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The red‐crowned crane (Grus japonensis), a Class I protected animal in China, inhabits Northeast Asia, including China, Russia, and Japan. As sex‐monomorphic birds, red‐crowned cranes cannot be directly distinguished between females and males through observation. Molecular methods are accurate and stable for sex identification in birds and are widely used in zoos and farms. With the development of isothermal techniques, recombinase‐aided amplification (RAA) has provided novel insights into bird sexing owing to its low equipment dependence and rapid amplification. Advancements in the Pyrococcus furiosus Argonaute (PfAgo) biosensor have facilitated clinical detection. In this study, an innovative sex identification system was developed by integrating RAA and PfAgo in red‐crowned cranes. The RAA‐PfAgo system identified both females and males with remarkable accuracy. Via proper design of primers set, gDNA and probe, the RAA‐PfAgo system can complete visual detection, with detection limits between 0.35 ng/μL and 0.035 ng/μL under optimal conditions. The test samples exhibited strong green fluorescence in females, whereas no fluorescence was observed in males under blue light. The results of RAA‐PfAgo in the field were consistent with those obtained using conventional PCR. This study provides a high degree of rapidity, accuracy, and sensitivity for the sex identification of red‐crowned cranes. This study presents an innovative approach for sex identification in the red‐crowned crane (Grus japonensis) by combining recombinase‐aided amplification (RAA) with the Pyrococcus furiosus Argonaute (PfAgo) biosensor. The RAA‐PfAgo system enables rapid, accurate, and highly sensitive molecular sexing of these sex‐monomorphic birds, with a key advantage being the visual detection of fluorescence. The system achieves a detection limit as low as 0.35 ng/μL and demonstrates consistent performance with traditional PCR methods.

The most prevalent viruses currently causing diarrhea are norovirus and rotavirus, and rapid and sensitive detection methods are essential for the early diagnosis of disease. The purpose of this study was to establish a sensitive single‐tube two‐stage nucleic acid amplification method—reverse transcription recombinase‐assisted PCR (RT‐RAP)—for simultaneous detection of norovirus GII and group A Rotavirus, with the first stage consisting of isothermal reverse transcription recombinase‐aided amplification (RT‐RAA) and the second stage consisting of qPCR (quantitative PCR). RT‐RAP is more sensitive than either RT‐RAA or qRT‐PCR (quantitative RT‐PCR) alone. And the addition of a barrier that can be disassembled after heating enabled the detection of samples within 1 h in a single closed tube. Sensitivity was 10 copies/reaction of norovirus (Novs) GII and group A rotavirus (RVA). In parallel, two hundred fecal specimens were used to evaluate the method and compare it with a commercial fluorescent quantitative RT‐PCR. The data showed kappa values of 0.957 and 0.98 (p < 0.05) for detecting Novs GII and RVA by the two methods, indicating the potential of the newly established assay to be applied to clinical and laboratory testing. A sensitive single‐tube two‐stage nucleic acid amplification method—reverse transcription recombinase‐assisted PCR (RT‐RAP)—for simultaneous detection of norovirus GII and group A Rotavirus with the first stage consisting of isothermal reverse transcription recombinase‐aided amplification (RT‐RAA) and the second stage consisting of qPCR (quantitative PCR).

[This corrects the article DOI: 10.3389/fcimb.2025.1631633.].

The WHO declared to eliminate hepatitis B virus (HBV) by 2030. However, an increasing number of patients are presenting with low-level viremia (LLV) with the widespread use of antiviral medications. The diagnostic efficiency and coverage area of HBV infection are low. Hence, this study intended to drive the HBV infection detection to effectively adaptable for any small to medium-sized laboratory or field survey. We established, optimized, and evaluated a colloidal gold test strip for detection of HBV DNA based on CRISPR/Cas13a combined with recombinase-aided amplification (RAA) technology. Furthermore, 180 HBV-infected patients (including patients with different viral loads, LLV patients and dynamic plasma samples of patients on antiviral therapy) were enrolled for clinical validation. The strip detection of HBV DNA was established based on RAA-CRISPR-Cas13a technology with a sensitivity of 10 1 copies/μL and a specificity of 100%. HBV DNA gradient concentration plasmids and clinical samples were effectively identified by this approach. The positive coincidence rate for LLV patients was 87%, while the negative coincidence rate was 100%. The positive coincidence rate reached 100% in LLV patients (viral loading >100 IU/mL). The sensitivity, specificity, positive predictive agreement (PPA) and negative predictive agreement (NPA) values of dynamic plasma detection in patients on antiviral therapy were 100%, 92.15%, 93.75%, and 100%, respectively. We develop rapid and portable RAA-CRISPR/Cas13a-based strip of HBV DNA detection for LLV patients. This study provides a visual and faster alternative to current PCR-based diagnosis for HBV infection.

Background Hepatitis B virus (HBV) infection is the major public health problem worldwide. In clinical practice, serological and molecular assays are the most commonly used diagnostic methods to detect HBV infection in clinical practices. Methods Here we present a rapid and sensitive recombinase aided amplification assay (RAA) to detect HBV at 39.0 °C for 30 min without DNA extraction from serum samples. The analytical sensitivity of RAA assay was 100 copies per reaction and showed no cross reaction with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The universality of RAA assay was validated by testing of 41 archived serum samples with predefined HBV genotypes (B, C and D). Results A total of 130 archived suspected HBV infected serum samples were detected by commercial qPCR with DNA extraction and RAA assay without DNA extraction (heat-treatment). Compared with qPCR assay as a reference, the RAA assay obtained 95.7% sensitivity and 100% specificity and a kappa value of 0.818. Conclusions We developed a rapid, convenient, highly sensitive and specific method to detect HBV without DNA extraction in clinical samples. This RAA method of HBV detection is very suitable for clinical testing.

