Antigen detection provides particularly valuable information for medical diagnoses;however,the current detection methods are less sensitive and accurate than nucleic acid analysis.The combination of CRISPR/Cas12a and ...Antigen detection provides particularly valuable information for medical diagnoses;however,the current detection methods are less sensitive and accurate than nucleic acid analysis.The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm,but sensitive sensing and stable amplification in antigen detection remain challenging.Here,we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy.Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive,fast,and stable antigen detection.In a demonstration of this method,the limit of detection was at the single virus level(0.17 fM,approximately two copies/μL)in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples.The entire procedure required only 20 min.Given our system’s simplicity and modular setup,we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.展开更多
目的建立基于荧光定量PCR(fluorescence quantitative PCR,FQ-PCR)技术的产前无创胎儿ABO、RhD及RhCE血型基因型联合检测方法,并评估其在胎儿新生儿溶血病(Hemolytic Disease of the Fetus and Newborn,HDFN)产前早期诊断中的临床价值...目的建立基于荧光定量PCR(fluorescence quantitative PCR,FQ-PCR)技术的产前无创胎儿ABO、RhD及RhCE血型基因型联合检测方法,并评估其在胎儿新生儿溶血病(Hemolytic Disease of the Fetus and Newborn,HDFN)产前早期诊断中的临床价值。方法前瞻性纳入2022年1月至2024年12月于本院产检的200例单胎妊娠高风险孕妇,分为ABO血型不合组100例、RhD血型不合组50例、RhCE血型不合组50例及对照组200例,采用FQ-PCR技术检测母体血浆中胎儿游离DNA(cff-DNA),靶向ABO系统(261delG、796C>A)、RHD基因外显子5/7及RhCE系统(C/c、E/e)关键位点。产后通过脐带血血清学验证血型及溶血三项检测(直接抗人球蛋白试验、游离抗体试验、抗体释放试验),评估检测一致性及高危因素。结果ABO、RhD及RhCE血型的检测符合率分别为98.0%(98/100)、100.0%(50/50)及96.0%(48/50)。ABO血型不合组HDFN发生率为69.0%(69/100),显著高于RhD血型不合组(10.0%,5/50)及RhCE血型不合组(2.0%,1/50)。多因素分析显示,母亲O型血(OR=3.021)、母亲RhD阴性(OR=5.253)及产妇年龄≥35岁(OR=1.950)为HDFN独立危险因素(P均<0.05)。结论产前无创多重血型抗原基因型联合检测可显著提升HDFN早期诊断效能,为高危孕妇提供精准预警。展开更多
目的建立巢式PCR技术检测RhD阴性孕妇血浆中游离胎儿DNA(cffDNA)的RHD基因型,以预测胎儿RhD血型。方法采用QIAamp DNA Blood Mini Kit提取32例RhD阴性孕妇血浆游离DNA,针对RHD外显子7和10分别设计外侧、内侧2组特异性引物,巢式PCR方法...目的建立巢式PCR技术检测RhD阴性孕妇血浆中游离胎儿DNA(cffDNA)的RHD基因型,以预测胎儿RhD血型。方法采用QIAamp DNA Blood Mini Kit提取32例RhD阴性孕妇血浆游离DNA,针对RHD外显子7和10分别设计外侧、内侧2组特异性引物,巢式PCR方法检测孕妇血浆游离胎儿DNA的RHD型,测序验证PCR产物的序列特异性。结果孕妇血浆游离DNA经巢式PCR扩增后,有27例成功扩增出RHD外显子7、10特异性条带,5例未检测到RHD基因特异性扩增,32例中有30例胎儿RHD型与出生后血型相符,检测准确率为93.1%。结论采用巢式PCR技术检测RhD阴性孕妇血浆游离胎儿DNA来判定胎儿RHD型,具有良好的准确性、敏感性和特异性,为RhD新生儿溶血病的早期诊断提供了一种新的、可靠的检测手段。展开更多
基金This work was supported by the National Key R&D program of China(2020YFA0907800)the National Natural Science Foundation of China(31922002,31720103901,31772242 and 31870040),the 111 Project(B18022)+1 种基金the Fundamental Research Funds for the Central Universities[22221818014]the Youth Innovation Promotion Association CAS(Y202027)to W.W and the Open Project Funding of the State Key Laboratory of Bioreactor Engineering.
文摘Antigen detection provides particularly valuable information for medical diagnoses;however,the current detection methods are less sensitive and accurate than nucleic acid analysis.The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm,but sensitive sensing and stable amplification in antigen detection remain challenging.Here,we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy.Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive,fast,and stable antigen detection.In a demonstration of this method,the limit of detection was at the single virus level(0.17 fM,approximately two copies/μL)in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples.The entire procedure required only 20 min.Given our system’s simplicity and modular setup,we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.
文摘目的建立基于荧光定量PCR(fluorescence quantitative PCR,FQ-PCR)技术的产前无创胎儿ABO、RhD及RhCE血型基因型联合检测方法,并评估其在胎儿新生儿溶血病(Hemolytic Disease of the Fetus and Newborn,HDFN)产前早期诊断中的临床价值。方法前瞻性纳入2022年1月至2024年12月于本院产检的200例单胎妊娠高风险孕妇,分为ABO血型不合组100例、RhD血型不合组50例、RhCE血型不合组50例及对照组200例,采用FQ-PCR技术检测母体血浆中胎儿游离DNA(cff-DNA),靶向ABO系统(261delG、796C>A)、RHD基因外显子5/7及RhCE系统(C/c、E/e)关键位点。产后通过脐带血血清学验证血型及溶血三项检测(直接抗人球蛋白试验、游离抗体试验、抗体释放试验),评估检测一致性及高危因素。结果ABO、RhD及RhCE血型的检测符合率分别为98.0%(98/100)、100.0%(50/50)及96.0%(48/50)。ABO血型不合组HDFN发生率为69.0%(69/100),显著高于RhD血型不合组(10.0%,5/50)及RhCE血型不合组(2.0%,1/50)。多因素分析显示,母亲O型血(OR=3.021)、母亲RhD阴性(OR=5.253)及产妇年龄≥35岁(OR=1.950)为HDFN独立危险因素(P均<0.05)。结论产前无创多重血型抗原基因型联合检测可显著提升HDFN早期诊断效能,为高危孕妇提供精准预警。
文摘目的建立巢式PCR技术检测RhD阴性孕妇血浆中游离胎儿DNA(cffDNA)的RHD基因型,以预测胎儿RhD血型。方法采用QIAamp DNA Blood Mini Kit提取32例RhD阴性孕妇血浆游离DNA,针对RHD外显子7和10分别设计外侧、内侧2组特异性引物,巢式PCR方法检测孕妇血浆游离胎儿DNA的RHD型,测序验证PCR产物的序列特异性。结果孕妇血浆游离DNA经巢式PCR扩增后,有27例成功扩增出RHD外显子7、10特异性条带,5例未检测到RHD基因特异性扩增,32例中有30例胎儿RHD型与出生后血型相符,检测准确率为93.1%。结论采用巢式PCR技术检测RhD阴性孕妇血浆游离胎儿DNA来判定胎儿RHD型,具有良好的准确性、敏感性和特异性,为RhD新生儿溶血病的早期诊断提供了一种新的、可靠的检测手段。
