The purpose of this study was to compare the detection of human papillomavirus (HPV) DNA with detection of mRNA. The study included 4,136 women >30 years of age. E6/E7 mRNA expression from the ...carcinogenic HPV types 16, 18, 31, 33, and 45 was detected by the PreTect HPV-Proofer assay, whereas the presence of HPV DNA was detected by Gp5+/6+ consensus PCR followed by type-specific PCR. A total of 4.0% had an abnormal cytologic diagnosis, 3.0% were positive by PreTect HPV-Proofer, 4.4% by type-specific PCR, and 10.4% by consensus PCR. For detection of HPV in high-grade squamous intraepithelial lesion (HSIL), no significant difference was observed between PreTect HPV-Proofer and consensus PCR. For women with a cytologic normal, atypical squamous cell of uncertain significance, and low-grade SIL diagnosis, the detection rate of HPV was significantly higher by Gp5+/6+ consensus PCR (P < 0.005) than by PreTect HPV-Proofer. Histology confirmed 14 of 23 cytologic HSIL as cervical intraepithelial neoplasia grade >2. Of these women, PreTect HPV-Proofer and type-specific PCR detected 12, whereas consensus PCR detected 13. In conclusion, for HSIL, detection of E6/E7 transcripts from HPV types 16, 18, 31, 33, and 45 are present to the same degree as DNA detected by consensus PCR. Equally important, only a small proportion of the HPV DNA-positive women with a normal, atypical squamous cell of uncertain significance or low-grade SIL diagnosis had a detectable mRNA expression. HPV E6/E7 mRNA detection by PreTect HPV-Proofer represents a new promising test as an adjunct to cytology.
The oncogenic potential of the human papillomavirus (HPV) early genes E6 and E7 is well established and a source of interest with regard to HPV testing for cervical carcinoma. Here we present a study ...performed with 204 histologically confirmed invasive cervical squamous cell carcinomas (SCCs) in which we evaluated the HPV E6 and E7 mRNA detection assay PreTect HPV-Proofer for detection of high-risk HPV types 16, 18, 31, 33, and 45. For further evaluation, detection of E6 and E7 mRNA from HPV types 35, 52, and 58 by real-time multiplex nucleic acid sequence-based amplification was also included. For comparison and to assess the overall prevalence of various HPV types, samples were also tested for HPV DNA by both consensus and type-specific PCR, reverse line blotting, sequencing, and in situ hybridization. The overall prevalence of HPV was 97%. HPV E6 and E7 transcripts were detected in 188 of 204 (92%) biopsy specimens, of which 181 contained one of the following HPV types: 16, 18, 31, 33, or 45. Consensus PCR and type-specific PCR detected HPV in 187 of 204 and 188 of 204 (92%) specimens, respectively. In conclusion, this study verifies the presence of HPV E6 and E7 mRNA in SCCs and demonstrates that HPV infections among Norwegian women with SCCs are limited mainly to the five high-risk types, 16, 18, 31, 33, and 45. This, together with the fact that PreTect HPV-Proofer detects the HPV oncogenic transcripts, suggests that the assay is a valuable approach in the field of HPV detection in cervical carcinoma.
Esox lucius (northern pike) is an invasive species in fresh water and causes extreme impacts in the local habitat. Northern pike easily replaces the local native species and disrupts the regional ...ecosystem. Traditionally, in connection with environmental monitoring, invasive species are identified using PCR through species-specific DNA. PCR involves many cycles of heating to amplify the target DNA and requires complex equipment; on the contrary, loop-mediated isothermal amplification (LAMP) entails isothermal amplification, which means the target needs to be heated to only one temperature between 60 and 65°C. In this study, the authors conducted a LAMP assay and a conventional PCR assay to determine which technique is less time consuming, more sensitive and reliable for use in real-time and on-site environmental monitoring. Mitochondrial gene cytochrome b, an essential factor in electron transport; histone (H2B), a nuclear DNA responsible for the chromatin structure; and glyceraldehyde 3-phosphate dehydrogenase involved in energy metabolism are taken as the reference genes for this article. The results show that LAMP is more sensitive and less time consuming than the conventional PCR, and thus it can be used for the detection of northern pike in aquatic ecosystems related to environmental monitoring.
In many ways, globalization is beneficial, but in one way, it promotes the spread of alien (invasive) species through international trade and transport. In different habitats, Esox lucius (northern ...pike) can be considered a regionally alien species, and this fish tends to establish a higher density population than desired in fresh water. Early identification of such invasive species using sensitive and quick methods is important to be able to take immediate measures and avoid environmental problems. Loop-mediated isothermal amplification (LAMP) has emerged as the best DNA/RNA detection technique, without any expensive equipment and could be used to detect environmental DNA (eDNA). However, the reagents for amplification are not stable at ambient temperature for field applications. Therefore, this work aims to lyophilize the entire reaction mixture as a single microbead, with enzyme, and LAMP primers towards the detection of mitochondrial cytochrome B (Cyt B), a housekeeping gene in Esox lucius. Analytical and molecular techniques were performed to characterize and validate the lyophilized beads, respectively. The lyophilized beads were stored at two different temperatures, at 20 °C and 4 °C, and tested for biological activity after different time intervals. The result shows that lyophilized beads are bioactive for almost 30 days when stored at 20 °C, while beads at 4 °C did not lose their bioactivity after storage for up to one year. This study will be particularly useful for conducting on-site LAMP analyses in the field, where resources for freezing and storage are limited.
