DPF3 polymorphisms increased the risk of pulmonary tuberculosis in the Northwest Chinese Han population.

PURPOSE: This study aimed to investigate the association between single nucleotide polymorphisms (SNPs) in DPF3 and susceptibility to pulmonary tuberculosis (PTB) in the Northwest Chinese Han population. METHODS: Genotyping of four DPF3 SNPs (rs10140566, rs75575287, rs202075571, and rs61986330) was performed using Agena MassARRAY from 488 PTB patients and 488 healthy controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression. Multifactor dimensionality reduction (MDR) analysis was employed to investigate the effect of SNP-SNP interactions on PTB risk. The GSE54992 dataset was analyzed using R software to ascertain DPF3 expression levels. RESULTS: Overall analysis revealed that rs202075571 (allele: OR = 1.31, p = 0.015; CC vs. TT: OR = 1.97, p = 0.049; dominant: OR = 1.33, p = 0.032) and rs61986330 (allele: OR = 1.38, p = 0.010; CA vs. CC: OR = 1.35, p = 0.044; dominant: OR = 1.40, p = 0.019) were associated with an increased PTB risk. Stratified analysis showed that rs10140566 was a PTB risk factor in females, those aged ≤40 and non-smokers, and rs202075571 was associated with PTB risk in individuals aged >40 and smokers, and rs61986330 was associated with PTB risk in males, those aged >40 and smokers. The four SNPs model demonstrated significant predictive potential for PTB risk. Furthermore, DPF3 exhibited higher expression in PTB compared to healthy controls. CONCLUSION: DPF3 polymorphisms (rs10140566, rs202075571, and rs61986330) are associated with an increased risk of PTB, providing valuable new insights into the mechanism of PTB.

Palm-Sized Lab-In-A-Magnetofluidic Tube Platform for Rapid and Sensitive Virus Detection.

Simple, sensitive, and accurate molecular diagnostics are critical for preventing rapid spread of infection and initiating early treatment of diseases. However, current molecular detection methods typically rely on extensive nucleic acid sample preparation and expensive instrumentation. Here, a simple, fully integrated, lab-in-a-magnetofluidic tube (LIAMT) platform is presented for "sample-to-result" molecular detection of virus. By leveraging magnetofluidic transport of micro/nano magnetic beads, the LIAMT device integrates viral lysis, nucleic acid extraction, isothermal amplification, and CRISPR detection within a single engineered microcentrifuge tube. To enable point-of-care molecular diagnostics, a palm-sized processor is developed for magnetofluidic separation, nucleic acid amplification, and visual fluorescence detection. The LIAMT platform is applied to detect SARS-CoV-2 and HIV viruses, achieving a detection sensitivity of 73.4 and 63.9 copies µL-1, respectively. Its clinical utility is further demonstrated by detecting SARS-CoV-2 and HIV in clinical samples. This simple, affordable, and portable LIAMT platform holds promise for rapid and sensitive molecular diagnostics of infectious diseases at the point-of-care.

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR-Cas12a.

The CRISPR-Cas12a system has emerged as a powerful tool for next-generation nucleic acid-based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA-enabled trans-cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full-size RNA targets, although LbCas12a exhibits weaker trans-cleavage activity than AsCas12a on both single-stranded DNA and RNA substrates. Remarkably, we find that the RNA-activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA-Sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM-less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double-stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a-based nucleic acid detection and further enhance the understanding of CRISPR-Cas biochemistry.

CRISPR-powered quantitative keyword search engine in DNA data storage.

Despite the growing interest of archiving information in synthetic DNA to confront data explosion, quantitatively querying the data stored in DNA is still a challenge. Herein, we present Search Enabled by Enzymatic Keyword Recognition (SEEKER), which utilizes CRISPR-Cas12a to rapidly generate visible fluorescence when a DNA target corresponding to the keyword of interest is present. SEEKER achieves quantitative text searching since the growth rate of fluorescence intensity is proportional to keyword frequency. Compatible with SEEKER, we develop non-collision grouping coding, which reduces the size of dictionary and enables lossless compression without disrupting the original order of texts. Using four queries, we correctly identify keywords in 40 files with a background of ~8000 irrelevant terms. Parallel searching with SEEKER can be performed on a 3D-printed microfluidic chip. Overall, SEEKER provides a quantitative approach to conducting parallel searching over the complete content stored in DNA with simple implementation and rapid result generation.

