Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes

Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.     

Locked nucleic acids for optimizing displacement probes for quantitative real-time PCR

Displacement probes have recently been described as a novel probe-based detection system for use in both quantitative real-time polymerase chain reaction (PCR) and single nucleotide polymorphism genotyping analysis. Previous reports have shown that shorter probes (23 mer) had improved detection sensitivity relative to longer probes (29 mer), with the likely reason for this effect being the improved hybridization kinetics of shorter probes. Sterically modified locked nucleic acids (LNAs) have been used to improve the design of a range of real-time PCR probes by raising the melting temperature (Tm) of the probe and enabling shorter probe designs to be considered. A displacement probe for gapdh was designed and tested successfully, and this probe was then redesigned with LNAs to an 11 mer probe. This probe showed increased detection sensitivity compared with the original 26 mer probe. To detect the widest range of displacement probe designs at maximum sensitivity, we have also developed a novel fluorescence capture two-step PCR protocol. This method produces enhanced probe quenching with a single standardized protocol ideal for high-throughput applications. The displacement probes tested produced sensitive and efficient quantitative analyses of template serial dilutions when compared with a range of commercially available predesigned real-time PCR detection systems, including TaqMan MGB probes, QuantiTect MGB probes, and LUX primers.     

TaqMan probe array for quantitative detection of DNA targets

To date real-time quantitative PCR and gene expression microarrays are the methods of choice for quantification of nucleic acids. Herein, we described a unique fluorescence resonance energy transfer-based microarray platform for real-time quantification of nucleic acid targets that combines advantages of both and reduces their limitations. A set of 3′ amino-modified TaqMan probes were designed and immobilized on a glass slide composing a regular microarray pattern, and used as probes in the consecutive PCR carried out on the surface. During the extension step of the PCR, 5′ nuclease activity of DNA polymerase will cleave quencher dyes of the immobilized probe in the presence of nucleic acids targets. The increase of fluorescence intensities generated by the change in physical distance between reporter fluorophore and quencher moiety of the probes were collected by a confocal scanner. Using this new approach we successfully monitored five different pathogenic genomic DNAs and analyzed the dynamic characteristics of fluorescence intensity changes on the TaqMan probe array. The results indicate that the TaqMan probe array on a planar glass slide monitors DNA targets with excellent specificity as well as high sensitivity. This set-up offers the great advantage of real-time quantitative detection of DNA targets in a parallel array format.     

分子信标DNA计算模型的研究进展与展望

由于分子信标具有结构简单,灵敏度高及反应迅速等优点,因此,利用分子信标进行数学问题的求解将成为可能.通过对分子信标的计算模型进行详细的介绍,并对分子信标的计算模型的研究思路进行了展望,据此思路,可以建立多种组合优化问题及逻辑门的分子信标计算模型.     

Structure-function relationships of shared-stem and conventional molecular beacons

Molecular beacons are oligonucleotide probes capable of forming a stem-loop hairpin structure with a reporter dye at one end and a quencher at the other end. Conventional molecular beacons are designed with a target-binding domain flanked by two complementary short arm sequences that are independent of the target sequence. Here we report the design of shared-stem molecular beacons with one arm participating in both stem formation when the beacon is closed and target hybridization when it is open. We performed a systematic study to compare the behavior of conventional and shared-stem molecular beacons by conducting thermodynamic and kinetic analyses. Shared-stem molecular beacons form more stable duplexes with target molecules than conventional molecular beacons; however, conventional molecular beacons may discriminate between targets with a higher specificity. For both conventional and shared-stem molecular beacons, increasing stem length enhanced the ability to differentiate between wild-type and mutant targets over a wider range of temperatures. Interestingly, probe-target hybridization kinetics were similar for both classes of molecular beacons and were influenced primarily by the length and sequence of the stem. These findings should enable better design of molecular beacons for various applications.     

Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5' nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >/=10(2) cells ml(-1) was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.     

Molecular beacons: fluorogenic probes for living cell study

Molecular beacons are a new class of fluorescent probes that can report the presence of specific nucleic acids with high sensitivity and excellent specificity. In addition to their current wide applications in monitoring the progress of polymerase chain reactions, their unique properties make them promising probes for the detection and visualization of target biomolecules in living cells. This article is focused on our recent research in exploring the potential of using molecular beacon for living-cell studies in three important areas: the monitoring of mRNA in living cells, the development of ultrasmall DNA/RNA biosensors, and the novel approach of combining molecular beacon's signal transduction mechanism with aptamer's specificity for real-time protein detection. These applications demonstrate molecular beacon's unique properties in bioanalysis and bioassay development.     

