of H. pylori, large-scale screening and guiding clinical medication.

Novel methods for the rapid and sensitive detection of Nipah virus based on a CRISPR/Cas12a system.

Nipah virus (NiV), a bat-borne zoonotic viral pathogen with high infectivity and lethality to humans, has caused severe outbreaks in several countries of Asia during the past two decades. Because of the worldwide distribution of the NiV natural reservoir, fruit bats, and lack of effective treatments or vaccines for NiV, routine surveillance and early detection are the key measures for containing NiV outbreaks and reducing its influence. In this study, we developed two rapid, sensitive and easy-to-conduct methods, RAA-CRISPR/Cas12a-FQ and RAA-CRISPR/Cas12a-FB, for NiV detection based on a recombinase-aided amplification (RAA) assay and a CRISPR/Cas12a system by utilizing dual-labeled fluorophore-quencher or fluorophore-biotin ssDNA probes. These two methods can be completed in 45 min and 55 min and achieve a limit of detection of 10 copies per μL and 100 copies per μL of NiV N DNA, respectively. In addition, they do not cross-react with nontarget nucleic acids extracted from the pathogens causing similar symptoms to NiV, showing high specificity for NiV N DNA detection. Meanwhile, they show satisfactory performance in the detection of spiked samples from pigs and humans. Collectively, the RAA-CRISPR/Cas12a-FQ and RAA-CRISPR/Cas12a-FB methods developed by us would be promising candidates for the early detection and routine surveillance of NiV in resource-poor areas and outdoors.

Rapid detection of avian leukemia virus using CRISPR/Cas13a based lateral flow dipstick.

Avian leukemia virus (ALV) is one of the main pathogens of poultry tumor diseases, and has caused significant economic losses to the poultry industry since its discovery. Therefore, establishing a rapid detection method is essential to effectively prevent and control the spread of ALV. In this study, specific CRISPR RNA (crRNA) and recombinase-aided amplification (RAA) primers with T7 promoter were designed based on the relatively conserved sequence of avian leukemia virus. When crRNA recognized the target sequence, Cas13a protein was activated to cut the reporting probes, and then the detection results were read by using lateral flow dipstick (LFD). The RAA-CRISPR/Cas13a-LFD reaction system was constructed. The RAA amplification time, Cas13a protein concentration, crRNA concentration and CRISPR reaction time were optimized to evaluate the specificity, sensitivity and reproducibility of the system. Finally, RAA-CRISPR/Cas13a-LFD method was compared with Polymerase chain reaction (PCR)-Agarose electrophoresis method and qPCR method in the detection of clinical samples, and the reliability of RAA-CRISPR/Cas13a-LFD method was evaluated. The results showed that the RAA-CRISPR/Cas13a-LFD method could effectively amplify the target gene at 37°C for 40 min, and the test results could be determined by LFD visual observation. The method had good specificity and no cross-reaction with Marek's disease virus (MDV), Fowl adenovirus (FAdV), Infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), Infectious laryngotracheitis virus (ILTV), and Infectious bronchitis virus (IBV). The minimum detection limit of the method was 100 copies/μL, and it had good repeatability and stability. The coincidence rate of clinical detection reached 97.69% and 99.23%. In summary, this study established a simple, efficient, accurate and visualized ALV detection method, which can be used for the prevention and rapid clinical diagnosis of avian leukosis (AL).

Development and application of a recombinase-aided amplification combined with a lateral flow dipstick assay for rapid visual detection of anguillid herpesvirus 1.

Eel (Anguilla sp.) is an important freshwater-cultured species with high economic value in China. Anguillid herpesvirus 1 (AngHV-1) has been proven to be the pathogen of "mucus sloughing and haemorrhagic septicaemia disease" in eels, resulting in significant mortality and substantial losses to the eel industry. Current diagnostic methods for detecting AngHV-1 are limited to laboratory-based tests, for example, conventional end-point PCR and qPCR. Therefore, there is an urgent need to develop an accurate, rapid, and simple detection method for on-site diagnosis of AngHV-1. In this study, we developed a recombinase-aided amplification combined lateral flow dipstick (RAA-LFD) assay for the detection of AngHV-1. The RAA-LFD assay can be performed within a temperature range of 18-45°C, with a reaction time of just 10 min for amplification. Importantly, the established RAA-LFD assay exhibited no reactivity with other common aquatic viral pathogens, indicating its high specificity. The limit of detection for this method is 102 copies of AngHV-1, which is more sensitive than the established conventional end-point PCR method similarly targeting ORF95. Clinical detection of the diseased samples demonstrated that the accuracy of RAA-LFD was significantly higher than that of the conventional end-point PCR. In conclusion, the developed RAA-LFD assay has proven to be a convenient, rapid, sensitive, and reliable tool for on-site diagnosis of AngHV-1. This advancement will be invaluable for the prevention and control of AngHV-1 in the eel farming industry.