Cryogel wound dressings based on natural polysaccharides perfectly adhere to irregular wounds for rapid haemostasis and easy disassembly.

Skin injuries can have unexpected surfaces, leading to uneven wound surfaces and inadequate dressing contact with these irregular surfaces. This can decrease the dressing's haemostatic action and increase the healing period. This study recommends the use of sticky and flexible cryogel coverings to promote faster haemostasis and efficiently handle uneven skin wounds. Alginate cryogels have a fast haemostatic effect and shape flexibility due to their macroporous structure. The material demonstrates potent antibacterial characteristics and enhances skin adherence by adding grafted chitosan with gallic acid. In irregular defect wound models, cryogels can cling closely to uneven damage surfaces due to their amorphous nature. Furthermore, their macroporous structure allows for quick haemostasis by quickly absorbing blood and wound exudate. After giving the dressing a thorough rinse, its adhesive strength reduces and it is simple to remove without causing any damage to the wound. Cryogel demonstrated faster haemostasis than gauze in a wound model on a rat tail, indicating that it has considerable potential for use as a wound dressing in the biomedical area.

4D Flow MRI of Portal Vein Hemodynamics in Healthy Volunteers and Patients with Chronic Liver Disease.

AIM: To identify age-matched healthy volunteers, non-cirrhotic chronic liver disease (CLD) and cirrhotic patients based on portal hemodynamic parameters using 4D flow MRI. METHODS: A total of 10 age-matched healthy volunteers and 69 CLD patients were enrolled and underwent 4D flow MRI prospectively. 4D flow MR images were processed by an MD in biomedical engineering working on the GTFlow platform. Portal hemodynamic parameters include net flow (mL/cycle), flow volume per second through the lumen (mL/sec), average flow velocity (cm/sec), and maximum flow velocity (cm/sec). The difference in portal hemodynamic parameters of 4D flow MRI was compared among healthy volunteers, non-cirrhotic CLD patients and patients with cirrhosis by one-way ANOVA or Kruskal-Wallis nonparametric test and post hoc tests. RESULTS: 10 CLD patients without cirrhosis and 56 patients with cirrhosis were eventually included, along with 10 healthy volunteers who were divided into three groups. 3 patients with cirrhosis whose image quality did not meet the requirements were excluded. There were no significant differences in portal hemodynamic parameters among the three groups except portal average velocity (P > 0.05). There was no statistical difference in all portal hemodynamic parameters of 4D flow MRI between healthy volunteers and patients with cirrhosis (P > 0.05). There were significant differences in portal average velocity between non-cirrhotic CLD patients, healthy volunteers and patients with cirrhosis, respectively (11.44±3.93 vs 8.10±2.66, P=0.013; 11.44±3.93 vs 8.60±2.22, P=0.007). CONCLUSION: Portal average velocity obtained by 4D flow MRI can be an auxiliary means to identify cirrhosis in patients with CLD.

CRISPR-Powered Aptasensor for Diagnostics of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia, characterized by the accumulation of amyloid beta (Aß) peptides in the brain. Here, we present a simple, rapid, and affordable CRISPR-powered aptasensor for the quantitative detection of Aß40 and Aß42 biomarkers in cerebrospinal fluid (CSF) samples, enabling early and accurate diagnostics of AD patients. The aptasensor couples the high specificity of aptamers for Aß biomarkers with CRISPR-Cas12a-based fluorescence detection. The CRISPR-powered aptasensor enables us to detect Aß40 and Aß42 in CSF samples within 60 min, achieving a detection sensitivity of 1 pg/mL and 0.1 pg/mL, respectively. To validate its clinical utility, we quantitatively detected Aß40 and Aß42 biomarkers in clinical CSF samples. Furthermore, by combining CSF Aß42 levels with the c(Aß42)/c(Aß40) ratio, we achieved an accurate diagnostic classification of AD patients and healthy individuals, showing superior performance over the conventional ELISA method. We believe that our innovative aptasensor approach holds promise for the early diagnostic classification of AD patients.

[Detection method of early heart valve diseases based on heart sound features].

