基于CRISPR/Cas13a系统的寨卡病毒检测方法的研究

摘要

研究背景和目的：

寨卡病毒（Zika Virus，ZIKV）作为虫媒病毒，可通过输血传播，对受血者健康构成重大威胁。现有检测方法存在局限性，如病毒分离耗时长，核酸检测易污染导致假阳性，抗原和抗体检测无法分型或存在交叉反应。因此，研发快速、准确的寨卡病毒检测技术迫在眉睫。CRISPR/Cas13a系统具有比传统技术更高的检测效率，其内嵌重组酶聚合酶扩增RAA技术，适用于低载量样本的检测。检测灵敏度和特异性得到同步提升。本研究旨在开发基于CRISPR/Cas13a系统的寨卡病毒检测方法，通过对病毒基因的特征性片段扩增获得大量检测模板，利用CRISPR特异性识别并激活Cas蛋白，然后Cas剪切核酸片段，激活信号探针，实现对低载量样本的一管化快速可视化检测。该方法缩短核酸反应至30 min，为现场快速筛查寨卡病毒提供技术可行性。

研究内容：

{C}（1）    RT-RAA反应的建立与优化

{C}（2）    CRISPR/Cas13a反应的建立与优化

{C}（3）    一管化RT-RAA-T7 -CRISPR/Cas13a检测方法的建立

{C}（4）    一管化RT-RAA-T7 -CRISPR/Cas13a检测方法的灵敏度与特异性评价

（5） 一管化RT-RAA-T7 -CRISPR/Cas13a检测方法和RT-PCR法的临床样本检测结果比较

研究方法：

（1）在NCBI上查找和筛选ZIKV保守序列，通过样本扩增与筛选，构建含有ZIKV病原体保守序列的标准质粒。

（2）以国家临检中心ZIKV标准品为模板，建立RT-RAA反应。通过将RT-RAA产物进行琼脂糖凝胶电泳，对RT-RAA引物和RT-RAA反应温度进行优化，选择明亮且单一的电泳条带对应的RT-RAA引物和RT-RAA反应温度作为后续反应条件。

（3）以国家临检中心ZIKV标准品为模板，建立CRISPR/Cas13a反应。通过荧光定量PCR仪记录实时荧光强度对crRNA、crRNA浓度、Cas13a酶浓度和ssRNA浓度进行优化，选择荧光强度值高对应的crRNA、crRNA浓度和Cas13a酶浓度作为后续反应条件。

（4）在管底加入上述优化后的CRISPR/Cas13a反应体系，再先后加入RT-RAA反应体系， T7转录体系和MgCl₂，37℃ 30min，从而触发CRISPR/Cas13a的切割体系，荧光定量PCR仪实时记录荧光强度，或用试纸条进行检测判读。

（5）ZIKV的一管化RT-RAA-T7 -CRISPR/Cas13a检测方法的特异性和灵敏度评价。以国家临检中心ZIKV标准品为阳性对照，ddH₂O为模板作阴性对照，检测其他10种病毒（HBV、HCV 、DENV-1、DENV-2、 SINV、PRV 、PPV 、EMCV 、 MVM、BVDV），通过上述步骤建立反应，从而评估该方法的特异性。以ZIKV标准品（4×10^4-0 copies/μL）为模板， 通过上述步骤建立反应，从而评估该方法的灵敏性。

（6）以本实验室扩增的ZIKV病毒液和血站的健康人血浆样本充分混合制作ZIKV临床血浆模拟样本。以ZIKV临床血浆模拟样本为待测样本，核酸提取后，同时用一管化RT-RAA-CRISPR/Cas13a检测方法和金标准RT-PCR方法检测，比较两种方法的检测结果，以评价本方法的临床效能。

研究结果：

（1） 通过样本扩增与筛选，构建了ZIKA的标准质粒。

（2） 筛选出了ZIKV的最优RT-RAA引物，且最优的RT-RAA反应条件为37 ℃ 30min。

（3） 筛选出了ZIKV的最优crRNA，且最优的CRISPR/Cas13a反应条件为150 nM crRNA、75 nM Cas13a,1.2 μM MgCl₂和100 nM ssRNA。

（4） 成功建立了ZIKV的一管化RT-RAA-T7-CRISPR/Cas13a可视化联合检测方法。

（5） ZIKV的一管化RT-RAA-T7-CRISPR/Cas13a检测方法的特异度良好，与其他病毒无交叉反应；ZIKV的一管化RT-RAA-T7-CRISPR/Cas13a检测方法的灵敏度达4 copies/μL。

