Статті в журналах: "Direct nucleic acid detection"

1

Zezza, Paola, María Isabel Lucío, Estrella Fernández, Ángel Maquieira, and María-José Bañuls. "Surface Micro-Patterned Biofunctionalized Hydrogel for Direct Nucleic Acid Hybridization Detection." Biosensors 13, no. 3 (February 23, 2023): 312. http://dx.doi.org/10.3390/bios13030312.

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The present research is focused on the development of a biofunctionalized hydrogel with a surface diffractive micropattern as a label-free biosensing platform. The biosensors described in this paper were fabricated with a holographic recording of polyethylene terephthalate (PET) surface micro-structures, which were then transferred into a hydrogel material. Acrylamide-based hydrogels were obtained with free radical polymerization, and propargyl acrylate was added as a comonomer, which allowed for covalent immobilization of thiolated oligonucleotide probes into the hydrogel network, via thiol-yne photoclick chemistry. The comonomer was shown to significantly contribute to the immobilization of the probes based on fluorescence imaging. Two different immobilization approaches were demonstrated: during or after hydrogel synthesis. The second approach showed better loading capacity of the bioreceptor groups. Diffraction efficiency measurements of hydrogel gratings at 532 nm showed a selective response reaching a limit of detection in the complementary DNA strand of 2.47 µM. The label-free biosensor as designed could significantly contribute to direct and accurate analysis in medical diagnosis as it is cheap, easy to fabricate, and works without the need for further reagents.

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Ouyang, Wei, and Jongyoon Han. "Universal amplification-free molecular diagnostics by billion-fold hierarchical nanofluidic concentration." Proceedings of the National Academy of Sciences 116, no. 33 (July 29, 2019): 16240–49. http://dx.doi.org/10.1073/pnas.1904513116.

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Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis.

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Iwanaga, Masanobu. "High-Sensitivity High-Throughput Detection of Nucleic Acid Targets on Metasurface Fluorescence Biosensors." Biosensors 11, no. 2 (January 27, 2021): 33. http://dx.doi.org/10.3390/bios11020033.

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Worldwide infection disease due to SARS-CoV-2 is tremendously affecting our daily lives. High-throughput detection methods for nucleic acids are emergently desired. Here, we show high-sensitivity and high-throughput metasurface fluorescence biosensors that are applicable for nucleic acid targets. The all-dielectric metasurface biosensors comprise silicon-on-insulator nanorod array and have prominent electromagnetic resonances enhancing fluorescence emission. For proof-of-concept experiment on the metasurface biosensors, we have conducted fluorescence detection of single-strand oligoDNAs, which model the partial sequences of SARS-CoV-2 RNA indicated by national infection institutes, and succeeded in the high-throughput detection at low concentrations on the order of 100 amol/mL without any amplification technique. As a direct detection method, the metasurface fluorescence biosensors exhibit high performance.

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Knight, Ivor T., Jocelyne DiRuggiero, and Rita R. Colwell. "Direct Detection of Enteropathogenic Bacteria in Estuarine Water Using Nucleic Acid Probes." Water Science and Technology 24, no. 2 (July 1, 1991): 261–66. http://dx.doi.org/10.2166/wst.1991.0070.

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Direct detection and enumeration of pathogenic bacteria, rather than indicator organisms, in aquatic environments is desirable but hindered by the difficulties of culturing and identifying specific pathogens from these environments. We have developed a method for concentrating bacteria from water samples and extracting their DNA and RNA for use as targets for pathogen-specific gene probes. The method has been used to detect and enumerate Salmonella spp. in estuarine water samples. The probe binds Salmonella DNA quantitatively, making it possible to estimate relative amounts of target in each sample. Salmonella spp. were detected in samples which yielded no Salmonella spp. using culturing. Since the probe method does not require culturing the target organism, both culturable and non-culturable forms are detected. We have also used polymerase chain reaction to amplify a region of the enterotoxin gene in enterotoxigenic Escherichiacoli and Vibriocholerae (ltx and ctx, respectively). The amplified products are then identified with ctx and ltx probes, making specific, highly sensitive detection possible.

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5

Kricka, Larry J., and Paolo Fortina. "Analytical Ancestry: “Firsts” in Fluorescent Labeling of Nucleosides, Nucleotides, and Nucleic Acids." Clinical Chemistry 55, no. 4 (April 1, 2009): 670–83. http://dx.doi.org/10.1373/clinchem.2008.116152.

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Abstract Background: The inherent fluorescent properties of nucleosides, nucleotides, and nucleic acids are limited, and thus the need has arisen for fluorescent labeling of these molecules for a variety of analytical applications. Content: This review traces the analytical ancestry of fluorescent labeling of nucleosides, nucleotides, and nucleic acids, with an emphasis on the first to publish or patent. The scope of labeling includes (a) direct labeling by covalent labeling of nucleic acids with a fluorescent label or noncovalent binding or intercalation of a fluorescent dye to nucleic acids and (b) indirect labeling via covalent attachment of a secondary label to a nucleic acid, and then binding this to a fluorescently labeled ligand binder. An alternative indirect strategy involves binding of a nucleic acid to a nucleic acid binder molecule (e.g., antibody, antibiotic, histone, antibody, nuclease) that is labeled with a fluorophore. Fluorescent labels for nucleic acids include organic fluorescent dyes, metal chelates, carbon nanotubes, quantum dots, gold particles, and fluorescent minerals. Summary: Fluorescently labeled nucleosides, nucleotides, and nucleic acids are important types of reagents for biological assay methods and underpin current methods of chromosome analysis, gel staining, DNA sequencing and quantitative PCR. Although these methods use predominantly organic fluorophores, new types of particulate fluorophores in the form of nanoparticles, nanorods, and nanotubes may provide the basis of a new generation of fluorescent labels and nucleic acid detection methods.

