探针浓度值对LSAW-bisPNA传感器检测系统的影响

目的探讨双体肽核酸（bisPNA）探针浓度值对漏声表面波（LSAW）-bisPNA基因传感器检测系统的影响。方法LSAW基因传感器表面分别固定终浓度为0.001、0.005、0.01、0.05、0.5、1.0、1.5、2.0、3.0、4.0μmol/L的bisPNA探针后,分别观察其与相应的HPV靶序列杂交时相位变化及反应所需时间。结果与HPV靶序列杂交所引起的相位变化值是先升后降,以1.0μmol/L探针浓度为分界线;杂交平衡时间上各组间未见明显的变化趋势。结论1.0μmol/L bisPNA探针固定浓度最为适宜。     

漏声表面波生物传感器检测系统构建时靶序列浓度的影响

目的 探讨新型的漏声表面波(LSAW)传感器检测系统构建时,人乳头状瘤病毒(HPV)靶序列浓度对其杂交效应和反应时间的影响.方法 LSAW传感器表面先固定上1.0 μmol/L的肽核酸(PNA)探针,观察终浓度为1 pg/L～1 mg/L的HPV靶序列DNA与探针进行杂交所引起的相位变化及反应所需时间,并对相位下降值与靶序列浓度做线性回归,确定其线性范围.结果 随着靶序列浓度从1 pg/L增加到1 mg/L,杂交反应引起的相位下降值呈先增加后趋于缓和的趋势,以100μg/L为分界线,而杂交平衡时间并没有显现出一定的变化趋势;在1 pg/L～100μg/L的HPV靶序列浓度范围内,相位变化与靶序列浓度的线性回归方程为△P=2.3392 1gC+14.584,相关系数r2=0.9622.结论 随着靶序列浓度的升高,杂交反应引起的相位下降值呈典型饱和曲线趋势,靶序列浓度的线性检测范围为:1 pg/L～100μg/L.     

离子强度对乙型脑炎病毒基因传感器检测系统的影响

目的 探讨磷酸盐缓冲液(PBS)中Na+浓度值对乙型脑炎病毒(JEV)基因传感器检测系统的影响.方法 漏声表面波(LSAW)基因传感器表面固定终浓度为1.0μmol/L的乙型脑炎病毒探针,然后分别在pH 7.6的10 mmol/L PBS缓冲液(含Na+浓度值0.1～0.6 mol/L)中和终浓度为1.0 μmol/L的JEV靶序列杂交,分别记录相位变化及反应所需时间.结果 杂交反应引起的相位变化是先上升后趋于缓和的趋势,以0.3 mol/L Na+浓度为分界线;杂交平衡时间同相位变化一致,当PBS中Na+浓度为0.3 mol/L,杂交反应的平衡时间为33 min.结论 随着Na+浓度的升高,杂交反应引起的相位变化呈典型的饱和曲线趋势,PBS中Na+浓度0.3 mol/L,杂交反应平衡时间33 min,是LSAW-JEV基因传感器杂交的最适反应条件.     

漏声表面波生物传感器不同检测方法的实验研究

目的构建双通道漏声表面波生物传感器液相检测技术平台。方法以人乳头状瘤病毒为检测对象,传感器检测系统运用相位、频率及延迟线3种检测方法对其进行实时检测;利用扫描隧道显微镜观察传感器裸金表面、固定巯基化探针以及核酸杂交后的金膜表面结构的微观变化。结果相位和频率两种检测方法较好;扫描隧道显微镜下可以看到裸金膜表面金原子排列有序;探针分子分布密度增加;杂交后寡核酸分子密度更为密集。结论成功构建了漏声表面波生物传感器液相检测技术平台并运用多种检测方法;扫描隧道显微镜可以形象直观地对传感器金膜表面、核酸杂交进行直接观察,结果进一步证实了实验结论的正确性。     

