Characterization and Validation of a Lyophilized Loop-Mediated Isothermal Amplification Method for the Detection of Esox lucius.

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.

Comparative evaluation of loop-mediated isothermal amplification and PCR for detection of Esox lucius housekeeping genes for use in on-site environmental monitoring.

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.

The potential of RNA as a target for national screening of pre-cancer.

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.

Unique human papillomavirus-type distribution in South African women with invasive cervical cancer and the effect of human immunodeficiency virus infection.

OBJECTIVES: Cervical cancer is the most common cause of cancer-related deaths among South African women. Viral types associated with cervical cancer may differ not only between countries and regions, but possibly also between human immunodeficiency virus (HIV)-infected and noninfected women. METHODS: In a population with high HIV prevalence, human papillomavirus (HPV)-type infections detected with DNA analyses were reported in a cohort of 299 women diagnosed with invasive cervical cancer. RESULTS: One hundred fifty-four women tested HIV negative, 77 tested HIV positive, and HIV status was unknown for 68 women. The mean age for HIV-positive women was 41.3 years, and that for HIV-negative women was 55.8 years (P < 0.001). Ninety-two percent of women tested HPV-DNA positive. Human papillomavirus types 16 and/or 18 were present in 62% of HIV-negative women and 65% of HIV-positive women. The 5 most common HPV types in HIV-positive women were, in decreasing frequency, HPV 16, 18, 45, 33, and 58. In HIV-negative women, the most common HPV types were HPV 16, 18, 35, and 45, followed by HPV 33 and 52. Human papillomavirus type 45 was more likely in the HIV positive compared with the HIV negative (odds ratio, 3.07; 95% confidence interval, 1.07-8.77). The HIV-positive women had more multiple high-risk HPV-type infections than did the HIV-negative women (27% vs 8%, P = 0.001). CONCLUSIONS: A high number of women in South Africa with cervical cancer are HIV positive. Without viral cross-protection, HPV vaccines should prevent around 65% of cervical cancers in this population. Human papillomavirus type 45 infection is significantly linked to HIV and important for future vaccine developments.

An efficient passive planar micromixer with ellipse-like micropillars for continuous mixing of human blood.

In this paper, a passive planar micromixer with ellipse-like micropillars is proposed to operate in the laminar flow regime for high mixing efficiency. With a splitting and recombination (SAR) concept, the diffusion distance of the fluids in a micromixer with ellipse-like micropillars was decreased. Thus, space usage for micromixer of an automatic sample collection system is also minimized. Numerical simulation was conducted to evaluate the performance of proposed micromixer by solving the governing Navier-Stokes equation and convection-diffusion equation. With software (COMSOL 4.3) for computational fluid dynamics (CFD) we simulated the mixing of fluids in a micromixer with ellipse-like micropillars and basic T-type mixer in a laminar flow regime. The efficiency of the proposed micromixer is shown in numerical results and is verified by measurement results.

Comparison of the sensitivities of three commercial assays for detection of the high risk HPV types 16, 18 and 45.

The relative and analytical sensitivity of the APTIMA HPV test (AHPV, broad-spectrum, target amplification) and the PreTect HPV-Proofer (type-specific, target amplification) for the detection of HPV mRNA in various cell lines was compared. Equivalent relative sensitivity for the HPV 16-containing cell lines (2.5 cells/ml with both CaSki and SiHa) was observed for the mRNA assays--and similar sensitivities were observed for the detection of HPV 18 (HeLa) and 45 (MS751); ranging from 2.5 cells/ml (Proofer) to 25 cells/ml (APTIMA). In relation to analytical sensitivity, again, the mRNA assays showed similar sensitivities to each other, ranging from 0.1 to 1 cell per reaction for APTIMA and 0.1 to 10 cells per reaction for PreTect HPV-Proofer (depending on cell line). Both mRNA assays consistently achieved a higher analytical sensitivity than a DNA based comparator--the Hybrid Capture 2 High-Risk HPV DNA test (hc2). This study indicates that mRNA tests had high analytical sensitivity, higher than a well established DNA-test based when using cell lines as target. Implications for clinical application are discussed.