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6

Uno, Takeshi, Toshihito Ohtake, Hitoshi Tabata, and Tomoji Kawai. "Direct Deoxyribonucleic Acid Detection Using Ion-Sensitive Field-Effect Transistors Based on Peptide Nucleic Acid." Japanese Journal of Applied Physics 43, No. 12B (November 19, 2004): L1584—L1587. http://dx.doi.org/10.1143/jjap.43.l1584.

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7

Faron, Matthew L., Nathan A. Ledeboer, Jessica Connolly, Paul A. Granato, Brenda R. Alkins, Jennifer Dien Bard, Judy A. Daly, Stephen Young, and Blake W. Buchan. "Clinical Evaluation and Cost Analysis of Great Basin Shiga Toxin Direct Molecular Assay for Detection of Shiga Toxin-Producing Escherichia coli in Diarrheal Stool Specimens." Journal of Clinical Microbiology 55, no. 2 (December 7, 2016): 519–25. http://dx.doi.org/10.1128/jcm.01939-16.

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ABSTRACTThe Shiga Toxin Direct molecular assay (ST Direct) relies on nucleic acid amplification and solid array-based amplicon detection to identify Shiga toxin-producingEscherichia coli(STEC) in preserved stool specimens. Genes encoding Shiga toxin (stx1andstx2), as well as theE. coliserotype O:157-specific markerrfbE, are simultaneously detected within 2 h. ST Direct was evaluated using 1,084 prospectively collected preserved stool specimens across five clinical centers. An additional 55 retrospectively collected, frozen specimens were included to increase the number of positive specimens evaluated. Results were compared to results from routine culture and an enzyme immunoassay (EIA) specific for the recovery and identification of STEC. ST Direct was found to be 93.2% sensitive and 99.3% specific for detection ofstx1andstx2and 95.7% sensitive and 99.3% specific for detection ofE. coliserotype O:157. All specimens with false-positive results were found to containstx1orstx2or were found to be positive for serotype O:157 when analyzed using alternative molecular methods. All 4 false-negativestx1orstx2results were reported for frozen, retrospectively tested specimens. In all cases, the specimens tested positive forstxby an alternative FDA-cleared nucleic acid amplification test (NAAT) but were negative forstx1andstx2following nucleic acid sequence analysis. Based on these data, culture and EIA-based methods for detection of STEC are only 33% sensitive compared to molecular tests. A retrospective cost analysis demonstrated 59% of the cost of routine stool culture to be attributable to the identification of STEC. Taken together, these data suggest that ST Direct may provide a cost-effective, rapid molecular alternative to routine culture for the identification of STEC in preserved stool specimens.

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8

Ji, Minghui, Yun Xia, Jacky Fong-Chuen Loo, Lang Li, Ho-Pui Ho, Jianan He, and Dayong Gu. "Automated multiplex nucleic acid tests for rapid detection of SARS-CoV-2, influenza A and B infection with direct reverse-transcription quantitative PCR (dirRT-qPCR) assay in a centrifugal microfluidic platform." RSC Advances 10, no. 56 (2020): 34088–98. http://dx.doi.org/10.1039/d0ra04507a.

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9

Baron, Ellen Jo, Fred C. Tenover, and Devasena Gnanashanmugam. "Direct Detection of Mycobacterium tuberculosis in Clinical Specimens Using Nucleic Acid Amplification Tests." Clinical Microbiology Newsletter 40, no. 13 (July 2018): 107–12. http://dx.doi.org/10.1016/j.clinmicnews.2018.06.003.

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10

Zhou, Yunying, Fengyan Pei, Mingyu Ji, Li Wang, Huailong Zhao, Huanjie Li, Weihua Yang, Qingxi Wang, Qianqian Zhao, and Yunshan Wang. "Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative." PLOS ONE 15, no. 11 (November 18, 2020): e0241469. http://dx.doi.org/10.1371/journal.pone.0241469.

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The early detection and differential diagnosis of respiratory infections increase the chances for successful control of COVID-19 disease. The nucleic acid RT-PCR test is regarded as the current standard for molecular diagnosis. However, the maximal specificity confirmation target ORF1ab gene is considered to be less sensitive than other targets in clinical application. In addition, recent evidence indicated that the initial missed diagnosis of asymptomatic patients with SARS-CoV-2 and discharged patients with “re-examination positive” might be due to low viral load, and the ability of rapid mutation of SARS-CoV-2 also increases the rate of false-negative results. Moreover, the mixed sample nucleic acid detection is helpful in seeking out the early community transmission of SARS-CoV-2 rapidly, but the detection kit needs ultra-high detection sensitivity. Herein, the lowest detection concentration of different nucleic acid detection kits was evaluated and compared to provide direct evidence for the selection of kits for mixed sample detection or make recommendations for the selection of validation kit, which is of great significance for the prevention and control of the current epidemic and the discharge criteria of low viral load patients.

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Regnault, Béatrice, Thomas Bigot, Laurence Ma, Philippe Pérot, Sarah Temmam, and Marc Eloit. "Deep Impact of Random Amplification and Library Construction Methods on Viral Metagenomics Results." Viruses 13, no. 2 (February 7, 2021): 253. http://dx.doi.org/10.3390/v13020253.

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Clinical metagenomics is a broad-range agnostic detection method of pathogens, including novel microorganisms. A major limit is the low pathogen load compared to the high background of host nucleic acids. To overcome this issue, several solutions exist, such as applying a very high depth of sequencing, or performing a relative enrichment of viral genomes associated with capsids. At the end, the quantity of total nucleic acids is often below the concentrations recommended by the manufacturers of library kits, which necessitates to random amplify nucleic acids. Using a pool of 26 viruses representative of viral diversity, we observed a deep impact of the nature of sample (total nucleic acids versus RNA only), the reverse transcription, the random amplification and library construction method on virus recovery. We further optimized the two most promising methods and assessed their performance with fully characterized reference virus stocks. Good genome coverage and limit of detection lower than 100 or 1000 genome copies per mL of plasma, depending on the genome viral type, were obtained from a three million reads dataset. Our study reveals that optimized random amplification is a technique of choice when insufficient amounts of nucleic acid are available for direct libraries constructions.

