Thermal Cycling Parameters Research Articles

A preliminary exploration for co-detecting RNA virus and STR type on capillary electrophoresis in forensic practice.

RNA virus infection such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection shows severe respiratory symptoms on human and could be an obvious individual characteristic for investigations in forensic science. As for biological samples suspected to contain RNA virus in forensic casework, it requires respective detection of viral RNA and human DNA: reverse transcriptase polymerase chain reaction and DNA type (short tandem repeat [STR] analysis). Capillary electrophoresis (CE) has been shown to be a versatile technique and used for a variety of applications, so we preliminarily explored the co-detection of RNA virus and STR type on CE by developing a system of co-detecting SARS-CoV-2 and STR type under ensuring both the efficiency of forensic DNA analysis and safety of the laboratory. This study investigated the development and validation of the system, including N and ORF1ab primer designs, polymerase chain reaction amplification, allelic ladder, CE detection, thermal cycling parameters, concordance, sensitivity, species specificity, precision, and contrived and real SARS-CoV-2 sample studies. Final results showed the system could simultaneously detect SARS-CoV-2 and STR type, further indicating that CE has possibilities in the multi-detection of RNA viruses/STR type to help to prompt individual characteristics (viral infection) and narrow the scope of investigation in forensic science.

Evaluation of Solid Particle Erosion of EB-PVD TBCs under Thermal Cycling Conditions Based on a Stochastic Approach

The solid particle erosion behavior of electron-beam physical vapor deposition (EB-PVD) thermal barrier coatings (TBCs) was numerically evaluated under thermal cycling conditions. The erosion rates were calculated based on the mechanics-based formulae where the model parameters are fitted to the temperature-process-dependent test data available in the literature. A stochastic approach was applied to simulate the erosion behavior toward service conditions. The mechanics-based formulae were then validated by experimentally measured temperature and sintering-dependent erosion rates from the literature. The pseudoductile erosion behavior is identified for silica particles in the EB-PVD topcoat (TC) erosion system above the intermediate temperatures (~220 °C) due to the softening of partial molten silica particles, thus leading to an increase in the cutting wear and a decrease in deformation wear. The erosion rates are found to decrease versus temperature but increase versus thermal cycles. Such erosion behavior could be attributed to propagation of sintering cracks induced at elevated temperatures. The parametric calculations show that both erosion and thermal cycling parameters have a profound effect on the erosion mechanism of EB-PVD TC. The erosion rate increases at higher solid particle velocity and accumulated mass but displays a pseudoductile erosion behavior versus variation of impacting angles. Two types of erosion mechanisms were evaluated under different thermal cycling conditions. Under the burner cycling test with a short high-temperature dwell period, the erosion mechanism of EB-PVD TBCs is governed by temperature, while under an isothermal cycling test with a high-temperature long dwell period, the erosion is determined by sintering time. The failure mechanisms of EB-PVD TBCs under solid particle erosion processes are discussed combining internal cracking within topcoat and external erosion on the surface of topcoat.

DSC Cycling Effects on Phase Transformation Temperatures of Micron and Submicron Grain Ni50.8Ti49.2 Microwires

The effect of thermal cycling parameters on the phase transformation temperatures of micron and submicron grain size recrystallized Ni–Ti microwires was investigated. The suppression of martensitic transformation by thermal cycling was found to enhance when combined with room temperature aging between the cycles and enhances even more when aged at elevated temperature of 100 °C. While aging at room temperature alone has no clear effect on the martensitic transformation, elevated temperature aging at 100 °C alone suppresses the martensitic transformation. All aforementioned effects were found to be stronger in large grain samples than in small grain samples. Martensitic transformation suppression in all cases was in line with the formation of Ni4Ti3 precursors in the form of 〈111〉B2 Ni clusters as concluded from the observed diffuse intensity in the electron diffraction patterns revealing short-range ordering enhancement. Performing thermal cycling in some different temperature ranges to separate the effect of martensitic transformation and high temperature range of DSC cycling revealed that both high temperature- and martensitic transformation-included cycles enhance the short-range ordering.

Process parameters and mechanical properties of SAPH440 steel in pulse arc welding with different heat inputs

Thermal cycling parameters of pulse arc welding for SAPH440 steel was experimentally determined for different heat inputs. The welding thermal cycle curves of the weld fusion and heat-affected zones were successfully constructed based on the thermocouple measurements through the back drilling hole. The hardness and tensile strength variations under different heat inputs were also analyzed. This provided a database for the determination and optimization of process parameters, improvement of microstructure and properties of weld fusion and heat affected zones, and overall quality of pulse arc welding of steels.