Probe design rules and effective enzymes for endonuclease-based detection of nucleic acids

Junction probe (JP) platform is an isothermal endonuclease-based detection assay for both RNA and DNA. Herein, we screen 31 REAse and identify effective restriction endonucleases that can be used for JP detection. Secondly, we investigate how different probe architectures affect JP cleavage rates and conclude that although molecular beacon (MB) JP probes give less background noise than linear JP probes, the cleavage of MB JP probes are slower than linear JP probes.     

Design and Development of a Multiplex Real-Time PCR Assay for Detection of HIV-1 and HCV Using Molecular Beacons

At least 10 million individuals worldwide are co-infected with immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). These two viruses are transmitted most primarily by exposure to infected blood or blood products. Various nucleic acid assays have been developed for diagnostics and therapeutic monitoring of infections. In the present study, a multiplex real-time PCR assay for simultaneous detection of HCV and HIV-1 using molecular beacons were designed and validated. A well-conserved region in the HIV-1 pol gene and 5′NCR of HCV genome were used for primers and molecular beacon design. The analysis of scalar concentrations of the samples indicated that this multiplex procedure detects at least 1,000 copies/ml of HIV-1 and 100 copies/ml of HCV with linear reference curve (R ² > 0.94). The results demonstrate that a specificity of 100 % and sensitivity of 96 % can be achieved. The analytical sensitivity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes only detected HIV-1 and all major variants of HCV. This assay may represent an alternative rapid and relatively inexpensive screening method for detection of HIV-1/HCV co-infection especially in blood screening.     

Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR

Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.     

Detection of Ralstonia solanacearum Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to ≥10² cells ml⁻¹ was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.     

Development of a real‐time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus

Aims: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real‐time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. Methods and Results: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real‐time PCR assay specifically detected bovine and porcine TTV DNA without cross‐amplification of other common pathogens. The assay was compared with conventional PCR and nested‐PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. Conclusions: The real‐time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. Significance and Impact of the Study: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri‐food continuum.     

分子信标探针用于PCR检测对虾白斑杆状病毒

将对虾白斑杆状病毒的一段特异性DNA设计成分子信标探针,用于该病毒的PCR检测.温度与荧光强度之间的关系表明,所设计探针的发夹既可以形成也可以打开,符合PCR对分子信标探针的要求.结果表明,在PCR同时加入分子信标探针不影响PCR扩增,分子信标探针只能与目的DNA杂交,具有较高的特异性.随着PCR循环数的增加以及含目的DNA的质粒拷贝数的增加,荧光强度都随之增强.     

双链探针实时荧光PCR核酸检测新技术研究*

目的:采用一种“双链探针”实时荧光PCR技术,提高HBV核酸检测灵敏度,并在同一反应管中实现代谢酶CYP2C19^*2基因型检测。方法:采用双链探针与TaqMan探针同时检测不同浓度HBV血清样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和结果统计分析;采用双链探针检测代谢酶CYP2C19^*2不同基因型样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和基因型确定。结果:不同浓度HBV血清样本检测,双链探针荧光本底低,检测灵敏度更高,与TaqMan探针检测结果相比,两者核酸检测Ct值存在显著性差异(P<0.05);双链探针检测36份样本的代谢酶CYP2C19^*2基因型,检测结果与Sanger测序结果完全一致。结论:双链探针实时荧光PCR检测技术可完成目的基因的高灵敏核酸检测,也可实现基因型分析。     

