高灵敏度电化学发光PCR方法检测基因点突变研究

近年来发展了一种用于定量检测基因点突变的电化学发光PCR方法。该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对待测基因进行PCR扩增;随后,采用特定的限制性内切酶对扩增产物进行酶切,由于野生型样品和突变型样品间存在酶切位点的变化,其中只有一种基因型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到样品池中;进行电化学发光检测,通过所得信号的有无可以判断其基因型。我们分别将该法用于Presenilin-1基因和H-ras癌基因的点突变检测,结果均可明显区分突变型样品和野生型样品。该法具有灵敏、快速、简便、安全等优点,是一种实用的基因点突变检测方法。     

A Whole-Cell Biosensor for the Detection of Gold

Geochemical exploration for gold (Au) is becoming increasingly important to the mining industry. Current processes for Au analyses require sampling materials to be taken from often remote localities. Samples are then transported to a laboratory equipped with suitable analytical facilities, such as Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) or Instrumental Neutron Activation Analysis (INAA). Determining the concentration of Au in samples may take several weeks, leading to long delays in exploration campaigns. Hence, a method for the on-site analysis of Au, such as a biosensor, will greatly benefit the exploration industry. The golTSB genes from Salmonella enterica serovar typhimurium are selectively induced by Au(I/III)-complexes. In the present study, the golTSB operon with a reporter gene, lacZ, was introduced into Escherichia coli. The induction of golTSB::lacZ with Au(I/III)-complexes was tested using a colorimetric β-galactosidase and an electrochemical assay. Measurements of the β-galactosidase activity for concentrations of both Au(I)- and Au(III)-complexes ranging from 0.1 to 5 µM (equivalent to 20 to 1000 ng g⁻¹ or parts-per-billion (ppb)) were accurately quantified. When testing the ability of the biosensor to detect Au(I/III)-complexes_(aq) in the presence of other metal ions (Ag(I), Cu(II), Fe(III), Ni(II), Co(II), Zn, As(III), Pb(II), Sb(III) or Bi(III)), cross-reactivity was observed, i.e. the amount of Au measured was either under- or over-estimated. To assess if the biosensor would work with natural samples, soils with different physiochemical properties were spiked with Au-complexes. Subsequently, a selective extraction using 1 M thiosulfate was applied to extract the Au. The results showed that Au could be measured in these extracts with the same accuracy as ICP-MS (P<0.05). This demonstrates that by combining selective extraction with the biosensor system the concentration of Au can be accurately measured, down to a quantification limit of 20 ppb (0.1 µM) and a detection limit of 2 ppb (0.01 µM).     

高灵敏度电化学发光PCR方法检测花椰菜花叶病毒35 S启动子

大多数转基因植物中使用花椰菜花叶病毒(cauliflower mosaic virus,CaMV)35 S作为启动子,因此可通过检测该启动子来判断植物样品中是否含有转基因成分。实验将高灵敏度电化学发光PCR方法用于检测转基因烟草中的CaMV35 S启动子,将该启动子的PCR产物与生物素标记的探针杂交,可以起到特异性筛选产物的作用;与发光标记物——三联吡啶钌标记的探针杂交,从而实现电化学发光检测。两种探针同时与待测样品的PCR产物进行杂交,进一步对样品进行特异性筛选,从而提高了检测的准确性,避免了假阳性结果的产生。实验结果表明:该法可以准确的区分待测样品中是否含有35 S启动子,从而区别转基因烟草和非转基因烟草。电化学发光PCR方法灵敏度高,可靠性强,操作简便,结果准确,有望成为一种高效的转基因植物检测方法。     

A Rapid and Specific Method for the Detection of Indole in Complex Biological Samples

Indole, a bacterial product of tryptophan degradation, has a variety of important applications in the pharmaceutical industry and is a biomarker in biological and clinical specimens. Yet, specific assays to quantitate indole are complex and require expensive equipment and a high level of training. Thus, indole in biological samples is often estimated using the simple and rapid Kovács assay, which nonspecifically detects a variety of commonly occurring indole analogs. We demonstrate here a sensitive, specific, and rapid method for measuring indole in complex biological samples using a specific reaction between unsubstituted indole and hydroxylamine. We compared the hydroxylamine-based indole assay (HIA) to the Kovács assay and confirmed that the two assays are capable of detecting microgram amounts of indole. However, the HIA is specific to indole and does not detect other naturally occurring indole analogs. We further demonstrated the utility of the HIA in measuring indole levels in clinically relevant biological materials, such as fecal samples and bacterial cultures. Mean and median fecal indole concentrations from 53 healthy adults were 2.59 mM and 2.73 mM, respectively, but varied widely (0.30 mM to 6.64 mM) among individuals. We also determined that enterotoxigenic Escherichia coli strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 produces 3.3 ± 0.22 mM indole during a 24-h period in the presence of 5 mM tryptophan. The sensitive and specific HIA should be of value in a variety of settings, such as the evaluation of various clinical samples and the study of indole-producing bacterial species in the gut microbiota.     

