Recombinase polymerase amplification combined with Pyrococcus furiosus Argonaute for fast Salmonella spp. testing in food safety.

Foodborne illness caused by Salmonella spp. is one of the most prevalent public health problems globally, which have brought immeasurable economic burden and social impact to countries around the world. Neither current nucleic acid amplification detection method nor standard culture method (2-3 days) are suitable for field detection in areas with a heavy burden of Salmonella spp. Here, we developed a highly sensitive and accurate assay for Salmonella spp. detection in less than 40 min. Specifically, the invA gene of Salmonella spp. was amplified by recombinase polymerase amplification (RPA), followed by Pyrococcus furiosus Argonaute (PfAgo)-based target sequence cleavage, which could be observed by a fluorescence reader or the naked eye. The assay offered the lowest detectable concentration of 1.05 × 101 colony forming units/mL (CFU/mL). This assay had strong specificity and high sensitivity for the detection of Salmonella spp. in field samples, which indicated the feasibility of this assay.

Design and development of a rapid meat detection system based on RPA-CRISPR/Cas12a-LFD.

In recent years, meat adulteration safety incidents have occurred frequently, triggering widespread attention and discussion. Although there are a variety of meat quality identification methods, conventional assays require high standards for personnel and experimental conditions and are not suitable for on-site testing. Therefore, there is an urgent need for a rapid, sensitive, high specificity and high sensitivity on-site meat detection method. This study is the first to apply RPA combined with CRISPR/Cas12a technology to the field of multiple meat identification. The system developed by parameter optimization can achieve specific detection of chicken, duck, beef, pork and lamb with a minimum target sequence copy number as low as 1 × 100 copies/µL for 60 min at a constant temperature. LFD test results can be directly observed with the naked eye, with the characteristics of fast, portable and simple operation, which is extremely in line with current needs. In conclusion, the meat identification RPA-CRISPR/Cas12a-LFD system established in this study has shown promising applications in the field of meat detection, with a profound impact on meat quality, and provides a model for other food safety control programs.

Development of a POCT detection platform based on a locked nucleic acid-enhanced ARMS-RPA-GoldMag lateral flow assay.

In this study, a novel genotyping point-of-care testing (POCT) rapid detection device, the locked nucleic acid (LNA)-amplification refractory mutation system (ARMS)-recombinase polymerase amplification (RPA)-GoldMag lateral flow assay (LFA) platform, was provided by mining and synthesis based on prior technology. Research methods based on system-integrated innovation and knowledge-integrated generation have become a new trend in technology development. Here, we exploit the combination of LNA-coupled ARMS-RPA and gold nanoparticle probe technology for detection signal amplification, thus pioneering a new tool for accurate, rapid, and cost-effective genotyping. We also performed SNP typing detection and clinical validation of this new assay platform using common glucose-6-phosphate dehydrogenase (G6PD) gene single nucleotide polymorphism (SNP) loci, and the results demonstrated the high sensitivity, specificity, stability, accuracy and feasibility of the LNA-ARMS-RPA-GoldMag lateral flow assay platform. It is hoped that this new technology will make a significant contribution to the field of POCT rapid diagnosis and aim to expand the application space, reflecting its clinical application value and development prospects.

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

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

A Rapid Antimicrobial Resistance Diagnostic Platform for Staphylococcus aureus Using Recombinase Polymerase Amplification.