COVID-19 was officially declared a global pandemic disease on 11 March 2020, with severe implications for healthcare systems, economic activity, and human life worldwide. Fast and sensitive amplification of the severe acute respiratory syndrome virus 2 (SARS-CoV-2) nucleic acids is critical for controlling the spread of this disease. Here, a real-time reverse transcription recombinase-aided amplification (RT-RAA) assay, targeting conserved positions in the nucleocapsid protein gene (N gene) of SARS-CoV-2, was successfully established for SARS-CoV-2. The assay was specific to SARS-CoV-2, and there was no cross-reaction with other important viruses. The sensitivity of the real-time RT-RAA assay was 142 copies per reaction at 95% probability. Furthermore, 100% concordance between the real-time RT-RAA and RT-qPCR assays was achieved after testing 72 clinical specimens. Further linear regression analysis indicated a significant correlation between the real-time RT-RAA and RT-qPCR assays with an R2 value of 0.8149 (p < 0.0001). In addition, the amplicons of the real-time RT-RAA assay could be directly visualized by a portable blue light instrument, making it suitable for the rapid amplification of SARS-CoV-2 in resource-limited settings. Therefore, the real-time RT-RAA method allows the specific, sensitive, simple, rapid, and reliable detection of SARS-CoV-2.

[This corrects the article DOI: 10.3389/fcimb.2024.1474676.].

Ribonucleic acid (RNA) viruses are one of the major classes of pathogens that cause human diseases. The conventional method to detect RNA viruses is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), but it has some limitations. It is expensive and time-consuming, with infrastructure and trained personnel requirements. Its high throughput requires sophisticated automation and large-scale infrastructure. Isothermal amplification methods have been explored as an alternative to address these challenges. These methods are rapid, user-friendly, low-cost, can be performed in less specialized settings, and are highly accurate for detecting RNA viruses. Microfluidic technology provides an ideal platform for performing virus diagnostic tests, including sample preparation, immunoassays, and nucleic acid-based assays. Among these techniques, nucleic acid isothermal amplification methods have been widely integrated with microfluidic platforms for RNA virus detection owing to their simplicity, sensitivity, selectivity, and short analysis time. This review summarizes some common isothermal amplification methods for RNA viruses. It also describes commercialized devices and kits that use isothermal amplification techniques for SARS-CoV-2 detection. Furthermore, the most recent applications of isothermal amplification-based microfluidic platforms for RNA virus detection are discussed in this article.

Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis (TB), is the leading cause of death due to a single infectious agent worldwide. Rapid and accurate diagnosis of MTB is critical for controlling TB especially in resource-limited countries, since any diagnosis delay increases the chances of transmission. Here, a real-time recombinase-aided amplification (RAA) assay targeting conserved positions in IS1081 gene of MTB, is successfully established to detect MTB. The intact workflow was completed within 30 min at 42 °C with no cross-reactivity observed for non-tuberculous mycobacteria and other clinical bacteria, and the detection limit for recombinant plasmid of MTB IS1081 was 163 copies/reaction at 95% probability, which was approximately 1.5-fold increase in analytical sensitivity for the detection of MTB, compared to conventional quantitative real-time PCR (qPCR; 244 copies/reaction). Furthermore, the result of clinical performance evaluation revealed an increased sensitivity of RAA assay relative to qPCR was majorly noted in the specimens with low bacteria loads. Our results demonstrate that the developed real-time RAA assay is a convenient, sensitive, and low-cost diagnostic tool for the rapid detection of MTB.

Molecular diagnostic methods largely rely on expensive equipment and complex operations, which makes it difficult to achieve rapid and low-cost detection. In recent years, the increasing demand for point-of-care testing has driven the rapid development of isothermal amplification techniques, due to their simplicity and low equipment requirements. However, complex nucleic acid extraction steps are still required before most isothermal amplification. In this study, we propose a nucleic acid extraction-free gene detection method: direct multiplex Recombinase Aided Amplification combined with reverse dot blot hybridization (dmRAA-RDB). This method can detect multiple targets simultaneously and offers advantages such as high sensitivity, high specificity, low cost, no need for expensive instruments, and visual detection. By pre-treating whole blood samples with sodium hydroxide solution, the samples can be directly used for isothermal amplification and combined with commercial reverse dot blot (RDB) technology to rapidly detect 17 types of β-thalassemia mutations. The experimental results demonstrated that the pre-treated whole blood samples allowed for the simultaneous, stable, and efficient enrichment of all three target fragments via direct multiplex isothermal amplification. This single-tube triple amplification strategy demonstrates significant innovation in the field of isothermal amplification. Moreover, the workflow is shortened by nearly half, and a side-by-side comparison of 60 clinical samples showed 100 % agreement with the commercial PCR-RDB kit. The dmRAA-RDB method offers a highly promising, innovative solution for large-scale, rapid, and low-cost β -thalassemia screening in primary-care and resource-limited settings, and opens a new avenue for detecting other mutant genes.