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Guatelli, J. C., T. R. Gingeras, and D. D. Richman. "Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection." Clinical Microbiology Reviews 2, no. 2 (April 1989): 217–26. http://dx.doi.org/10.1128/cmr.2.2.217.

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The enzymatic amplification of specific nucleic acid sequences in vitro has revolutionized the use of nucleic acid hybridization assays for viral detection. With this method, the copy number of a pathogen-specific sequence is increased several orders of magnitude before detection is attempted. The sensitivity and specificity of detection are thus markedly improved. Mullis and Faloona devised the first method of sequence amplification in vitro, the polymerase chain reaction (K.B. Mullis and F.A. Faloona, Methods Enzymol. 155:355-350, 1987). By this method, synthetic oligonucleotide primers direct repeated, target-specific, deoxyribonucleic acid-synthetic reactions, resulting in an exponential increase in the amount of the specific target sequence. The application of sequence amplification to viral detection was initially performed with human immunodeficiency virus type 1 and human T-cell lymphoma virus type I. In principle, however, this approach can be applied to the detection of any deoxyribonucleic or ribonucleic acid virus; the only requirement is that sufficient nucleotide sequence data exist to allow the synthesis of target-specific oligonucleotide primers. The use of target amplification in vitro will permit a variety of studies of viral pathogenesis which have not been feasible because of the low copy number of the viral nucleic acids in infected material. This approach is particularly applicable to the study of human retroviral infections, which are chronic and persistent and are characterized by low titers of virus in tissues. In addition, target amplification in vitro will facilitate the development of new methods of sequence detection, which will be useful for rapid viral diagnosis in the clinical laboratory.

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13

Ostroff, Rachel M., Deborah Hopkins, Ayla B. Haeberli, Wahab Baouchi, and Barry Polisky. "Thin Film Biosensor for Rapid Visual Detection of Nucleic Acid Targets." Clinical Chemistry 45, no. 9 (September 1, 1999): 1659–64. http://dx.doi.org/10.1093/clinchem/45.9.1659.

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Abstract Background: We have developed a silicon-based biosensor that generates a visual signal in response to nucleic acid targets. Methods: In this system, capture oligonucleotide probes are immobilized on the surface of the biosensor. Interaction of the capture probes with a complementary target and a biotinylated detector oligonucleotide allows initiation of formation of an organic thin film on the biosensor. Thin film formation is completed by enzymatic activity of peroxidase conjugated to an anti-biotin antibody. Peroxidase catalyzes deposition of an insoluble product onto the silicon surface, generating a uniform thin film. The increased thickness on the surface alters the perceived color of the biosensor through changes in the interference patterns of reflected light from the surface, causing a color change from gold to purple. Results: The biosensor results may be evaluated by direct visual inspection or quantified by ellipsometry. Results are obtained in 25 min with a detection limit of 5 pmol/L (150 amol/sample). Selectivity of the biosensor is demonstrated by discrimination of single nucleotide mismatches. Multitarget arrays are also analyzed with the thin film biosensor, and the system is capable of detecting targets from human serum and urine. Conclusions: The biosensor surface is inexpensive to produce, and the assay format is simple and rapid. The thin film biosensor is adaptable to a wide variety of nucleic acid detection applications, including rapid diagnostic testing for infectious disease panels, antibiotic resistance panels, or allelic discrimination of specific genetic markers.

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Kabza, Adam M., and Jonathan T. Sczepanski. "l-DNA-Based Catalytic Hairpin Assembly Circuit." Molecules 25, no. 4 (February 20, 2020): 947. http://dx.doi.org/10.3390/molecules25040947.

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Isothermal, enzyme-free amplification methods based on DNA strand-displacement reactions show great promise for applications in biosensing and disease diagnostics but operating such systems within biological environments remains extremely challenging due to the susceptibility of DNA to nuclease degradation. Here, we report a catalytic hairpin assembly (CHA) circuit constructed from nuclease-resistant l-DNA that is capable of unimpeded signal amplification in the presence of 10% fetal bovine serum (FBS). The superior biostability of the l-DNA CHA circuit relative to its native d-DNA counterpart was clearly demonstrated through a direct comparison of the two systems (d versus l) under various conditions. Importantly, we show that the l-CHA circuit can be sequence-specifically interfaced with an endogenous d-nucleic acid biomarker via an achiral peptide nucleic acid (PNA) intermediary, enabling catalytic detection of the target in FBS. Overall, this work establishes a blueprint for the detection of low-abundance nucleic acids in harsh biological environments and provides further impetus for the construction of DNA nanotechnology using l-oligonucleotides.

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Lamture, Jagannath B., Kenneth LBeattie, Barry E. Burke, Mitchell D. Eggers, Dan J. Ehrlich, Rick Fowler, Mark A. Hollis, et al. "Direct detection of nucleic acid hybridization on the surface of a charge coupled device." Nucleic Acids Research 22, no. 11 (1994): 2121–25. http://dx.doi.org/10.1093/nar/22.11.2121.

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Roth, Johanna M., Laura de Bes, Patrick Sawa, George Omweri, Victor Osoti, Boris Oberheitmann, Henk D. F. H. Schallig, and Pètra F. Mens. "Plasmodium Detection and Differentiation by Direct-on-Blood PCR Nucleic Acid Lateral Flow Immunoassay." Journal of Molecular Diagnostics 20, no. 1 (January 2018): 78–86. http://dx.doi.org/10.1016/j.jmoldx.2017.09.004.