（6）  一管化RT-RAA-T7-CRISPR/Cas13a检测方法检测45份ZIKV临床模拟血浆样本，其中ZIKV阳性样本30份，ZIKV阴性样本15份，结果显示检出率为100%。RT-PCR检测结果显示病毒上清低于10³ copies/mL无法检出。

研究结论：

本研究建立的一管化RT-RAA-T7-CRISPR/Cas13a检测方法特异性强、灵敏度高，能可视化检测ZIKV，可用于资源紧缺地区和现场检测，在开发下一代核酸检测生物传感器方面的具有巨大潜力。

 

短波紫外线灭活血管性血友病因子制品中病毒的实验研究

摘要

研究背景和目的：

血管性血友病因子(von Willebrand factor, vWF)是血浆中关键的凝血辅助因子,通过介导血小板黏附及稳定血管性血友病因子Ⅷ参与止血过程,是血浆衍生制剂和重组蛋白药物的核心成分。然而,vWF制品的生产和应用存在病毒污染的生物安全风险。人细小病毒B19以及肝炎病毒等病原体可能通过原料血浆或生产环节引入,导致交叉感染风险增加。尽管当前病毒灭活工艺如热处理、低pH孵育已广泛用于血液制品安全控制,但其对vWF结构和功能的潜在破坏如多聚体解离、凝血活性丧失限制了临床应用的安全性与有效性。例如,热灭活法(60℃,30 min)可导致 vWF瑞斯托霉素辅因子活性(ristocetin cofactor ac tivity, RCoA)下降40%以上,而S/D(有机溶剂/ 去污剂)法对灭活包膜病毒非常有效,无包膜病毒不受影响,且其中的有机溶剂/去污剂必须去除,可能引入残留毒性。因此,开发1种既能高效灭活病毒又不显著影响vWF功能的替代技术具有重要科学和临床价值。

短波紫外线(ultraviolet-C,UVC,190~280 nm) 通过诱导病毒核酸形成嘧啶二聚体抑制复制,且对蛋白质结构干扰较小,已应用于血小板的病毒灭活。然而,其灭活效率受病毒衣壳结构、核酸类 型及样品透明度影响显著。例如,细小病毒B19因无包膜且衣壳致密,对UVC的敏感性较腺病毒 低2~3个对数级。此外,UVC照射剂量需精准 控制,剂量不足导致灭活不完全,过量则可能引发 光氧化反应破坏vWF的活性。本研究以vWF制品 为样品,使用UVC辐照仪(波长254 nm)对指示病毒进行照射灭活,探究不同照射剂量对病毒的灭活效果及vWF成分的影响。通过梯度实验筛选最佳照射参数,旨在建立兼顾安全性与有效性的UVC 辐照灭活方案。

研究内容：

(1) vWF抗原水平（vWF:Ag）与功能活性（vWF:CBA）的辐照剂量效应分析

(2) UVC辐照剂量对四种指示病毒的灭活效能评估

(3)病毒灭活与vWF功能保留的平衡优化

研究方法：

(1) UVC辐照剂量梯度灭活实验

(2) vWF抗原(vWF:Ag)和vWF胶原结合活性( vWF:CBA)检测

(3) 病毒灭活与vWF功能保留的剂量效应分析

研究结果：

(1) UVC辐照剂量大于100 J/m²时，可使4种指示病毒病毒滴度下降≥4.0 logs。

(2) WF抗原在UVC辐照剂量100、125、150 、175和200 J/m²时，保留率可分别达到93.67%、91.72%、93.54%、79.05%和85.50%；

(3) vWF胶原结合活性在UVC辐照剂量100、125、150 、175和200 J/m²时，保留率可分别达到81.11%、87.81%、86.15%、73.73%和73.72%。

研究结论：

综合病毒灭活效果与vWF制品功能保留，兼顾安全性与有效性，UVC 辐照仪的最佳照射剂量为100~150 J/m²。

 

CRISPR/Cas13a-Coupled RT-RAA: A High-Sensitivity Method for Zika Virus RNA Detection