Enzymatic signal amplification of molecular beacons for sensitive DNA detection

Molecular beacons represent a new family of fluorescent probes for nucleic acids, and have found broad applications in recent years due to their unique advantages over traditional probes. Detection of nucleic acids using molecular beacons has been based on hybridization between target molecules and molecular beacons in a 1:1 stoichiometric ratio. The stoichiometric hybridization, however, puts an intrinsic limitation on detection sensitivity, because one target molecule converts only one beacon molecule to its fluorescent form. To increase the detection sensitivity, a conventional strategy has been target amplification through polymerase chain reaction. Instead of target amplification, here we introduce a scheme of signal amplification, nicking enzyme signal amplification, to increase the detection sensitivity of molecular beacons. The mechanism of the signal amplification lies in target-dependent cleavage of molecular beacons by a DNA nicking enzyme, through which one target DNA can open many beacon molecules, giving rise to amplification of fluorescent signal. Our results indicate that one target DNA leads to cleavage of hundreds of beacon molecules, increasing detection sensitivity by nearly three orders of magnitude. We designed two versions of signal amplification. The basic version, though simple, requires that nicking enzyme recognition sequence be present in the target DNA. The extended version allows detection of target of any sequence by incorporating rolling circle amplification. Moreover, the extended version provides one additional level of signal amplification, bringing the detection limit down to tens of femtomolar, nearly five orders of magnitude lower than that of conventional hybridization assay.     

Development of a TaqMan PCR assay for the detection of Bonamia species

The development of molecular diagnostic assays with increased sensitivity compared with conventional histological techniques is highly desirable for effective management of bonamiosis in cultured oyster stocks and wild populations. A real-time TaqMan PCR assay was developed for the specific detection of Bonamia species in infected oyster tissues. The TaqMan assay was shown to be significantly more sensitive than histopathology. Although a real-time TaqMan PCR assay is comparable with conventional PCR in terms of sensitivity, it offers the advantages that it is a rapid test and has a very low risk of sample cross-contamination. Furthermore, it can be optimised to quantify the parasite load in samples. The assay detected Bonamia isolates from Australia, New Zealand, Europe, Canada, Chile and the USA and therefore demonstrated genus specificity as tested in this study.     

Evaluation of methods for improved detection of Cryptosporidium spp. in mussels (Mytilus californianus)

Bivalve molluscs concentrate Cryptosporidium oocysts from fecal-contaminated aquatic environments and are therefore useful in monitoring water quality. A real-time TaqMan polymerase chain reaction (PCR) system was developed to allow for large scale quantitative detection of Cryptosporidium spp. in mussels (Mytilus californianus). The TaqMan sensitivity and specificity were compared to conventional PCR and direct immunofluorescent antibody (DFA) assays, with and without immunomagnetic separation (IMS), to identify the best method for parasite detection in mussel hemolymph, gill washings and digestive glands. TaqMan PCR and two conventional PCR systems all detected 1 or more oocysts spiked into 1 ml hemolymph samples. The minimum oocyst detection limit in spiked 5 ml gill wash and 1 g digestive gland samples tested by TaqMan PCR and DFA was 100 oocysts, with a 1 log(10) improvement when samples were first processed by IMS. For tank exposed mussels, TaqMan and conventional PCR methods detected C. parvum in <5% of hemolymph samples. No gill washings from these same mussels tested positive by TaqMan PCR or DFA analysis even with IMS concentration. All methods detected the highest prevalence of C. parvum-positive samples in digestive gland tissues of exposed mussels. In conclusion, the most sensitive method for the detection of C. parvum in oocyst-exposed mussels was IMS concentration with DFA detection: 80% of individual and 100% of pooled digestive gland samples tested positive. TaqMan PCR was comparable to conventional PCR for detection of C. parvum oocysts in mussels and additionally allowed for automated testing, high throughput, and semi-quantitative results.     

Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples

Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the β-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower C _t values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier C _t values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits.     

The method of single-nucleotide variations detection using capillary electrophoresis and molecular beacons

We demonstrate that single-nucleotide variations in a DNA sequence can be detected using capillary electrophoresis (CE) and molecular beacons (MBs). In this method, the region surrounding the site of a nucleotide variation was amplified in a polymerase chain reaction, then hybridize PCR products with each of MBs. The sequences of the PCR products are different at the site of 2,044 in exon of interleukin (IL)-13 which to be identified. Through denaturation, the PCR product became single strand and hybridized with the completely complementary MB. The MB-target duplexes were separated using CE and solution-based fluorescence techniques. The results show that in each reaction a fluorescent response was elicited from the molecular beacon which was perfectly complementary to the amplified DNA, but not from the other MB whose probe sequence mismatched the target sequence. The method of CE based on MBs is able to identify single-nucleotide variations in a DNA sequence and can discriminate the genotyping of the SNP between the homo- and heteroduplexes of DNA fragments.