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Статті в журналах з теми "Droplet Recombinase Polymerase Amplification"

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Автор: Grafiati

Опубліковано: 6 вересня 2023

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1

Schuler, Friedrich, Frank Schwemmer, Martin Trotter, Simon Wadle, Roland Zengerle, Felix von Stetten, and Nils Paust. "Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA." Lab on a Chip 15, no. 13 (2015): 2759–66. http://dx.doi.org/10.1039/c5lc00291e.

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2

Cui, Johnson Q., Frank X. Liu, Hojeong Park, Ka Wai Chan, Tyler Leung, Ben Zhong Tang, and Shuhuai Yao. "Droplet digital recombinase polymerase amplification (ddRPA) reaction unlocking via picoinjection." Biosensors and Bioelectronics 202 (April 2022): 114019. http://dx.doi.org/10.1016/j.bios.2022.114019.

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3

Ibekwe, Mark A., Shelton E. Murinda, Stanley Park, Amarachukwu Obayiuwana, Marcia A. Murry, Gregory Schwartz, and Trygve Lundquist. "Comparative Use of Quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), and Recombinase Polymerase Amplification (RPA) in the Detection of Shiga Toxin-Producing E. coli (STEC) in Environmental Samples." Water 12, no. 12 (December 13, 2020): 3507. http://dx.doi.org/10.3390/w12123507.

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E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was <10 CFU/mL, while ddPCR showed quantification from 1 to 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.

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4

Chowdhury, Rajashree, Prakash Ghosh, Md Anik Ashfaq Khan, Faria Hossain, Khaledul Faisal, Rupen Nath, James Baker, et al. "Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis." Tropical Medicine and Infectious Disease 5, no. 2 (June 5, 2020): 95. http://dx.doi.org/10.3390/tropicalmed5020095.

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To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

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5

Daher, Rana K., Gale Stewart, Maurice Boissinot, and Michel G. Bergeron. "Isothermal Recombinase Polymerase Amplification Assay Applied to the Detection of Group B Streptococci in Vaginal/Anal Samples." Clinical Chemistry 60, no. 4 (April 1, 2014): 660–66. http://dx.doi.org/10.1373/clinchem.2013.213504.

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Abstract BACKGROUND Group B streptococcal infections are the leading cause of sepsis and meningitis in newborns. A rapid and reliable method for the detection of this pathogen at the time of delivery is needed for the early treatment of neonates. Isothermal amplification techniques such as recombinase polymerase amplification have advantages relative to PCR in terms of the speed of reaction and simplicity. METHODS We studied the clinical performance of recombinase polymerase amplification for the screening of group B streptococci in vaginal/anal samples from 50 pregnant women. We also compared the limit of detection and the analytical specificity of this isothermal assay to real-time PCR (RT-PCR). RESULTS Compared to RT-PCR, the recombinase polymerase amplification assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The limit of detection was 98 genome copies and the analytical specificity was 100% for a panel of 15 bacterial and/or fungal strains naturally found in the vaginal/anal flora. Time-to-result for the recombinase polymerase amplification assay was <20 min compared to 45 min for the RT-PCR assay; a positive sample could be detected as early as 8 min. CONCLUSIONS We demonstrate the potential of isothermal recombinase polymerase amplification assay as a clinically useful molecular diagnostic tool that is simple and faster than PCR/RT-PCR. Recombinase polymerase amplification offers great potential for nucleic acid–based diagnostics at the point of care.

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6

Daher, Rana K., Gale Stewart, Maurice Boissinot, and Michel G. Bergeron. "Recombinase Polymerase Amplification for Diagnostic Applications." Clinical Chemistry 62, no. 7 (July 1, 2016): 947–58. http://dx.doi.org/10.1373/clinchem.2015.245829.

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Abstract BACKGROUND First introduced in 2006, recombinase polymerase amplification (RPA) has stirred great interest, as evidenced by 75 publications as of October 2015, with 56 of them just in the last 2 years. The widespread adoption of this isothermal molecular tool in many diagnostic fields represents an affordable (approximately 4.3 USD per test), simple (few and easy hands-on steps), fast (results within 5–20 min), and sensitive (single target copy number detected) method for the identification of pathogens and the detection of single nucleotide polymorphisms in human cancers and genetically modified organisms. CONTENT This review summarizes the current knowledge on RPA. The molecular diagnostics of various RNA/DNA pathogens is discussed while highlighting recent applications in clinical settings with focus on point-of-care (POC) bioassays and on automated fluidic platforms. The strengths and limitations of this isothermal method are also addressed. SUMMARY RPA is becoming a molecular tool of choice for the rapid, specific, and cost-effective identification of pathogens. Owing to minimal sample-preparation requirements, low operation temperature (25–42 °C), and commercial availability of freeze-dried reagents, this method has been applied outside laboratory settings, in remote areas, and interestingly, onboard automated sample-to-answer microfluidic devices. RPA is undoubtedly a promising isothermal molecular technique for clinical microbiology laboratories and emergence response in clinical settings.