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Kuralay, Filiz, Susana Campuzano, David A. Haake, and Joseph Wang. "Highly sensitive disposable nucleic acid biosensors for direct bioelectronic detection in raw biological samples." Talanta 85, no. 3 (September 2011): 1330–37. http://dx.doi.org/10.1016/j.talanta.2011.06.012.

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Syed, Zia. "Ultra-Low Detection of Infectious Disease Nucleic Acid Biomarkers By a Visual Colorimetric Sensor." ECS Meeting Abstracts MA2022-02, no. 51 (October 9, 2022): 1988. http://dx.doi.org/10.1149/ma2022-02511988mtgabs.

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The gold standard method for detecting nucleic acid markers is the polymerase chain reaction. However, this method demands high technical skills, labor-intensive, time-consuming, and expensive. Many resource-limited countries around the globe do not have access to such laboratory-based PCR tests. To address the global test needs of virus and other pathogen infections, as well as address the early biomarker detection of diseases. We have developed an affordable nanoparticle colorimetric sensor approach. In our system, the virus RNA marker is captured by the complementary oligonucleotide attached to low fouling gold-coated iron oxide nanoparticles. Further specificity is achieved through a second complementary hybridization probe conjugated with streptavidin-horseradish peroxidase labels. Visual colorimetry is performed using a chromogenic substrate allowing the visual conversion of the colorless solution to a blue color confirming a positive test. The intensity of the color is in direct correlation to the concentration of the virus RNA marker. We have successfully demonstrated attomolar detection in buffer and picomolar detection in real undiluted biofluids such as in neat human serum. Our ongoing efforts focus on saliva-based non-invasive detection with ultra-low capabilities, optimization of the assay, repeatability, and expanding this work for a multiplex detection format

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19

Ho, Hoang A., Kim Doré,, Maurice Boissinot, Michel G. Bergeron, Robert M. Tanguay, Denis Boudreau, and Mario Leclerc. "Direct Molecular Detection of Nucleic Acids by Fluorescence Signal Amplification." Journal of the American Chemical Society 127, no. 36 (September 2005): 12673–76. http://dx.doi.org/10.1021/ja053417j.

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20

Sawata, Shinya, Eriko Kai, Kazunori Ikebukuro, Tetsuya Iida, Takeshi Honda, and Isao Karube. "Application of peptide nucleic acid to the direct detection of deoxyribonucleic acid amplified by polymerase chain reaction." Biosensors and Bioelectronics 14, no. 4 (April 1999): 397–404. http://dx.doi.org/10.1016/s0956-5663(99)00018-4.

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21

Schroeder, Megan E., Mangkey A. Bounpheng, Sandy Rodgers, Rocky J. Baker, Wendy Black, Hemant Naikare, Binu Velayudhan, Loyd Sneed, Barbara Szonyi, and Alfonso Clavijo. "Development and performance evaluation of calf diarrhea pathogen nucleic acid purification and detection workflow." Journal of Veterinary Diagnostic Investigation 24, no. 5 (August 8, 2012): 945–53. http://dx.doi.org/10.1177/1040638712456976.

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Calf diarrhea (scours) is a primary cause of illness and death in young calves. Significant economic losses associated with this disease include morbidity, mortality, and direct cost of treatment. Multiple pathogens are responsible for infectious diarrhea, including, but not limited to, Bovine coronavirus (BCV), bovine Rotavirus A (BRV), and Cryptosporidium spp. Identification and isolation of carrier calves are essential for disease management. Texas Veterinary Medical Diagnostic Laboratory current methods for calf diarrhea pathogen identification include electron microscopy (EM) for BCV and BRV and a direct fluorescent antibody test (DFAT) for organism detection of Cryptosporidium spp. A workflow was developed consisting of an optimized fecal nucleic acid purification and multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) for single tube concurrent detection of BCV, BRV, and Cryptosporidium spp., and an internal control to monitor nucleic acid purification efficacy and PCR reagent functionality. In “spike-in” experiments using serial dilutions of each pathogen, the analytical sensitivity was determined to be <10 TCID50/ml for BCV and BRV, and <20 oocysts for Cryptosporidium spp. Analytical specificity was confirmed using Canine and Feline coronavirus, Giardia spp., and noninfected bovine purified nucleic acid. Diagnostic sensitivity was ≥98% for all pathogens when compared with respective traditional methods. The results demonstrate that the newly developed assay can purify and subsequently detect BCV, BRV, and Cryptosporidium spp. concurrently in a single PCR, enabling simplified and streamlined calf diarrhea pathogen identification.

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Tomanovic, Snezana, and Slobodanka Djukic. "Classical and molecular methods for diagnosis of Chlamydia trachomatis infections." Medical review 64, no. 9-10 (2011): 477–80. http://dx.doi.org/10.2298/mpns1110477t.

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Introduction. Genital Chlamydia trachomatis infection is the leading cause of bacterial sexually transmitted diseases in industrial countries, particularly among young people. The consequences of chlamydial infections may involve pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility. Methods. Available tests for detection of chlamydia in men and women include culture in tissue culture cells, direct immunofluorescence test, enzyme immune assay, nucelic acid probe hibridization and polymerase chain reaction. Nucleic acid amplification tests use different ribonucleic and deoxyribonucleic acid regions as target molecules for amplifying Chlamydia trachomatis ribonucleic/deoxyribonucleic acid in clinical samples. Nucleic acid amplification tests are more sensitive than non-nucleic acid amplification tests. Conclusion. Although screening programmes exist in a number of countries, the continuously increasing prevalence of chlamydial infections demonstrates the necessity for defining the best method for the diagnosis and the population for screening.

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Sridapan, Thanawat, and Theerapong Krajaejun. "Nucleic Acid-Based Detection of Pythium insidiosum: A Systematic Review." Journal of Fungi 9, no. 1 (December 23, 2022): 27. http://dx.doi.org/10.3390/jof9010027.

