Polymerase Chain Reaction Reaction Mixture Research Articles

Screening for Group A Streptococcal Disease via Solid-State Nanopore Detection of PCR Amplicons.

Single-molecule detection methods are becoming increasingly important for diagnostic applications. Practical early detection of disease requires sensitivity down to the level of single copies of the targeted biomarkers. Of the candidate technologies that can address this need, solid-state nanopores show great promise as digital sensors for single-molecule detection. Here, we present work detailing the use of solid-state nanopores as downstream sensors for a polymerase chain reaction (PCR)-based assay targeting group A streptococcus (strep A), which can be readily extended to detect any pathogen that can be identified with a short nucleic acid sequence. We demonstrate that with some simple modifications to the standard PCR reaction mixture, nanopores can be used to reliably identify strep A in clinical samples. We also discuss methodological best practices for both adapting PCR-based assays to solid-state nanopore readout and analytical approaches by which to decide on sample status.

Detection of mecA and panton-valentine leukocidin genes in methicillin resistant staphylococcus aureus isolated from various clinical samples in a tertiary care hospital

Detection of mecA and panton-valentine leukocidin genes in methicillin resistant staphylococcus aureus isolated from various clinical samples in a tertiary care hospital - IJMR- Print ISSN No: - 2394-546X Online ISSN No:- 2394-5478 Article DOI No:- 10.18231/2394-5478.2019.0014, Indian Journal of Microbiology Research-I

Development of a sequence‐characterized amplified region marker using inter‐simple sequence repeats for detection of Puccinia striiformis f. sp. tritici

AbstractStripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most devastating wheat disease in China. Early and accurate detection of the pathogens would facilitate effective control of the diseases. DNA‐based methods now provide essential tools for accurate plant disease diagnosis. In this study, inter‐simple sequence repeats (ISSR) technique has been successfully applied to develop a sequence‐characterized amplified region (SCAR) marker for diagnosis of stripe rust of wheat and detection of Pst. In this study, one fragment unique to Pst was identified by ISSR and then sequenced. Based on the specific fragment, a pair of SCAR primers (616AF/616AR) was designed to amplify a 299‐bp DNA fragment within the sequenced region. The primers can amplify a unique DNA fragment for all tested isolates of Pst but not for the other pathogens of wheat leaves and the uninfected leaves. The polymerase chain reaction (PCR) assay could detect as low as 0.1 ng of genomic DNA in a 25.0 μl PCR reaction mixture and detect the pathogen from asymptomatic wheat leaves inoculated with Pst under glasshouse conditions.

Handheld energy-efficient magneto-optical real-time quantitative PCR device for target DNA enrichment and quantification

A PCR provides not only valuable genetic information that enables precise differential disease diagnosis but also quantitative data to assess various clinical states. We report the successful integration of novel dual-mode magnetic Fe3O4 nanoclusters that deliver photothermal conversion. The clusters can be excited with pulsed laser light for precision thermal cycle modulation to develop an ultrafast quantitative PCR system. Traditional PCR heats and cools the sample from outside the PCR tube; the heat needs to pass through the heat block, tube wall and transfer to the DNA through water molecules. In contrast, our system uses nanoparticles inside the liquid phase as numerous ‘nanoheaters’; thus the thermal transfer between particles and adjacent water or DNA molecules becomes extremely efficient because of proximity at the molecular level. Moreover, the defective mitochondrial DNA from cybrid cell lines of a patient with chronic progressive external ophthalmoplegia syndrome, a mitochondrial disease, was efficaciously detected. The system has a simple design, is extremely energy efficient and is faster than traditional qPCR. Our finding provides new insight into rapid and accurate quantitative diagnostics for future point-of-care applications. Iron nanoparticles play double duty as ‘nanoheaters’ for DNA amplification and as molecular manipulators in a new point-of-care prototype. Polymerase chain reaction (PCR) devices create multiple copies of small DNA strands for genetic testing, but usually require bulky heating and cooling mechanisms. Dar-Bin Shieh and colleagues from National Cheng Kung University in Taiwan have found that tiny clusters of iron oxide nanocubes can improve heat transfer in PCR devices by entering the liquid phase next to the target DNA. When irradiated by laser light, the iron nanoclusters release heat to precisely modulate DNA temperatures. Furthermore, functionalizing the nanoparticles with nucleotide probes enables them to attach to and extract specific disease-indicating mitochondria using magnetic fields. The portable, battery-powered prototype can wirelessly interface with tablet computers and is twice as efficient at isolating mitochondria as commercial kits. A novel dual-mode magnetic Fe3O4 nanoclusters was developed that exhibit efficient photothermal conversion and superparamagnetism. The particles served as numerous ‘nanoheaters’ in a PCR reaction mixture while enabling effective magnetic-based macromolecular manipulation. The thermal transfer between particles and adjacent water and DNA molecules thus become extremely efficient because of molecular-level proximity and allowed rapid amplification cycle progression. The defective mitochondrial DNA from cybrid cell lines of a patient with chronic progressive external ophthalmoplegia syndrome was efficaciously quantified by fluorescence intensity detection. Our finding provides new insight into rapid and accurate quantitative DNA diagnostics for future point-of-care applications.