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7

Tomar, Saurabh, Barbora Lavickova, and Carlotta Guiducci. "Recombinase polymerase amplification in minimally buffered conditions." Biosensors and Bioelectronics 198 (February 2022): 113802. http://dx.doi.org/10.1016/j.bios.2021.113802.

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8

Nair, Gayatri, Juan David Ramírez, A. Clinton White, Alejandro Castellanos-Gonzalez, A. Elizabeth Pinilla, Mauricio Rebolledo, M. Consuelo López, R. Rebecca Richards-Kortum, and Zachary Crannell. "Detection of Entamoeba histolytica by Recombinase Polymerase Amplification." American Journal of Tropical Medicine and Hygiene 93, no. 3 (September 2, 2015): 591–95. http://dx.doi.org/10.4269/ajtmh.15-0276.

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9

Higgins, Matthew, Matt Ravenhall, Daniel Ward, Jody Phelan, Amy Ibrahim, Matthew S. Forrest, Taane G. Clark, and Susana Campino. "PrimedRPA: primer design for recombinase polymerase amplification assays." Bioinformatics 35, no. 4 (August 8, 2018): 682–84. http://dx.doi.org/10.1093/bioinformatics/bty701.

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10

Lobato, Ivan Magriñá, and Ciara K. O'Sullivan. "Recombinase polymerase amplification: Basics, applications and recent advances." TrAC Trends in Analytical Chemistry 98 (January 2018): 19–35. http://dx.doi.org/10.1016/j.trac.2017.10.015.

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11

Wang, Jianchang, Jinfeng Wang, Libing Liu, Ruiwen Li, and Wanzhe Yuan. "Rapid detection ofPorcine circovirus 2by recombinase polymerase amplification." Journal of Veterinary Diagnostic Investigation 28, no. 5 (August 19, 2016): 574–78. http://dx.doi.org/10.1177/1040638716654201.

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12

Khmeleva, S. A., G. R. Kutdusova, I. F. Duskaev, K. G. Ptitsyn, V. E. Kuznetsova, V. E. Kuznetsova, S. A. Lapa, A. V. Chudinov, and S. P. Radko. "DEOXYURIDINE TRIPHOSPHATES MODIFIED WITH AROMATIC GROUPS OF TYROSINE OR TRYPTOPHAN FOR DIRECT ELECTROCHEMICAL DETERMINATION OF DOUBLE-STRANDED DNA AMPLIFICATION PRODUCTS." http://eng.biomos.ru/conference/articles.htm 1, no. 19 (2021): 248–50. http://dx.doi.org/10.37747/2312-640x-2021-19-248-250.

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A set of deoxyuridine triphosphates modified with aromatic groups of tyrosine or tryptophan was studied as substrates for amplification (by polymerase chain reaction or recombinase polymerase amplification) and as carriers of an electroactive ‘label’ for direct electrochemical detection of double-stranded DNA.

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13

Choi, Goro, Jae Hwan Jung, Byung Hyun Park, Seung Jun Oh, Ji Hyun Seo, Jong Seob Choi, Do Hyun Kim, and Tae Seok Seo. "A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria." Lab on a Chip 16, no. 12 (2016): 2309–16. http://dx.doi.org/10.1039/c6lc00329j.

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14

Wan Rasni, Wan Hawa Najibah, Nazariyah Yahaya, and Maryam Mohamed Rehan. "Recombinase Polymerase Amplification and Their Application in Phytopathogen Detection." Malaysian Journal of Science Health & Technology 8, no. 2 (May 16, 2022): 14–24. http://dx.doi.org/10.33102/2022254.