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Pythiosis, a life-threatening infectious condition caused by Pythium insidiosum, has been increasingly reported in humans and animals worldwide. Antifungal drugs usually fail to control the pathogen. The surgical removal of an infected organ is the treatment of choice. Many affected patients die due to advanced infection. A timely and accurate diagnosis could lead to a better prognosis in pythiosis patients and save their lives. Although a standard culture method is available in microbiological laboratories, it is time-consuming, laborious, and insensitive for P. insidiosum identification. Immunological assays have been developed to improve the diagnosis of pythiosis. However, immunological methods are commercially unavailable and primarily detect anti-P. insidiosum antibodies, which constitute indirect evidence of pythiosis, making it challenging to differentiate a past from a recent infection. Moreover, such immunological tests cannot diagnose patients with a local infection, such as in the eye. Nucleic acid-based tests (NATs) are efficient for the direct and rapid detection of P. insidiosum DNA in trace-amount or culture-negative specimens. The reagents and equipment required for NATs are usually available in molecular diagnostic laboratories. Herein, we provide a systematic review to comprehensively present the principal and clinical usages, advantages, and limitations of such NATs in the detection of P. insidiosum. Various NATs have been established to detect P. insidiosum, which can be classified into amplification-based (i.e., PCR assays, isothermal tests, and next-generation sequencing methods) and non-amplification-based (i.e., DNA hybridization) techniques. This concise review on NATs constitutes an up-to-date reference with which healthcare professionals can learn about and decide upon which detection method is suitable for their respective laboratory environments.

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Courtney, Samantha J., Zachary R. Stromberg, and Jessica Z. Kubicek-Sutherland. "Nucleic Acid-Based Sensing Techniques for Diagnostics and Surveillance of Influenza." Biosensors 11, no. 2 (February 12, 2021): 47. http://dx.doi.org/10.3390/bios11020047.

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Influenza virus poses a threat to global health by causing seasonal outbreaks as well as three pandemics in the 20th century. In humans, disease is primarily caused by influenza A and B viruses, while influenza C virus causes mild disease mostly in children. Influenza D is an emerging virus found in cattle and pigs. To mitigate the morbidity and mortality associated with influenza, rapid and accurate diagnostic tests need to be deployed. However, the high genetic diversity displayed by influenza viruses presents a challenge to the development of a robust diagnostic test. Nucleic acid-based tests are more accurate than rapid antigen tests for influenza and are therefore better candidates to be used in both diagnostic and surveillance applications. Here, we review various nucleic acid-based techniques that have been applied towards the detection of influenza viruses in order to evaluate their utility as both diagnostic and surveillance tools. We discuss both traditional as well as novel methods to detect influenza viruses by covering techniques that require nucleic acid amplification or direct detection of viral RNA as well as comparing advantages and limitations for each method. There has been substantial progress in the development of nucleic acid-based sensing techniques for the detection of influenza virus. However, there is still an urgent need for a rapid and reliable influenza diagnostic test that can be used at point-of-care in order to enhance responsiveness to both seasonal and pandemic influenza outbreaks.

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Tanke, H. J., J. Vrolijk, and A. K. Raap. "Image analysis of fish stained specimens: A new diagnostic tool in genetics and oncology." Proceedings, annual meeting, Electron Microscopy Society of America 52 (1994): 78–79. http://dx.doi.org/10.1017/s0424820100168128.

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In situ hybridization allows the detection of specific nucleic acid sequences in morphologically intact cells and chromosomes. Presently, fluorescence in situ hybridization (FISH) has reached a high detection sensitivity (defined as the smallest DNA target detectable, high DNA resolution (defined as the smallest distance in kilobasepairs between two DNA targets that can be resolved microscopically and a high multiplicity (defined as the number of different probes that can be identified simultaneously. The current status of FISH methodology:*several direct and indirect nucleic acid modifications*sensitivity: unique DNA sequences 1-5 kb*DNA resolution: metaphase ~ > 1-3 Mbp (light microscopy) interphase ~50 kbp DNA halos ~ 1 kbp*multiplicity: at least 12Progress in in situ hybridization and related technology has caused molecular cytogenetics to become an established. In particular the ability of ISH techniques to detect chromosomes and/or chromosome parts in interphase nuclei (interphase cytogenetics) has significantly contributed to this acceptance of ISH, since this technique provides statistically reliable means to study the genetic composition of cells that can not, or only by complicated techniques, be brought in mitosis.

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Mahony, James B. "Detection of Respiratory Viruses by Molecular Methods." Clinical Microbiology Reviews 21, no. 4 (October 2008): 716–47. http://dx.doi.org/10.1128/cmr.00037-07.

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SUMMARY Clinical laboratories historically diagnose seven or eight respiratory virus infections using a combination of techniques including enzyme immunoassay, direct fluorescent antibody staining, cell culture, and nucleic acid amplification tests. With the discovery of six new respiratory viruses since 2000, laboratories are faced with the challenge of detecting up to 19 different viruses that cause acute respiratory disease of both the upper and lower respiratory tracts. The application of nucleic acid amplification technology, particularly multiplex PCR coupled with fluidic or fixed microarrays, provides an important new approach for the detection of multiple respiratory viruses in a single test. These multiplex amplification tests provide a sensitive and comprehensive approach for the diagnosis of respiratory tract infections in individual hospitalized patients and the identification of the etiological agent in outbreaks of respiratory tract infection in the community. This review describes the molecular methods used to detect respiratory viruses and discusses the contribution that molecular testing, especially multiplex PCR, has made to our ability to detect respiratory viruses and to increase our understanding of the roles of various viral agents in acute respiratory disease.