Heart valve disease (HVD) is one of the common cardiovascular diseases. Heart sound is an important physiological signal for diagnosing HVDs. This paper proposed a model based on combination of basic component features and envelope autocorrelation features to detect early HVDs. Initially, heart sound signals lasting 5 minutes were denoised by empirical mode decomposition (EMD) algorithm and segmented. Then the basic component features and envelope autocorrelation features of heart sound segments were extracted to construct heart sound feature set. Then the max-relevance and min-redundancy (MRMR) algorithm was utilized to select the optimal mixed feature subset. Finally, decision tree, support vector machine (SVM) and k-nearest neighbor (KNN) classifiers were trained to detect the early HVDs from the normal heart sounds and obtained the best accuracy of 99.9% in clinical database. Normal valve, abnormal semilunar valve and abnormal atrioventricular valve heart sounds were classified and the best accuracy was 99.8%. Moreover, normal valve, single-valve abnormal and multi-valve abnormal heart sounds were classified and the best accuracy was 98.2%. In public database, this method also obtained the good overall accuracy. The result demonstrated this proposed method had important value for the clinical diagnosis of early HVDs.

RClaNet: An explainable Alzheimer's disease diagnosis framework by joint registration and classification.

Alzheimer's disease (AD) is a degenerative mental disorder of the central nervous system that affects people's ability of daily life. Unfortunately, there is currently no known cure for AD. Thus, the early detection of AD plays a key role in preventing and controlling its progression. Magnetic resonance imaging (MRI)-based measures of cerebral atrophy are regarded as valid markers of the AD state. As one of representative methods for measuring brain atrophy, image registration technique has been widely adopted for AD diagnosis. However, AD detection is sensitive to the accuracy of image registration. To address this problem, an AD assistant diagnosis framework based on joint registration and classification is proposed. Specifically, in order to capture more local deformation information, we propose a novel patch-based joint brain image registration and classification network (RClaNet) to estimate the local dense deformation fields (DDF) and disease risk probability maps that explain high-risk areas for AD patients. RClaNet consists of a registration network and a classification network, in which the deformation field from registration network is fed into the classification network to enhance the prediction accuracy of the disease. Then, the exponential distance weighting method is used to obtain the global DDF and the global disease risk probability map, and it can remove grid-like artifacts by uniformly weighting method. Finally, the global classification network uses the global disease risk probability map for the early detection of AD. We evaluate the proposed method on the OASIS-3, AIBL and ADNI datasets, and experimental results show that the proposed RClaNet achieves superior registration performances than several state-of-the-art methods. Early diagnosis of AD using the global disease risk probability map also yielded competitive results. To demonstrate the generality, we also evaluate the proposed method on a COVID-19 dataset and achieve decent registration and classification results. These experiments prove that the deformation information in the registration process can be used to characterize subtle changes of degenerative diseases and further assist clinicians in diagnosis.

Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA.

Nucleic acid detection powered by CRISPR technology provides a rapid, sensitive, and deployable approach to molecular diagnostics. While exciting, there remain challenges limiting its practical applications, such as the need for pre-amplification and the lack of quantitative ability. Here, we develop an asymmetric CRISPR assay for cascade signal amplification detection of nucleic acids by leveraging the asymmetric trans-cleavage behavior of competitive crRNA. We discover that the competitive reaction between a full-sized crRNA and split crRNA for CRISPR-Cas12a can induce cascade signal amplification, significantly improving the target detection signal. In addition, we find that CRISPR-Cas12a can recognize fragmented RNA/DNA targets, enabling direct RNA detection by Cas12a. Based on these findings, we apply our asymmetric CRISPR assay to quantitatively detect microRNA without the need for pre-amplification, achieving a detection sensitivity of 856 aM. Moreover, using this method, we analyze and quantify miR-19a biomarker in plasma samples from bladder cancer patients. This asymmetric CRISPR assay has the potential to be widely applied for simple and sensitive nucleic acid detection in various diagnostic settings.

Is a soft tissue reinforcing mesh necessary in immediate prosthetic breast reconstruction for early breast cancer in patients with low-volume breast? A single-center, retrospective Chinese clinical study.