Rapid Visual Detection of Glaesserella parasuis with a Real-Time Recombinase-Aided Amplification Assay

Glaesserella parasuis is a specific bacterial pathogen of Glässer’s disease which causes significant economic losses to the swine industry. Dependable and rapid detection of G. parasuis is crucial to prevent and control Glässer’s disease outbreaks. In this study, a recombinase-aided amplification (RAA) assay based on the infB gene was developed to rapid detect G. parasuis. The novel method performs isothermal detection at 42°C for real-time analysis or visualization and data analysis (RAA-VDA). The developed assay showed high specificity for G. parasuis detection without cross-reactions to other clinically important swine pathogens. The analytical sensitivity of real-time RAA was 67.17 copies per reaction with 95% reliability, which was comparable to the G. parasuis quantitative real-time PCR (qPCR). However, the detection limit of RAA-VDA was 142.43 copies per reaction with 95% reliability. The coefficient of variation analysis of the intrabatch and interbatch experimental replicate results were less than 4.30% and 6.74%, respectively, indicating the real-time RAA assay had high repeatability and reproducibility. A total of 108 clinical tissue samples were used to evaluate the clinical diagnostic performance. The diagnostic accordance rates of qPCR with real-time RAA and RAA-VDA were 100% and 98.15% (106/108), respectively. This system combined instrumental analysis and visualized analysis to accomplish a new try for rapid detection of G. parasuis in clinical practice.

Development and performance of recombinase-aided amplification (RAA) assay for detecting Schistosoma haematobium DNA in urine samples

Rapid diagnosis of urogenital schistosomiasis caused by Schistosoma haematobium requires an accurate and timely assay, especially for low-intensity S. haematobium infection cases and in non-endemic areas. The mitochondrial cytochrome c oxidase 1 (cox1) gene fragment of S. haematobium was selected as detection target as this short fragment, which can be rapidly sequenced and yet possess good diagnostic resolution. A pair of primers and a fluorescent probe were designed according to the principle of recombinase-aided amplification (RAA), which was subsequently optimized and applied as an S. haematobium-specific RAA assay. Its diagnostic performance was validated for sensitivity and specificity in comparison to microscopy-based egg counting after urine filtration. The RAA assay could detect as little as 10 copies/μL of S. haematobium recombinant plasmid, and no cross-reactions were observed with S. mansoni, S. japonicum, Ancylostoma duodenale, Clonorchis sinensis, Echinococcus granulosus, or Ascaris lumbricoides. This test can be conducted at 39 °C and the whole RAA reaction can be completed within 20 min. The validation of the RAA assay showed that it had 100 % consistency with urine-egg microscopy, as it does not require an elaborate reading tool, is simple to use, and should be useful for field diagnostics and point-of-care applications.

Rapid detection of H5 subtype avian influenza virus using CRISPR Cas13a based-lateral flow dipstick

Due to its high mortality rate, highly pathogenic avian influenza (HPAI), a notifiable animal illness designated by the World Organisation for Animal Health (WOAH), has caused enormous financial losses to the poultry sector. The H5 subtype of avian influenza virus (H5-AIV) is regarded as the most common highly pathogenic avian influenza virus (HPAIV) that threatens public health and safety. Virus isolation and reverse transcription quantitative PCR (RT-qPCR) are usually used to detect H5-AIV and are important for the timely diagnosis and control of H5-AIV. However, these methods are time-consuming and require a significant amount of effort. In this study, we established a recombinase-aided amplification (RAA) combined with CRISPR-Cas13a and lateral flow dipstick (LFD) assay for the detection of H5-AIV. The results showed that the process can be completed within 40 min at 37°C. The method had a detection limit of 0.1 copy/μL, which was comparable to the RT-qPCR. There was no cross-reactivity with H3-AIV, H7-AIV, H9-AIV, H10-AIV, IBV, NDV, RVA and DAstV. The kappa value of RT-RAA-Cas13a-LFD and RT-qPCR in 380 clinical samples was 0.89 (κ>0.75). In conclusion, we established a convenient, efficient and accurate method to detect H5-AIV, and the results can be visualized and interpreted using LFD, which can be adapted to the needs of grassroots laboratories and field-deployable assays. This approach provides a new perspective for clinical H5-AIV diagnosis and has great potential for application in clinical quarantine of the poultry farming.

