鼠疫耶尔森菌重组酶聚合酶核酸扩增荧光检测方法的建立

目的 建立一种简单,快速的重组酶聚合酶核酸扩增技术检测鼠疫耶尔森氏菌的方法.方法 基于耶尔森氏菌的特异性序列设计引物及探针;通过对不同温度的检测来优化反应温度;通过对不同菌株的DNA模板进行检测评价该方法的特异性;通过对不同稀释浓度的样本DNA模板进行检测来确定灵敏性;通过模拟样品进行应用评价.结果 基于鼠疫耶尔森菌的...     

五种出血热病毒重组酶聚合酶等温扩增方法的建立

目的 建立五种出血热相关病毒,即扎伊尔型埃博拉病毒、苏丹型埃博拉病毒、马尔堡病毒、拉沙病毒和黄热病毒的一步法重组酶聚合酶等温扩增(RPA)技术,为病原体的确定提供现场快速检测方法.方法 通过基因组序列分析,选择每种病毒的特异核酸片段作为检测靶基因,针对靶基因序列设计RPA检测的引物和探针;取系列稀释的模板基因进行RPA检测,确定其灵敏性;取出血热相关病毒核酸为模板进行RPA检测,确定其特异性;验证不同温度反应条件(37~42℃)对检测结果的影响.结果 五种出血热病毒的RPA反应体系均可有效扩增靶基因,灵敏性为1.5×102~1.5×103copies/反应,与其他出血热相关病毒的核酸无交叉反应;RPA实验在37~42℃均可实现靶基因的有效扩增.结论 建立了五种出血热病毒的RPA等温扩增体系,该体系反应快速,温度范围宽,适用于现场快速检测.     

基于上转发光免疫层析技术的常见食源性致病菌快速检测方法研究与评价

目的：研制针对4种常见食源性致病菌：甲型副伤寒沙门菌（ Salmonella paratyphi A）、乙型副伤寒沙门菌（Salmonella paratyphi B）、大肠埃希菌O157∶H7（Escherichia coli O157∶H7）、副溶血弧菌（Vibrio parahaemolyticus）的基于上转发光免疫层析技术（ UPT－LF）的快速定量检测试纸,并对其检测性能进行评价。方法以上转发光纳米颗粒（ UCP－NPs）作为示踪物,基于双抗体夹心检测模型,研制针对上述4种靶标菌的UPT－LF试纸;以4种靶标菌的系列浓度标准菌悬液作为标准样品,评价试纸的敏感性、特异性、线性和精密性,并进行模拟染菌食品的检测,评价模拟样品阳性检出率。结果针对4种靶标菌的UPT－LF试纸均具有良好的线性,相关系数r为0．985～0．996;检测敏感性达105～106 CFU／ml,与其他近缘菌株无交叉反应;对乳制品、饮料、小食品、水产、肉类等细菌污染的食品检出率较高。结论该研究建立的对4种常见食源性致病菌进行快速检测的UPT－LF,简便快速,具有良好的敏感性、特异性和线性定量能力,操作性能可满足食品安全检测的要求。     

Rapid and direct detection of male DNA by recombinase polymerase amplification assay

Screening of male DNA is important in forensic investigations, especially sexual assault cases. Quantitative real-time polymerase chain reaction (qPCR) is widely used for the detection of male DNA. However, the use of this technique as a screening tool is time-consuming and labor-intensive. In this study, we established a recombinase polymerase amplification (RPA) assay targeting the multicopy loci on the Y-chromosome for the rapid detection of male DNA (referred to as Y-RPA). The Y-RPA assay was able to detect male DNA in less than 20 min with a sensitivity of 0.025–0.005 ng/µL. Additionally, the Y-RPA assay was highly tolerant to inhibitors; male DNA was detectable in the presence of up to 1000 ng/µL humic acid, 250 µM indigo carmine, and 500 µM hematin. Then, considering its tolerance to inhibitors, we examined the feasibility of the direct Y-RPA assay. The alkaline lysis protocol (addition of sodium hydroxide and heating at 95 °C for 5 min) was employed for preparing the DNA template. The Y-RPA assay successfully detected male DNA using crude DNA extracted from blood, saliva, and semen samples. This approach enabled the screening of male DNA within approximately 30 min (5 min for lysis and 20 min for Y-RPA). These findings suggest that the Y-RPA assay is a promising screening tool for the rapid, simple, and efficient detection of male DNA.     

