Lateral Flow Strip Research Articles

Rapid and sensitive detection of two fungal pathogens in soybeans using the recombinase polymerase amplification/CRISPR-Cas12a method for potential on-site disease diagnosis.

Diaporthe aspalathi and Diaporthe caulivora are two of the fungal pathogens causing soybean stem canker (SSC) in soybean, which is one of the most widespread diseases in soybean growing regions and can cause 100% loss of yield. Current methods for the detection of fungal pathogens, including morphological identification and molecular detection, are mostly limited by the need for professional laboratories and staff. To develop a detection method for potential on-site diagnosis for two of the fungal pathogens causing SSC, we designed a rapid assay combining recombinase polymerase amplification (RPA) and CRISPR-Cas12a-based diagnostics to specifically detect D. aspalathi and D. caulivora. The translation elongation factor 1-alpha gene was employed as the target gene to evaluate the specificity and sensitivity of this assay. The RPA/CRISPR-Cas12a system has excellent specificity to distinguish D. aspalathi and D. caulivora from closely related species. The sensitivities of RPA/CRISPR-Cas12a-based fluorescence detection and lateral flow assay for D. aspalathi and D. caulivora are 14.5 copies and 24.6 copies, respectively. This assay can detect hyphae in inoculated soybean stems at 12 days after inoculation and has a recovery as high as 86% for hyphae-spiked soybean seed powder. The total time from DNA extraction to detection was not more than 60 min. The method developed for rapid detection of plant pathogens includes DNA extraction with magnetic beads or rapid DNA extraction, isothermal nucleic acid amplification at 39 °C, CRISPR-Cas12a cleavage reaction at 37 °C, and lateral flow assay or endpoint fluorescence visualization at room temperature. The RPA and CRISPR-Cas12a reagents can be preloaded in the microcentrifuge tube to simplify the procedures in the field. Both RPA and CRISPR-Cas12a reaction can be realized on a portable incubator, and the results are visualized using lateral flow strips or portable flashlight. This method requires minimal equipment and operator training, and has promising applications for rapid on-site disease screening, port inspection, or controlling fungal pathogen transmission in crop. © 2023 Society of Chemical Industry.

Rapid Visual Detection of African Swine Fever Virus with a CRISPR/Cas12a Lateral Flow Strip Based on Structural Protein Gene D117L

African swine fever virus (ASFV) is a large double-stranded DNA virus that is highly infectious and seriously affects domestic pigs and wild boars. African swine fever (ASF) has caused huge economic losses to endemic countries and regions. At present, there is still a lack of effective vaccines and therapeutics. Therefore, rapid and accurate detection is essential for the prevention and control of ASF. The portable DNA endonuclease (Cas12a)-mediated lateral flow strip detection method (Cas12a-LFS) combined with recombinant polymerase amplification (RPA) has been gradually recognized as effective for virus detection including ASFV. In this study, based on the ASFV structural protein p17 gene (D117L), an RPA-Cas12a-LFS detection method was established. The detection method exhibits a sensitivity of up to two gene copies and has no cross-reaction with nine other swine viruses. Thus, the method is highly sensitive and specific. In 68 clinical samples, the coincidence rate of the p17 strip was 100%, compared to the traditional quantitative PCR (qPCR). In conclusion, we have developed a simple, rapid, sensitive, and specific ASFV visual detection method and demonstrated the potential of on-site detection of ASFV.

Rapid RNase H-dependent PCR lateral flow assay for the detection of red snapper

Red snapper (Lutjanus campechanus or Lutjanus purpureus) is one of the most commercially important and commonly mislabeled seafood species in the United States. Existing molecular methods for species identification are expensive and require being sent to an off-site laboratory, which takes multiple days to process. In this study, we have standardized a rapid rhPCR-coupled lateral flow assay for the identification of red snapper species. Our assay involved a simple heat-lysis DNA extraction procedure and a multiplex PCR reaction, consisting of a red snapper single nucleotide polymorphism (SNP)-specific rhPCR primer, thermotolerant RNase H2 enzyme, and an internal amplification control (IAC) for their onsite identification. Amplicons generated in the PCR reaction were detected using dual target lateral flow strips. The standardized assay was validated with 108 barcoded fish samples. Samples identified as Lutjanus campechanus or L. purpureus by DNA barcoding formed three distinct bands, while other fish species formed only two bands on the lateral flow strips. A minimum of 0.37 ng of crude DNA was detectable in the rhPCR reaction and generated a visible band on the lateral flow dipstick. The assay showed 100% specificity and took 90–120 min to complete. The assay generated results that can be used to authenticate red snapper at the wholesale and retail levels of seafood processing and commerce. Our results confirm the ability of rhPCR-lateral flow assays to be an alternative tool for onsite species authentication for the seafood industry.