It is controversial as to whether soft tissue reinforcement mesh should be used for immediate prosthetic breast reconstruction after nipple-sparing mastectomy for low-volume breast early breast cancer (LVBEBC) in Chinese adult women. We collected data on 89 patients with LVBEBC who underwent such a surgery and divided them into two groups: 39 patients in the totally subpectoral prosthesis-only breast reconstruction group (simple group) and 50 patients in the prosthesis-combined titanium-coated polypropylene mesh (TCPM) group (or the so-called "dual plane" or "mesh-assisted partially subpectoral breast reconstruction group") (combined group). The results demonstrated no difference in operative time, intraoperative bleeding, and postoperative complications between the two groups; however, total drainage volume and extubation time were less and shorter, respectively, in the combined group. The median follow-up time was 18.6 months without local recurrence or distant metastasis in both groups. At 24 months after surgery, the excellent and good rates of breast reconstruction were higher in the combined group. However, patients' BMI, breast morphology, and breast volume of 300 mL or more had an effect on the shape of the reconstructed breast; in addition, in patients with higher BMI, conical breast morphology, and breast volume over 300 mL, the shape of the breast was more perfect with the prosthesis combined with TCPM reconstruction.Trial registration: This retrospective study was "retrospectively registered" in the Sixth Affiliated Hospital of South China University of Technology of China on March 15, 2022 (No. 2022018) and in the National Medical Research Registry filing system of China ( https://www.medicalresearch.org.cn ) (No. MR-44-22-003618).

Simultaneous removal of antibiotics and nitrogen by microbial fuel cell-constructed wetlands: Microbial response and carbon-nitrogen metabolism pathways.

Microbial fuel cell-constructed wetlands (MFC-CWs) are attracted extensive attention due to their simultaneous removal performance during the co-occurrence of various pollutants in wastewater. This study explored the performance and mechanisms on the simultaneous removal of antibiotics and nitrogen from MFC-CWs which packed with coke (MFC-CW (C)) and quartz sand (MFC-CW (Q)) substrate. Results showed that removal of sulfamethoxazole (93.60 %), COD (77.94 %), NH4+-N (79.89 %), NO3-- N (82.67 %), and TN (70.29 %) significantly enhanced by MFC-CW (C) due to the enhancement of relative abundance of membrane transport, amino acid metabolism and carbohydrate metabolism pathways. The results indicated that coke substrate can generate more electric energy in MFC-CW. Firmicutes (18.56-30.82 %), Proteobacteria (23.33-45.76 %), and Bacteroidetes (17.1-27.85 %) were dominant phyla in the MFC-CWs. MFC-CW (C) posed significant effects on the microbial diversity and structure, which motivated the functional microbes involved in the transformation of antibiotics and nitrogen and bioelectricity generation. Given the overall performance of MFC-CW, packing with cost-effective substrate to electrode region of MFC-CWs was found to be an effective strategy for simultaneously removing antibiotics and nitrogen in the wastewater treatment.

CRISPR-powered Biosensing Platform for Quantitative Detection of Alpha-fetoprotein by a Personal Glucose Meter.

Alpha-fetoprotein (AFP) is an important protein biomarker of liver cancer, as its serum levels are highly correlated with the progression of disease. Conventional immunoassays for AFP detection rely on enzyme-linked immunosorbent assay analyses with expensive and bulky equipment. Here, we developed a simple, affordable, and portable CRISPR-powered personal glucose meter biosensing platform for quantitative detection of the AFP biomarker in serum samples. The biosensor takes advantage of the excellent affinity of aptamer to AFP and the collateral cleavage activity of CRISPR-Cas12a, enabling sensitive and specific CRISPR-powered protein biomarker detection. To enable point-of-care testing, we coupled invertase-catalyzed glucose production with the glucose biosensing technology to quantify AFP. Using the developed biosensing platform, we quantitatively detected AFP biomarker in spiked human serum samples with a detection sensitivity of down to 10 ng/mL. Further, we successfully applied the biosensor to detect AFP in clinical serum samples from patients with liver cancer, achieving comparable performance to the conventional assay. Therefore, this novel CRISPR-powered personal glucose meter biosensor provides a simple yet powerful alternative for detecting AFP and potentially other tumor biomarkers at the point of care.

Association between GLS Gene Polymorphisms and the Susceptibility to Lung Cancer in the Chinese Han Population.