乙型肝炎病毒靶序列浓度对新型肽核酸压电基因传感器阵列的影响

目的探索乙型肝炎病毒(HBV)靶序列浓度值对肽核酸压电基因传感器阵列杂交效应的影响.方法压电基因传感器阵列表面先固定上1.5μmol/L的bis-PNA探针,观察终浓度为1pg/L至100μg/L的HBV DNA与探针,进行杂交所引起的频率变化及反应所需时间.并对频率下降值与靶序列浓度做线性回归,确定其线性范围.结果靶序列浓度为1pg/L时,传感器未能检测出任何频率的改变.随着浓度从10 pg/L增加到100μg/L,杂交反应引起的频率下降值呈先增加后趋于缓和的趋势,以10μg/L为分界线,而杂交平衡时间并没有显现出一定的变化趋势.在10pg/L到10μg/L的浓度范围内,浓度与频率下降值的线性回归方程为lgC=-2.7455 0.0691×△F,相关系数r=0.9923.结论随着靶序列浓度的升高,杂交反应引起的频率下降值呈典型饱和曲线趋势,靶序列浓度的线性检测范围为10pg/L～10μg/L.     

长、短链探针以链延伸反应提高基因传感器检测MRSA灵敏度的实验研究

目的：利用链延伸反应的原理延长引物链以提高石英谐振式基因传感器表面的质量负载，从而提高传感器检测MRSA的灵敏度。方法：以不对称PCR的反应产物做为长链探针，人工合成的寡核苷酸片段为短链探针，在基因传感器阵列表面分别固定上金黄色葡萄球菌mecA和femA的长链及短链探针共4种探针片段，与相应的靶序列杂交后，加入Klenow酶，37℃进行链延伸反应1h。结果：长、短链探针均有效地固定了传感器表面。mecA,femA短链探针在链延伸反应前的检测灵敏度为0.5nmol/L，但经链延伸反应后检测灵敏提高到了0.05nmol/L；而长链探针却无法与相应的基因组靶序列发生杂交。结论：短链探针链延伸反应有效地提高了石英谐振式基因传感器的灵敏度，但该法不适用于链探针。     

基于自组装单分子层技术的压电石英晶体DNA传感器固定方法的研究

目的 探讨一种高效的压电石英晶体DNA传感器固定的方法。方法 分别采用常规自组装法（未还原探针法）与探针预先经过还原的自组装法（还原探针法），将针对铜绿假单胞菌的探针固定在DNA传感器的金膜表面，比较不同探针浓度下，探针固定和杂交反应所引起的频率的变化以及所用的时间。结果 与未还原探针法相比，还原探针法具有探针固定量大、反应速度快、频率下降明显等优点。结论 还原探针法提高了DNA探针的固定效率，优于未还原探针法。     

漏声表面波生物传感器直接快速检测病原体DNA的实验研究

目的应用漏声表面波生物传感器直接检测从临床标本中提取的HPV基因组DNA。方法选取36例临床确诊为宫颈癌的患者,提取其生殖器分泌物的基因组DNA,并经荧光定量PCR检测为HPV阳性的标本,以14例健康志愿者为阴性对照;利用漏声表面波生物传感器检测临床样本,并与荧光定量PCR法进行方法学比较。结果传感器法检测35例标本为阳性,阳性率为97.22%,而荧光定量PCR法检测36例均为阳性;14例阴性对照标本中,两种方法的检测结果均为阴性;传感器法的检测限为1.21pg/L。结论传感器法与荧光定量PCR法差异无统计学意义,一致性较好,且漏声表面波生物传感器实现了对临床标本中HPV基因组DNA的直接快速检测。     

基因芯片技术(下)

基因芯片测序采用的是杂交测序(SBH)的原理.该技术包括:末知序列的DNA与大量的短链寡核苷酸探针杂交;所形成完整的双链体的鉴定与分析.如对于一种12碱基的靶基因片段,假设其核苷酸顺序为5'-AGCCTAGCTGAA-3'.用它与一组8个碱基完全排列组合的寡核苷酸探针群混合杂交.在这一组由总数为48=65536种8个碱基的寡核苷酸组成的完全探针群中,仅有5种会同靶基因片段形成完全互补的双链分子.     