Towards a "Sample-In, Answer-Out" Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System.

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.

Design and optimization of non-clogging counter-flow microconcentrator for enriching epidermoid cervical carcinoma cells.

Clogging failure is common for microfilters in living cells concentration; for instance, the CaSki Cell-lines (Epidermoid cervical carcinoma cells) utilizing the flat membrane structure. In order to avoid the clogging, counter-flow concentration units with turbine blade-like micropillar are proposed in microconcentrator design. Due to the unusual geometrical-profiles and extraordinary microfluidic performance, the cells blocking does not occur even at permeate entrances. A counter-flow microconcentrator was designed, with both processing layer and collecting layer arranged in terms of the fractal based honeycomb structure. The device was optimized by coupling Artificial Neuron Network (ANN) and Computational Fluid Dynamics (CFD). The excellent concentration ratio of a final microconcentrator was presented in numerical results.

Fully integrated micro-separator with soft-magnetic micro-pillar arrays for filtrating lymphocytes.

A fully integrated micro-separator with soft-magnetic micro-pillar arrays has been developed, which merely employs one independent Lab-On-Chip to realize the lymphocytes isolation from the human whole blood. The simulation, fabrication and experiment are executed to realize this novel microseparator. The simulation results show that, the soft-magnetic micro-pillars array can amplify and redistribute the electromagnetic field generated by the microcoils. The tests certify desirable separation efficiency can be realized using this new separator at low current. No extra cooling system is required for such a micro-separator. This micro-separator can also be used to separate other target cells or particles with the same principle.

E6/E7 mRNA expression analysis: a test for the objective assessment of cervical adenocarcinoma in clinical prognostic procedure.

Detection of E6/E7 mRNA expression using the real-time nucleic acid sequence-based amplification assay (NASBA) PreTect HPV-Proofer was compared with results of human papillomavirus (HPV) DNA detection in 98 paraffin-embedded samples from patients with cervical adenocarcinoma. HR-HPV DNA was detected in 61 (62%), while HR-HPV E6/E7 mRNA was detected in 63 (64%) of the samples. Correlation between results from DNA analyses and the E6/E7 mRNA assay showed consistent results in 87% of samples (47 of 54). The results from these two methods in detecting presence of HPV infection of any type agreed in 77%. Overall agreement between the methods was seen in 82 of the 98 cases (84%). When evaluating change in sensitivity for detection of HPV positives by adding more HPV types to the HPV DNA assay, maximum sensitivity was reached by targeting four HPV types. The coverage of HPV DNA presence was 76.9%, while the E6/E7 mRNA assay achieved a maximum coverage of 80.8% using only three HPV types. Thus, E6/E7 oncogene expression analysis may provide a more objective test for assessment of neoplastic glandular cells. Further studies may reveal whether the clinical performance of the E6/E7 mRNA assay will be of prognostic value in management of cervical adenocarcinoma.

Comparison of HPV detection technologies: Hybrid capture 2, PreTect HPV-Proofer and analysis of HPV DNA viral load in HPV16, HPV18 and HPV33 E6/E7 mRNA positive specimens.

Human papillomavirus (HPV) testing using molecular methods in liquid based cytology (LBC) specimens may be useful as an adjunct to cervical screening by cytology. We compared the positivity rate of the commercially available HPV DNA method hybrid capture 2 (hc2) and the commercially available E6/E7 mRNA method PreTect HPV-Proofer in cytological specimens (n=299). LBC specimens collected (n=299) represented the following cervical cytological disease categories: Normal (n=60), borderline nuclear abnormalities (BNA) (n=34), CIN1 (n=121), CIN2 (n=60), CIN3 (n=24). Overall, 69% (205/299) of the cases were positive by hc2 and 38% (112/299) of the cases were positive by PreTect HPV-Proofer. Concordance rates between the two tests were highest in the high-grade cytology cases (CIN2: 67% and CIN3: 83%) and the normal cytology cases (88%) and lowest in the BNA and CIN1 categories (56% and 52%). HPV DNA viral load analyses were carried out on HPV16 (n=55), HPV18 (n=9) and HPV33 (n=13) samples that were positive by PreTect HPV-Proofer. The sensitivity and specificity of PreTect HPV-Proofer and the hc2 DNA test for the detection of high-grade cytology (i.e. CIN2+) were 71.4% and 75.8% vs 100% and 43.7%, respectively. The relatively low detection rate observed by PreTect HPV-Proofer in the whole range of cytological positive cases, combined with a relatively higher specificity and PPV, suggests that PreTect HPV-Proofer may be more useful than hc2 for triage and in predicting high-grade disease.