Preliminary application of recombinase -aided amplification in detection of Clonorchis sinensis metacercariae in freshwater fish

To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay. Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples. Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ2 = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ2 = 11.20, P < 0.05) and fluorescent RAA assay (χ2 = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05). Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.

Development and pre-clinical evaluation of a Zika virus diagnostic for low resource settings.

Zika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks. With the majority of cases reported in low-resource locations, simple, low-cost detection methods are considered highly desirable. Here we have developed a sensitive and specific ZIKV diagnostic using reverse transcription recombinase-aided amplification (RT-RAA) coupled with lateral flow detection (LFD) targeting a highly conserved region of the ZIKV NS1 gene. We show our rapid, isothermal-ZIKV-diagnostic (Iso-ZIKV-Dx) can detect 500 copies of synthetic ZIKV RNA/μL in under 30 min at a constant 39°C. Using simulated urine samples, we observed that Iso-ZIKV-Dx also detects as low as 34.28 RNA copies/reaction of ZIKV (MR766 strain). Specificity testing confirmed that our test does not detect any co-circulating flaviviruses (dengue, West Nile, Japanese encephalitis, Murray Valley encephalitis and yellow fever viruses) or chikungunya virus. Sample processing results show complete inactivation of ZIKV (MR766 strain) in 5 min at room temperature using our novel viral RNA sample preparation reagent. Furthermore, lateral flow strips testing demonstrates positive diagnoses in as little as 5 min in running buffer. Contrary to conventional RT-qPCR, our Iso-ZIKV-Dx does not require expensive machinery, specialised laboratory settings or extensively trained personnel. Pre-clinical evaluation demonstrates that our test exhibits robust, in-field capabilities without compromising sensitivity or specificity. When compared to the gold-standard RT-qPCR, our Iso-ZIKV-Dx test offers an array of applications that extend beyond diagnostics alone, including potential for surveillance and monitoring of ZIKV vector competency.

Detection of Clostridium perfringens Using Novel Methods Based on Recombinase-Aided Amplification Assay-Assisted CRISPR/Cas12a System

Clostridium perfringens is a highly versatile pathogen of humans and animals. Rapid and sensitive detection methods for C. perfringens are urgently needed for the timely implementation of control. In this study, to provide novel promising methods for the detection of C. perfringens, two rapid, sensitive, and instrument-free C. perfringens detection methods based on recombinase-aided amplification (RAA) assay and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (CRISPR/Cas12a) system were developed depending on fluorescence signal (RAA-CRISPR/Cas12a-FL) and lateral flow strip (RAA-CRISPR/Cas12a-LFS), respectively. The limit of detection of the RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12a-LFS methods is 2 copies and 20 copies of C. perfringens genomic DNA per reaction, respectively, and the whole process can be completed in 1 hr. Moreover, these two methods show no cross-reactivity with nontarget bacteria, which were used as a negative control to evaluate the specificity of two developed methods in the detection of C. perfringens and have 100% consistent with real-time polymerase chain reaction tests for 12 clinical samples collected from 2 Chinese Milu at Beijing Milu Ecological Research Center and 6 spiked samples from human blood and stool. Overall, the constructed C. perfringens detection methods, RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12a-LFS, have great potential as a novel detection scheme for the early diagnosis of C. perfringens infection in humans and animals.

Recombinase-aided amplification coupled with lateral flow dipstick for efficient and accurate detection of Bombyx mori nucleopolyhedrovirus.

The infection of Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the main causes of economic losses in sericulture. Thus, it is essential to establish rapid and effective method for BmNPV detection. In the present study, we have developed a recombinase-aided amplification (RAA) to amplify the BmNPV genomic DNA at 37°C within 30min, and achieved a rapid detection method by coupling with a lateral flow dipstick (LFD). The RAA-LFD method had a satisfactory detection limit of 6 copies/μL of recombinant plasmid pMD19-T-IE1, and BmNPV infection of silkworm can be detected 12h post-infection. This method was highly specific for BmNPV, and without cross-reactivity to other silkworm pathogens. In contrast to conventional polymerase chain reaction (PCR), the RAA-LFD assay showed higher sensitivity, cost-saving, and especially is apt to on-site detection of BmNPV infection in the sericulture production.

