Closed-tube Format Research Articles

Development of a one-pot and sequence-specific LAMP assay based on the self-quenching probe integrated by the complementary oligonucleotide for an enhanced fluorescence quenching

Single-modified fluorogenic primer (Sfp) enables accurate identification of LAMP amplicons without being affected by non-specific products. However, the fluorescence self-quenching by nucleobases for Sfp is generally of low efficiency, and the high background signal makes it a great challenge to achieve visual inspection with naked eyes. In the present study, the oligonucleotide (Ao) complementary to Sfp was designed, which would hybridize to Sfp and dramatically heighten the quenching effect, leading to a low background signal in negative reaction. Instead, for positive reaction, Sfp is incorporated into the double-stranded amplicons, resulting in dequenching and consequently, enhanced fluorescence. The detection scheme can be further improved by a dual-color fluorescence strategy, allowing visual detection of 1 pg rainbow trout DNA in a closed-tube format within 30 min. Therefore, our LAMP-Ao-Sfp assay represents a useful tool for rapid and sensitive detection, and can serve as a reliable method for on-site detection in low-resource settings.

An improved visual closed tube Loop mediated isothermal amplification (LAMP) assay for rapid identification of orf virus in sheep and goats.

The orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry-over contamination associated with LAMP as open tube format prevents the assay applicability as point of care test in field diagnostic settings. In this study, the B2L gene based LAMP assay was optimized in a closed tube format using hydroxynaphthol blue (HNB) and calcein as pre-addition dyes and it has shown a clear positive and negative signal at 60 °C using 4 and 5 mM concentrations of MgSO4 respectively for these dyes. Optimitimzed assay that could reveal the result within one hour is highly specific and senstive with a limit of detection at 12.5 femtogram of viral genomic DNA or ~85 virus genome equivalent. This improved method prevented the cross-contamination of future LAMP reactions in the laboratory without compromising diagnostic sensitivity (100%) and specificity (100%) when compared to open tube system. This closed tube LAMP method has potential to act as a simple visual detection assay for the rapid and specific diagnosis of ORFV in sheep and goats.

CUT-LAMP: Contamination-Free Loop-Mediated Isothermal Amplification Based on the CRISPR/Cas9 Cleavage.

Loop-mediated isothermal amplification (LAMP) is a sensitive and widely used gene amplification technique. However, high amplification efficiency and amplification products containing multiple inverted repeats make the LAMP reaction extremely vulnerable to false-positive amplification caused by contamination. Herein, a contamination-free LAMP (CUT-LAMP) assisted by the CRISPR/Cas9 cleavage with superior reliability and durability has been reported. The core of CUT-LAMP is the engineering of the forward or backward inner primer in the target-independent region, which makes the LAMP products contain a protospacer adjacent motif (PAM) site for the CRISPR/Cas9 recognition. For the CUT-LAMP reaction, cross-contamination can be efficiently cleaved by the corresponding Cas9/sgRNA, but the target gene can get rid of digestion due to the lack of a PAM site near the recognition region. CUT-LAMP shows impressive contamination resistance but does not significantly increase procedure complexity; thus, it represents a simple and versatile toolkit facilitating the adoption by open- and closed-tube detection format.

Asymmetric mutant-enriched polymerase chain reaction and quantitative DNA melting analysis of KRAS mutation in colorectal cancer

Identification of mutant genes in tumor tissues and blood plasma (solid and liquid biopsy samples, respectively) is a necessity for individualized treatment of cancer patients. Here we report the use of a novel mutant-enriched PCR - quantitative DNA melting curve analysis (mePCR-qDMA) with TaqMan probes. The TaqMan probes served as blocking agents during PCR and as hybridization probes during DNA melting curve analyses. The end-point measurement of melt peaks areas by PeakFit software, a nonlinear iterative curve-fitting program, permitted quantification of the mutant/wild-type allele ratios. Approximately 6% and 0.1% of mutant KRAS allele in an excess of wild-type allele is detected with the standard and mePCR-qDMA processes, respectively. The application of the approach was tested for detecting the KRAS codon 12/13 mutation in paired tumor and blood plasma samples from 20 colorectal cancer patients. KRAS mutants were detected in 7 and 18 FFPE tumor samples, and in 3 and 7 plasma samples by the standard and mePCR-qDMA process, respectively. The results were confirmed by Sanger sequencing. This simple, rapid, cost-effective, and quantitative method carried out in a closed-tube format could be applied for the clinical analyses of other cancer genes.