PreTect HPV-Proofer: real-time detection and typing of E6/E7 mRNA from carcinogenic human papillomaviruses.

Monitoring human papillomavirus (HPV) E6/E7 mRNA expression may provide an accurate and informative diagnostic approach for detection of oncogene activity related to the development of severe dysplasia or cervical carcinoma. A multiplex nucleic acid sequence based amplification (NASBA) assay, utilizing molecular beacon probes for real-time detection was developed for the identification of E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45. The assay is called PreTect HPV-Proofer and this report describes the development and the analytical performance of the assay. The reproducibility of PreTect HPV-Proofer with regard to a positive result was found to be between 96 and 100%, depending on HPV type. The melting temperature for the different molecular beacons was in the range of 48-55 degrees C, indicating conformational stability, i.e. the molecular beacons will not get activated by the 41 degrees C annealing temperature, but will be activated by the annealing to the target itself. The limit of detection for HPV 16 was ten SiHa or CaSki cells and for HPV 18 one HeLa cell. No cross reactivity was observed with E6/E7 mRNA from the other tested HPV types. mRNA from cervical cells was also successfully amplified after more than one year of storage. In conclusion, the PreTect HPV-Proofer assay, individually identifying E6/E7 mRNA expression from five carcinogenic HPV types, is a reproducible assay that may serve as a valuable tool in monitoring HPV infections producing proteins with a transforming potential.

Microchips for the diagnosis of cervical cancer.

Cancer affects more people than any other disease. About one-third of the world's population is likely to get this diagnosis during their lifetime. Currently, the diagnostic methods for cancer detection are based on visual inspection. The lack of high analytical and clinical specificity and sensitivity makes these methods in many cases inferior to recently developed molecular methods. The increased clinical specificity and sensitivity of these new molecular approaches have great benefits, such as the possibility of implementing the molecular methods in miniaturized systems and enabling easier and faster point-of-care or bedside diagnostics. This chapter provides an introduction to performing clinical trials, screening, and molecular diagnostics against cancer-related markers. In addition, an example of molecular diagnosis of cervical cancer within a microsystem concept will be presented.

Expression of E6/E7 mRNA from 'high risk' human papillomavirus in relation to CIN grade, viral load and p16INK4a.

Detection of E6/E7 mRNA expression with real-time nucleic acid sequence-based amplification assay (NASBA) method (PreTect HPV-Proofer) from high-risk types of human papillomaviruses (HR-HPV) were compared with the presence of viral load, determined with quantitative real-time PCR in 80 cervical samples. Results regarding positivity and typing were in agreement using the two methods. However, there was no correlation between viral loads for HPV 16 or 18/45 and oncogene expression. Among 15 women with low grade atypia detected at a population-based cytology screening, and scored as 'within normal limits' according to histopathology, 14% were positive for oncogene expression, whereas 71% were HR-HPV positive. A correlation was observed between HR-HPV oncogene expression and high scores of p16(INK4a) positivity. Since HPV-Proofer detects full-length E6/E7 mRNA, a positive result should correlate with presence of integrated HPV, loss of HPV replication and stabilized E6/E7 full-length mRNA expression. Such expression from integrated HR-HPV generates a high and stable expression of full-length E6 proteins, which explains why a positive HPV-Proofer result was independent of viral load and correlate with high expression of p16(INK4a). Thus, E6/E7 oncogene expression analysis yielded information, which is consistent with and will complement the results from a real-time PCR method in a clinical prognostic procedure.