Abstract

Background and Aims

Zika virus (ZIKV), an arbovirus transmitted by Aedes species mosquitoes, represents a critical health threat to transfusion recipients due to its capacity for blood-borne transmission. Current diagnostic methodologies exhibit limitations, including prolonged turnaround times for virus isolation, false-positive nucleic acid test results caused by contamination risks, and the inability of antigen/antibody detection systems to achieve serotype differentiation or resolve cross-reactivity issues. Consequently, there is an urgent need to develop rapid and accurate detection technologies for Zika virus. The CRISPR/Cas13a system demonstrates enhanced detection efficiency compared to conventional approaches through its integrated reverse-transcription recombinase-aided amplification (RT-RAA) technology, which is specifically optimized for low viral load specimens. This system simultaneously achieves improvements in both detection sensitivity and specificity. This study aims to establish a CRISPR/Cas13a-based detection method f or ZIKV. The protocol employs amplification of conserved viral gene fragments to generate sufficient template material, followed by CRISPR-mediated specific recognition and activation of Cas13a protein. Subsequent cleavage of target nucleic acid sequences by Cas13a triggers signal probe activation, enabling rapid visual detection within a single-tube format. This optimized workflow reduces nucleic acid amplification time to 30 minutes while maintaining high performance, demonstrating technical feasibility for point-of-care screening of Zika virus in resource-limited settings.

Research Objectives

(1) Establishment and Optimization of RT-RAA Reaction
(2) Establishment and Optimization of CRISPR/Cas13a Reaction
(3) Development of One-Tube Integrated RT-RAA-T7-CRISPR/Cas13a Detection Assay
(4) Evaluation of Sensitivity and Specificity for the One-Tube Integrated RT-RAA-T7-CRISPR/Cas13a Detection Assay
(5) Comparative Analysis of Clinical Sample Detection Results between the One-Tube Integrated RT-RAA-T7-CRISPR/Cas13a Assay and Conventional RT-PCR Method

Methods

(1) Identification and Screening of ZIKV Conserved Sequences
Conserved sequences of Zika virus (ZIKV) were identified and screened from the NCBI database. Standard plasmids containing ZIKV pathogen conserved sequences were constructed through PCR amplification and subsequent cloning.

(2) Establishment and Optimization of RT-RAA Assay
The RT-RAA assay was established using the ZIKV standard sample from the National Center for Clinical Laboratories (NCCL). Agarose gel electrophoresis was performed to analyze RT-RAA products. Primer pairs and reaction temperatures were optimized based on the intensity and specificity of DNA bands. Final conditions were selected based on bright and distinct electrophoretic bands.

(3) Establishment and Optimization of CRISPR/Cas13a Assay
The CRISPR/Cas13a assay was optimized using the NCCL ZIKV standard sample. Real-time fluorescence intensity was recorded via a qPCR instrument to evaluate crRNA sequences, crRNA concentration, Cas13a enzyme concentration, and single-stranded RNA (ssRNA) concentration. Parameters were selected based on the highest fluorescence intensity.

(4) Development of One-Tube Integrated RT-RAA-T7-CRISPR/Cas13a Assay
The optimized CRISPR/Cas13a reaction system was added to the bottom of a tube, followed by sequential addition of the RT-RAA reaction mixture, T7 transcription reagents, and MgCl₂. The reaction was incubated at 37°C for 30 minutes to activate the CRISPR/Cas13a cleavage system. End-point fluorescence was visually observed under real-time fluorescence signals were recorded using a qPCR instrument. Alternatively, lateral flow strip detection was performed.

(5) Evaluation of Specificity and Sensitivity
Specificity: The one-tube RT-RAA-T7-CRISPR/Cas13a assay was validated using the NCCL ZIKV standard as a positive control and ddH₂O as a negative control. Cross-reactivity was assessed by testing 10 other viruses (HBV, HCV, DENV-1, DENV-2, SINV, PRV, PPV, EMCV, MVM, BVDV).
Sensitivity: Serial dilutions of the ZIKV standard (4×10^4-0 copies/μL) were tested to determine the limit of detection (LOD).

(6) Clinical Performance Evaluation

Simulated clinical plasma samples were prepared by mixing laboratory-amplified ZIKV viral RNA with healthy donor plasma. Nucleic acids were extracted from these samples and analyzed in parallel using the one-tube RT-RAA-T7-CRISPR/Cas13a assay and the gold-standard RT-PCR method. Concordance rates and clinical sensitivity/specificity were calculated to evaluate the assay’s performance.

Results

(1) A standard plasmid containing ZIKV sequences was successfully constructed through PCR amplification and subsequent screening of clinical samples.

(2) The optimal RT-RAA primers for ZIKV were identified, with the ideal reaction conditions determined as 37°C for 30 minutes.