A mechanistic model of damage evolution in lead free solder joints under combinations of vibration and thermal cycling with varying amplitudes

A broad range of electronics applications involve the long-term exposure to combinations of thermal excursions and vibration with constantly varying amplitudes, and the ultimate life is very often limited by fatigue of the solder joints. The assessment of this is usually based on a combination of simplified accelerated tests and the assumption of simple acceleration factor expressions or models. Neither of the latter can, however, even account for observed effects of accelerated test parameters. To make matters worse common constitutive relations may be strongly misleading. Also, current damage accumulation rules cannot account for major interactions between thermal cycling and vibration or even just for ongoing variations in vibration amplitudes or thermal cycling parameters, respectively. Even if we are ‘just’ aiming to decide between alternative material, design, or process parameters, or to compare a new product to a previous (supposedly similar) one, we should be concerned with the relative performance in service and not just in accelerated tests.The main objective of the present work is to combine two recent mechanistic models into a more comprehensive one that includes interactions between effects of thermal cycling and vibration with varying amplitudes. Important parameters in the resulting model still remain to be determined but a detailed understanding of the underlying mechanisms and the consequences of their interactions for realistic Tin-Silver-Copper (SnAgCu) joints allows for the prevention of surprises, the definition of appropriate test protocols, and the proper interpretation of results. This includes the identification of ‘worst case’ real life scenarios to test for, as well as justification for the use of shear testing in thermal cycling when event detection is not an option. Extension of the model to new alloys is briefly discussed as well.

Effect of thermal cycling process parameters on recrystallization kinetics for processing of fine-grained pure copper

ABSTRACTThe present work developed a two-step thermal cycling technique for processing of fine-grained pure copper for improved properties. This included initial annealing of specimens followed by heavy cold rolling and a series of heating and cooling cycles. The study investigated the important microstructural changes occurring in the cold deformed grains in the absence of phase transformations. A major interest of the present research was to closely describe the effect of thermal cycling parameters on the recrystallization and grain growth kinetics for processing of fine-grained structure. The study determined the optimum values of process parameters for the developed thermal cycling route including the extent of cold deformation, annealing temperatures, holding periods, and the number of thermal cycles. The thermal cycling process produced closely distributed fine grains with bi-modal microstructure leading to increased hardness and strength without any significant loss in electrical conductivity.

Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR) is facilitated by leveraging droplet microfluidic (DMF) system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points). This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs). The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind) panels of extracted patient samples with acceptably high PCR efficiency (>94%). The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.

Developmental validation of the GlobalFiler(®) express kit, a 24-marker STR assay, on the RapidHIT(®) System.

Rapid DNA typing provides a transformative solution to help forensic laboratories and law enforcement agencies solve and prevent crimes. The RapidHIT(®) System is a fully integrated instrument with a simplified user interface enabling an operator to run the system and obtain a DNA profile from a sample in less than two hours. The integration and developmental validation of the NDIS-approved 24 loci GlobalFiler(®) Express kit expands the capabilities of the RapidHIT System to increase discrimination power, reduce adventitious matches, and improve cross-border data sharing capabilities. Developmental validation studies were performed according to the SWGDAM guidelines and tested several critical areas of performance including three sensitivity studies, inhibited samples, thermal cycling parameters, and cross-contamination. Validation studies indicate that the optimized PCR parameters and sensitivity of the system is capable of generating STR profiles from buccal or blood swab reference samples. Results were concordant with genotypes produced using standard bench thermal cyclers and capillary electrophoresis platforms. Furthermore, swabs can be retrieved from the system and re-run or reprocessed with traditional bench chemistries, e.g. Y-STRs, to gain additional information. Our results demonstrate that the GlobalFiler Express assay run on the RapidHIT System is reliable for generating profiles from reference samples after forensic review.

The Influence of Nucleotide Sequence and Temperature on the Activity of Thermostable DNA Polymerases

Extension rates of a thermostable, deletion-mutant polymerase were measured from 50°C to 90°C using a fluorescence activity assay adapted for real-time PCR instruments. Substrates with a common hairpin (6-base loop and a 14-bp stem) were synthesized with different 10-base homopolymer tails. Rates for A, C, G, T, and 7-deaza-G incorporation at 75°C were 81, 150, 214, 46, and 120 seconds(-1). Rates for U were half as fast as T and did not increase with increasing concentration. Hairpin substrates with 25-base tails from 0% to 100% GC content had maximal extension rates near 60% GC and were predicted from the template sequence and mononucleotide incorporation rates to within 30% for most sequences. Addition of dimethyl sulfoxide at 7.5% increased rates to within 1% to 17% of prediction for templates with 40% to 90% GC. When secondary structure was designed into the template region, extension rates decreased. Oligonucleotide probes reduced extension rates by 65% (5'-3' exo-) and 70% (5'-3' exo+). When using a separate primer and a linear template to form a polymerase substrate, rates were dependent on both the primer melting temperature (Tm) and the annealing/extension temperature. Maximum rates were observed from Tm to Tm - 5°C with little extension by Tm + 5°C. Defining the influence of sequence and temperature on polymerase extension will enable more rapid and efficient PCR.