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DNA identification method is indispensable for the detection of a plant pathogen. However, established techniques, though reliable, requires advanced equipment, and their application outside specialized laboratories is limited. Along with the advancement of molecular techniques, several isothermal amplification methods, including Recombinase Polymerase Amplification (RPA), has been developed in this study. In fact, RPA is a rapid and sensitive amplification method, operating optimally at 37-42 degree celcius for 15 to 30 minutes with minimal sample preparation, and can amplify as low as 1-10 target copies. Furthermore, RPA has been a favourable method for the detection of plant pathogens due to its advantageous parameters. This review presents the current knowledge of RPA and its application in plant pathogen detection.

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15

Shahrajabian, Mohamad H., Wenli Sun, and Qi Cheng. "Different Methods for Molecular and Rapid Detection of Human Novel Coronavirus." Current Pharmaceutical Design 27, no. 25 (September 15, 2021): 2893–903. http://dx.doi.org/10.2174/1381612827666210604114411.

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Introduction: While PCR has been recognized as one of the appropriate ways to diagnose infectious diseases, Loop-mediated isothermal amplification (LAMP) which is a nucleic acid amplification method, can be considered as an alternative to PCR, and it is faster, cost-effective, and easier to perform than nested PCR. Patients and Methods: Keywords were searched in PubMed/MEDLINE, Scopus and Institute for Scientific Information Web of Science, as well as the search engine of Google Scholar. Keywords included PCR, LAMP, RAA, RPA, Virus and COVID-19. Results: LAMP technology has been extensively applied for the detection of human pathogenic bacteria, crop pests, pathogenic organisms and components in meat products. A new isotheral method, Recombinase polymerase amplification (RPA), can amplify the DNA as well as RPA. RPA involves benefits of isothermal PCR as well as simplicity and rapid amplification. Recombinase aided amplification (RAA) assay has been favorably used in the detection of bacterial and viral pathogens and solved the technical difficulties posed by DNA amplification methods because it does not need thermal denaturation of the template and involves a low and constant temperature. Conclusions: Reverse transcription polymerase chain reaction, digital PCR, LAMP, nicking endonuclease amplification reaction, recombinase polymerase amplification, and clustered regularly interspaced short palindromic repeats are different nucleic acid amplification tests of COVID-19. LAMP methods can be more specific than qPCR and immunoassays. The LAMP assay can be applied for rapid detection of SARS-CoV, MERS-CoV, SARS-CoV-2, and influenza, because LAMP is a highly sensitive and specific DNA/RNA amplification technique.

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16

Lei, Rong, Zhengyue Yan, Fan Hu, Shuifang Zhu, Yufen Xiong, and Xiaohong Fan. "Rapid identification of quarantine invasiveSolanum elaeagnifoliumby real-time, isothermal recombinase polymerase amplification assay." RSC Advances 7, no. 83 (2017): 52573–80. http://dx.doi.org/10.1039/c7ra10781a.

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17

Wang, Ying, Xiangdong Li, Dongmei Xi, and Xiaoqiang Wang. "Visual detection of Fusarium proliferatum based on asymmetric recombinase polymerase amplification and hemin/G-quadruplex DNAzyme." RSC Advances 9, no. 64 (2019): 37144–47. http://dx.doi.org/10.1039/c9ra05709a.

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18

Boyle, David S., Ruth McNerney, Hwee Teng Low, Brandon Troy Leader, Ailyn C. Pérez-Osorio, Jessica C. Meyer, Denise M. O'Sullivan, David G. Brooks, Olaf Piepenburg, and Matthew S. Forrest. "Rapid Detection of Mycobacterium tuberculosis by Recombinase Polymerase Amplification." PLoS ONE 9, no. 8 (August 13, 2014): e103091. http://dx.doi.org/10.1371/journal.pone.0103091.

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19

Crannell, Zachary, Alejandro Castellanos-Gonzalez, Gayatri Nair, Rojelio Mejia, A. Clinton White, and Rebecca Richards-Kortum. "Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa." Analytical Chemistry 88, no. 3 (January 12, 2016): 1610–16. http://dx.doi.org/10.1021/acs.analchem.5b03267.

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20

Castellanos-Gonzalez, A., A. C. White, P. Melby, and B. Travi. "Molecular diagnosis of protozoan parasites by Recombinase Polymerase Amplification." Acta Tropica 182 (June 2018): 4–11. http://dx.doi.org/10.1016/j.actatropica.2018.02.002.

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21

Xu, Jialiang, Xiaoxun Wang, Liya Yang, Biao Kan, and Xin Lu. "Rapid detection of mcr-1 by recombinase polymerase amplification." Journal of Medical Microbiology 67, no. 12 (December 1, 2018): 1682–88. http://dx.doi.org/10.1099/jmm.0.000865.

