新型压电石英DNA传感器微阵列的构建及其在血液感染病原菌快速检测中的应用
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摘要
背景近年来,随着抗肿瘤药物、免疫抑制剂等药物使用和人工瓣膜、人工关节、留置
静脉导管等侵入性操作的增加,各种介入性诊治手段应用范围的日益扩大以及人群老龄化和免疫力低下的人群的出现,血液感染在医院中的发生比率呈逐年递增趋势,严重威胁着人类健康。致病菌的检出是诊断血液感染的唯一方法。目前,临床上血液感染致病菌普遍采用的检测方法主要有两大类:一类是培养鉴定法,包括血液细菌培养及生化反应等一系列过程,通常需要3～5天,耗时长、操作繁琐;另一类是以免疫学方法为代表的快速检测方法,如抗血清凝集试验、协同凝集试验、酶联免疫法等,但是这些方法存在着敏感度低和特异性差等问题,常常导致误诊和漏诊,远远不能满足临床血液感染诊断的需要。16S rDNA是编码原核生物核糖体小亚基rRNA(16SrRNA)的基因,有“细菌化石”之称,是近年来细菌分类和鉴定研究中的热点,为病原菌的分子生物学快速检测供了新的理想靶点。
压电石英传感器,又被称为石英晶振微天平(quartz crystal microbalance,QCM),是新发展起来的生物诊断技术,它综合应用了信息学、电子学、机械学、分子生物学等多学科技术,利用生物反应过程中传感器表面质量负载发生的微小变化所引起的振荡频率的变化,对与传感器表面生物敏感膜发生反应的生物学大分子进行分析。近年来,国内外陆续有应用压电石英传感器检测病原微生物的报道,但是这些压电石英DNA传感器的检测灵敏度尚未达到临床检测的要求,还局限于单一菌种检测的实验研究。
因此,结合本课题组前期的工作基础,本课题确定了五种临床常见血液感染致病菌:大肠埃希菌、铜绿假单胞菌、金黄色葡萄球菌、表皮葡萄球菌、肺炎克雷伯氏菌为研究对象,充分利用细菌16S rDNA序列的分子生物学特点,分别将酶生物信号放大系统和纳米金磁微粒信号放大系统引入压电石英DNA传感器,构建新型压电石英DNA传感器微阵列,进而建立一种操作简单、敏感度高、特异性高的血液感染病原菌快速检测方法,期望为压电石英DNA传感器微阵列进一步应用于普通实验室甚至野战、自然灾害等特殊领域提供坚实的实验基础。
方法
1.在前期研究基础上,对压电石英DNA传感器检测平台的硬件设施和软件设施进行改进,增加控温系统,加强电磁屏蔽,采用Visual C++编程语言升级频率分析软件为PESA 4.0,对传感器微阵列的稳定性、重复性进行评价。对自组装膜法进行改进,采用二硫代苏糖醇(DTT)将巯基修饰的的寡核苷酸探针还原后再进行自组装法固定,探讨探针固定影响因素,确定最适反应条件。
2.利用Vector NTI 9.0软件套组对目标病原菌的16S rDNA序列进行分析,确定16 S rDNA保守序列区域和可变序列区域;利用Primer Premier 5.0软件,针对16S rDNA保守区域序列,设计适用于目标病原菌的PCR扩增通用引物;利用Array Designer 4.0软件设计针对各靶病原菌16 S rDNA可变区域序列的特异性寡核苷酸探针。探讨杂交反应各影响因素,优化反应体系,初步建立血液感染常见病原菌压电石英DNA传感器检测方法。
3.分别建立纳米金磁微粒信号放大系统和酶生物信号放大系统:应用特异性探针与生物素修饰靶序列复合体捕获链霉亲和素修饰的纳米金磁微粒,增加石英晶振表面质量负载;应用生物素和链霉亲和素之间的特异性结合及辣根过氧化物酶催化DAB生成不可溶沉淀附着于金膜表面,增加石英晶振表面质量负载,放大压电生物传感器检测信号,优化信号放大系统反应条件。将基于两种信号放大系统的压电石英DNA传感器用于病原菌检测,评价两种信号放大系统的可行性和有效性。建立基于压电传感器单元的EIS检测系统,应用EIS方法进一步验证压电石英传感器检测方法及信号放大系统的各步骤的真实性和可靠性。
4.以临床108例血培养标本为检测对象,对两种血液标本中细菌基因组DNA的提取方法进行比较,并以血培养鉴定法为参照方法,对建立的压电石英DNA传感器微阵列检测方法进行方法学比较和评估,确定临床样本检测步骤。
结果
1.成功构建新一代小型化压电石英传感器检测仪,检测仪规格(25.0±1.0)×(35.0±1.0)×(15.0±1.0)cm(长×宽×高)。检测系统稳定性良好,10分钟检测气相频率变化为±1Hz,液相条件下频率变化为±1Hz。邻位检测池插拔,不会对其它已经稳定谐振的传感器造成影响,三通道间基本不存在干扰,达到了阵列化的要求。还原巯基化探针法法显著提高了探针固定效率(p<0.05),最适探针固定浓度为2μmol/l。