BACKGROUND: Lung cancer is one of the most serious malignant tumors endangering human health and life. This study focused on evaluating the association between single nucleotide polymorphisms (SNPs) of the glutaminase (GLS) and lung cancer susceptibility in the Chinese Han population. METHODS: A total of 684 lung cancer patients and 684 healthy individuals were enrolled. Five GLS SNPs (rs143584207 C/A, rs117985587 T/C, rs74271715 G/T, rs2355570 G/A, and rs6713444 A/G) were screened as candidate genetic loci. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to assess the association between GLS SNPs and lung cancer susceptibility. False-positive report probability (FPRP) analysis further verified whether the positive results deserved attention. Finally, the multi-factor dimensionality reduction (MDR) method was applied to analyze the interactions between SNPs. RESULTS: The overall analysis revealed that GLS rs143584207 and rs6713444 were significantly associated with lung cancer susceptibility. The subgroup and clinical information analyses further revealed that GLS rs143584207 and rs6713444 could remarkably reduce lung cancer susceptibility in different subgroups (age >60, females, body mass index (BMI) <24, and lung adenocarcinoma). Rs143584207 could significantly reduce lung cancer susceptibility in non-smokers. Additionally, rs6713444 also had a protective effect on patients with advanced lung cancer. CONCLUSIONS: Our study indicated that GLS rs143584207 and rs6713444 could strikingly reduce lung cancer susceptibility in the Chinese Han population, which will give a new direction for the timely treatment of lung cancer.

CRISPR gel: A one-pot biosensing platform for rapid and sensitive detection of HIV viral RNA.

CRISPR technology has recently emerged as a powerful biosensing tool for sensitive and specific nucleic acid detection when coupled with isothermal amplification (e.g., recombinase polymerase amplification (RPA)). However, it remains a challenge to incorporate isothermal amplification into CRISPR detection in a one-pot system due to their poor compatibility. Here, we developed a simple CRISPR gel biosensing platform for human immunodeficiency virus (HIV) RNA detection by combining reverse transcription-recombinase polymerase amplification (RT-RPA) reaction solution with a CRISPR gel. In our CRISPR gel biosensing platform, CRISPR-Cas12a enzymes are embedded into the agarose gel, providing a spatially separated but connected reaction interface with the RT-RPA reaction solution. During isothermal incubation, the RT-RPA amplification occurs initially on the CRISPR gel. When RPA products are sufficiently amplified and reach the CRISPR gel, the CRISPR reaction occurs in the whole tube. With the CRISPR gel biosensing platform, we successfully detected down to 30 copies of HIV RNA per test within 30 min. Furthermore, we validated its clinical utility by detecting HIV clinical plasma samples, achieving superior performance compared with the real-time RT-PCR method. Thus, our one-pot CRISPR gel biosensing platform demonstrates great potential for rapid and sensitive molecular detection of HIV and other pathogens at the point of care.

Bilateral multicenter pseudohemangiomatous interstitial hyperplasia of the breast: a case report.

Bilateral multicenter breast pseudohemangiomatous stromal hyperplasia (PASH) is a rare, benign breast disease. Here, we report on a female patient with bilateral multicenter PASH who underwent a mastectomy and prosthetic reconstruction. The surgery was successful, and no recurrence was observed during the 18 months of follow up.

Bioinspired CRISPR-Mediated Cascade Reaction Biosensor for Molecular Detection of HIV Using a Glucose Meter.

HIV molecular detection plays a significant role in early diagnosis and antiretroviral therapy for HIV patients. CRISPR technology has recently emerged as a powerful tool for highly sensitive and specific nucleic acid based molecular detection when used in combination with isothermal amplification. However, it remains a challenge to improve the compatibility of such a multienzyme reaction system for simple and sensitive molecular detection. Inspired by the multicompartment structures in a living cell, we present a nanoporous membrane-separated (compartmentalized), artificial, cascade reaction system to improve the compatibility of a CRISPR-mediated multienzyme reaction. We further integrated the multienzyme cascade reaction system with a microfluidic platform and glucose biosensing technology to develop a bioinspired, CRISPR-mediated cascade reaction (CRISPR-MCR) biosensor, enabling HIV molecular detection by a simple glucose meter, analogous to diabetes home testing. We applied the bioinspired CRISPR-MCR biosensor to detect HIV DNA and HIV RNA, achieving a detection sensitivity of 43 copies and 200 copies per test, respectively. Further, we successfully validated the bioinspired biosensor by testing clinical plasma samples of HIV, demonstrating its great application potential for point-of-care testing of HIV virus and other pathogens at home or in resource-limited settings.