Establishment of an ultrasensitive and visual detection platform for Neospora caninum based-on the RPA-CRISPR/Cas12a system

Neospora caninum is a protozoan parasite that causes neosporosis in cattle, and leads to a high rate of abortion and severe financial losses. Rapid and accurate detection is particularly important for preventing and controlling neosporosis. In our research, a highly effective diagnostic technique based on the RPA-CRISPR/Cas system was created to successfully identify N. caninum against the Nc5 gene, fluorescent reporter system and the lateral flow strip (LFS) biosensor were exploited to display results. The specificity and sensitivity of the PRA-CRISPR/Cas12a assay were evaluated. We discovered that it was highly specific and did not react with any other pathogens. The limit of detection (LOD) for this technology was as low as one parasite per milliliter when employing the fluorescent reporter system, and was approximately ten parasites per milliliter based on the LFS biosensor and under blue or UV light. Meanwhile, the placental tissue samples were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (59.4 %, 19/32). The canine feces were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (8.6 %, 6/70). The RPA-CRISPR/Cas12a detection procedure was successfully finished in within 90 min and offers advantages of high sensitivity and specificity, speed and low cost. The technique was better suitable for extensive neosporosis screening in non-laboratory and resource-constrained locations. This study provided a new strategy for more rapid and portable identification of N. caninum.

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.

Rapid detection of avian influenza virus based on CRISPR-Cas12a

BackgroundAvian influenza (AI) is a disease caused by the avian influenza virus (AIV). These viruses spread naturally among wild aquatic birds worldwide and infect domestic poultry, other birds, and other animal species. Currently, real-time reverse transcription polymerase chain reaction (rRT-PCR) is mainly used to detect the presence of pathogens and has good sensitivity and specificity. However, the diagnosis requires sophisticated instruments under laboratory conditions, which significantly limits point-of-care testing (POCT). Rapid, reliable, non-lab-equipment-reliant, sensitive, and specific diagnostic tests are urgently needed for rapid clinical detection and diagnosis. Our study aimed to develop a reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR method which improves on these limitations.MethodsThe Cas12a protein was purified by affinity chromatography with Ni-agarose resin and observed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Specific CRISPR RNA (crRNA) and primers targeting the M and NP genes of the AIV were designed and screened. By combining RT-RPA with the Cas12a/crRNA trans-cleavage system, a detection system that uses fluorescence readouts under blue light or lateral flow strips was established. Sensitivity assays were performed using a tenfold dilution series of plasmids and RNA of the M and NP genes as templates. The specificity of this method was determined using H1–H16 subtype AIVs and other avian pathogens, such as newcastle disease virus (NDV), infectious bursal disease virus (IBDV), and infectious bronchitis virus (IBV).ResultsThe results showed that the method was able to detect AIV and that the detection limit can reach 6.7 copies/μL and 12 copies/μL for the M and NP gene, respectively. In addition, this assay showed no cross-reactivity with other avian-derived RNA viruses such as NDV, IBDV, and IBV. Moreover, the detection system presented 97.5% consistency and agreement with rRT-PCR and virus isolation for detecting samples from poultry. This portable and accurate method has great potential for AIV detection in the field.ConclusionAn RT-RPA/CRISPR method was developed for rapid, sensitive detection of AIV. The new system presents a good potential as an accurate, user-friendly, and inexpensive platform for point-of-care testing applications.

LAMP assay coupled with a CRISPR/Cas12a system for the rapid and ultrasensitive detection of porcine circovirus-like virus in the field.