Characterization of Paracoccidioides brasiliensis by FT-IR spectroscopy and nanotechnology

Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, is a dimorphic fungus existing as mycelia in the environment (or at 25°C in vitro) and as yeast cells in the human host (or at 37°C in vitro). Because mycological examination of lesions in patients frequently is unable to show the presence of the fungus and serological tests can misdiagnose the disease with other mycosis, the development of new approach’s for molecular identification of P. brasiliensis spurges is needed. This study describes the use of a gold nanoprobe of a known gene sequence of P. brasiliensis as a molecular tool to identify P. brasiliensis by regular polymerase chain reaction (PCR) associated with a colorimetric methods. This approach is suitable for testing in remote areas because it does not require any further step than gene amplification, being safer and cheaper than electrophoresis methods. The proposed test showed a color change of the PCR reaction mixture from red to blue in negative samples, whereas the solution remains red in positive samples. We also performed a Fourier Transform Infrared (FT-IR) Spectroscopy analysis to characterize and compare the chemical composition between yeast and mycelia forms, which revealed biochemical differences between these two forms. The analysis of the spectra showed that differences were distributed in chemical bonds of proteins, lipids and carbohydrates. The most prominent difference between both forms was vibration modes related to 1,3-β-glucan usually found in mycelia and 1,3-α-glucan found in yeasts and also chitin forms. In this work, we introduce FT-IR as a new method suitable to reveal overall differences that biochemically distinguish each form of P. brasiliensis that could be additionally used to discriminate biochemical differences among a single form under distinct environmental conditions.

Genetic diversity among different physiological traits of Sorghum bicolor cultivars of subtropical origin.

The genetic diversity of Saudi locally growing sorghum (Sorghum bicolor) cultivars has not been thoroughly characterized. To understand the genomic patterns of diversification in Saudi sorghum cultivars (N = 7), random amplified polymorphic DNA (RAPD) was used as a rapid, inexpensive method for providing information regarding genomic variability below the species level. Six commercially available primers were initially used to select a single primer based on availability, universality, and its use with standard polymerase chain reaction (PCR) conditions. PCR-amplified molecular markers were reproducibly detected in Saudi cultivars. The single primer 2 produced clear bands and revealed variability among the cultivars. Seven tested cultivars were categorized into 2 major groups, indicating 2 genomogroups for the Saudi-cultivars. Five cultivars (S2, S3, S4, S5, and S6) showed identical banding patterns and were grouped in the same clade, although their panicles varied in size, shape, and color. Two cultivars (S1 and S7) showed different banding patterns. In this study, a single primer (P2) was used to demonstrate the effectiveness of genotype detection among sorghum cultivars. This is the first report describing genetic variation among S. bicolor cultivars in Saudi Arabia. The commercial primer (P2) and PCR reaction mixture used in this study are readily available and can be used in sorghum improvement programs.

Bias toward a 1:1 ratio in primer-introduced restriction analysis PCR: mechanism and minimization.

Primer-introduced restriction analysis is widely used in molecular genetics. However, several studies have reported inconsistent data regarding sequencing, mainly among heterozygous samples. This discrepancy may be related to the bias towards a 1:1 ratio typically observed in heterozygous digestion products. In this study, we investigated the mechanism and minimization of this observed bias. Three mismatched polymerase chain reaction (PCR) models were analyzed by testing different PCR conditions and reaction mixtures. For EPHX1 gene rs1051740 single-nucleotide polymorphism PCR, DNA concentration, denaturation and elongation time, annealing temperature, and cycle number significantly influenced product ratios. For SERPINA1 gene PIMmalton deletion (ΔPhe52) and CHRNA3 gene rs1051730 single-nucleotide polymorphism PCRs, significant bias fluctuations were observed only for the annealing temperature and cycle number conditions. The relevance of these results to the amplification efficiency parameter is discussed. Rather than reducing the observed bias, our data provide evidence of a counterbalance for preferential amplification, depending on cycle number, annealing temperature, and amplification efficiency alteration. Our results are relevant for application to primer-introduced restriction analysis PCR assays.