Development of a Highly Sensitive and Specific Method for Detection of Circulating Tumor Cells Harboring Somatic Mutations in Non-Small-Cell Lung Cancer Patients

Background

Oncogenic mutations are powerful predictive biomarkers for molecularly targeted cancer therapies. For mutation detection patients have to undergo invasive tumor biopsies. Alternatively, archival samples are used which may no longer reflect the actual tumor status. Circulating tumor cells (CTC) could serve as an alternative platform to detect somatic mutations in cancer patients. We sought to develop a sensitive and specific assay to detect mutations in the EGFR gene in CTC from lung cancer patients.

Methods

We developed a novel assay based on real-time polymerase chain reaction (PCR) and melting curve analysis to detect activating EGFR mutations in blood cell fractions enriched in CTC. Non-small-cell lung cancer (NSCLC) was chosen as disease model with reportedly very low CTC counts. The assay was prospectively validated in samples from patients with EGFR-mutant and EGFR-wild type NSCLC treated within a randomized clinical trial. Sequential analyses were conducted to monitor CTC signals during therapy and correlate mutation detection in CTC with treatment outcome.

Results

Assay sensitivity was optimized to enable detection of a single EGFR-mutant CTC/mL peripheral blood. CTC were detected in pretreatment blood samples from all 8 EGFR-mutant lung cancer patients studied. Loss of EGFR-mutant CTC signals correlated with treatment response, and its reoccurrence preceded relapse.

Conclusions

Despite low abundance of CTC in NSCLC oncogenic mutations can be reproducibly detected by applying an unbiased CTC enrichment strategy and highly sensitive PCR and melting curve analysis. This strategy may enable non-invasive, specific biomarker diagnostics and monitoring in patients undergoing targeted cancer therapies.     

Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical,Food, and Environmental Samples

Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.Botulinum neurotoxins (BoNTs) are the most toxic agents known, and as little as 30 ng neurotoxin is potentially lethal to humans (36). These toxins are responsible for botulism, a disease characterized by flaccid paralysis. Seven antigenically distinct BoNTs are known (types A to G), and BoNT types A, B, E, and F are the principal types associated with human botulism (37). Significant sequence diversity and antigenically variable subtypes have recently been reported for the type A, B, and E neurotoxin genes (14, 22, 23, 42).Apart from the species Clostridium botulinum, which itself consists of four phylogenetically distinct groups of organisms, some strains of other clostridia, namely Clostridium butyricum and Clostridium baratii, are also known to produce BoNTs (2, 4, 7, 13, 20, 26, 34, 44). Also, strains that produce two toxins and strains carrying silent toxin genes have been reported (8, 22, 24, 39). Due to the great physiological variation of the BoNT-producing clostridia, their isolation and identification cannot depend solely on biochemical characteristics (32). Indeed, the standard culture methods take into consideration only C. botulinum and not C. baratii and C. butyricum, and identification and confirmation require detection of BoNT by a standard mouse bioassay (SMB) (12). The SMB is highly sensitive and specific but also expensive, time-consuming, and undesirable because of the use of experimental animals. Detection of neurotoxin gene fragments by PCR is a rapid alternative method for detection and typing of BoNT-producing clostridia (3). Different PCR methods have been described for detecting neurotoxin type A-, B-, E-, and F-producing clostridia (9, 15-18, 21, 40, 41).A previously described multiplex PCR method able to simultaneously detect type A, B, E, and F neurotoxin genes is a useful tool for rapid detection of the BoNT-producing clostridia (31). While this method generally has a high level of inclusivity for detection of type B, E, and F neurotoxin genes, limitations for detection of the recently described subtype A2, A3, and A4 strains have been identified (6, 28). To increase the efficiency of this multiplex PCR method, new primers were designed to detect genes for all identified type A neurotoxin subtypes (19). Additionally, an internal amplification control (IAC) was added according to ISO 22174/2005. The specificity and selectivity of this multiplex PCR method were evaluated in comparison with an SMB (12) using target and nontarget strains, and the robustness was assessed using clinical, food, and environmental samples. Moreover, to evaluate the applicability of this multiplex PCR method, a survey with food and environmental samples was performed in a German food control laboratory.     

Detection of Diverse New Francisella-Like Bacteria in Environmental Samples

Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.     

一种转基因植物高特异定性检测方法

近年来,植物基因工程技术的迅速发展使得转基因植物及其产品与人们生活越来越密切.与此同时,转基因植物检测技术不断丰富和发展,主要是在整合、转录、翻译等不同水平上检测转基因产物的存在.但各种方法都存在不同的缺点,如缺乏特异性、假阳性较高,操作繁琐、成本高等.利用TAIL-PCR(thermal asym-metric interlaced PCR)和融合克隆结合建立一种简单、快速、特异性高的转基因植物检测方法,以两个转基因植株(C3-3株系和TB-5株系)为例,通过TAIL-PCR获得其侧翼序列后设计特异引物,并用于特定转基因植株的筛选.     