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Tarim, E. Alperay, Betul Karakuzu, Cemre Oksuz, Oyku Sarigil, Melike Kizilkaya, Mahmoud Khatib A. A. Al-Ruweidi, Huseyin Cagatay Yalcin, Engin Ozcivici, and H. Cumhur Tekin. "Microfluidic-based virus detection methods for respiratory diseases." Emergent Materials 4, no. 1 (February 2021): 143–68. http://dx.doi.org/10.1007/s42247-021-00169-7.

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AbstractWith the recent SARS-CoV-2 outbreak, the importance of rapid and direct detection of respiratory disease viruses has been well recognized. The detection of these viruses with novel technologies is vital in timely prevention and treatment strategies for epidemics and pandemics. Respiratory viruses can be detected from saliva, swab samples, nasal fluid, and blood, and collected samples can be analyzed by various techniques. Conventional methods for virus detection are based on techniques relying on cell culture, antigen-antibody interactions, and nucleic acids. However, these methods require trained personnel as well as expensive equipment. Microfluidic technologies, on the other hand, are one of the most accurate and specific methods to directly detect respiratory tract viruses. During viral infections, the production of detectable amounts of relevant antibodies takes a few days to weeks, hampering the aim of prevention. Alternatively, nucleic acid–based methods can directly detect the virus-specific RNA or DNA region, even before the immune response. There are numerous methods to detect respiratory viruses, but direct detection techniques have higher specificity and sensitivity than other techniques. This review aims to summarize the methods and technologies developed for microfluidic-based direct detection of viruses that cause respiratory infection using different detection techniques. Microfluidics enables the use of minimal sample volumes and thereby leading to a time, cost, and labor effective operation. Microfluidic-based detection technologies provide affordable, portable, rapid, and sensitive analysis of intact virus or virus genetic material, which is very important in pandemic and epidemic events to control outbreaks with an effective diagnosis.

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Jacobsen, Carsten S., Julia R. De Lipthay, Mikkel Bender, Line Fredslund, Anders R. Johnsen, and Kaare Johnsen. "Direct analysis of microbial populations in soil and freshwater aquifers using nucleic acid based techniques." Geological Survey of Denmark and Greenland (GEUS) Bulletin 4 (July 20, 2004): 33–36. http://dx.doi.org/10.34194/geusb.v4.4777.

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The first DNA-based methods for direct quantification of soil protozoa, and a DNA-based quantification method to describe the spread of phenanthrene-degrading bacteria in soil and freshwater aquifers, have recently been developed at the BIOPRO Research Centre at the Geological Survey of Denmark and Greenland (GEUS). Well-known genes for phenoxyalcanoic acid degradation have been used to monitor the in situ degradation of phenoxyalcanoic acid pesticides. Studies have been initiated on the short-lived mRNA molecules that are expected to provide a shortcut to the understanding of low, yet important, microbial activity in geological samples. This article reviews recent developments in techniques based on analysis of nucleic acids from soils and aquifers. Analytical work has been carried out mainly on soil samples from a former asphalt production plant at Ringe (Fig. 1). The Ringe plant constitutes one of the most polluted industrial sites in Denmark, and is a priority site of studies by the BIOPRO Research Centre. Although rich in carbon, the Ringe subsoil is an oligotrophic environment due to the high content of polycyclic aromatic hydrocarbons (PAH). This is an environment where the supply of nutrients to microorganisms is low, leading to slow growth, low total numbers of microorganisms and small cells. To study microbial communities of oligotrophic environments, analytical methods with low detection limits are needed. Until recently, microorganisms of natural environments were mainly studied by cultivation-dependent methods. However, microorganisms that can be cultured on agar plates are now known to represent only a small fraction of the total microbial community. Modern methods, therefore, need to be based on the detection of biomolecules in the microorganisms rather than being dependent on growth of the microorganisms. The best available techniques are based on DNA and RNA molecules (Fig. 2), which due to their high level of resolution allow closely related organisms or functional genes to be distinguished. In the following review, examples are given of applications of these nucleic acid based methods.

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Preston, David R., G. Rasul Chaudhry, and Samuel R. Farrah. "Detection and identification of poliovirus in environmental samples using nucleic acid hybridization." Canadian Journal of Microbiology 36, no. 9 (September 1, 1990): 664–69. http://dx.doi.org/10.1139/m90-113.

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A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage [Formula: see text]. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples. Key words: nucleic acid hybridization, polio virus, water, dot blot.

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Sethi, Komal, Gabrielle P. Dailey, Osama K. Zahid, Ethan W. Taylor, Jan A. Ruzicka, and Adam R. Hall. "Direct Detection of Conserved Viral Sequences and Other Nucleic Acid Motifs with Solid-State Nanopores." ACS Nano 15, no. 5 (April 29, 2021): 8474–83. http://dx.doi.org/10.1021/acsnano.0c10887.

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Kong, Derong, Xuejun Wang, Chenjian Gu, Mingquan Guo, Yao Wang, Zhaolin Ai, Shen Zhang, et al. "Direct SARS-CoV-2 Nucleic Acid Detection by Y-Shaped DNA Dual-Probe Transistor Assay." Journal of the American Chemical Society 143, no. 41 (October 8, 2021): 17004–14. http://dx.doi.org/10.1021/jacs.1c06325.

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32

Jenison, Robert D., Richard Bucala, Diana Maul, and David C. Ward. "Thin-film technology for direct visual detection of nucleic acid sequences: applications in clinical research." Expert Review of Molecular Diagnostics 6, no. 1 (January 2006): 89–99. http://dx.doi.org/10.1586/14737159.6.1.89.

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Rodríguez López, Carlos M., Beatriz Guzmán Asenjo, Amanda J. Lloyd, and Mike J. Wilkinson. "Direct Detection and Quantification of Methylation in Nucleic Acid Sequences Using High-Resolution Melting Analysis." Analytical Chemistry 82, no. 21 (November 2010): 9100–9108. http://dx.doi.org/10.1021/ac1024057.