Development of a recombinase-aided amplification combined with a lateral flow dipstick assay for rapid detection of H7 subtype avian influenza virus.

Avian influenza viruses (AIV) pose a significant persistent threat to the public health and safety. It is estimated that there have been over 100 outbreaks caused by various H7 subtypes of avian influenza viruses (AIV-H7) worldwide, resulting in over 33 million deaths of poultry. In this study, we developed a recombinase-aided amplification combined with a lateral flow dipstick assay for the detection of hemagglutinin (HA) genes to provide technical support for rapid clinical detection of AIV-H7. The results showed that the assay can complete the reaction within 30 min at a temperature of 39°C. Specificity tests demonstrated that there was no cross-reactivity with other common poultry pathogens, including Newcastle disease virus (NDV) and infections bronchitis virus (IBV). The detection limit of this assay was 1 × 101 copies/μL, while RT-qPCR method was 1 × 101 copies/μL, and RT-PCR was 1 × 102 copies/μL. The κ value of the RT-RAA-LFD and RT-PCR assay in 132 avian clinical samples was 0.9169 (p < 0.001). These results indicated that the developed RT-RAA-LFD assay had good specificity, sensitivity, stability and repeatability and may be used for rapid detection of AIV-H7 in clinical diagnosis.

One-Step Reverse-Transcription Recombinase-Aided Amplification CRISPR/Cas12a-Based Lateral Flow Assay for Fast Field Screening and Accurate Differentiation of Four Major Tobamoviruses Infecting Tomato and Pepper.

Several tobamoviruses cause substantial economic losses to tomato and pepper crops globally, especially the pepper mild mosaic virus (PMMoV), tomato brown rugose fruit virus (ToBRFV), tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV). A fast and accurate detection method is essential for virus identification. An all-in-one reaction method combining a one-step reverse-transcription recombinase-aided amplification (RT-RAA) and CRISPR/Cas12a-based lateral flow assay in one mixture was developed to rapidly screen and accurately differentiate among these four tobamoviruses for field detection in tomato and pepper plants. With a generic RT-RAA primer set and a mix of four specific crRNAs, along with a portable metal incubator and the use of a crude extraction method, this method screened for PMMoV, ToBRFV, ToMV, and ToMMV concurrently in less than 1 h, enabling field workers to take action immediately. The accurate differentiation of these four viruses could be achieved by later adding a single specific crRNA.

Development of a recombinase-aided amplification method combined with lateral flow dipstick assay to detect Porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is a pathogenic virus that suppresses the immune system of pigs, impacting their health and causing economic losses. Rapid diagnostic tools for early detection of PCV2 are critical to disease prevention and control. Several molecular techniques have been established for detecting PCV2 but costly equipment and time-consuming methods are unsuitable for field inspection. In this study, we developed a recombinase-aided amplification combined with lateral flow dipstick (RAA-LFD) assay to compare with polymerase chain reaction (PCR) and quantitative PCR (qPCR) in detecting PCV2 in suspected field samples. To amplify RAA products, 15 primer pairs were designed from the conserved region of the open reading frame (ORF) 1 gene based on multiple alignments of eight PCV2 genotypes. The most efficient primer pair and conditions for the RAA-LFD assay were tested and selected. Limit of detection, repeatability, and reproducibility were determined using the constructed plasmid. DNA was extracted from positive samples for specificity testing as well as from 100 field samples to compare the detection of the RAA-LFD assay with PCR and qPCR. The F1/R1 primer pair was chosen and labeled with fluorescein isothiocyanate at the 5' end of the forward primer and with biotin at the 5' end of the reverse primer. The limit of detection of the RAA-LFD assay was 10 copies/μL at 38°C for 30 min. The RAA-LFD assay was repeatable and reproducible, with no cross-reaction with PCV3, Actinobacillus pleuropneumoniae, Porcine epidemic diarrhea virus, Classical swine fever virus, Porcine reproductive and respiratory syndrome virus - North America strain (PRRSV-US) and Porcine reproductive and respiratory syndrome virus - European strain (PRRSV-EU). Based on testing with 100 samples, the developed RAA showed 100% specificity and 90.56% and 85.71% sensitivity when compared to PCR and qPCR, respectively Cohen's kappa coefficients showed a good agreement with the established techniques. The RAA-LFD assay targeting the ORF1 gene was highly sensitive, specific, quick, and simple to perform in the field.