Evaluation of efficiency of hemi-nested PCR assay for the detection of ‘Candidatus Liberibacter’ infecting citrus

The present study reports the development and evaluation of a hemi-nested polymerase chain reaction (hnPCR) assay for the efficient detection of ‘Candidatus Liberibacter’ infecting citrus plants. A set of new primers was designed by alignment of nucleotide sequences of the β-operon (rplKAJL-rpoBC) ribosomal protein genes from all the known ‘Candi-datus Liberibacter asiaticus’ isolates reported in Genbank. Hemi-nested PCR reaction components and thermal cycling parameters were optimized and reaction conditions were standardized. Sequencing of the PCR products from hemi nested-PCR reactions confirmed the specificity of new primer pairs for ‘Candidatus Liberibacter asiaticus’. The reliability and sensitivity of hnPCR was evaluated by comparing it to real-time PCR.

Thermal Cycling Reliability of Sn-Ag-Cu Solder Interconnections. Part 1: Effects of Test Parameters

The work presented in part 1 of this study focuses on identifying the effects of thermal cycling test parameters on the lifetime of ball grid array (BGA) component boards. Detailed understanding about the effects of the thermal cycling parameters is essential because it provides means to develop more efficient and meaningful methods of reliability assessment for electronic products. The study was carried out with a single package type (BGA with 144 solder balls), printed wiring board (eight-layer build-up FR4 structure), and solder interconnection composition (Sn-3.1Ag-0.5Cu) to ensure that individual test results would be comparable with each other. The effects of (i) temperature difference (ΔT), (ii) lower dwell temperature and lower dwell time, (iii) mean temperature, (iv) dwell time, and (v) ramp rate were evaluated. Based on the characteristic lifetimes, the thermal cycling profiles were categorized into three lifetime groups: (i) highly accelerated conditions, (ii) moderately accelerated conditions, and (iii) mildly/nonaccelerated conditions. Thus, one might be tempted to use the highly accelerated conditions to produce lifetime statistics as quickly as possible. However, to do this one needs to know that the failure mechanisms do not change from one lifetime group to another and that the failure mechanisms correlate with real-use failures. Therefore, in part 2 the observed differences in component board lifetimes will be explained by studying the failure mechanisms that take place in the three lifetime groups.

A Porcine Model of Familial Adenomatous Polyposis

We created gene-targeted pigs with mutations in the adenomatous polyposis coli (APC) gene (APC) that are orthologous to those responsible for human familial adenomatous polyposis (FAP). One-year-old pigs with the APC(1311) mutation (orthologous to human APC(1309)) have aberrant crypt foci and low- and high-grade dysplastic adenomas in the large intestine, similar to the precancerous lesions that develop in patients with FAP. Dysplastic adenomas accumulate β-catenin and lose heterozygosity of APC. This large-animal, genetic model of FAP will be useful in the development of diagnostics and therapeutics for colorectal cancer. DNA sequence data: NCBI accession number GU951771.

Specific detection of all members of the Venezuelan Equine Encephalitis complex: Development of a RT-Nested PCR

Venezuelan Equine Encephalitis (VEE) complex belongs to alphavirus genus in the family Togaviridae. Several species of this complex are pathogenic to humans. VEE infections can produce severe or mild disease, and many cases remain undiagnosed. A specific and sensitive reverse transcriptase nested polymerase chain reaction (RT-Nested PCR) method was developed for the detection of all VEE subtypes, including Rio Negro Virus (RNV) (subtype VI), which circulates only in Argentina. Degenerated primers were designed and thermal cycling parameters were standardized. This technique is suitable for rapid and specific detection of these viruses, and may be useful for diagnosis and surveillance.