Antimicrobial resistance (AMR) has posed a global threat to public health. The Staphylococcus aureus strains have especially developed AMR to practically all antimicrobial medications. There is an unmet need for rapid and accurate detection of the S. aureus AMR. In this study, we developed two versions of recombinase polymerase amplification (RPA), the fluorescent signal monitoring and lateral flow dipstick, for detecting the clinically relevant AMR genes retained by S. aureus isolates and simultaneously identifying such isolates at the species level. The sensitivity and specificity were validated with clinical samples. Our results showed that this RPA tool was able to detect antibiotic resistance for all the 54 collected S. aureus isolates with high sensitivity, specificity, and accuracy (all higher than 92%). Moreover, results of the RPA tool are 100% consistent with that of PCR. In sum, we successfully developed a rapid and accurate AMR diagnostic platform for S. aureus. The RPA might be used as an effective diagnostic test in clinical microbiology laboratories to improve the design and application of antibiotic therapy. IMPORTANCE Staphylococcus aureus is a species of Staphylococcus and belongs to Gram-positive. Meanwhile, S. aureus remains one of the most common nosocomial and community-acquired infections, causing blood flow, skin, soft tissue, and lower respiratory tract infections. The identification of the particular nuc gene and the other eight genes of drug-resistant S. aureus can reliably and quickly diagnose the illness, allowing doctors to prescribe treatment regimens sooner. The detection target in this work is a particular gene of S. aureus, and a POCT is built to simultaneously recognize S. aureus and analyze genes representing four common antibiotic families. We developed and assessed a rapid and on-site diagnostic platform for the specific and sensitive detection of S. aureus. This method allows the determination of S. aureus infection and 10 different AMR genes representing four different families of antibiotics within 40 min. It was easily adaptable in low-resource circumstances and professional-lacking circumstances. It should be supported in overcoming the continuous difficulty of drug-resistant S. aureus infections, which is a shortage of diagnostic tools that can swiftly detect infectious bacteria and numerous antibiotic resistance indicators.

Molecular Surveillance of Artemisinin-Based Combination Therapies Resistance in Plasmodium falciparum Parasites from Bioko Island, Equatorial Guinea.

Artemisinin-based combination therapies (ACTs) resistance has emerged and could be diffusing in Africa. As an offshore island on the African continent, the island of Bioko in Equatorial Guinea is considered severely affected and resistant to drug-resistant Plasmodium falciparum malaria. However, the spatial and temporal distribution remain unclear. Molecular monitoring targeting the Pfcrt, Pfk13, Pfpm2, and Pfmdr1 genes was conducted to provide insight into the impact of current antimalarial drug resistance on the island. Furthermore, polymorphic characteristics, haplotype network, and the effect of natural selection of the Pfk13 gene were evaluated. A total of 152 Plasmodium falciparum samples (collected from 2017 to 2019) were analyzed for copy number variation of the Pfpm2 gene and Pfk13, Pfcrt, and Pfmdr1 mutations. Statistical analysis of Pfk13 sequences was performed following different evolutionary models using 96 Bioko sequences and 1322 global sequences. The results showed that the prevalence of Pfk13, Pfcrt, and Pfmdr1 mutations was 73.68%, 78.29%, and 75.66%, respectively. Large proportions of isolates with multiple copies of Pfpm2 were observed (67.86%). In Bioko parasites, the genetic diversity of Pfk13 was low, and purifying selection was suggested by Tajima's D test (-1.644, P > 0.05) and the dN/dS test (-0.0004438, P > 0.05). The extended haplotype homozygosity analysis revealed that Pfk13_K189T, although most frequent in Africa, has not yet conferred a selective advantage for parasitic survival. The results suggested that the implementation of continuous drug monitoring on Bioko Island is an essential measure. IMPORTANCE Malaria, one of the tropical parasitic diseases with a high transmission rate in Bioko Island, Equatorial Guinea, especially caused by P. falciparum is highly prevalent in this region and is commonly treated locally with ACTs. The declining antimalarial susceptibility of artemisinin-based drugs suggested that resistance to artemisinin and its derivatives is developing in P. falciparum. Copy number variants in Pfpm2 and genetic polymorphisms in Pfk13, Pfcrt, and Pfmdr1 can be used as risk assessment indicators to track the development and spread of drug resistance. This study reported for the first time the molecular surveillance of Pfpm2, Pfcrt, Pfk13, and Pfmdr1 genes in Bioko Island from 2017 to 2019 to assess the possible risk of local drug-resistant P. falciparum.

Simultaneous detection of G6PD mutations using SNPscan in a multiethnic minority area of Southwestern China.