多聚T尾型探针在压电石英晶体微天平DNA传感器检测铜绿假单胞菌中的实验研究

目的 构建一种压电石英晶体微天平(QCM)DNA传感器多聚T尾型探针敏感膜制备新方法,同时对其重要影响因素进行优化探讨.方法 设计一种多聚T尾型探针,固定在压电QCM DNA传感器晶振表面,对探针固定浓度.杂交温度和杂交时间等因素进行实验研究,进一步将构建的多聚T尾型探针DNA传感器和普通探针DNA传感器用于铜绿假单胞菌检测,比较两种传感器的频率响应性能,同时对该压电QCM DNA传感器的再生性能进行研究.结果 探针最佳固定浓度为2.0 μmol/L,最适杂交温度为40℃,最适杂交时间为90 min;多聚T尾型探针DNA传感器的响应性能显著大于普通探针DNA传感器(P<0.05),可以重复使用7次.结论 基于多聚T尾型探针的压电QCM DNA传感器,具有杂交效率高、技术难度低、频率响应性能高等优点,在压电QCM DNA传感器基因检测领域有较好的应用前景.     

肽核酸生物传感器直接检测临床标本中提取的病毒基因组DNA

目的 以肽核酸生物传感器检测系统直接检测从临床标本中提取的乙型肝炎病毒基因组DNA。方法 抽取63例临床免疫学检测证实为乙型肝炎病毒(HBV)感染者标本血清的DNA，以及15例免疫学检测全阴的标本血清中的DNA，分别用肽核酸生物传感器法以及定量PCR法检测，比较两种方法的优劣并确定传感器法的检测限。结果 传感器法检测60例标本阳性，阳性率为95．24％，而定量PCR法检测63例均为阳性；15例阴性血清两种方法检测均为阴性；传感器法的检测限为8．6pg／L；两种方法检测HBV差异无显著性，但传感器检测法所用时间更短，且无需进行探针的标记。结论 利用肽核酸生物传感器成功地绕过了PCR扩增而直接检测出了临床标本中的HBV基因组DNA。     

蛋白质缺乏对小鼠胸腺细胞花生凝集素受体的影响

本文用异硫氰酸荧光素标记花生凝集素(FITC-PNA)测定蛋白质缺乏小鼠胸腺细胞表面花生凝集素(PNA)受体的变化。结果表明,给予缺蛋白饲料后,小鼠体重不断减轻,胸腺组织逐渐萎缩,PNA阳性细胞数减少,荧光反应强度减弱。小鼠体重下降与胸腺细胞表面PNA受体的减少呈显著正相关(P<0.01)。提示蛋白质缺乏可对小鼠胸腺细胞的分化、成熟产生不良影响。     

基于HRP-DAB信号放大系统的压电石英晶体DNA传感器微阵列检测表皮葡萄球菌的研究

目的在已构建的压电压电石英晶体DNA传感器检测系统中引入HRP-DAB生物信号放大系统,提高传感器的检测灵敏度,并基于该系统建立一种表皮葡萄球菌快速检测方法。方法采用自组装技术在压电石英晶体DNA传感器上固定针对表皮葡萄球菌的寡核苷酸探针,将掺入生物素(biotin)-dUTP的表皮葡萄球菌靶DNA与之进行杂交反应,加入HRP-链亲和素(streptavidin)、DAB工作液,观察反应频率变化;对35份表皮葡萄球菌感染的临床标本进行检测,并与传统培养法进行比较。结果该压电石英晶体DNA传感器微阵列检测表皮葡萄球菌的灵敏度为95.5%,特异度为84.6%,正确诊断指数为0.801,检出限为2×102CFU/ml,与血培养法差异无统计学意义(P>0.05)。结论基于HRP-DAB信号放大系统的压电石英晶体DNA传感器微阵列,有效的提高了压电石英晶体传感器检测的灵敏度,可用于临床病原菌感染的快速检测,具有较高的临床推广价值。     

A fluorescent based PCR assay for the detection and quantitation of Giardia duodenalis genotypes in mixed populations.

A method based on the polymerase chain reaction has been developed for differentiating between genotypically and phenotypically distinct strains of Giardia duodenalis and quantifying the amount of initial template of the different genotypes in mixed populations. The assay relies on a sequence-specific probe, labelled with two fluorescent dyes, designed to bind within the small subunit ribosomal (SSU) RNA gene. This target region is amplified by primers specific for either Group 1 or Group 2-type isolates of G. duodenalis and the probe binds within the primer-targeted region. This quantitative method takes advantage of the 5' nuclease activity of Taq DNA polymerase, which, on encountering a probe bound within the target DNA sequence cleaves it, causing it to become dissociated from the template. When the two fluorescent dyes bound to the probe are in close proximity (when the probe is intact), the interaction of the two dyes prevents the reporter dye from fluorescing. However, during the extension phase of amplification, the activity of the DNA polymerase causes the dyes to become separated and hence the reporter dye increases its fluorescent intensity. This release of fluorescence is directly related to the amplified amount of target template. This assay was developed with the aim of providing a unique method with which to investigate interactions within mixed populations of genetically distinct strains of G. duodenalis.     