Presence of E6 and E7 mRNA from human papillomavirus types 16, 18, 31, 33, and 45 in the majority of cervical carcinomas.

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.

Parallel nanoliter detection of cancer markers using polymer microchips.

A general multipurpose microchip technology platform for point-of-care diagnostics has been developed. Real-time nucleic acid sequence-based amplification (NASBA) for detection of artificial human papilloma virus (HPV) 16 sequences and SiHa cell line samples was successfully performed in cyclic olefin copolymer (COC) microchips, incorporating supply channels and parallel reaction channels. Samples were distributed into 10 parallel reaction channels, and signals were simultaneously detected in 80 nl volumes. With a custom-made optical detection unit, the system reached a sensitivity limit of 10(-6) microM for artificial HPV 16 sequences, and 20 cells microl(-1) for the SiHa cell line. This is comparable to the detection limit of conventional readers, and clinical testing of biological samples in polymer microchips using NASBA is therefore possible.

Comparison of human papillomavirus messenger RNA and DNA detection: a cross-sectional study of 4,136 women >30 years of age with a 2-year follow-up of high-grade squamous intraepithelial lesion.

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.

Predicting CIN2+ when detecting HPV mRNA and DNA by PreTect HPV-proofer and consensus PCR: A 2-year follow-up of women with ASCUS or LSIL Pap smear.

It has been suggested that human papillomavirus (HPV) testing improves follow-up of atypical cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) in cervical cancer screening programs. To evaluate the prognostic value of including HPV testing as an adjunct to cytology, we carried out a 2-year follow-up study of 77 women with ASCUS or LSIL Papanicolaou (Pap) smear in the Norwegian Cervical Cancer Screening Program (NCCSP) for detection of histological cervical intraepithelial neoplasia (CIN) 2+. The study includes a comparison between viral mRNA and DNA detection. PreTect HPV-Proofer was used for HPV E6/E7 mRNA detection from the 5 high-risk types 16, 18, 31, 33 and 45, and Gp5+/6+ consensus PCR was used for HPV DNA detection. Twice as many women were positive for HPV DNA (54.6%) than for HPV mRNA (23.4%). PreTect HPV-Proofer and consensus PCR had a sensitivity of 85.7% (95% confidence interval [CI] = 42.1-99.6) for detecting CIN2+ during follow-up. The specificity was significantly higher for PreTect HPV-Proofer, 84.9% (95% CI = 73.9-92.5), than for consensus PCR, 50.0% (95% CI = 37.4-62.6). PreTect HPV-Proofer positive women were 69.8 times (95% CI = 4.3-1137.3) more likely to be diagnosed with CIN2+ within 2 years than PreTect HPV-Proofer negative women. Consensus PCR-positive women were 5.7 times (95% CI = 0.6-52.0) more likely to be diagnosed with CIN2+ within 2 years than PCR-negative women. With equal sensitivity and higher specificity than consensus PCR, the PreTect HPV-Proofer might offer an improvement for the triage of women with ASCUS or LSIL Pap smear.

Real-time nucleic acid sequence-based amplification in nanoliter volumes.

Real-time nucleic acid sequence-based amplification (NASBA) is an isothermal method specifically designed for amplification of RNA. Fluorescent molecular beacon probes enable real-time monitoring of the amplification process. Successful identification, utilizing the real-time NASBA technology, was performed on a microchip with oligonucleotides at a concentration of 1.0 and 0.1 microM, in 10- and 50-nL reaction chambers, respectively. The microchip was developed in a silicon-glass structure. An instrument providing thermal control and an optical detection system was built for amplification readout. Experimental results demonstrate distinct amplification processes. Miniaturized real-time NASBA in microchips makes high-throughput diagnostics of bacteria, viruses, and cancer markers possible, at reduced cost and without contamination.