2.利用设计的通用引物可以一次性完成所有靶细菌16Sr DNA序列的顺利扩增,电泳条带明亮清晰,具有较好的重复性。自行设计的探针特异性强、可靠性好,探针间Tm值相差仅为0.5℃,基本上具有相同的杂交条件,可用于传感器微阵列系统的检测。检测最适杂交温度为40℃,杂交检测时间为90min。
3.建立的两种信号放大系统均可有效的提高信噪比,提高检测的灵敏度,基于纳米金磁微粒信号放大系统的压电石英DNA传感器可以检测到2.0×103 CFU/ml的病原菌,基于酶生物信号放大系统的压电石英DNA传感器可以检测到2.0×102 CFU/ml的病原菌,与阴性对照和空白对照检测信号比较,均有显著性差异(p<0.01)。由于酶生物信号放大系统所引起的频率变化值是纳米金磁微粒引起的频率变化值的2.1倍,因此,选择酶生物信号放大系统为压电石英DNA传感器检测血液感染病原菌的信号放大方法。
4.对两种血液标本中病原菌基因组DNA提取方法比较结果显示,碱裂解法操作简便、污染机会少,而且效果好,能裂解所研究的所有菌种,PCR产物电泳条带清晰明亮,无拖尾现象,可作为有效的样本前处理方法。检测的108例临床样本中,其中102例传感器检测结果,与临床检测结果相符合,灵敏度为90.2%,特异度为98.5%,可靠性为90%。
结论:
1.我们所构建的新型压电石英DNA传感器微阵列,降低了温度和电磁效应对检测稳定性的影响,极大的减少了各检测通道之间的信号干扰,从而达到了传感器阵列化的要求,为DNA检测提供了背景噪音低、性能稳定的微阵列检测平台。
2.自行设计的16Sr DNA通用引物可以一次性对所有靶细菌16Sr DNA序列的顺利扩增;设计的寡核苷酸探针特异性好,具有相同的杂交条件,较大的增加了传感器微阵列的检测通量,简化了操作步骤。
3.酶生物放大系统和纳米金磁微粒检测系统可显著增加压电传感器检测DNA靶序列时的频率变化,有效提高压电石英传感器的信噪比,显著提高检测的灵敏度,为压电生物传感器检测生物大分子提供了可靠的信号放大方法,在生物传感器领域有较大的应用潜力。
4.构建的压电石英DNA传感器微阵列,可以在4小时内同时检测五种血液感染病原菌,检测方法具有较高的灵敏度、特异性和准确性,技术稳定简单易于掌握,成本低廉。为临床病原菌的快速检测乃至其他基因检测提供一种新思路,有望克服临床常规检测方法存在的问题,满足临床快速诊断的迫切需求,具有广阔的应用前景。
Background
Bacteremia-induced severe sepsis is the leading cause of morbidity and mortality worldwide, particularly in patients who are immunocompromised. In last decades, various techniques have been developed for the clinical diagnosis of bloodstream infections, such as conventional blood culture techniques, enzyme-linked immunosorbent assay, immunoassay, sequence analysis, and so on. These conventional methods are generally reliable but time-consuming, laborious and expensive. Therefore, it is desirable to explore some simple, sensitive, and low-cost diagnostic methods to detect pathogenic bacteria in clinical blood samples. 16S rRNA sequence analysis has been used to clarify the taxonomic affinities of a wide range of taxa and as a powerful tool for the detection of pathogenic bacteria.