Multiplex bacteria detection using one-pot CRISPR/Cas13a-based droplet microfluidics

High-throughput detection of bacteria at low levels is critical in public health, food safety, and first response. Herein, for the first time, we present a platform based on droplet microfluidics coupling with the recombinase aided amplification (RAA)-assisted one-pot clustered regularly interspaced short palindromic repeats together with CRISPR-associated proteins 13a (CRISPR/Cas13a) assay, and droplet encoding strategy for accurate and sensitive determination of nucleic acids from various foodborne pathogens. The workflow takes full advantage of CRISPR/Cas13a signal amplification and droplet confinement effects, which enhances the detection sensitivity and enables end-point quantitation. Meanwhile, by varying the color of droplets, the number of bacteria detected at the same time is greatly improved. It possesses the capability to simultaneously detect seven different types of foodborne pathogens. Notably, the system is also applied to real food samples with satisfactory results. Overall, in view of superiorities in high sensitivity, outstanding selectivity, and large-scale multiplexing, the one-pot CRISPR/Cas13a-based droplet microfluidic system could be expanded and universalized for identifying other bacteria.

FAdV-4 Promotes Expression of Multiple Cytokines and Inhibits the Proliferation of aHEV in LMH Cells.

Single or mixed infections of multiple pathogens such as avian hepatitis E virus (aHEV) and avian leukosis virus subgroup J (ALV-J) have been detected in numerous laying hens with severe liver injury in China. Thus, aHEV and immunosuppressive viruses are speculated to cause co-infections. In this study, co-infection with aHEV and fowl adenovirus (FAdV) was confirmed by nested RT-PCR and recombinase-aided amplification combined with gene sequencing in two flocks with severe liver injury. Subsequently, the two reference strains, aHEV and FAdV-4, were inoculated into LMH cells to identify their co-infection potential. Confocal microscopy revealed aHEV and FAdV-4 co-infected LMH cells. In addition, the replication dynamics of aHEV and FAdV-4 along with the expression levels of immuno-cytokines were measured. The results indicated colocalization of aHEV and FAdV-4 and inhibition of viral replication in LMH cells. The transcription levels of MDA5, Mx, OASL, and IFN-α were significantly upregulated in LMH cells, whereas those of immune-related factors induced by FAdV-4 were downregulated upon FAdV-4 and aHEV co-infection. These results confirmed the co-infection of aHEV and FAdV-4 in vitro and prompted the antagonistic pathogenic effects of FAdV-4 and aHEV, thereby providing novel insights into the counterbalancing effects of these viruses.

Establishment of two assays based on reverse transcription recombinase-aided amplification technology for rapid detection of H5 subtype avian influenza virus.

The avian influenza virus (AIV), a member of the type A influenza virus group, is harbored by avian hosts. The highly pathogenic H5 subtype of AIV has inflicted significant financial damage on the poultry industry and also presents a possible pandemic hazard to the worldwide human population. There is an urgent need for a detection method that is both rapid and accurate in identifying H5-AIV. In this study, two assays based on reverse transcription recombinase-aided amplification technology (RT-RAA) have been developed to detect H5-AIV, namely, real-time fluorescence and reverse transcription recombinase-aided amplification (RF-RT-RAA) and reverse transcription recombinase-aided amplification combined lateral flow dipstick (RT-RAA-LFD). The completion of the two methods can be achieved in 30 min, and there was no cross-reaction with the nucleic acid of other avian pathogens. The results indicated that the lowest detectable limit of RF-RT-RAA and RT-RAA-LFD for H5-AIV detection was 1 copy/μL, which was 100 times higher than that of RT-PCR (102 copies/μL). A total of 376 samples, consisting of 350 clinical and 26 experimental samples, underwent testing utilizing virus isolation, RF-RT-RAA and RT-RAA-LFD methods, respectively. The results demonstrated a high degree of consistency between those assays. In summary, both the RF-RT-RAA and RT-RAA-LFD methods demonstrated excellent specificity and sensitivity, making them ideal for use in both laboratory and field settings.IMPORTANCEAvian influenza virus (AIV) subtype H5 is a highly contagious zoonotic disease and a serious threat to the farming industry and public health. Traditional detection methods, including virus isolation and real-time PCR, require tertiary biological laboratories and are time-consuming and complex to perform, making it difficult to rapidly diagnose H5 subtype avian influenza viruses. In this study, we successfully developed two methods, namely, RF-RT-RAA and RT-RAA-LFD, for rapid detection of H5-AIV. The assays are characterized by their high specificity, sensitivity, and user-friendliness. Moreover, the results of the reaction can be visually assessed, which are suitable for both laboratory testing and grassroots farm screening for H5-AIV.

