Virology Journal
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2025
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IF 3.8
Jinlei Guo,Yunfeng Shan,Ge Hu,Xiaojuan Zhu,Kangchen Zhao,Tao Wu,Qiao Qiao,Ying Chi,Lunbiao Cui,Yiyue Ge
Background Monkeypox virus (MPXV) infection has garnered significant global attention due to its rising incidence and substantial public health implications. A rapid, sensitive, and accurate diagnostic method is urgently required to enable early intervention and effective management of MPXV outbreaks. Results In this study, we developed a novel one-step assay that integrates loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b in one-pot for the detection of MPXV. The entire detection process did not require opening the lid of the reaction tube and could be completed within 40 min using extracted viral nucleic acids, which is faster than real-time quantitative PCR (qPCR). And the results could be interpreted through either real-time fluorescence or naked-eye visualization. The limit of detection (LOD) of the assay was demonstrated to be 6.5 copies per reaction and no cross-reactivity with other pathogens such as HSV, EBV, CVA16, EV-A71, and MV was found. Furthermore, when evaluated using 113 clinical samples, the assay achieved 100% sensitivity (71/71) and 100% specificity (42/42) compared to the qPCR. Conclusions In resource-limited settings, our method requires only a portable heat block or water bath and a blue light or ultraviolet flashlight for visual detection of MPXV, making it highly accessible. The integration of LAMP and CRISPR/Cas12b provides a robust, user-friendly platform for point-of-care testing, with promising potential for the rapid molecular diagnosis of infectious diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s12985-025-02780-0.
研究领域:
医学
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分子生物学
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传染病学
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DOI
PubMed
高影响因子
Frontiers in Cellular and Infection Microbiology
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2025
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IF 4.8
Yizhuo Zhang,Luqin Lv,Su Xu,Yijian Chen
Introduction: Clostridioides difficile (C. difficile), a human pathogen that causes diarrhea and colon lesions, has garnered widespread attention. Rapid and accurate detection of bacterial virulence factors is essential for the diagnosis of C. difficile infection (CDI). To date, numerous laboratory tests have been developed; however, none fully meet the combined requirements of speed, cost-effectiveness, portability, sensitivity, and specificity. Molecular diagnostic technologies based on CRISPR-Cas systems have provided a promising solution to this challenge. Nonetheless, the limited compatibility between pre-amplification and CRISPR cleavage, coupled with the inherent selectivity of CRISPR systems for protospacer adjacent motif (PAM) sequences near the target site, poses additional constraints on the broader adoption of this approach.Methods: Here, we developed PAM-unconventional, One-step LAMP/CRISPR-Cas12b (POLC) detection platforms for the toxin-encoding genes tcdA and tcdB of C. difficile.Results: The POLC platforms operated at 60 °C, enabling result interpretation either through fluorescence intensity measurements or direct visualization under UV light. The limits of detection (LoDs) ranged from 3 to 14 copies/μL using a fluorescence reader and from 6 to 18 copies/μL via direct observation. Compared to qPCR, which typically requires over an hour, the POLC platforms reduced the detection time to approximately 40 minutes. Each reaction cost approximately USD 6.5, offering a substantial cost saving compared to qPCR-based commercial kits (over USD 10 per test). In clinical validation with 55 fecal samples, the tcdA POLC assay achieved 86.4% sensitivity and 84.8% specificity, while the tcdB POLC assay demonstrated 96.6% sensitivity and 100% specificity, using qPCR as the reference standard.Discussion: Our research presents innovative CRISPR-based one-step nucleic acid detection platforms that eliminate canonical PAM sequence requirements. These platforms exhibit high sensitivity and specificity while achieving rapid detection under simple conditions, making them promising candidates for clinical diagnostics and point-of-care testing (POCT).