Evaluation of high-resolution melting analysis for spa-typing of methicillin-resistant and -susceptible Staphylococcus aureus isolates.

Molecular characterization of Staphylococcus aureus isolates from nosocomial and community-acquired infections using accurate, reproducible, and rapid typing methods is essential for the fast identification of prevalent and epidemic strains. Although sequence-based spa typing is highly effective, PCR-based techniques (such as high-resolution melting curve analysis, HRM) are simpler, less expensive, faster, and can be performed in a single and closed-tube assay format, thereby reducing the risk of contamination. A total of 51 methicillin-resistant S. aureus (MRSA) (n = 26) and methicillin-sensitive S. aureus (MSSA) (n = 25) isolates from Karaj (n = 10) and Yasuj (n = 41), Iran, were subjected to HRM. All selected isolates were identified by the standard spa-typing method. Among the 51 tested isolates, 11 genotype profiles were distinguished from 12 spa types. Strains t1077 and t1816 exhibited the highest and lowest melting temperatures (81.8°C and 79.4°C), with 46.7% and 39.8% G + C contents, respectively. Strains t706 and t1816, with almost identical G + C contents, had the same HRM genotypes, but their curves differed due to different G + C distributions. Four standard spa types (strains t030, t037, t701 and t5598) were differentiated correctly and their melting temperatures were 81.2°C, 81.4°C, 80.4°C and 80.1°C, respectively. We demonstrated that HRM profiling is a rapid method which enables the accurate screening of certain strains (especially the endemic ones), and may be used for bacterial surveillance. However, it cannot replace sequence-based spa typing, especially for newly emerging spa types, and therefore cannot be used as a standardized global method.

TaqMan probes as blocking agents for enriched PCR amplification and DNA melting analysis of mutant genes.

Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ~5% and 15%-20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR. Here, we demonstrate that, under specially selected conditions of asymmetric PCR, TaqMan probes can play the role of blocking agents. Preferential blocking of the wild-type allele brings about enriched amplification of mutant alleles. As a result, an ~10-fold increase in the detection sensitivity for mutant BRAF and NRAS genes was achieved.

Scanning for KRAS, NRAS, BRAF, and PIK3CA mutations by DNA melting analysis with TaqMan probes

Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin-fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.

TriXY—Homogeneous genetic sexing of highly degraded forensic samples including hair shafts

Sexing of biological evidence is an important aspect in forensic investigations. A routinely used molecular-genetic approach to this endeavour is the amelogenin sex test, which is integrated in most commercially available polymerase chain reaction (PCR) kits for human identification. However, this assay is not entirely effective in respect to highly degraded DNA samples. This study presents a homogeneous PCR assay for robust sex diagnosis, especially for the analysis of severely fragmented DNA. The introduced triplex for the X and Y chromosome (TriXY) is based on real-time PCR amplification of short intergenic sequences (<50bp) on both gonosomes. Subsequent PCR product examination and molecular-genetic sex-assignment rely on high-resolution melting (HRM) curve analysis. TriXY was optimized using commercially available multi-donor human DNA preparations of either male or female origin and successfully evaluated on challenging samples, including 46 ancient DNA specimens from archaeological excavations and a total of 16 DNA samples extracted from different segments of eight hair shafts of male and female donors. Additionally, sensitivity and cross-species amplification were examined to further test the assay’s utility in forensic investigations. TriXY's closed-tube format avoids post-PCR sample manipulations and, therefore, distinctly reduces the risk of PCR product carry-over contamination and sample mix-up, while reducing labour and financial expenses at the same time. The method is sensitive down to the DNA content of approximately two diploid cells and has proven highly useful on severely fragmented and low quantity ancient DNA samples. Furthermore, it even allowed for sexing of proximal hair shafts with very good results. In summary, TriXY facilitates highly sensitive, rapid, and costeffective genetic sex-determination. It outperforms existing sexing methods both in terms of sensitivity and minimum required template molecule lengths. Therefore, we feel confident that TriXY will prove to be a reliable addition to the toolbox currently used for sex-typing in forensic genetics and other fields of research.