Doing more with less: Implementing direct amplification with the PowerPlex ® 18D System

Time and labor savings continue to be key goals for database and paternity labs. Increasing sample numbers and tightening government budgets necessitate that database laboratories seek process improvements. Similarly, efficiency gains are key for commercial paternity testing organizations to remain competitive. Recently, the STR-typing community has begun to adopt the concept of “direct amplification” from unpurified samples on FTA ® cards. Significant time and labor savings can be achieved by eliminating DNA purification and amplifying samples directly from buccal or blood samples on FTA ® cards. Another time-consuming process is the PCR itself—often three hours or more. The duration of this step often dictates daily workflow for laboratories, and when combined with DNA purification and capillary electrophoresis, the process often requires more than one day. Although thermal cycling is a hands-off step, decreasing sample time on an instrument with rapid amplification allows samples to be processed in a single day. The PowerPlex ® 18D System allows direct amplification and reduced thermal cycling time to provide a simpler and faster genotyping system for database and paternity laboratories. Additionally, the 18 loci amplified represent all markers commonly typed in CODIS laboratories. A validation of the PowerPlex ® 18D System was completed according to SWGDAM guidelines [1] to demonstrate effectiveness and performance of the assay. This testing included sensitivity, concordance, variation of thermal cycling parameters, variation of reaction component concentrations, and tolerance to contaminating substances. The results support that the PowerPlex ® 18D System is a robust and reliable identification assay fit for human identification purposes and the kit has subsequently been approved for use by NDIS CODIS-participating laboratories.

Molecular markers for circulating tumor cells in breast cancer.

223 Background: Recently, we identified a six gene panel (CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8) for the RT-qPCR based detection of circulating tumor cells (CTC) in breast cancer patients. The aim of the present study was to evaluate the gene panel in further blood samples. Methods: Blood samples were taken from breast cancer patients with metastatic disease (MBC, N=10) or with no evidence of disease (NED, N=30). Putative CTC were enriched by Oncoquick density gradient centrifugation. Total RNA was isolated with RNeasy Micro Kit (QIAgen). Template cDNA was generated with M-MLV Reverse Transcriptase, RNase H Minus (Promega) and random nonamers as primers. RT-qPCR was performed in duplicate reactions using TaqMan Assays (Applied Biosystems) with default thermal cycling parameters. Raw data were analyzed with the AB7900 Sequence Detection Software version 2.2.2 using automatic baseline correction and manual cycle threshold setting. Gene expression was normalized to GAPDH expression. A threshold value TX for each gene X was set at two standard deviations above the mean dCtX value in the healthy control group. A patient was defined as CTC-positive, if at least one gene marker was over-expressed compared to the defined threshold. Results: The gene panel consisting of CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8 identified 4/11 MBC but only 5/27 NED patients as CTC positive (p=0.163). By adding known CTC markers (SCGB2A2, TFF1, FXYD3, AGR2, S100A18, and EPCAM) to the panel, 7/11 MBC but only 6/27 NED patients were CTC positive (p=0.018). The presence of CTC in NED patients correlated with pN staging (p=0.026). Only one out of the six CTC positive NED patients relapsed within the observation period (median 35 months, range 25-39 months from blood sampling). We observed no correlation of CTC positivity and recurrence in NED patients. Conclusions: The sensitivity of the RT-qPCR based CTC detection in breast cancer patients may be enhanced by adding known CTC markers (SCGB2A2, TFF1, FXYD3, AGR2, S100A18, and EPCAM) to the six gene panel (CCNE2, DKFZp762E1312, EMP2, MAL2, PPIC, and SLC6A8). Longer follow-up times are needed to evaluate the predictive value of the gene markers on survival.

Development and Validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: A Multiplex Assay for the Direct Amplification of Single-Source Samples*,†

The AmpFℓSTR(®) Identifiler(®) Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single-source blood and buccal samples on FTA(®) card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay's sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA(®) cards, and the assay's specificity was verified by establishing minimal cross-reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA(®) substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler(®) Direct Kit for forensic standards and database samples genotyping.

A Sensitive and Reliable RT-Nested PCR Assay for Detection of Citrus tristeza Virus from Naturally Infected Citrus Plants

A specific and sensitive reverse transcriptase-nested polymerase chain reaction assay (RT-nPCR) was developed for the detection of Citrus tristeza virus (CTV) from naturally infected citrus samples. Two sets of primer pairs were designed by alignment of nucleotide sequences available in GenBank database for different genotypes of CTV. RT-nPCR reaction components and thermal cycling parameters were optimized and reaction conditions were standardized. Sequencing of the PCR products from direct and nested-PCR reactions confirmed the specificity of both primer pairs. Presence of CTV specific amplicons in asymptomatic samples which were collected from diseased orchards indicated the sensitivity of the test. As RT-nPCR technique, developed in the present study, is specific and efficient in detecting CTV, this could be envisioned for diagnostic applications and surveillance.