Because of sensitivity, simplicity and cost-effectiveness, piezoelectric quartz crystal microbalance (QCM) biosensors have showed attractive roles in various molecular analysis techniques Recent years, piezoelectric biosensor is becoming a focus in biomolecule detection field. It could detect many biomolecular real-timely, sensitively and specifically, such as DNA, antibody, enzyme, bacterium, based on different recognition elements. In recently research, it had been used in many research fields such as molecular recognition, clinical diagnosis, molecular filming, surface property research, poison research and so forth. However, for most of the previously reported nucleic acid biosensors for bacterial detection, their sensitivity and detection limit of are still difficult to meet the demand of clinical analysis.
In this study, a QCM DNA biosensor array was developed to rapidly detect 5 common pathogenic bacteria which are the major causes of bloodstream infections. Moreover, in order to improve the detection sensitivity of QCM nucleic acid biosensors, two signal amplification strategies have been developed, which includ enzyme and nanoparticles signal amplification. The sensitivity and specificity of the QCM system were evaluated. The application of the QCM system was tested in real clinical blood samples. This study lays the groundwork for applying this QCM DNA biosensor array in the labs and for rapid detection of pathogenic bacteria.
Methods
1. The QCM DNA biosensor array was constructed which includes electronic oscillation circuit, voltage stabilizer, thermal controller system, and 2×5 detection wells. The new software PESA 4.0 was designed based on Visual C++, which was interfaced with the QCM detector. The stability of the new system was tested in gas phase and liquid phase individually. The deoxidized thiol-modified probe immobilization was developed,and the experimental conditions of the immobilization methodwere optimized.
2. The universial primers were designed to be complementary to the conserved regions of bacteria 16S rRNA gene using Primer Premier 5.0 software. The specific oligonucleotide probes were designed to be complementary to the highly variable regions of 16S rRNA gene in target bacteria by Array Designer 4.0, which include Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis, Escherichia coli. The experimental conditions for the biosensoring system were optimized.
3. Enzyme and nanoparticles signal amplification strategies were developed. The sensitivity and specificity of the current QCM biosensor system based on these two signal amplification methods were tested. In addition, Faradaic impedance spectra measurements were carried out to demonstrate the alteration of the interfacial properties of the QCM electrodes during the signal amplification process.
4. To demonstrate the use of the biosensing system for the identification of pathogenic bacteria, 108 blood culture samples were detected individually using the QCM nucleic acid array and the conventional microbiological techniques. Chelex 100 and alkali lysis method were investigated. The clinical sensitivity and specificity of the current QCM biosensor array system for bacteria detections of clinical samples were also tested.
Results
1. The size of the new biosensor array has been minimized to (25.0±1.0)×(35.0±1.0)×(15.0±1.0)cm(L×W×H).Moreover, the QCM biosensor array has strong vibration ability and table frequency in liquid phase. In ten minutes, the changes of frequency were less than±1Hz both in gas phase and liquid phase. The deoxidization of thiol-modified probes significantly increased immobilization efficiency (p<0.05) and we selected the probe concentration of 2.0μM as the optimal immobilization concentrations for the following experiments.