In vitro growth kinetics, differentiation and morphological characterisation of Tunisian Leishmania infantum parasites

In this study, a negative peanut agglutinin (PNA) selection was used as a marker for promastigote differentiation to compare the in vitro growth and differentiation kinetics of two visceral and two cutaneous Leishmania (Leishmania) infantum parasites. All parasites had different growth and differentiation kinetics. Cultures initiated with PNA(+) parasites purified during the early stationary phase (Day 4), when PNA(-) (non-agglutinating) parasites peaked, yielded a high PNA(-) percent. Further morphological analysis at this time point showed that 60-86% of PNA(+) forms were procyclics, whilst PNA(-) forms were composed of 53-71% leptomonads. Nectomonads were present both in PNA(-) and PNA(+) promastigote fractions at nearly equivalent proportions, suggesting that they constitute a transition state in the Leishmania development process, with a fraction of them sharing common constituents of the surface coat with procyclics and the other with leptomonads. Obtaining a high density of promastigotes undergoing developmental differentiation may be useful for further molecular and biochemical identification of developmental stage-specific markers.     

Diagnosis of Plasmodium vivax malaria using a specific deoxyribonucleic acid probe

A deoxyribonucleic acid (DNA) probe which specifically distinguishes Plasmodium vivax from P. falciparum malaria has been derived from a P. vivax genomic DNA library. This probe, VPL101, consists of 3.2 kilobase pairs and does not hybridize with up to 6 micrograms of human or P. falciparum DNA. VPL101 contains at least two copies of a 205 base pair repeat sequence. The subcloned repeat probe, VPL101/5, reacted with 73 of 76 microscopically diagnosed P. vivax samples but not with any of 17 human DNA samples or any of 8 P. falciparum DNA samples from cultured parasites. It was possible to detect P. vivax in mixed infections in which only P. falciparum parasites were identifiable by microscopy. This P. vivax DNA probe provides a useful epidemiological tool for malaria control programmes.     

DNA生物传感器检测电路的设计

设计一种用于检测某种DNA杂交反应的石英晶体传感器及其放大、显示电路。将单链DNA作为探针点在电极表面,测量晶振频率的改变量,并设计出后续处理电路。该设计能实时动态显示出DNA杂交反应中频率的变化。     

Specific detection of Salmonella enterica serotype Enteritidis using the polymerase chain reaction.

An assay was developed for the specific detection of Salmonella enterica serotype Enteritidis, using a novel application of the polymerase chain reaction (PCR). This PCR assay is based on the mismatch amplification mutation assay, an allele-specific reaction, and can discriminate Enteritidis from all other salmonella. PCR primers were selected to amplify a 351-base pair (bp) DNA fragment from the salmonella plasmid virulence A (spv A) gene of Enteritidis. A single base difference at position 272 is present between the nucleotide sequence of the spvA gene of Enteritidis and other salmonellae. The downstream PCR primer, that encompasses position 272 of the Enteritidis spvA gene, was designed to contain a single base mismatch at the penultimate position, resulting in a 1-base mismatch with Enteritidis and a 2-base mismatch with other salmonellae that harbour the virulence plasmid. The upstream primer was completely homologous with the region immediately 5'' to the spvA gene. When these primers were used and the annealing and extension reactions were performed at the same temperature, the PCR assay was specific for Enteritidis; no PCR product was detected for 40 other serotypes and 28 different genera examined. In pure culture, 120 colony forming units (c.f.u.) could be detected; a PCR product was observed from template derived from a 5 h enrichment broth culture of chicken seeded with 1 c.f.u. per gram of Enteritidis. This PCR assay is specific, reproducible, and less time consuming than the standard bacteriological methods used to detect Enteritidis.