Development of specific and rapid detection of human DNA by recombinase polymerase amplification assay for forensic analysis

The determination of human-derived samples is very important in forensic investigations and case investigation in order to determine vital information on the suspect and the case. In this study, we established a recombinase polymerase amplification (RPA) assay for rapid identification of human-derived components. The sensitivity of the assay was 0.003125 ng, with excellent species specificity, and human-derived DNA could be detected in the presence of non-human-derived components at a ratio of 1:1000. Moreover, the RPA assay had a strong tolerance to inhibitors, in the presence of 800 ng/μL humic acid, 400 ng/μL tannic acid, and 8000 ng/μL collagen. In forensic investigation, common body fluids (blood, saliva, semen, vaginal secretions) are all applicable, and the presence of DNA can be detected from samples after simple alkaline lysis, which greatly shortens the detection time. Four simulation and case samples (aged bones, aged bloodstains, hair, touch DNA) were also successfully applied. The above research results show that the RPA assay constructed in this study can be fully applied to forensic medicine to provide high sensitivity and applicability detection methods.     

基于上转发光免疫层析技术快速检测单核细胞增生李斯特菌

目的：建立可快速检测单核细胞增生李斯特菌（ Listeria monocytogenes, LM）的上转发光免疫层析技术（ up-converting phosphor technology based lateral flow assay , UPT-LF）,即LM-UPT-LF。方法针对LM特异的p60蛋白制备单克隆抗体,并与上转发光纳米颗粒（ up-converting phosphor nanoparticles , UCP-NPs）共价偶联作为结合物,采用双抗体夹心免疫层析模式建立LM-UPT-LF,并对其敏感性与特异性进行评价。结果建立了可快速检测LM的LM-UPT-LF平台,在样本＜10 cfu、10～99 cfu、100～1000 cfu绝对LM污染量条件下可分别在培养20、18、16 h检出阳性。其他13种食源性致病菌在高浓度（109 cfu／ml）时无非特异性交叉。结论所建立的LM-UPT-LF操作简便快速,具有良好的敏感性和特异性,适用于一线病原调查与检测。     

蓖麻毒素单抗制备及上转发光免疫层析定量检测方法研究

目的：建立一种可快速、精确地对蓖麻毒素（ RT）进行定量检测的上转发光免疫检测技术（ UPT-LF）,即RT-UPT-LF。方法采用杂交瘤细胞技术制备蓖麻毒素单克隆抗体并对效价进行评价;采用效价最高的4种单抗分别与上转发光纳米颗粒（ UCP-NP）共价偶联,分别作为检测带,进而两两配伍,确定最优的配伍组合条件建立RT-UPT-LF方法;对RT-UPT-LF的敏感性、精密性、定量能力和特异性进行评价。结果与结论所建立的RT-UPT-LF方法可在15 min内完成对蓖麻毒素的检测,灵敏度可达0．5 ng／ml,定量范围为0．5～1000 ng／ml,与其他高浓度毒素无非特异反应,该法为蓖麻毒素检测提供了一种新手段。     

聚合酶螺旋反应快速检测甲型H1N1流感病毒

目的 建立利用聚合酶螺旋反应(PSR)快速检测甲型H1N1流感病毒的方法.方法 针对甲型H1N1流感病毒的特异性HA基因设计了6套引物,通过实时浊度法和显色法两种方法判断结果.结果与结论 从6套引物中筛选出了最佳引物,并确定最佳温度为65℃;进一步实验表明最佳引物能特异性地检测H1N1病毒,与14种其他呼吸道病原核酸无交叉反应,敏感性达到100拷贝,与PCR敏感性一致.所建立的方法简单快速、特异性强、敏感性高,适合现场和基层单位应用推广.