A recent outbreak of porcine circovirus-like virus (PCLV), a virus that may be associated with porcine diarrhea, has been reported in swine herds in China. The virus is spreading rapidly, causing huge economic losses to the swine farming industry. To achieve the rapid, inexpensive, and sensitive detection of PCLV, we combined loop-mediated isothermal amplification (LAMP) and the CRISPR/Cas12a system, whose fluorescence intensity readout can detect PCLV ORF4 gene levels as low as 10 copies. To overcome the need for sophisticated equipment, lateral flow strip reading technology was introduced for the first time in a LAMP-Cas12a-based system to detect PCLV. The lateral flow strip (LFS) results were readout by the naked eye, and the method was highly sensitive with a detection limit of 10 copies, with a detection time of about 60 min. In addition, the method is highly specific and has no cross-reactivity with other related viruses. In conclusion, LAMP-CRISPR/Cas12a-based assays have the advantages of rapidity, accuracy, portability, low cost, and visualization of the results. They therefore have great potential, especially for areas where specialized equipment is lacking, and can expect to be an ideal method for early diagnosis and on-site detection of PCLV.

TASER: A flexible approach for nucleic acid detection using a molecular converter

Advancing expeditious and highly responsive in vitro diagnostic techniques is paramount to ensure the efficacy of disease prevention and control measures. Although polymerase chain reaction (PCR) has historically been regarded as the preferred technique for nucleic acid detection, its application is constrained by the need for thermal cycling equipment. While isothermal amplification methods do address some of the challenges associated with PCR, the approach of quantifying amplicons has limitations in the development of highly accessible and flexible measurement of nucleic acids. In this study, we employed the translational machinery as a biological module to transform target nucleic acids into a variety of proteins. These proteins were subsequently detectable using various equipment and devices commonly accessible in various settings. These include luminometers, fluorometers, spectrophotometers, lateral flow strip sensors, and glucose meters. This approach termed target-assisted synthesis of enzyme reporters (TASER) is based on straightforward mechanisms that transform target nucleic acids into easily quantifiable proteins, thereby offering a versatile pathway for highly adaptable and cost-effective nucleic acid testing.

Rapid, sensitive, and convenient detection of Plasmodium falciparum infection based on CRISPR and its application in detection of asymptomatic infection

Rapid and convenient detection of the Plasmodium in clinically diagnosed individuals and asymptomatically infected populations is essential for global malaria eradication, especially in malaria-endemic African countries where medical equipment and professionals are relatively deficient. Here, we described a CRISPR-based diagnostic for the detection of Plasmodium falciparum, the deadliest and most prevalent species of malaria parasite in Africa, via lateral flow strip readout without the need of nucleic acid extraction. The assay exhibited 100% sensitivity on clinical samples (5 P falciparum) and significant consistency with qPCR test on asymptomatic infection samples (49 P falciparum and 51 non-P. falciparum, Kappa=0.839). An artemisinin-resistant P. falciparum strain and 4 other laboratory-cultured strains can also be detected through this assay, whereas no cross-reactivity with Plasmodium vivax was observed. A 0.001% parasitaemia (corresponding to ∼60 parasites/μL) below the “low parasite density” test threshold (200 parasites/µL) is detectable. Our study demonstrated that direct malaria detection using whole blood on the spot and the detection of both clinical and asymptomatic infections of P. falciparum are feasible. This method is expected to be employed for clinical testing and large-scale community screening in Africa and possibly other places, contributing to the accurate diagnosis and control of malaria.