Single restriction enzyme-assisted megaprimer polymerase chain reaction to fuse two DNA sequences on separate cloning vectors

We describe a simple and versatile method to fuse two DNA sequences on separate cloning vectors in a single polymerase chain reaction (PCR). The method, termed restriction enzyme-assisted megaprimer PCR (REM–PCR), requires that the two cloning vectors share a common sequence and that the DNA sequences to be fused are cloned in the same orientation with respect to the common sequence. Fusion of the two sequences is achieved by mutual priming at the common sequence between two DNA fragments that were generated by restriction enzyme and linearly amplified by repetitive priming in the PCR reaction mixture.

Enhanced Expression of Keratinocyte Growth Factor and Its Receptor Correlates with Venous Invasion in Pancreatic Cancer

Keratinocyte growth factor (KGF) and KGF receptor (KGFR) have been implicated in cancer growth as well as tissue development and repair. In this study, we examined whether KGF and KGFR have a role in human pancreatic ductal adenocarcinoma (PDAC). KGFR mRNA was expressed in eight pancreatic cancer cell lines, whereas the KGF mRNA was detected in seven of the cell lines and was absent in MIA PaCa-2 cells. KGFR and KGF immunoreactivity were localized in the cancer cells in 41.5 and 34.0% of patients, respectively. There was a significant correlation between KGFR or KGF immunoreactivity and venous invasion and a significant correlation between the presence of both markers and venous invasion, vascular endothelial growth factor (VEGF)-A expression, and poor prognosis. Exogenous KGF increased VEGF-A expression and release in MIA PaCa-2 cells, and PANC-1 cells stably transfected to overexpress KGF-exhibited increased VEGF-A expression. Moreover, short hairpin-KGFR transfection in MIA PaCa-2 cells reduced the stimulatory effect of exogenous KGF on VEGF-A expression. Short hairpin-KGF transfection in KLM-1 cells reduced VEGF-A expression in the cells. KGFR and KGF may act to promote venous invasion and tumor angiogenesis in PDAC, raising the possibility that they may serve as novel therapeutic targets in anti-angiogenic strategies in PDAC.

A simple device using magnetic transportation for droplet-based PCR

The Polymerase chain reaction (PCR) was successfully and rapidly performed in a simple reaction device devoid of channels, pumps, valves, or other control elements used in conventional lab-on-a-chip technology. The basic concept of this device is the transportation of aqueous droplets containing hydrophilic magnetic beads in a flat-bottomed, tray-type reactor filled with silicone oil. The whole droplets sink to the bottom of the reactor because their specific gravity is greater than that of the silicone oil used here. The droplets follow the movement of a magnet located underneath the reactor. The notable advantage of the droplet-based PCR is the ability to switch rapidly the proposed reaction temperature by moving the droplets to the required temperature zones in the temperature gradient. The droplet-based reciprocative thermal cycling was performed by moving the droplets composed of PCR reaction mixture to the designated temperature zones on a linear temperature gradient from 50 degrees C to 94 degrees C generated on the flat bottom plate of the tray reactor. Using human-derived DNA containing the mitochondria genes as the amplification targets, the droplet-based PCR with magnetic reciprocative thermal cycling successfully provided the five PCR products ranging from 126 to 1,219 bp in 11 min with 30 cycles. More remarkably, the human genomic gene amplification targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was accomplished rapidly in 3.6 min with 40 cycles.

Comprehensive Arrayed Primer Extension Array for the Detection of 59 Sequence Variants in 15 Conditions Prevalent Among the (Ashkenazi) Jewish Population

In the Ashkenazi Jewish population, serious and lethal genetic conditions occur with relatively high frequency. A single test that encompasses the majority of population-specific mutations is not currently available. For comprehensive carrier screening and molecular diagnostic purposes, we developed a population-specific and inclusive microarray. The arrayed primer extension genotyping microarray carries 59 sequence variant detection sites, of which 53 are detectable bi-directionally. These sites represent the most common variants in Tay-Sachs disease, Bloom syndrome, Canavan disease, Niemann-Pick A, familial dysautonomia, torsion dystonia, mucolipidosis type IV, Fanconi anemia, Gaucher disease, factor XI deficiency, glycogen storage disease type 1a, maple syrup urine disease, nonsyndromic sensorineural hearing loss, familial Mediterranean fever, and glycogen storage disease type III. Several mutations in the selected disorders that are not prevalent per se in the Ashkenazi Jewish populations, as well pseudodeficiency alleles, are also included in the array. The initial technical evaluation of this microarray demonstrates that it is comprehensive, robust, sensitive, specific, and easily modifiable. This cost-effective array is based on a diversely applied platform technology and is suitable for both carrier screening and disease detection in Ashkenazi and Sephardic Jewish populations.