2. The experiments showed these target bacterial could be successfully amplified by one-time PCR with the common primers. The results also showed that the premiers were in good repetitivety. The designed probes have strong specificity and great reliability. In addition, the difference of Tm value between the probes was only 0.5℃, hence they had the almost same hybridization conditions and could meet the requirements of the biosensor array detections. The optimal hybridization temperature is 40℃experiments and the optimal hybridization time is 90 min.
3. The use of both Au nanoparticles and enzyme effectively amplified the signals in frequency shifts due to the relatively large mass compared to DNA targets, and resulted in a improved detection limit of 2.0×103 CFU/ml and 2.0×102 CFU/ml, respectively. The frequency shift caused by the Au nanoparticles method(271.6±12.1Hz) was significantly larger than that caused by enzyme method (127.5±6.5Hz) (P<0.01). Therefore, enzyme method was used in subsequent experiments.
4. The alkali lysis method was the most sensitive, reproducible, simple and cost-effective extraction method to extract bacteria DNA in clinical blood cultures. Compared with conventional microbiological method, the diagnostic sensitivity of the assays resulted in 90.2 % and the specificity resulted in 98.5 %. This test system allows identification of 5 bacteria within ca. 4 h.
Conclusions
1. The developed QCM DNA biosensor array has strong vibration ability and table frequency in liquid phase, and it is simple and cost-effectiveness. The new QCM DNA biosensor system can provide a reliable plateform for gene detection.
2. The designed universial primers and specific oligonucleotides can increase the detection flux of the current QCM DNA biosensor array system and simplify the process of the identification of pathogenic bacteria.
3. Both enzyme signal amplification and Au nanoparticles amplification system could detect target DNA specifically and amplify the piezoelectric signal significantly. It has great versatility and can be used in different types of piezoelectric biosensors in detecting tiny mass biomolecular.
4. For clinical blood sample detection, the developed QCM DNA biosensor array has good clinical sensitivity and specificity compared with the conventional microbiological method, which suggest that this QCM nucleic acid biosensor array with enzyme amplification method can be expected to be a potential clinical diagnosis method for rapid detection of pathogenic bacteria causing bloodstream infections or other microorganisms in clinical samples.