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Wang, S. X., and L. Tay. "Evaluation of Three Nucleic Acid Amplification Methods for Direct Detection of Mycobacterium tuberculosis Complex in Respiratory Specimens." Journal of Clinical Microbiology 37, no. 6 (1999): 1932–34. http://dx.doi.org/10.1128/jcm.37.6.1932-1934.1999.

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Two hundred thirty respiratory specimens from 230 patients were analyzed by using COBAS AMPLICOR PCR, Amplified Mycobacterium tuberculosis Direct Test, and ligase chain reaction methods. Results were compared with those of smear microscopy and radiometric culture (Bactec) methods. No significant differences were observed among the results of the three methods, which are acceptable for direct detection of M. tuberculosis complex in respiratory specimens.

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Toranzos, Gary A., and Abdiel J. Alvarez. "Solid-phase polymerase chain reaction: applications for direct detection of enteric pathogens in waters." Canadian Journal of Microbiology 38, no. 5 (May 1, 1992): 365–69. http://dx.doi.org/10.1139/m92-062.

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The techniques in current use for detection of pathogens in environmental samples are restricted to those organisms whose replication in either culture media or cell culture is feasible. These methods lack the selectivity and sensitivity necessary for their unequivocal detection and identification. We have developed an assay for the detection of bacterial cells in large volumes of water. Low concentrations of cells containing target sequences were concentrated on membrane filters and were subjected to amplification directly using a stepwise polymerase chain reaction. This procedure, together with nucleic acid probes, has enhanced the limit of detection to the level of a single bacterial cell. This technique could be used for the detection of any bacteria or virus in water or air. Key words: polymerase chain reaction, waterborne pathogens, water.

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Brunauer, Anna, René D. Verboket, Daniel M. Kainz, Felix von Stetten, and Susanna M. Früh. "Rapid Detection of Pathogens in Wound Exudate via Nucleic Acid Lateral Flow Immunoassay." Biosensors 11, no. 3 (March 6, 2021): 74. http://dx.doi.org/10.3390/bios11030074.

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The rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 105 CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy.

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Kher, Rajesh, and Robert Bacallao. "Direct in situ reverse transcriptase-polymerase chain reaction." American Journal of Physiology-Cell Physiology 281, no. 2 (August 1, 2001): C726—C732. http://dx.doi.org/10.1152/ajpcell.2001.281.2.c726.

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In situ hybridization has been used for localization of specific nucleic acid sequences at the cellular level despite providing relatively low-detection sensitivity. In situ reverse transcriptase-polymerase chain reactions (RT-PCR) enhance sensitivity and thus enable localization of low-abundance mRNA in a cell. However, the available methods are fraught with problems of nonspecific amplifications as a result of mispriming and/or amplification from partially digested residual genomic DNA in tissue. Herein, we demonstrate that nonspecific background amplification can be eliminated by pretreatment of samples with restriction enzymes before DNase I digestion. Primers tagged with a far-red shifted fluorescent dye such as Cy5 in PCR reactions allow identification of target mRNA by fluorescence microscopy. These novel modifications lead to increased specificity and rapid in situ detection of cellular mRNA and thus may be used for pathological diagnosis.

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Ling, Kai, Hongyan Jiang, Xue Huang, Yang Li, Juanjuan Lin, and Fu-Rong Li. "Direct chemiluminescence detection of circulating microRNAs in serum samples using a single-strand specific nuclease-distinguishing nucleic acid hybrid system." Chemical Communications 54, no. 15 (2018): 1909–12. http://dx.doi.org/10.1039/c7cc09087k.

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We developed a microplate-based enhanced chemiluminescence system for the direct detection of circulating miRNAs. The system exhibited a high target sensitivity and specificity, with a detection limit of 3.02 fM.

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39

Fourie, Nicolaas H., Sarah K. Abey, Eric Ferguson, Natnael Kenea, Ana F. Diallo, Chang-Hee Kim, and Wendy A. Henderson. "Direct Nucleic Acid Detection of Diarrheal Pathogens in Stool Using an Antibody-Free Lateral Flow Assay." Gastroenterology 152, no. 5 (April 2017): S817. http://dx.doi.org/10.1016/s0016-5085(17)32826-3.

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40

Hamilton, Jennifer R., Elizabeth C. Stahl, Connor A. Tsuchida, Enrique Lin-Shiao, C. Kimberly Tsui, Kathleen Pestal, Holly K. Gildea, et al. "Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples." PLOS ONE 16, no. 8 (August 5, 2021): e0255690. http://dx.doi.org/10.1371/journal.pone.0255690.

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Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

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Aliberti, A., A. M. Cusano, E. Battista, F. Causa, and P. A. Netti. "High sensitive and direct fluorescence detection of single viral DNA sequences by integration of double strand probes onto microgels particles." Analyst 141, no. 4 (2016): 1250–56. http://dx.doi.org/10.1039/c5an02001h.

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42

Hubbard, Roger A. "Human Papillomavirus Testing Methods." Archives of Pathology & Laboratory Medicine 127, no. 8 (August 1, 2003): 940–45. http://dx.doi.org/10.5858/2003-127-940-hptm.

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Abstract Testing for human papillomavirus (HPV) relies exclusively on techniques of molecular biology using nucleic acid probes. Tests for HPV using nucleic acid probes have been commercially available since the late 1980s, but early tests were cumbersome, involving the use of nucleic acid probes labeled with radioactive phosphorus (32P). These early HPV tests did not achieve widespread use because they did not detect all oncogenic HPV genotypes. The current commercial HPV detection kit, Digene's Hybrid Capture 2 kit, detects virtually all high-risk oncogenic HPV types, as well as most low-risk nononcogenic HPV genotypes. The Hybrid Capture 2 test format is a proprietary nucleic acid hybridization signal amplification system owned by Digene Corporation. Virtually all test formats for DNA sequence analysis are amenable to applications intended to detect and perhaps quantify the various HPV genotypes. These methods can involve direct hybridization with complementary DNA probes, such as Southern blotting or in situ hybridization, signal amplification, such as the Hybrid Capture 2 method or target nucleic acid amplification, most notably the polymerase chain reaction (PCR). Polymerase chain reaction has been used for HPV detection, genotyping, and viral load determination. General or consensus primer–mediated PCR assays have enabled screening for a broad spectrum of HPV types in clinical specimens using a single PCR reaction. Following amplification using consensus primers, individual HPV genotypes are identified using a variety of methods. Using consensus primers in a test format known as real-time quantitative PCR (RQ-PCR), it is possible to generate viral load (concentration) data from reaction curves generated by monitoring PCR reaction kinetics in real time.