CRISPR/Cas13a-Assisted accurate and portable hepatitis D virus RNA detection

ABSTRACT Background & Aims Hepatitis delta virus (HDV) infection accelerates the progression of chronic hepatitis B virus (HBV) infection, posing a large economic and health burden to patients. At present, there remains a lack of accurate and portable detection methods for HDV RNA. Here, we aim to establish a convenient, rapid, highly sensitive and specific method to detect HDV RNA using CRISPR–Cas13a technology. Methods We established fluorescence (F) and lateral flow strip (L) assays based on CRISPR–Cas13a combined with RT–PCR and RT-RAA for HDV RNA detection, respectively. we conducted a cohort study of 144 patients with HDV-IgG positive to evaluate the CRISPR–Cas13a diagnostic performance for identifying HDV in clinical samples, compared to RT–qPCR and RT-ddPCR. Results For synthetic HDV RNA plasmids, the sensitivity of RT–PCR-CRISPR-based fluorescence assays was 1 copy/μL, higher than that of RT–qPCR (10 copies/μL) and RT-ddPCR (10 copies/μL); for HDV RNA-positive samples, the sensitivity of RT-RAA-CRISPR-based fluorescence and lateral flow strip assays was 10 copies/μL, as low as that of RT–qPCR and RT-ddPCR, and the assay took only approximately 85 min. Additionally, the positivity rates of anti-HDV IgG-positive samples detected by the RT–qPCR, RT-ddPCR, RT–PCR-CRISPR fluorescence and RT-RAA-CRISPR lateral flow strip methods were 66.7% (96/144), 76.4% (110/144), 81.9% (118/144), and 72.2% (104/144), respectively. Conclusions We developed a highly sensitive and specific, as well as a portable and easy CRISPR-based assay for the detection of HDV RNA, which could be a prospective measure for monitoring the development of HDV infection and evaluating the therapeutic effect.

Development of an RPA-based CRISPR/Cas12a assay in combination with a lateral flow strip for rapid detection of toxigenic Fusarium verticillioides in maize

Fusarium verticillioides is an important phytopathogenic fungus that poses a threat to maize yield and quality in global maize-growing regions by causing Fusarium ear and stalk rot. The fungus is known to produce fumonisins, which are toxic secondary metabolites and have been associated with high incidences of esophageal cancer. The FUM1 gene is responsible for producing a crucial polyketide synthase required for fumonisin biosynthesis and is present in all pathogenic strains of F. verticillioides. This study aims to develop a rapid and accurate detection assay for F. verticillioides based on the FUM1 gene, by utilizing Chelex-100 resin for DNA extraction, recombinase polymerase amplification coupled with CRISPR/Cas12a cleavage and lateral flow detection (RPA-Cas12a-LFD) assay. The developed RPA-Cas12a-LFD assay exhibited remarkable specificity for F. verticillioides, and the lowest limit of detection was 2 ag DNA of F. verticillioides. The entire diagnostic process was completed in just 73 min, including sample DNA extraction, RPA reaction, Cas12a cleavage, and result readout. Furthermore, RPA-Cas12a-LFD assay was found to be equivalent to single-spore isolation and partial translation elongation factor 1α gene (TEF-1α) sequencing in identifying diseased samples in the field. In summary, this accurate and portable detection equipment has great potential for detecting and recognizing F. verticillioides, especially in areas where sophisticated lab equipment is not available.

Recognition-Activated Primer-Mediated Exponential Rolling Circle Amplification for Signal Probe Production and Ultrasensitive Visual Detection of Ochratoxin A with Nucleic Acid Lateral Flow Strips.

We proposed a visual strategy for rapid and ultrasensitive detection of ochratoxin A (OTA) by integration of primer-mediated exponential rolling circle amplification (P-ERCA) with a designed nucleic acid lateral flow strip (LFS). The recognition component was preimmobilized in the tube by hybridization between the immobilized functionalized aptamer and complementary ssDNA. Recognition of OTA induces the release of complementary ssDNA from the tube, which will also act as the primer of the designed P-ERCA. Three nicking sites on the template P-ERCA could contribute to the production of enormous signal probes based on the simultaneous amplification-nicking model, which can be visually measured directly with the constructed nucleic acid LFS. Importantly, the nicked signal probe can also act as the trigger of the new-round RCA, achieving exponential growth of signal probes for measurement and signal enhancement. Taking advantage of the extraordinary amplification efficiency of P-ERCA and the simplicity of LFS, this P-ERCA-LFS method demonstrates ultrasensitive detection of OTA with a visual limit of detection as low as 100 fg/mL for qualitative screening and a limit of detection of 35 fg/mL for semiquantitative analysis. This designed strategy could also be utilized as a universal method for detection of other chemical analytes with the replacement of the aptamer for recognition, and the nucleic acid LFS unit could also be a useful protocol for direct ssDNA analysis.