静脉导管等侵入性操作的增加,各种介入性诊治手段应用范围的日益扩大以及人群老龄化和免疫力低下的人群的出现,血液感染在医院中的发生比率呈逐年递增趋势,严重威胁着人类健康。致病菌的检出是诊断血液感染的唯一方法。目前,临床上血液感染致病菌普遍采用的检测方法主要有两大类:一类是培养鉴定法,包括血液细菌培养及生化反应等一系列过程,通常需要3～5天,耗时长、操作繁琐;另一类是以免疫学方法为代表的快速检测方法,如抗血清凝集试验、协同凝集试验、酶联免疫法等,但是这些方法存在着敏感度低和特异性差等问题,常常导致误诊和漏诊,远远不能满足临床血液感染诊断的需要。16S rDNA是编码原核生物核糖体小亚基rRNA(16SrRNA)的基因,有“细菌化石”之称,是近年来细菌分类和鉴定研究中的热点,为病原菌的分子生物学快速检测供了新的理想靶点。
压电石英传感器,又被称为石英晶振微天平(quartz crystal microbalance,QCM),是新发展起来的生物诊断技术,它综合应用了信息学、电子学、机械学、分子生物学等多学科技术,利用生物反应过程中传感器表面质量负载发生的微小变化所引起的振荡频率的变化,对与传感器表面生物敏感膜发生反应的生物学大分子进行分析。近年来,国内外陆续有应用压电石英传感器检测病原微生物的报道,但是这些压电石英DNA传感器的检测灵敏度尚未达到临床检测的要求,还局限于单一菌种检测的实验研究。
因此,结合本课题组前期的工作基础,本课题确定了五种临床常见血液感染致病菌:大肠埃希菌、铜绿假单胞菌、金黄色葡萄球菌、表皮葡萄球菌、肺炎克雷伯氏菌为研究对象,充分利用细菌16S rDNA序列的分子生物学特点,分别将酶生物信号放大系统和纳米金磁微粒信号放大系统引入压电石英DNA传感器,构建新型压电石英DNA传感器微阵列,进而建立一种操作简单、敏感度高、特异性高的血液感染病原菌快速检测方法,期望为压电石英DNA传感器微阵列进一步应用于普通实验室甚至野战、自然灾害等特殊领域提供坚实的实验基础。
方法
1.在前期研究基础上,对压电石英DNA传感器检测平台的硬件设施和软件设施进行改进,增加控温系统,加强电磁屏蔽,采用Visual C++编程语言升级频率分析软件为PESA 4.0,对传感器微阵列的稳定性、重复性进行评价。对自组装膜法进行改进,采用二硫代苏糖醇(DTT)将巯基修饰的的寡核苷酸探针还原后再进行自组装法固定,探讨探针固定影响因素,确定最适反应条件。
2.利用Vector NTI 9.0软件套组对目标病原菌的16S rDNA序列进行分析,确定16 S rDNA保守序列区域和可变序列区域;利用Primer Premier 5.0软件,针对16S rDNA保守区域序列,设计适用于目标病原菌的PCR扩增通用引物;利用Array Designer 4.0软件设计针对各靶病原菌16 S rDNA可变区域序列的特异性寡核苷酸探针。探讨杂交反应各影响因素,优化反应体系,初步建立血液感染常见病原菌压电石英DNA传感器检测方法。
3.分别建立纳米金磁微粒信号放大系统和酶生物信号放大系统:应用特异性探针与生物素修饰靶序列复合体捕获链霉亲和素修饰的纳米金磁微粒,增加石英晶振表面质量负载;应用生物素和链霉亲和素之间的特异性结合及辣根过氧化物酶催化DAB生成不可溶沉淀附着于金膜表面,增加石英晶振表面质量负载,放大压电生物传感器检测信号,优化信号放大系统反应条件。将基于两种信号放大系统的压电石英DNA传感器用于病原菌检测,评价两种信号放大系统的可行性和有效性。建立基于压电传感器单元的EIS检测系统,应用EIS方法进一步验证压电石英传感器检测方法及信号放大系统的各步骤的真实性和可靠性。
4.以临床108例血培养标本为检测对象,对两种血液标本中细菌基因组DNA的提取方法进行比较,并以血培养鉴定法为参照方法,对建立的压电石英DNA传感器微阵列检测方法进行方法学比较和评估,确定临床样本检测步骤。
结果
1.成功构建新一代小型化压电石英传感器检测仪,检测仪规格(25.0±1.0)×(35.0±1.0)×(15.0±1.0)cm(长×宽×高)。检测系统稳定性良好,10分钟检测气相频率变化为±1Hz,液相条件下频率变化为±1Hz。邻位检测池插拔,不会对其它已经稳定谐振的传感器造成影响,三通道间基本不存在干扰,达到了阵列化的要求。还原巯基化探针法法显著提高了探针固定效率(p<0.05),最适探针固定浓度为2μmol/l。
2.利用设计的通用引物可以一次性完成所有靶细菌16Sr DNA序列的顺利扩增,电泳条带明亮清晰,具有较好的重复性。自行设计的探针特异性强、可靠性好,探针间Tm值相差仅为0.5℃,基本上具有相同的杂交条件,可用于传感器微阵列系统的检测。检测最适杂交温度为40℃,杂交检测时间为90min。
3.建立的两种信号放大系统均可有效的提高信噪比,提高检测的灵敏度,基于纳米金磁微粒信号放大系统的压电石英DNA传感器可以检测到2.0×103 CFU/ml的病原菌,基于酶生物信号放大系统的压电石英DNA传感器可以检测到2.0×102 CFU/ml的病原菌,与阴性对照和空白对照检测信号比较,均有显著性差异(p<0.01)。由于酶生物信号放大系统所引起的频率变化值是纳米金磁微粒引起的频率变化值的2.1倍,因此,选择酶生物信号放大系统为压电石英DNA传感器检测血液感染病原菌的信号放大方法。
4.对两种血液标本中病原菌基因组DNA提取方法比较结果显示,碱裂解法操作简便、污染机会少,而且效果好,能裂解所研究的所有菌种,PCR产物电泳条带清晰明亮,无拖尾现象,可作为有效的样本前处理方法。检测的108例临床样本中,其中102例传感器检测结果,与临床检测结果相符合,灵敏度为90.2%,特异度为98.5%,可靠性为90%。
结论:
1.我们所构建的新型压电石英DNA传感器微阵列,降低了温度和电磁效应对检测稳定性的影响,极大的减少了各检测通道之间的信号干扰,从而达到了传感器阵列化的要求,为DNA检测提供了背景噪音低、性能稳定的微阵列检测平台。