(3) The optimal crRNA for ZIKV was selected, and the CRISPR/Cas13a reaction parameters were established as 150 nM crRNA, 75 nM Cas13a, 1.2 μM MgCl₂, and 100 nM ssRNA.

(4) A one-tube integrated RT-RAA-T7-CRISPR/Cas13a visual detection assay was successfully developed for ZIKV.

(5) The one-tube RT-RAA-T7-CRISPR/Cas13a assay demonstrated excellent specificity, showing no cross-reactivity with other viruses. The sensitivity of the assay reached 4 copies/μL.

(6) The one-tube RT-RAA-T7-CRISPR/Cas13a assay was used to test 45 clinical simulated plasma samples (30 ZIKV-positive and 15 ZIKV-negative). The detection rate was 100%. In contrast, RT-PCR failed to detect viral loads below 10³ copies/mL.

Conclusions
The one-tube integrated RT-RAA-T7-CRISPR/Cas13a detection method established in this study exhibits high specificity and sensitivity, enabling visual detection of ZIKV. It is particularly suitable for resource-limited settings and field applications, demonstrating significant potential for advancing next-generation biosensors in nucleic acid-based diagnostics.

Viral Inactivation in von Willebrand Factor Preparations via UVC Irradiation: An Experimental Approach

Abstract

Background and Aims

Von Willebrand factor (vWF), a critical coagulation cofactor in plasma, mediates platelet adhesion and stabilizes von Willebrand factor VIII, playing a central role in hemostasis. As a core component of plasma-derived therapeutics and recombinant protein drugs, vWF products face biosafety risks associated with viral contamination. Pathogens such as human parvovirus B19 and hepatitis viruses may be introduced through source plasma or manufacturing processes, increasing the risk of cross-infection. Although established viral inactivation methods like heat treatment and low-pH incubation are widely used for blood product safety control, their potential damage to vWF structure and function—such as multimer dissociation and loss of coagulation activity—limits clinical safety and efficacy. For instance, heat inactivation (60°C for 30 minutes) causes a >40% reduction in vWF ristocetin cofactor activity (RCoA), while solvent/detergent (S/D) treatment effectively inactivates enveloped viruses but fails against non-enveloped viruses. Additionally, residual toxicity may arise from incomplete removal of solvents/detergents. Therefore, developing an alternative technology that achieves efficient viral inactivation without significantly compromising vWF function holds significant scientific and clinical value.

Ultraviolet-C (UVC, 190–280 nm) irradiation inhibits viral replication by inducing pyrimidine dimer formation in viral nucleic acids, with minimal interference in protein structure. This method has been applied to platelet viral inactivation. However, its efficacy depends on viral capsid structure, nucleic acid type, and sample transparency. For example, non-enveloped parvovirus B19 exhibits 2–3 log lower sensitivity to UVC compared to adenoviruses due to its dense capsid. Furthermore, UVC irradiation dose must be precisely controlled: insufficient doses lead to incomplete inactivation, while excessive doses may induce photooxidative damage to vWF activity. This study investigates UVC irradiation (254 nm wavelength) for inactivating indicator viruses in vWF products. Gradient experiments were conducted to optimize irradiation parameters, aiming to establish a UVC-based protocol that balances safety and efficacy.

Research Objectives

(1) Dose-dependent analysis of vWF antigen levels (vWF:Ag) and functional activity (vWF:CBA)

(2) Evaluation of UVC irradiation efficacy against four indicator viruses across dose gradients

(3) Balanced optimization of viral inactivation efficiency and vWF functional preservation

Methods

(1) Dose-response inactivation assay for UVC irradiation across gradient doses

(2) Detection of vWF antigen (vWF:Ag) and collagen-binding activity (vWF:CBA)

(3) Dose-response analysis of viral inactivation efficiency and vWF functional preservation

Results

(1) When UVC irradiation doses exceeded 100 J/m², a ≥4.0 log₁₀ reduction in viral titers was observed for all four indicator viruses (PPV, EMCV, PRV, and VSV).

(2) The retention rates of vWF antigen (vWF:Ag) were 93.67%, 91.72%, 93.54%, 79.05%, and 85.50% at UVC doses of 100, 125, 150, 175, and 200 J/m², respectively.

(3) Correspondingly, the retention rates of vWF collagen-binding activity (vWF:CBA) were 81.11%, 87.81%, 86.15%, 73.73%, and 73.72% under identical dose conditions.

Conclusions

Considering both inactivation efficacy and functional preservation while ensuring safety and effectiveness, 100–150 J/m² represents the optimal inactivation dose.