Objectives: Baise, a multiethnic inhabited area of southwestern China, is a historical malaria-endemic area with a high prevalence of G6PD deficiency. However, few studies of G6PD deficiency have been conducted in this region. Therefore, we performed a genetic analysis of G6PD deficiency in the Baise population from January 2020 to June 2021. Methods: A SNPscan assay was developed to simultaneously detect 33 common Chinese G6PD mutations. 30 G6PD-deficient samples were used for the method's validation. Then, a total of 709 suspected G6PD-deficient samples collated from the Baise population were evaluated for G6PD status, type of mutation and effect of mutations. Results: The SNPscan test had a sensitivity of 100% [95% confidence interval (CI): 94.87%-100%] and a specificity of 100% (95% CI: 87.66%-100%) for identifying G6PD mutations. A total of fifteen mutations were identified from 76.72% (544/709) of the samples. The most common mutation was discovered to be G6PD Kaiping (24.12%), followed by G6PD Canton (17.91%), and G6PD Gaohe (11.28%). We compared the G6PD mutation spectrum among Zhuang, Han and other Southeast Asian populations, and the Zhuang population's mutation distribution was quite similar to that in the Han population. Conclusion: This study provided a detailed G6PD mutation spectrum in Baise of southwestern China and will be valuable for the diagnosis and research of G6PD deficiency in this area. Furthermore, the SNPscan assay could be used to quickly diagnose these G6PD mutations accurately.

A visual method to detect meat adulteration by recombinase polymerase amplification combined with lateral flow dipstick.

Determining the animal source in meat and meat products is crucial to prevent meat adulteration and fraud. Conventional methods require considerable operator skills, expensive instruments and are unable to provide fast mobile on-site detection systems to detect contamination of meat products. We developed a visual method based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) to identify beef (Bos taurus), sheep (Ovis aries), pork (Sus scrofa), duck (Anas platyrhynchos) and chicken (Gallus gallus). The reaction was completed within 20 min. The results were determined by the naked eye. The detection limits of the RPA-LFD assays for duck, beef, sheep, chicken and pork were 101/µL, 102/µL, 102/µL, 101/µL and 101/µL, respectively. Furthermore, the RPA-LFD assays could differentiate species in boiled, microwaved, pressure-cooked or fried samples. These RPA-LFD assays represent a rapid, mobile detection system for determining meat product contamination.

Genetic diversity and natural selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium falciparum on Bioko Island, Equatorial Guinea and global comparative analysis.

BACKGROUND: Thrombospondin-related adhesive protein (TRAP) is a transmembrane protein that plays a crucial role during the invasion of Plasmodium falciparum into liver cells. As a potential malaria vaccine candidate, the genetic diversity and natural selection of PfTRAP was assessed and the global PfTRAP polymorphism pattern was described. METHODS: 153 blood spot samples from Bioko malaria patients were collected during 2016-2018 and the target TRAP gene was amplified. Together with the sequences from database, nucleotide diversity and natural selection analysis, and the structural prediction were preformed using bioinformatical tools. RESULTS: A total of 119 Bioko PfTRAP sequences were amplified successfully. On Bioko Island, PfTRAP shows its high degree of genetic diversity and heterogeneity, with π value for 0.01046 and Hd for 0.99. The value of dN-dS (6.2231, p < 0.05) hinted at natural selection of PfTRAP on Bioko Island. Globally, the African PfTRAPs showed more diverse than the Asian ones, and significant genetic differentiation was discovered by the fixation index between African and Asian countries (Fst > 0.15, p < 0.05). 667 Asian isolates clustered in 136 haplotypes and 739 African isolates clustered in 528 haplotypes by network analysis. The mutations I116T, L221I, Y128F, G228V and P299S were predicted as probably damaging by PolyPhen online service, while mutations L49V, R285G, R285S, P299S and K421N would lead to a significant increase of free energy difference (ΔΔG > 1) indicated a destabilization of protein structure. CONCLUSIONS: Evidences in the present investigation supported that PfTRAP gene from Bioko Island and other malaria endemic countries is highly polymorphic (especially at T cell epitopes), which provided the genetic information background for developing an PfTRAP-based universal effective vaccine. Moreover, some mutations have been shown to be detrimental to the protein structure or function and deserve further study and continuous monitoring.