2.自行设计的16Sr DNA通用引物可以一次性对所有靶细菌16Sr DNA序列的顺利扩增;设计的寡核苷酸探针特异性好,具有相同的杂交条件,较大的增加了传感器微阵列的检测通量,简化了操作步骤。
3.酶生物放大系统和纳米金磁微粒检测系统可显著增加压电传感器检测DNA靶序列时的频率变化,有效提高压电石英传感器的信噪比,显著提高检测的灵敏度,为压电生物传感器检测生物大分子提供了可靠的信号放大方法,在生物传感器领域有较大的应用潜力。
4.构建的压电石英DNA传感器微阵列,可以在4小时内同时检测五种血液感染病原菌,检测方法具有较高的灵敏度、特异性和准确性,技术稳定简单易于掌握,成本低廉。为临床病原菌的快速检测乃至其他基因检测提供一种新思路,有望克服临床常规检测方法存在的问题,满足临床快速诊断的迫切需求,具有广阔的应用前景。
Background
Bacteremia-induced severe sepsis is the leading cause of morbidity and mortality worldwide, particularly in patients who are immunocompromised. In last decades, various techniques have been developed for the clinical diagnosis of bloodstream infections, such as conventional blood culture techniques, enzyme-linked immunosorbent assay, immunoassay, sequence analysis, and so on. These conventional methods are generally reliable but time-consuming, laborious and expensive. Therefore, it is desirable to explore some simple, sensitive, and low-cost diagnostic methods to detect pathogenic bacteria in clinical blood samples. 16S rRNA sequence analysis has been used to clarify the taxonomic affinities of a wide range of taxa and as a powerful tool for the detection of pathogenic bacteria.
Because of sensitivity, simplicity and cost-effectiveness, piezoelectric quartz crystal microbalance (QCM) biosensors have showed attractive roles in various molecular analysis techniques Recent years, piezoelectric biosensor is becoming a focus in biomolecule detection field. It could detect many biomolecular real-timely, sensitively and specifically, such as DNA, antibody, enzyme, bacterium, based on different recognition elements. In recently research, it had been used in many research fields such as molecular recognition, clinical diagnosis, molecular filming, surface property research, poison research and so forth. However, for most of the previously reported nucleic acid biosensors for bacterial detection, their sensitivity and detection limit of are still difficult to meet the demand of clinical analysis.
In this study, a QCM DNA biosensor array was developed to rapidly detect 5 common pathogenic bacteria which are the major causes of bloodstream infections. Moreover, in order to improve the detection sensitivity of QCM nucleic acid biosensors, two signal amplification strategies have been developed, which includ enzyme and nanoparticles signal amplification. The sensitivity and specificity of the QCM system were evaluated. The application of the QCM system was tested in real clinical blood samples. This study lays the groundwork for applying this QCM DNA biosensor array in the labs and for rapid detection of pathogenic bacteria.