A fluorescence sensing array based on photonic crystals and RAA for sensitive and high-throughput detection of Salmonella in foods

Salmonella can cause serious foodborne diseases, which is a major concern for global public health. The sensitive and fast analysis of Salmonella is of importance for food safety. Herein, we developed a fluorescence sensing array based on photonic crystals (PCs) and recombinase-aided amplification (RAA) for high-throughput and high-sensitive analysis of Salmonella in foods. In this assay, RAA was employed for powerful amplification of the target DNA of Salmonella, and the generated dsDNA amplicon was embedded with nucleic acid dye SYBR Green I (SGI), emitting fluorescence. Furthermore, the fluorescence signal was physically enhanced by PCs array; meanwhile, the high-throughput and sensitive detection was realized by microplate reader. Because of the isothermal reaction and lyophilized regent of RAA, the proposed array possessed gentle operation and stable amplification performance. Besides, a small volume of loading samples with 2 μL was required on the PCs array for powerful fluorescence enhancement. Due to the synergistic signal amplification effect of RAA and photonic crystals, a low limit of detection (LOD) of 10 CFU/mL was achieved. Besides, the proposed array exhibited excellent performance with 100% accuracy in 20 spiked food samples. Therefore, the proposed method with the advantages of sensitivity and high throughput showed great application potential.

A Novel RAA Combined Test Strip Method Based on Dual Gene Targets for Pathogenic Vibrio vulnificus in Aquatic Products.

Vibrio vulnificus can cause disease in aquatic animals and humans, therefore, rapid and simple field detection of pathogenic V. vulnificus is important for early disease prevention. In this study, a novel recombinase-aided amplification (RAA) combined test strip with double T-lines (RAA-TS-DTL) was developed for the rapid detection of V. vulnificus in aquatic products. Pathogenic V. vulnificus was detected using the virulence vvhA gene and the housekeeping gene gyrB gene as the dual target of the test strip. The RAA-TS-DTL method showed 100% specificity for V. vulnificus, and no cross-reaction was observed with Vibrio spp. or other bacteria (n = 14). Furthermore, sensitive detection of V. vulnificus in oysters was achieved. The LODs of the gyrB and vvhA genes were 6 CFU/mL and 23 CFU/mL, respectively, which was about five times higher than that of the commercial test strip. The method was validated with spiked samples (n = 60) of fish, shrimp and oyster. The consistency between RAA-TS-DTL and the traditional culture method was 97.9%. In addition, the entire process of detection, including preparation of the sample, could be completed within 50 min. Our results indicated that the developed RAA-TS-DTL was a reliable and useful tool for rapid screening or on-site detection of pathogenic V. vulnificus in aquatic products and aquaculture water.

Visual detection of fowl adenovirus serotype 4 via a portable CRISPR/Cas13a-based lateral flow assay

ABSTRACT The widespread occurrence of fowl adenovirus serotype 4 (FAdV-4)-induced hepatitis-hydropericardium syndrome (HHS) has led to significant economic losses for the poultry industry. A sensitive, accurate, and practical FAdV-4 diagnostic approach is urgently required to limit the incidence of the disease. In the present study, a practical method for detecting FAdV-4 was developed using the CRISPR/Cas13a system and recombinase-aided amplification. The approach was based on 37°C isothermal detection with visible results being achieved. The detection limit of the target gene with this approach was only 101 copies/μl, making it very sensitive and specific. Clinical samples fared well when tested with the Cas13a detection method. For identifying FAdV-4, this novel detection approach was found to be sensitive, specific, and effective. RESEARCH HIGHLIGHTS First study using the CRISPR/Cas13a-based lateral flow detection assay for FAdV-4 detection. The results can be observed by the naked eye. The developed assay could provide an alternative tool for detection of FAdV-4 with minimal equipment.