研究领域:
医学诊断
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分子生物学
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DOI
PubMed
高影响因子
TALANTA
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2022
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IF 6.556
Yaqin Zhang,Xiangyu Quan,Yingchun Li,Hangyu Guo,Fange Kong,Jiahui Lu,Lirong Teng,Jiasi Wang,Di Wang
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins-based platforms have been used for the detection of pathogens. However, in further applications and research, due to multiple steps needed, many methods showed an increased risk of cross-reactivity. The thermostable Cas12b enables the combination of isothermal amplification and CRISPR-mediated detection, which could decrease the risk of cross-contamination. In this study, we developed a portable and specific diagnostic method that combined the gold nanoparticle (AuNP) with thermal stable CRISPR/Cas12b-enhanced reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is called SCAN, to distinguish the N gene of SARS-CoV-2 from flu gene. We validated our method using RNA from cells transfected by plasmids. We could easily distinguish the positive results by the naked eye based on the strong molar absorption coefficient of AuNP. Moreover, SCAN has the potential for high-throughput tests owing to its convenient operation. In sum, SCAN has broken the site and equipment restrictions of traditional detection methods and could be applied outside of hospitals and clinical laboratories, greatly expanding the test of COVID-19.
研究领域:
分子生物学
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生物技术
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病毒学
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诊断医学
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DOI
高影响因子
Analytical Methods
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2025
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IF 2.6
Chen Lu,Jiayue Chen,Shengsheng Zhang,Yaru Tian,Xinyu Ying,Dan Zhang,Zhidan Luo,Xinxin Si,Ming Li
Two single nucleotide polymorphisms (SNPs) in the human SLCO1B1 gene, c.388A>G (rs2306283) and c.521T>C (rs4149056), are independent determinants of the efficacy and side effects of statin drugs. Multinational clinical guidelines recommend testing for SLCO1B1 genotypes before the initial use of statins. Current SLCO1B1 SNP identification methods, primarily based on quantitative fluorescence PCR, rely on expensive equipment, are time-consuming, and require cold-chain storage for reagents, making them unsuitable for use in resource-limited healthcare settings. In this study, we developed a CRISPR/Cas12b-based amplification-free genotyping technique for SLCO1B1 SNPs. Within 30 minutes of the isothermal reaction, genotyping of the c.388A>G and c.521T>C SNPs in the SLCO1B1 gene can be observed by the naked eyes under blue light. Additionally, maltodextrin was identified as an effective vitrification stabilizer for the CRISPR/Cas12b premix. A low-cost vitrification process was optimized to prepare a glass like solid reagent via room-temperature vacuum drying. The vitrified CRISPR/Cas12b reagent retained approximately 88% of its activity after 30 days of storage at 37 °C, eliminating the need for cold-chain storage and allowing for long-term preservation at room temperature. This vitrified CRISPR/Cas12b based rapid SNP detection technique is especially suitable for genotyping drug metabolism genes in primary healthcare settings, providing effective guidance for precision medicine in clinical practice.
研究领域:
分子生物学
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医学遗传学
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精准医疗
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2024
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IF 5.7
Xiao Han,Minghui Lu,Yaru Zhang,Xinru Liu,Qiang Zhang,Xue Bai,Shuli Man,Liangjuan Zhao,Long Ma
Genetically modified crops (GMCs) have been discussed due to unknown safety, and thus, it is imperative to develop an effective detection technology. CRISPR/Cas is deemed a burgeoning technology for nucleic acid detection. Herein, we developed a novel detection method for the first time, which combined thermostable Cas12b with loop-mediated isothermal amplification (LAMP), to detect genetically modified (GM) soybeans in a customized one-pot vessel. In our method, LAMP-specific primers were used to amplify the cauliflower mosaic virus 35S promoter (CaMV35S) of the GM soybean samples. The corresponding amplicons activated the trans-cleavage activity of Cas12b, which resulted in the change of fluorescence intensity. The proposed bioassay was capable of detecting synthetic plasmid DNA samples down to 10 copies/μL, and as few as 0.05% transgenic contents could be detected in less than 40 min. This work presented an original detection method for GMCs, which performed rapid, on-site, and deployable detection.
研究领域:
植物学
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分子生物学
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生物技术
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DOI
高影响因子
危险品化学品经营许可证（带存储）