Methods
1. The QCM DNA biosensor array was constructed which includes electronic oscillation circuit, voltage stabilizer, thermal controller system, and 2×5 detection wells. The new software PESA 4.0 was designed based on Visual C++, which was interfaced with the QCM detector. The stability of the new system was tested in gas phase and liquid phase individually. The deoxidized thiol-modified probe immobilization was developed,and the experimental conditions of the immobilization methodwere optimized.
2. The universial primers were designed to be complementary to the conserved regions of bacteria 16S rRNA gene using Primer Premier 5.0 software. The specific oligonucleotide probes were designed to be complementary to the highly variable regions of 16S rRNA gene in target bacteria by Array Designer 4.0, which include Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis, Escherichia coli. The experimental conditions for the biosensoring system were optimized.
3. Enzyme and nanoparticles signal amplification strategies were developed. The sensitivity and specificity of the current QCM biosensor system based on these two signal amplification methods were tested. In addition, Faradaic impedance spectra measurements were carried out to demonstrate the alteration of the interfacial properties of the QCM electrodes during the signal amplification process.
4. To demonstrate the use of the biosensing system for the identification of pathogenic bacteria, 108 blood culture samples were detected individually using the QCM nucleic acid array and the conventional microbiological techniques. Chelex 100 and alkali lysis method were investigated. The clinical sensitivity and specificity of the current QCM biosensor array system for bacteria detections of clinical samples were also tested.
Results
1. The size of the new biosensor array has been minimized to (25.0±1.0)×(35.0±1.0)×(15.0±1.0)cm(L×W×H).Moreover, the QCM biosensor array has strong vibration ability and table frequency in liquid phase. In ten minutes, the changes of frequency were less than±1Hz both in gas phase and liquid phase. The deoxidization of thiol-modified probes significantly increased immobilization efficiency (p<0.05) and we selected the probe concentration of 2.0μM as the optimal immobilization concentrations for the following experiments.
2. The experiments showed these target bacterial could be successfully amplified by one-time PCR with the common primers. The results also showed that the premiers were in good repetitivety. The designed probes have strong specificity and great reliability. In addition, the difference of Tm value between the probes was only 0.5℃, hence they had the almost same hybridization conditions and could meet the requirements of the biosensor array detections. The optimal hybridization temperature is 40℃experiments and the optimal hybridization time is 90 min.
3. The use of both Au nanoparticles and enzyme effectively amplified the signals in frequency shifts due to the relatively large mass compared to DNA targets, and resulted in a improved detection limit of 2.0×103 CFU/ml and 2.0×102 CFU/ml, respectively. The frequency shift caused by the Au nanoparticles method(271.6±12.1Hz) was significantly larger than that caused by enzyme method (127.5±6.5Hz) (P<0.01). Therefore, enzyme method was used in subsequent experiments.
4. The alkali lysis method was the most sensitive, reproducible, simple and cost-effective extraction method to extract bacteria DNA in clinical blood cultures. Compared with conventional microbiological method, the diagnostic sensitivity of the assays resulted in 90.2 % and the specificity resulted in 98.5 %. This test system allows identification of 5 bacteria within ca. 4 h.
Conclusions
1. The developed QCM DNA biosensor array has strong vibration ability and table frequency in liquid phase, and it is simple and cost-effectiveness. The new QCM DNA biosensor system can provide a reliable plateform for gene detection.
2. The designed universial primers and specific oligonucleotides can increase the detection flux of the current QCM DNA biosensor array system and simplify the process of the identification of pathogenic bacteria.
3. Both enzyme signal amplification and Au nanoparticles amplification system could detect target DNA specifically and amplify the piezoelectric signal significantly. It has great versatility and can be used in different types of piezoelectric biosensors in detecting tiny mass biomolecular.
4. For clinical blood sample detection, the developed QCM DNA biosensor array has good clinical sensitivity and specificity compared with the conventional microbiological method, which suggest that this QCM nucleic acid biosensor array with enzyme amplification method can be expected to be a potential clinical diagnosis method for rapid detection of pathogenic bacteria causing bloodstream infections or other microorganisms in clinical samples.
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