Paper Lateral Flow Strips Based on Gold Nanorods for Ultrasensitive Detection of Traumatic Brain Injury Biomarkers

Paper Lateral Flow Strips Based on Gold Nanorods for Ultrasensitive Detection of Traumatic Brain Injury Biomarkers

Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay.

Melioidosis is a severe and often fatal infectious disease caused by Burkholderia pseudomallei, which poses significant challenges due to its high mortality rate and frequent misdiagnosis. To effectively treat infected patients, especially in epidemic regions like Hainan with a high resistance rate to ceftazidime (CAZ), early diagnosis and resistance testing are critical. Here, we designed a strategy that combines RNase-HII-dependent PCR with lateral flow strip assay (LFSA) for simultaneous detection of B. pseudomallei and its CAZ resistance mutation. In this strategy, we utilized rhPCR technology to design specific primers with a C3-spacer modification. These primers target a 115-base pair region within the ORF2 of the B. pseudomallei type III secretion system gene cluster, as well as the P174L mutation in the penA gene, which is a major mutation associated with CAZ resistance in Hainan. The primers contain complementary RNA bases that can be hydrolyzed only in the presence of thermostable RNase-HII enzymes, facilitating subsequent PCR reactions. Additionally, we introduced two pairs of universal probes carrying fluorescein isothiocyanate (FITC) and biotin, as well as digoxin and biotin. The sequences of these universal probes align with specific regions of the primers, resulting in target products labeled with different tags. The resulting amplification products are then subjected to LFSA, where the presence of specific markers is visually interpreted based on color development. These markers flow to test line 1 (T1) and are captured by a fixed anti-FITC antibody, generating a red line that indicates the presence of B. pseudomallei. Similarly, the occurrence of a red line on test line 2 (T2), which is fixed with anti-digoxin antibodies, indicates the presence of the P174L mutation in B. pseudomallei. This protocol serves as an excellent tool for the rapid diagnosis of melioidosis and provides a basis for selecting appropriate antibiotic medication. It has the potential to revolutionize the diagnosis of melioidosis, particularly in resource-limited settings. Furthermore, this technique holds promise as a versatile tool for disease diagnosis in general. IMPORTANCE This study focused on the development of a reaction system using rhPCR to amplify a specific gene, ORF2, of B. pseudomallei and to identify the P174L mutation associated with increased drug resistance to ceftazidime (CAZ). The system incorporated universal primer probes and a simple temperature cycle reaction. The amplified products were then analyzed using lateral flow strip assay (LFSA) for strain identification and mutation interpretation. The developed system provides a reliable basis for diagnosing melioidosis and selecting appropriate drugs. Its potential impact is particularly significant in resource-limited settings where access to advanced diagnostic techniques is limited. This platform stands out for its simplicity, convenience, sensitivity, specificity, and portability. It shows promise as a point-of-care testing method for detecting single nucleotide polymorphism in genes associated with other diseases. By leveraging the advantages of this platform, researchers and healthcare professionals can potentially expand its use beyond melioidosis and apply it to the rapid detection of genetic variations in other disease-related genes.

Rapid and visual detection of grapevine fleck virus using recombinase polymerase amplification combined with lateral flow strips

AbstractGrapevine flack virus (GFkV) causes fleck disease and leads to severe symptoms when the infection occurs with multiple viruses. Restricting the propagation of GFkV‐infected grapevines is the ideal management strategy to control the spread of this virus. Therefore, the development of rapid detection methods for GFkV is urgently required. In the present study, we developed a rapid and reliable assay based on the reverse transcription‐recombinase polymerase amplification (RT‐RPA) combined with lateral flow strips (LFS) using sequence‐specific primers and a probe‐based coat protein sequences for the GFkV detection in grapevines. The GFkV RT‐RPA‐LFS assay was optimized at 38°C for 10 min and an extra 5 min LFS incubation time. The optimized RT‐RPA‐LFS assay had similar sensitivity to RT‐PCR and showed no cross‐reaction with major viruses infecting grapevines in Korea. The assay was successfully validated for GFkV detection in grapevine field samples. This study provides a reliable technique for rapidly detecting GFkV as an alternative diagnostic approach for producing virus‐free grapevine nurseries.