Whole national screening of pre-cancer is done only in some few countries, dominated by The Netherlands, Denmark, UK, Norway and Finland. These national screenings are done combining national cancer ...registry, national public health and national medical bodies or hospitals. Until some few years ago national screening was only done using morphological or visual methods or technology. Today a number of molecular methods have been implemented to serve these national screening programs. Based on all the discussions within this review, it is clear that the main driving engine and the cause of cervical pre-cancer and the main cause of invasive cervical cancer is the expression of E6 and E7 oncoproteins from HPV 16, 18, 31, 33 and 45. However, the main challenge is the role of morphology or imaging-based diagnosis in the original definition of pre-cancer disease. This definition is not based on the cause of cervical precancer but based on a complex, subjective, morphological observations. The difference between these two definitions are discussed in this review. The unique discovery done while validating the first standardized detection technology used against mRNA, confirmed that the presence of both abnormal E6 and/or E7 mRNA and protein is the cause of cervical pre-cancer or severe neoplasia and the main cause of invasive cervical cancer. This confirmation was evident even though all these studies were disturbed by the above defined biases from morphology or imaging-based diagnosis. The use of the screening target that cause stable and high expression of the most carcinogenic compounds ever discovered, must cause a more accurate screening program. A number of studies have proved that the detection of E6/E7 mRNA followed-up by indirect or direct treatment in a well-organized national screening program, would reduce the incidence of cervical cancer. This review discusses the main studies involved in the scientific, clinical evaluation and how this unique technology could be used as a new medical gold standard for national screening of cervical pre-cancer.
Black silicon has become a versatile substrate for photoelectric and photothermic application due to its excellent light trapping ability. However, subsequent material engineering can conflict with ...its intrinsic anti-reflective behavior. In this paper, we present a two-step etching approach to create novel black silicon combining ordered micropores and disordered nanopores. The larger pores increase the optical path length and provide space to load functional materials, while the smaller ones facilitate coupling between the incident solar radiation and material. The proposed etching method increases the light-trapping ability of conventional nanostructured black silicon and achieves enhanced absorption in a broad spectrum. Besides, loading Au nanoparticles further improve the light absorbance in the near-infrared range of 1100–1700 nm. The intrinsic SiOx-rich layer on silicon surface produced during SF6/O2 plasma etching leads to photothermic conversion being the dominant dissipation route for the light energy, thereby yielding outstanding performance in photothermic and photo-thermal-electric demonstrations. This work establishes a robust relationship between three-dimensional structure and light-trapping property of black silicon and provides insights into substrate designs that facilitate the light conversion.
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•Novel black silicon with ordered micropores and disordered nanopores is developed through a two-step etching process.•Light-trapping ability is enhanced in the wavelength range of 220–1700 nm.•The boosted photothermic, and photo-thermal-electric conversion proves that the two-step etching strategy works.
Highlights • The SAR structures result in the reduction of the diffusion distance of two fluids. • The mixing strength is increased when one stream is injected into the other. • Fluids flow faster in ...SAR mixer for rapid mixing and mixing efficiency.
The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The ...automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n=28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.
•Novel design of shifted trapezoidal blades (STB) micromixer.•High mixing efficiency (>80%) for all Reynolds number in the range 0.5≤Re≤100.•Highest efficiency (∼95%), with a pressure drop of ∼32kPa, ...achieved when Re=40.•Easy-to-fabricate using only one photolithography step and small-footprint.
In this paper, a novel micromixer design, called the shifted trapezoidal blades (STB) micromixer, has been designed and fabricated to provide high mixing efficiency even at low Reynolds number (Re) based on the combination of several mixing principles, including vortices, transversal flows and chaotic advection. Although the STB micromixer has 3D geometrical structure, it can be easily fabricated by one-step photolithography technique, using only one mask and two inclined exposures and an aluminum base with 15° inclines. We conducted intensive numerical study to evaluate the performance of the proposed STB micromixer using COMSOL Multiphysics package with a wide range of Reynolds number from 0.5 to 100. We have fabricated STB micromixers for testing and verification. Both experiment and simulation results demonstrated that the STB micromixer had stable mixing efficiency of 80% or above for Reynolds number values in the range from 0.5 to 100. The most effective mixing performance was achieved at Re=40 in which the STB micromixer had the highest mixing efficiency value (95%) and a moderate pressure drop ΔP=30.27kPa. The proposed STB micromixer provided better mixing performance and smaller footprint compared to the previous micromixers presented in literatures. With a high mixing efficiency and the advantage of being easy to fabricate, the STB micromixer can be utilized in various microfluidic, point-of-care, point-of-need, central automatic diagnosis, and pre-treatment systems including sensor control systems.
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