A New Sensitive, Whole-Cell Hybridization Technique for Detection of Bacteria Involving a Biotinylated Oligonucleotide Probe Targeting rRNA and Tyramide Signal Amplification

A tyramide signal amplification system with biotinylated oligonucleotide probes and streptavidin-horseradish peroxidase was used to increase the sensitivity of fluorescent in situ hybridization techniques. When applied to both gram-negative and -positive bacteria immobilized on glass slides, a 7- to 12-fold amplification of the fluorescence signal was observed relative to that of cells hybridized with fluorescently monolabeled probes. A large proportion (62 to 78%) of bacteria could be detected under starvation conditions and in natural samples from the marine environment. This amplification procedure allows new investigations in marine oligotrophic ecosystems and water quality control.     

高灵敏荧光探针用于肿瘤 Ramos 细胞的快速检测

本文以聚苯乙烯纳米微球为载体,基于适体特异性识别和 DNA 杂交原理,组装了一种 DNA-CdTe 量子点纳米线,制备了具有较高荧光强度的复合型荧光探针,并成功用于 Ramos 细胞的荧光成像。该探针可以用于特异性识别肿瘤细胞,在荧光成像中信号强灵敏度高,为肿瘤细胞的检测提供一种新方法。     

Highly Sensitive Polarimetric Sensor Based on Fano Resonance for DNA Hybridization Detection

Plasmonics - Surface plasmon resonance (SPR) sensor based on reflectivity measurement has been widely studied for its convenient detection, high sensitivity, and real-time functions. However, the...     

Transfection of RNA from Organ Samples of Infected Animals Represents a Highly Sensitive Method for Virus Detection and Recovery of Classical Swine Fever Virus

Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities.     

Highly Specific Culture-Independent Detection of YGNGV Motif-Containing Pediocin-Producing Strains

A novel method based on (1) initial microbiological screening and (2) a highly specific PCR is described for selection of strains expressing YGNGV motif-containing pediocin. Initial screening is carried out using spot on the lawn assay for selection of acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity producing strains. This is followed by highly specific PCR for amplification of 406-bp fragment using forward primer: 5′-tggccaatatcattggtggt-3′ targeting signal peptide sequence of pediocin structural gene and reverse primer: 5′-ctactaacgcttggctggca-3′ encoding N-terminus of immunity gene. The assay was validated with Pediococcus pentosaceus NCDC273 and Pediococcus acidilactici NCDC252 using (1) digestion of amplified 406-bp fragment with HindIII restriction enzyme-producing two restriction fragments of expected sizes (227 and 179 bp), (2) nucleotide sequencing of 406-bp fragment from both strains found these pediocins identical to pediocin PA-1/AcH and (3) identification of both pediocins as pediocin PA-1 at protein level using RP-HPLC. The assay was used for screening six strains (3 pediococci, 2 lactobacilli and an Enterococcus faecium) producing acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity. This resulted in the detection of three new strains (P. pentosaceus NCDC35, E. faecium NCDC124 and Lactobacillus plantarum NCDC20) producing YGNGV motif-containing pediocins.     

用环介导等温扩增技术快速检测粪便样本中的沙门菌

目的:建立快速检测粪便样本中的沙门菌的环介导等温扩增技术(LAMP),并着重在灵敏度和特异性方面对此方法进行评价。方法:利用LAMP针对沙门菌特定基因invA(靶基因)设计的6条特异引物,通过引物特异性识别特定基因invA上的8个独立区域来快速检测沙门菌;LAMP反应过程中会产生白色沉淀焦磷酸镁,故可以通过监测浊度来判定反应结果。结果:实时浊度仪监测反应结果表明,LAMP反应在60~65℃等温条件下50 min内完成;如果在反应前添加羟基萘酚兰,蓝色阳性结果很明显区别于紫色阴性结果;LAMP的最低检出限为6.97 pg/μL,PCR为69.7pg/μL,LAMP方法的检测灵敏度是PCR的10倍,且具有良好的特异性。结论:LAMP方法用于快速检测沙门菌,具有检测过程简单、实验装置简便、反应结果肉眼可辨、灵敏度高、特异性强的特点,对非沙门菌菌株的结果呈阴性,表明引物设计有很好的特异性。对粪便样本进行检测,发现具有同样的敏感性和特异性。这表明LAMP法是潜在的和有价值的在粪便样本中直接检测沙门菌的方法,具有快速、简便、低成本的特点。LAMP法适用于快速临床诊断。     

A Highly Sensitive Chelator for Metal Staining, Bromopyridylazo-Diethylaminophenol

2-(5-Bromo-2-pyridylazo)-5-diethylaminophenol has been found to be capable of staining very small amounts of metals, particularly copper, iron, lead, and zinc, in tissue. The differentiation of a target metal from interfering metals is achieved by using masking agents, polyphosphates, and potassium cyanide.