Mistyping of Angiotensinogen M235T Alleles

Conflicting results are to be found in the literature on the relationship between the M235T polymorphism of the angiotensinogen (AGT) gene and hypertension. The controversy may be due to insufficient numbers of subjects, the variability of the inclusion criteria and the different genotype analysis methods used. We have experienced that the most frequently used, original polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method involves significant uncertainties when the TT genotype is determined, independently of the restriction digestion. To make the determination more accurate, we improved the PCR by designing a new antisense primer containing only one mismatch instead of the two in the original protocol and also by adding DMSO to the PCR reaction mixture. The original and our improved methods were compared by using DNA from 123 patients: parallel determinations resulted in values of 33 MM, 90 MT and 0 TT with the original method and of 33 MM, 56 MT and 34 TT with the improved RFLP protocol. In summary, a plausible explanation for some of the conflicting data published on AGT M235T polymorphism may be that inaccuracies arose during the determination of the genotype.

Identification of Functional Genetic Variants in and Their Association With Risk of Esophageal Cancer

Identification of Functional Genetic Variants in and Their Association With Risk of Esophageal Cancer

Identification of Functional Genetic Variants in Cyclooxygenase-2 and Their Association With Risk of Esophageal Cancer

Identification of Functional Genetic Variants in Cyclooxygenase-2 and Their Association With Risk of Esophageal Cancer

Specific detection of a basidiomycete, Phlebia brevispora associated with butt rot of Chamaecyparis obtusa, by PCR-based analysis

In order to monitor the basidiomycetous fungus Phlebia brevispora isolated from butt rot of Chamaecyparis obtusa (Japanese cypress) in 1997 in Nagasaki Prefecture, a sensitive polymerase chain reaction (PCR)-based assay was developed to specifically detect the fungus on-site. A species-specific primer for P. brevispora was derived from the internal transcribed spacer (ITS) region (containing 5.8S ribosomal DNA, ITS1 and ITS2) sequences of the fungus. The PCR assay was able to detect down to 1 fg DNA (per 1 µl PCR reaction mixture) and down to 0.2 mg mycelium of P. brevispora (per 1 g of decayed wood). The samples for on-site monitoring were collected in 2002 from the decayed tree stump in which P. brevispora had first been isolated. From the decayed tree tissue, P. brevispora could be detected by PCR assay even when its mycelium could not isolated from the tree tissue by culturing. This indicates that the PCR amplification using the specific primer developed here is a useful method for monitoring P. brevispora on-site.

Single-stranded DNA-binding protein facilitates gel-free analysis of polymerase chain reaction products in capillary electrophoresis

It has been recently demonstrated that single-stranded DNA-binding protein (SSB) can facilitate quantitative analyses of DNA, RNA, and proteins in gel-free capillary electrophoresis (CE). Here, we report the application of SSB-mediated gel-free CE for analyses of polymerase chain reaction (PCR) products. The unique ability of SSB to bind ssDNA but not double-stranded DNA (dsDNA) allows efficient separation of three types of DNA molecules in the PCR reaction mixture: primers, products (amplified templates), and by-products, which originate from non-specific DNA hybridization. SSB-mediated gel-free CE analysis of PCR products combines simplicity, high sensitivity, and outstanding quantitative capabilities. The ability of the method to distinguish between products and by-products makes this method an indispensable tool in preparative PCR (e.g., in the development of nucleotide aptamers).

Increased Susceptibility of Mice Lacking Clara Cell 10-kDa Protein to Lung Tumorigenesis by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, a Potent Carcinogen in Cigarette Smoke

Ninety percent of all human lung cancers are related to cigarette smoking. Both tobacco smoke and lung tumorigenesis are associated with drastically reduced levels of Clara cell 10-kDa protein (CC10), a multifunctional secreted protein, naturally produced by the airway epithelia of virtually all mammals. We previously reported that the expression of CC10 is markedly reduced in animals exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK, a potent carcinogen in tobacco smoke. Furthermore, it has been reported that CC10 expression, induced in certain tumor cells, reverses the transformed phenotype. We demonstrate here that NNK exposure of CC10-knock-out (CC10-KO) mice causes a significantly higher incidence of airway epithelial hyperplasia and lung adenomas compared with wild type (WT) littermates (30% CC10-KO versus 5% WT, p = 0.041). We also found that compared with NNK-treated WT mice, CC10-KO mice manifest increased frequency of K-ras mutation, elevated level of Fas ligand (FasL) expression, and increased MAPK/Erk phosphorylation, all of which are considered predisposing events in NNK-induced lung tumorigenesis. We propose that CC10 has a protective role against NNK-induced lung tumorigenesis mediated via down-regulation of the above-mentioned predisposing events.