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43

Price, Alexander M., Robert T. Steinbock, Chao Di, Katharina E. Hayer, Yize Li, Christin Herrmann, Nicholas A. Parenti, Jillian N. Whelan, Susan R. Weiss, and Matthew D. Weitzman. "Adenovirus prevents dsRNA formation by promoting efficient splicing of viral RNA." Nucleic Acids Research 50, no. 3 (October 21, 2021): 1201–20. http://dx.doi.org/10.1093/nar/gkab896.

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Abstract Eukaryotic cells recognize intracellular pathogens through pattern recognition receptors, including sensors of aberrant nucleic acid structures. Sensors of double-stranded RNA (dsRNA) are known to detect replication intermediates of RNA viruses. It has long been suggested that annealing of mRNA from symmetrical transcription of both top and bottom strands of DNA virus genomes can produce dsRNA during infection. Supporting this hypothesis, nearly all DNA viruses encode inhibitors of dsRNA-recognition pathways. However, direct evidence that DNA viruses produce dsRNA is lacking. Contrary to dogma, we show that the nuclear-replicating DNA virus adenovirus (AdV) does not produce detectable levels of dsRNA during infection. In contrast, abundant dsRNA is detected within the nucleus of cells infected with AdV mutants defective for viral RNA processing. In the presence of nuclear dsRNA, the cytoplasmic dsRNA sensor PKR is relocalized and activated within the nucleus. Accumulation of viral dsRNA occurs in the late phase of infection, when unspliced viral transcripts form intron/exon base pairs between top and bottom strand transcripts. We propose that DNA viruses actively limit dsRNA formation by promoting efficient splicing and mRNA processing, thus avoiding detection and restriction by host innate immune sensors of pathogenic nucleic acids.

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Wee, Soon Keong, Suppiah Paramalingam Sivalingam, and Eric Peng Huat Yap. "Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler." Genes 11, no. 6 (June 18, 2020): 664. http://dx.doi.org/10.3390/genes11060664.

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There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.

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Radwanska, M., S. Magez, H. Perry-O'Keefe, H. Stender, J. Coull, J. M. Sternberg, P. Buscher, and J. J. Hyldig-Nielsen. "Direct Detection and Identification of African Trypanosomes by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes." Journal of Clinical Microbiology 40, no. 11 (November 1, 2002): 4295–97. http://dx.doi.org/10.1128/jcm.40.11.4295-4297.2002.

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Holfelder, M., U. Eigner, A. M. Turnwald, W. Witte, M. Weizenegger, and A. Fahr. "Direct detection of methicillin-resistant Staphylococcus aureus in clinical specimens by a nucleic acid-based hybridisation assay." Clinical Microbiology and Infection 12, no. 12 (December 2006): 1163–67. http://dx.doi.org/10.1111/j.1469-0691.2006.01547.x.

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Uno, Takeshi, Hitoshi Tabata, and Tomoji Kawai. "Peptide−Nucleic Acid-Modified Ion-Sensitive Field-Effect Transistor-Based Biosensor for Direct Detection of DNA Hybridization." Analytical Chemistry 79, no. 1 (January 2007): 52–59. http://dx.doi.org/10.1021/ac060273y.

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Zhu, Lixuan, Zhihe Qing, Lina Hou, Sheng Yang, Zhen Zou, Zhong Cao, and Ronghua Yang. "Direct Detection of Nucleic Acid with Minimizing Background and Improving Sensitivity Based on a Conformation-Discriminating Indicator." ACS Sensors 2, no. 8 (August 8, 2017): 1198–204. http://dx.doi.org/10.1021/acssensors.7b00349.

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49

Lee, Jieon, Il-Soo Park, Euihan Jung, Younghoon Lee, and Dal-Hee Min. "Direct, sequence-specific detection of dsDNA based on peptide nucleic acid and graphene oxide without requiring denaturation." Biosensors and Bioelectronics 62 (December 2014): 140–44. http://dx.doi.org/10.1016/j.bios.2014.06.028.

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50

Zehnder, James, Reuel Van Atta, Carol Jones, Howard Sussman, and Michael Wood. "Cross-linking hybridization assay for direct detection of factor V Leiden mutation." Clinical Chemistry 43, no. 9 (September 1, 1997): 1703–8. http://dx.doi.org/10.1093/clinchem/43.9.1703.

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Abstract A nucleic acid photocross-linking technology was used in the development of a direct assay for factor V Leiden, a point mutation in the factor V gene (G1691A) that is the most common inherited risk factor for thrombosis. This cross-linking hybridization assay included two allele-specific capture probes and six signal-generating reporter probes; all were modified with a photoactivated cross-linking compound. By using two different capture probes complementary to a 16-base sequence at the factor V Leiden mutation site, but differing in the nucleotide opposite the mutation site (C vs T), wild-type and factor V Leiden alleles were differentiated in purified DNA specimens. The assay was also successfully applied to genomic DNA in leukocytes isolated from whole blood; the factor V status of 122 patients as determined by this method was in complete concordance with a standard PCR-based assay and clearly discriminated between healthy individuals and factor V Leiden heterozygotes.

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