Clinical Evaluation of the Luminex NxTAG Respiratory Pathogen Panel.

An evaluation of the Luminex NxTAG Respiratory Pathogen Panel was performed on 404 clinical respiratory specimens. Clinical sensitivities and specificities of the assay compared to those of the reference methods were 80.0% to 100.0% and 98.9% to 100.0%, respectively. Correct genotyping information was provided for 95.5% of influenza virus A specimens. The closed-tube format of the assay simplified the workflow and minimized carryover contamination.

Diagnostic accuracy of high resolution melting analysis for detection of KRAS mutations: a systematic review and meta-analysis.

Increasing evidence points to a negative correlation between KRAS mutations and patients' responses to anti-EGFR monoclonal antibody treatment. Therefore, patients must undergo KRAS mutation detection to be eligible for treatment. High resolution melting analysis (HRM) is gaining increasing attention in KRAS mutation detection. However, its accuracy has not been systematically evaluated. We conducted a meta-analysis of published articles, involving 13 articles with 1,520 samples, to assess its diagnostic accuracy compared with DNA sequencing. The quality of included articles was assessed using the revised Quality Assessment for Studies of Diagnostic Accuracy (QUADAS-2) tools. Random effects models were applied to analyze the performance of pooled characteristics. The overall sensitivity and specificity of HRM were 0.99 (95% confidence interval [CI]: 0.98–1.00) and 0.96 (95%CI: 0.94–0.97), respectively. The area under the summary receiver operating characteristic curve was 0.996. High sensitivity and specificity, less labor, rapid turn-around and the closed-tube format of HRM make it an attractive choice for rapid detection of KRAS mutations in clinical practice. The burden of DNA sequencing can be reduced dramatically by the implementation of HRM, but positive results still need to be sequenced for diagnostic confirmation.

Real-time reverse transcriptase PCR for the detection of bluetongue virus.

In recent years, real-time reverse transcription polymerase chain reaction (rRT-PCR) has become one of the most widely used methods for the diagnosis of infectious pathogens. The combined properties of high sensitivity, specificity, and speed, along with a low contamination risk, have made real-time PCR technology a highly attractive alternative to more conventional diagnostic methods. Numerous robust rRT-PCR systems have been developed and validated for important epizootic diseases of livestock, and in this chapter we describe an rRT-PCR protocol for the detection of bluetongue virus. The assay uses oligonucleotide primers to specifically amplify target regions of the viral genome and a dual-labeled fluorogenic (TaqMan®) probe which allows for the assay to be performed in a closed-tube format, thus minimizing the potential for cross-contamination.

One-step, codominant detection of imidazolinone resistance mutations in weedy rice (Oryza sativa L.)

article i nfo Background: Weedy rice (Oryza sativa L.) is a noxious form of cultivated rice (O. sativa L.) associated with intensive rice production and dry seeding. A cost-efficient strategy to control this weed is the Clearfield rice production system, which combines imidazolinone herbicides with mutant imidazolinone-resistant rice varieties.However,imidazolinone resistance mutationscan beintrogressed inweedy rice populations bynatural outcrossing, reducing the life span of the Clearfield technology. Timely and accurate detection of imidazolinone resistance mutations in weedy rice may contribute to avoiding the multiplication and dispersion of resistant weeds and to protect the Clearfield system. Thus, highly sensitive and specific methods with high throughput and low cost are needed. KBioscience's Allele Specific PCR (KASP) is a codominant, competitive allele-specific PCR-based genotyping method. KASP enables both alleles to be detected in a single reaction in a closed-tube format. The aim of this work is to assess the suitability and validity of the KASP method for detection in weedy rice of the three imidazolinone resistance mutations reported to date in rice. Results: Validation wascarriedoutbydetermining the analyticalperformance of thenew method andcomparing it with conventional allele-specific PCR, when genotyping sets of cultivated and weedy rice samples. The conventional technique had a specificity of 0.97 and a sensibility of 0.95, whereas for the KASP method, both parameters were 1.00. Conclusions: The new method has equal accuracy while being more informative and saving time and resources compared with conventional methods, which make it suitable for monitoring imidazolinone-resistant weedy rice in Clearfield rice fields.