Liquid-phase hybridization based PCR-ELISA for detection of genetically modified organisms in food

Polymerase chain reaction (PCR) screening for presence of transgenic components in food is becoming a routine method in modern food analysis. To develop a high throughput method for quantitation of the PCR products is needed for automatic industry analysis. Here we described an in situ liquid-phase hybridization (LPH) for PCR-enzyme linked immunoabsorbent assays (PCR-ELISA) that was widely used for quantitation of PCR products. In LPH-PCR-ELISA, the biotinylated PCR product was hybridized with digoxigenin-labeled probes in the PCR reaction mixture immediately after PCR cycles and the hybridizations was incorporated into the PCR program. Subsequent enzyme conversion of substrate gave distinct OD values when detecting samples with genetically modified organisms (GMOs) labels in the different concentrations. The described method enabled a fast, specific, and accurate detection of GMOs components in food products and thus can be developed to a full-automatic method for routine analysis of raw and processed food products in large sample number.

Evaluation of Methods To Improve Detection of Escherichia coli O157:H7 in Fresh Produce by Multiplex Polymerase Chain Reaction

Multiplex polymerase chain reaction (PCR) analysis was used to detect two genes encoding Shiga-like toxins (stx1 and stx2) and a universal Escherichia coli gene (gadA/B) in fresh produce spiked with E. coli O157:H7. Current U.S. Food and Drug Administration procedures for the analysis of fresh produce include the use of the rinsate from an initial rinse for the analysis of several potential pathogens, including E. coli O157:H7. In this study, several procedures were evaluated for their ability to increase the sensitivity of PCR analysis of rinsates from 15 types of produce. The procedures evaluated included the preliminary clarification and concentration of templates by centrifugation and the treatment of templates with compounds reported to facilitate nucleic acid amplification, including polyvinlypolypyrrolidone (PVPP), nonfat dry milk (NFDM), and InstaGene. The preliminary concentration of rinsates resulted in moderate improvements in detection sensitivity. The use of PVPP-treated templates in PCR reaction mixtures did not further improve sensitivity, but the inclusion of NFDM-treated templates increased sensitivity by an order of magnitude for 12 rinsates. The incorporation of InstaGene also improved the detection capability of the analysis; this procedure yielded the strongest gel bands for eight rinsates. However, for four other rinsates, the use of this reagent decreased sensitivity; these four rinsates were those for the produce varieties with the largest surface areas and were the most turbid rinsates. The use of facilitating compounds to block PCR inhibition may enable an analysis for Shiga toxin–producing E. coli in fresh produce to be completed in 1 to 2 days, rather than the 5 days required for current methods.

Single-tube two-round polymerase chain reaction using the LightCycler instrument.

For many diagnostic applications, the specificity and sensitivity of polymerase chain reaction (PCR) is markedly enhanced by applying two rounds of PCR with nested or semi-nested pairs of primers. In two-round PCR protocols on the LightCycler instrument, amplification products must be collected from the capillaries by centrifugation, a procedure thought to be particularly prone to product carry-over. Development of a technique to perform two-round PCR with the LightCycler instrument in a single closed capillary. Silicone oil was used to separate the second-round primers from first-round PCR mixture during the first-round PCR. The feasibility of the principle was demonstrated using a semi-nested primer system for the PCR analysis of genomic DNA. The first-round PCR reaction mixture was loaded into the capillary and covered by oil. Then, the second-round PCR reaction mixture was layered on top of it. PCR was run in two rounds separated by a centrifugation step that combined the second-round PCR mixture with the first-round products. Amplified products were visualized by fluorescence melting curve analysis. When a dilution series of genomic DNA was used for the single-capillary two-round PCR, 0.1 ng of DNA could consistently be detected. This was a 10-fold increase of sensitivity in comparison with single-round PCR. With the new technique, the first-round reaction mixture was sufficiently separated from second-round primers by the oil layer. Two-round PCR on the LightCycler using a single closed capillary excluded the possibility of amplification product carry-over. This new technique can easily be adapted for numerous applications, and should show feasibility for many nested primer PCR applications currently in use to the clinical detection of virus-derived DNA.