Comparison of single-pore non-liposuction near-infrared laparoscopy with conventional open surgery for axillary sentinel lymph node biopsy in patients with early breast cancer: a single-center, small-sample retrospective study.

BACKGROUND: This study aimed to compare the effects of single-pore non-liposuction near-infrared (NIR) endoscopic surgery and traditional open surgery for axillary sentinel lymph node biopsy (SLNB) in patients with early breast cancer (EBC). METHODS: The clinical pathological data of 61 patients with EBC who underwent axillary SLNB using indocyanine green (ICG) combined with carbon nanoparticle suspension (CNS) were retrospectively collected. Thirty patients received SLNB through single-pore non-liposuction NIR endoscopic surgery (endoscopic group), and the remaining 31 received SLNB through open-incision surgery (open group). The success rate, operation time, volume of intraoperative bleeding, postoperative axillary drainage, axillary extubation time, and the occurrence of postoperative complications were compared between the groups along with the total number of sentinel lymph nodes (SLNs), luminous SLNs, stained SLNs, and the pathological positivity rate of the SLNs. RESULTS: All patients underwent SLNB with a 100% success rate. SLNB operation times of the endoscopic group were longer than those of the open group (t = 3.963, P = 0.000), and the volume of axillary drainage was inferior (t = 3.035, P = 0.004). However, there were no differences in the intraoperative bleeding volumes, axillary extubation times, and postoperative complications (P > 0.05). In the Open group, the mean number of SLNs was 5.12 ± 2.16, and the pathological positivity rate was 13.53%; in the Endoscopic group, these numbers were 4.89 ± 1.73 and 12.39%. The mean number of SLNs detected (t = 0.458, P = 0.649) and the pathological positivity rates (χ2 = 0.058, P = 0.810) did not differ between the two groups. All 61 patients were followed for a median of 14.6 months. There were no local recurrences or distant metastases. CONCLUSIONS: Our single-center results reveal that single-hole non-liposuction NIR endoscopic axillary SLNB is not inferior to open SLNB and may be an appropriate option for patients with early breast cancer who desire breast preservation with fewer incisions. TRIAL REGISTRATION: This retrospective study was "retrospectively registered" at the Sixth Affiliated Hospital of South China University of Technology (no. 2020105) and in National Medical Research Registration and Archival Information System ( https://www.medicalresearch.org.cn , number: MR-44-21-004727).

Single-tube one-step gel-based RT-RPA/PCR for highly sensitive molecular detection of HIV.

We developed a single-tube one-step gel-based reverse transcription-recombinase polymerase amplification (RT-RPA)/polymerase chain reaction (PCR) (termed "SOG RT-RPA/PCR") to detect the human immunodeficiency virus (HIV). To improve the assay sensitivity, the RNA template is pre-amplified by RT-RPA prior to PCR. To simplify the detection process and shorten the assay time, we embedded PCR reagents into agarose gel, constructing it to physically separate the reagents from the RT-RPA reaction solution in a single tube. Due to the thermodynamic properties of agarose, the RT-RPA reaction first occurs independently on top of the PCR gel at a low temperature (e.g., 39 °C) during the SOG RT-RPA/PCR assay. Then, the RPA amplicons directly serve as the template for the second PCR amplification reaction, which begins when the PCR agarose dissolves due to the elevated reaction temperature, eliminating the need for multiple manual operations and amplicon transfer. With our SOG RT-RPA/PCR assay, we could detect 6.3 copies of HIV RNA per test, which is a 10-fold higher sensitivity than that of standalone real-time RT-PCR and RT-RPA. In addition, due to the high amplification efficiency of RPA, the SOG RT-RPA/PCR assay shows stronger fluorescence detection signals and a shorter detection time compared to the standalone real-time RT-PCR assay. Furthermore, we detected HIV viral RNA in clinical plasma samples and validated the superior performance of our assay. Thus, the SOG RT-RPA/PCR assay offers a powerful method for simple, rapid, and highly sensitive nucleic acid-based molecular detection of infectious diseases.