Scanning of mutations in short amplicons: Optimization of DNA melting method

The high resolution melting analysis (HRMA) is a new highly efficient method for genotyping and mutation scanning. HRMA is conducted immediately after PCR in closed-tube format, which enables the high throughput of the method. However, the closed-tube format makes HRMA dependent on the conditions of PCR and, thus, limits its capabilities. The open-tube format, which we have already developed (postamplification shortening of amplicons and optimized composition of ion medium), is applicable to the scanning of mutations of the K-RAS oncogene in tumor tissue and formalin-fixed paraffin-embedded samples. It is found that the open-tube format of DNA melting significantly increases the sensitivity of finding mutant alleles when using instruments both with and without an HRMA module. The higher sensitivity of the DNA melting compared to “Sanger” sequencing allows one to decrease the number of false-negative results of the mutation test, which is highly important for some forms of cancer.

Silica‐Modified Oligonucleotide−Gold Nanoparticle Conjugate Enables Closed‐Tube Colorimetric Polymerase Chain Reaction

A facile silica coating significantly enhances the thermal stability and polymerase chain reaction (PCR) compatibility of oligonucleotide-gold nanoparticle conjugates, thus enabling colorimetric detection of PCR results in a closed-tube format. This method is specific, sensitive, and generally applicable. Its simplicity, visual readout, and carryover contamination-free features hold promise for point-of-care or on-site DNA testing.

DNA melting analysis: Application of the “open tube” format for detection of mutant KRAS

High-resolution melting (HRM) analysis is a very effective method for genotyping and mutation scanning that is usually performed just after PCR amplification (the “closed tube” format). Though simple and convenient, the closed tube format makes the HRM dependent on the PCR mix, not generally optimal for DNA melting analysis. Here, the “open tube” format, namely the post-PCR optimization procedure (amplicon shortening and solution chemistry modification), is proposed. As a result, mutation scanning of short amplicons becomes feasible on a standard real-time PCR instrument (not primarily designed for HRM) using SYBR Green I. This approach has allowed us to considerably enhance the sensitivity of detecting mutant KRAS using both low- and high-resolution systems (the Bio-Rad iQ5–SYBR Green I and Bio-Rad CFX96–EvaGreen, respectively). The open tube format, though more laborious than the closed tube one, can be used in situations when maximal sensitivity of the method is needed. It also permits standardization of DNA melting experiments and the introduction of instruments of a “lower level” into the range of those suitable for mutation scanning.

Surpassing Specificity Limits of Nucleic Acid Probes via Cooperativity

The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.

Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci

BackgroundThe oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes.ResultsTwo novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the A. astaci strain Gb04. The primary amino acid sequence of these chitinase genes, termed CHI2 and CHI3, is composed of an N-terminal signal peptide directing the post-translational transport of the protein into the extracellular space, the catalytic GH18 domain, a proline-, serine-, and threonine-rich domain and a C-terminal cysteine-rich putative chitin-binding site. The A. astaci mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state CHI2 and CHI3 mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for CHI2 and CHI3, respectively, is in accordance with the multifunctionality of GH18 family members. To identify A. astaci-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes A. frigidophilus, A. invadans, A. helicoides, A. laevis, A. repetans, Achlya racemosa, Leptolegnia caudata, and Saprolegnia parasitica, as well as in the relevant fungi Fusarium solani and Trichosporon cutaneum. An A. astaci-specific primer pair targeting the novel genes CHI2 and CHI3 as well as CHI1 - a third GH18 family member - was multiplexed with primers targeting the 5.8S rRNA used as an endogenous control. A species was typed unambiguously as A. astaci if two peaks were concomitantly detected by melting curve analysis (MCA). For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe based real-time PCR (qPCR) assay was developed. It targets the same chitinase genes and allows quantification down to 25 target sequences.ConclusionThe simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. The homogenous closed-tube format, reduced detection time, higher specificity, and the considerably reduced chance of false negative detection achieved by targeting multiple genes (CHI1, CHI2, CHI3, and the endogenous control) at least two of which are subject to high functional constraint, are the major advantages of this multiplex assay compared to other diagnostic methods.Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission.