Direct amplification of STRs from blood or buccal cell samples

Forensic databasing laboratories routinely analyze blood or buccal cell samples deposited on FTA ® paper. Prior to PCR amplification of the STRs, the FTA ® samples must undergo multi-step sample purification protocols to remove the PCR inhibitors present within the sample and from the FTA ® paper. The multi-step sample purification protocols are laborious, time-consuming and increase the potential for sample cross-contamination. To eliminate the need for DNA purification, we conducted studies to optimize the PCR buffer and thermal cycling parameters to allow for direct amplification of STRs from blood or buccal samples on FTA ® paper. We evaluated the effect of various factors on the DNA profile including: FTA ® disc size, blood sample load variation, and buffer formulation. The new STR assay enables the direct amplification of DNA from single source samples on FTA ® discs without sample purification. The new STR assay improves the workflow by eliminating tedious steps and minimizing sample handling. Furthermore, the new STR assay reduces cost by eliminating the need for purification reagents and expensive robots.

Thermal Fatigue of SnPb and SAC Resistor Joints: Analysis of Stress-Strain as a Function of Cycle Parameters

Accelerated thermal cycling (ATC) has been widely used in the microelectronics industry for reliability assessment. The relative effects of thermal cycling parameters (temperature range, dwell time, and ramp rate) and the failure mechanisms they induce have been the subject of many studies; however, uncertainty remains, particularly regarding the role of a very high ramp rate such as encountered in a thermal shock chamber. In the present research, thermomechanical finite-element (FE) models were used to analyze the stress/strain response of a resistor test vehicle during ATC testing under three accelerated test conditions: two ramp rates (14 degC/min and 95 degC/min) and two temperature ranges (DeltaT=0degC-100degC and -40degC-125degC), denoted 14-100 (ramp rate-temperature range), 95-100, and 95-165. The temperature gradients through the thickness of the assemblies were measured during the ATC test with the high ramp rate and were used to create an FE model that included transient stresses and strains. The effect of the transient temperature gradients during thermal shock was found to be negligible in these resistor joints. The FE models were then used to simulate the above three ATC test conditions with either SnPb or Pb-free (SAC) solders and were compared with previously published thermal fatigue lives for this resistor test vehicle. For both SnPb and SAC resistors, the maximum total solder strains (sum of elastic, plastic, and creep) and strain energy dissipation per cycle predicted by the FE models in the 95-165 test condition were much greater than those in either the 14-100 or 95-100 test conditions, which produced almost identical strains and energy dissipation. In all cases, the strain energy density dissipation per cycle due to creep was much larger than that due to plastic deformation. The trend of these results was in accordance with the ATC tests, which showed that the thermal cycling lives decreased in the same order for both the SAC and SnPb solders; i.e., the fatigue life decreased as the predicted total strain increased

Contribution of Sensory Neurons to Sex Difference in the Development of Stress-Induced Gastric Mucosal Injury in Mice

Sensory neurons play a critical role in reducing stress-induced gastric mucosal injury by releasing calcitonin gene-related peptide (CGRP) through an increase in gastric mucosal levels of prostacyclin (PGI(2)). Because estrogen enhances nerve growth factor-mediated CGRP production in sensory neurons, we hypothesized that stress-induced gastric mucosal injury occurs less in females than in males. Gastric ulcer index, gastric myeloperoxidase activity, and gastric tissue levels of CGRP and 6-keto-PGF(1alpha), a stable metabolite of PGI(2), were determined in male and female wild-type (CGRP(+/+)) mice and CGRP knockout (CGRP(-/-)) mice subjected to water-immersion restraint stress. In CGRP(+/+) mice, ulcer index and myeloperoxidase activities were lower and gastric tissue levels of CGRP and 6-keto-PGF(1alpha) were higher in female mice than in male mice, but there were no such sex differences in CGRP(-/-)mice. Sex differences in CGRP(+/+) mice were eliminated by pretreatment with SB366791 (500 microg/kg intraperitoneally), a vanilloid receptor antagonist, and by ovariectomy. Reversal of sex differences by ovariectomy was not observed in female CGRP(+/+) mice with estradiol replacement (1 mg . kg(-1). wk(-1) for 3 weeks). Levels of CGRP messenger RNA in dorsal root ganglion neurons isolated from female CGRP(+/+) mice were decreased by ovariectomy, and these decreases were reversed by estradiol replacement. Estrogen-mediated increases in CGRP levels in sensory neurons might contribute to reduce stress-induced gastric mucosal injury by attenuating inflammatory responses. This might at least partly explain the sex difference observed in the development of stress-induced gastric mucosal injury in mice.