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 Cryptosporidium spp. in diarrheic cattle feces by isothermal recombinase polymerase amplification assays

As a zoonotic parasite, Cryptosporidium spp. could cause severe diarrhea mainly in calves and children globally. Monitoring and prevention of Cryptosporidium spp.’s prevalence is of great significance in both economy and public health aspects. In this study, specific primers and probes were designed within the conserved region of 18S rRNA gene for Cryptosporidium spp. and recombinase polymerase amplification assays based on the fluorescence monitoring (real-time RPA) as well as combined with a lateral flow strip (LFS RPA) were developed. Both of the two RPA assays allowed the exponential amplification of the target fragment within 20 min. After incubation on a metal bath at 42 °C, the LFS RPA results were displayed on the lateral flow strip within 5 min while real-time RPA allowed the real-time observation of the results in Genie III at 39 °C. The RPA assays showed high specificity for Cryptosporidium spp. without any cross-reaction with other tested pathogens causing diarrhea in cattle. With the recombinant plasmid DNA containing the 18S rRNA gene of Cryptosporidium spp. serving as a template, the limit of detection for real-time RPA and LFS RPA assays were 14.6 and 12.7 copies/reaction, respectively. Moreover, the RPA assays were validated by testing diarrheic cattle fecal samples and compared with a real-time PCR. The positive ratio of Cryptosporidium spp. was 24.04 % (44/183) and 26.23 % (48/183) in both RPA assays and real-time PCR assay, respectively, and the kappa coefficient value was 0.942. The diagnostic specificity and diagnostic sensitivity of both RPA assays were 100 % and 91.67 %, respectively. Forty-one of 48 positive samples were successfully sequenced and four Cryptosporidium species were detected, including C. parvum (n = 20), C. andersoni (n = 17), C. bovis (n = 3) and C. ryanae (n = 1). The developed RPA assays are easy to operate and faster to obtain the detection results, and they are suiting for the point-of-care detection and facilitating the prevention and control of Cryptosporidium spp. infections.

Development of Recombinase Polymerase Amplification Combined with Lateral Flow Strips for Rapid Detection of Cowpea Mild Mottle Virus.

Cowpea mild mottle virus (CPMMV) is a global plant virus that poses a threat to the production and quality of legume crops. Early and accurate diagnosis is essential for effective managing CPMMV outbreaks. With the advancement in isothermal recombinase polymerase amplification and lateral flow strips technologies, more rapid and sensitive methods have become available for detecting this pathogen. In this study, we have developed a reverse transcription recombinase polymerase amplification combined with lateral flow strips (RT-RPA-LFS) method for the detection of CPMMV, specifically targeting the CPMMV coat protein (CP) gene. The RT-RPA-LFS assay only requires 20 min at 40°C and demonstrates high specificity. Its detection limit was 10 copies/μl, which is approximately up to 100 times more sensitive than RT-PCR on agarose gel electrophoresis. The developed RT-RPA-LFS method offers a rapid, convenient, and sensitive approach for field detection of CPMMV, which contribute to controlling the spread of the virus.

Accuracy and stability enhanced honey authenticity visual tracing method via false positive-eradicating PCR assisted nucleic acid-capturing lateral flow strip

Honey authenticity guarantee is crucial for consumer health and fair-trading commerce. New visual false-positive-free molecular lateral flow strip (LFS), termed 5′-3′ exonuclease activity -directed false positive-eradicating PCR assisted lateral flow strip (FPE-PCR-LFS) was developed. This FPE-PCR-LFS explored the availability of using a signal-probe as the mediator to integrate the efficient amplification module with visual LFS module. With the genomic DNA extracted from target honey, the designed signal probe would be hydrolyzed and exhausted by the 5′-3′ exonuclease activity of Taq DNA polymerase in the amplification process. The hydrolyzed signal probe would not be recognized and capture on the T line with only C line of LFS, reflecting the authenticity of the tested honey. And as low as 0.5% authenticity can be accurately identified in commercial honey samples. Significantly, the false-positive-interference was successfully eradicated for the final visual results judgement, which would greatly widen the application of molecular PCR-LFS in various fields.