PCR-Based Methods for Detecting Single-Locus DNA Methylation Biomarkers in Cancer Diagnostics, Prognostics, and Response to Treatment

DNA methylation is a highly characterized epigenetic modification of the human genome that is implicated in cancer. The altered DNA methylation patterns found in cancer cells include not only global hypomethylation but also discrete hypermethylation of specific genes. In particular, numerous tumor suppressor genes undergo epigenetic silencing because of hypermethylated promoter regions. Some of these genes are considered promising DNA methylation biomarkers for early cancer diagnostics, and some have been shown to be valuable for predicting prognosis or the response to therapy. PCR-based methods that use sodium bisulfite-treated DNA as a template are generally accepted as the most analytically sensitive and specific techniques for analyzing DNA methylation at single loci. A number of new methods, such as methylation-specific fluorescent amplicon generation (MS-FLAG), methylation-sensitive high-resolution melting (MS-HRM), and sensitive melting analysis after real-time methylation-specific PCR (SMART-MSP), now complement the traditional PCR-based methods and promise to be valuable diagnostic tools. In particular, the HRM technique shows great potential as a diagnostic tool because of its closed-tube format and cost-effectiveness. Numerous traditional and new PCR-based methods have been developed for detecting DNA methylation at single loci. All have characteristic advantages and disadvantages, particularly with regard to use in clinical settings.

Single-Step Scalable-Throughput Molecular Screening for Huntington Disease

Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an unstable expansion of the CAG trinucleotide repeat in exon 1 of the HTT (huntingtin) gene and typically has an adult onset. Molecular diagnosis and screening for HD currently involve separate amplification and detection steps. We evaluated a novel, rapid microplate-based screening method for HD that combines the amplification and detection procedures in a single-step, closed-tube format. We carried out both the PCR for the HTT CAG-repeat region and the subsequent automated melting-curve analysis of the amplicon in the same wells on the plate. To establish cutoff melting temperatures (T(m)s) for each allelic class, we used a panel of reference DNA samples of known CAG-repeat sizes that represent a range of HTT alleles [normal (< or =26 repeats), intermediate (27-35 repeats), reduced penetrance expanded (36-39 repeats), and fully penetrant expanded (> or =40 repeats)]. We also measured well-to-well variation in T(m) across the thermal block and validated cutoff T(m)s with DNA samples from 5 different populations. We also conducted a blinded validation analysis of clinical samples from an additional 40 HD-affected and 30 unaffected individuals. We observed a strong correlation between CAG-repeat size and amplicon T(m) among the reference DNA samples. Use of the T(m) cutoffs we established revealed that 5 samples from unaffected individuals had been misclassified as affected (1.1% false-positive rate). All samples from HD-affected and unaffected individuals were correctly identified in the blinded analysis. This simple and scalable homogeneous assay may serve as a convenient, rapid, and accurate screen to detect the presence of pathologic expanded HD alleles in symptomatic patients.

High-Resolution DNA Melting Analysis for Simple and Efficient Molecular Diagnostics

High-resolution melting of DNA is a simple solution for genotyping, mutation scanning and sequence matching. The melting profile of a PCR product depends on its GC content, length, sequence and heterozygosity and is best monitored with saturating dyes that fluoresce in the presence of double-stranded DNA. Genotyping of most variants is possible by the melting temperature of the PCR products, while all variants can be genotyped with unlabeled probes. Mutation scanning and sequence matching depend on sequence differences that result in heteroduplexes that change the shape of the melting curve. High-resolution DNA melting has several advantages over other genotyping and scanning methods, including an inexpensive closed tube format that is homogenous, accurate and rapid. Owing to its simplicity and speed, the method is a good fit for personalized medicine as a rapid, inexpensive method to predict therapeutic response.