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22

Behrmann, Ole, Iris Bachmann, Frank Hufert, and Gregory Dame. "Schnellnachweis von SARS-CoV-2 mit recombinase polymerase amplification." BIOspektrum 26, no. 6 (October 14, 2020): 624–27. http://dx.doi.org/10.1007/s12268-020-1458-3.

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Abstract The COVID-19 pandemic highlights the need for fast and simple assays for nucleic acid detection. As an isothermal alternative to RT-qPCR, we outline the development of a detection scheme for SARS-CoV-2 RNA based on reverse transcription recombinase polymerase amplification (RT-RPA) technology. RPA uses recombination proteins in combination with a DNA polymerase for rapid amplification of target DNA at a constant temperature (39–42 °C) within 10 to 20 minutes and can be monitored in real-time with fluorescent probes.

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23

del Río, Jonathan Sabaté, Ivan Magriñà Lobato, Olena Mayboroda, Ioanis Katakis, and Ciara K. O’Sullivan. "Enhanced solid-phase recombinase polymerase amplification and electrochemical detection." Analytical and Bioanalytical Chemistry 409, no. 12 (March 2, 2017): 3261–69. http://dx.doi.org/10.1007/s00216-017-0269-y.

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24

Lee, Jeewon, Sunghoon Heo, and Duhee Bang. "Applying a Linear Amplification Strategy to Recombinase Polymerase Amplification for Uniform DNA Library Amplification." ACS Omega 4, no. 22 (November 12, 2019): 19953–58. http://dx.doi.org/10.1021/acsomega.9b02886.

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25

Liu, Dan, Haicong Shen, Yuqian Zhang, Danyu Shen, Mingyang Zhu, Yanling Song, Zhi Zhu, and Chaoyong Yang. "A microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay for rapid COVID-19 detection." Lab on a Chip 21, no. 10 (2021): 2019–26. http://dx.doi.org/10.1039/d0lc01222j.

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26

Yang, Huan-Lan, Shuang Wei, Ravi Gooneratne, Anthony N. Mutukumira, Xue-Jun Ma, Shu-Ze Tang, and Xi-Yang Wu. "Development of a recombinase polymerase amplification assay forVibrio parahaemolyticusdetection with an internal amplification control." Canadian Journal of Microbiology 64, no. 4 (April 2018): 223–30. http://dx.doi.org/10.1139/cjm-2017-0504.

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Анотація:

A novel RPA–IAC assay using recombinase polymerase and an internal amplification control (IAC) for Vibrio parahaemolyticus detection was developed. Specific primers were designed based on the coding sequence for the toxR gene in V. parahaemolyticus. The recombinase polymerase amplification (RPA) reaction was conducted at a constant low temperature of 37 °C for 20 min. Assay specificity was validated by using 63 Vibrio strains and 10 non-Vibrio bacterial species. In addition, a competitive IAC was employed to avoid false-negative results, which co-amplified simultaneously with the target sequence. The sensitivity of the assay was determined as 3 × 103CFU/mL, which is decidedly more sensitive than the established PCR method. This method was then used to test seafood samples that were collected from local markets. Seven out of 53 different raw seafoods were detected as V. parahaemolyticus-positive, which were consistent with those obtained using traditional culturing method and biochemical assay. This novel RPA–IAC assay provides a rapid, specific, sensitive, and more convenient detection method for V. parahaemolyticus.

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27

Eid, Charbel, and Juan G. Santiago. "Assay for Listeria monocytogenes cells in whole blood using isotachophoresis and recombinase polymerase amplification." Analyst 142, no. 1 (2017): 48–54. http://dx.doi.org/10.1039/c6an02119k.

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28

Li, Jia, Joanne Macdonald, and Felix von Stetten. "Correction: Review: a comprehensive summary of a decade development of the recombinase polymerase amplification." Analyst 145, no. 5 (2020): 1950–60. http://dx.doi.org/10.1039/c9an90127b.

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29

Kutdusova, G., and E. Suprun. "DEOXYURIDINE TRIPHOSPHATES MODIFIED WITH AROMATIC TYROSINE GROUPS FOR ELECTROCHEMICAL DETERMINATION OF RECOMBINASE POLYMERASE AMPLIFICATION PRODUCTS OF DNA." http://eng.biomos.ru/conference/articles.htm 1, no. 19 (2021): 236–38. http://dx.doi.org/10.37747/2312-640x-2021-19-236-238.

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Deoxyuridine triphosphates modified with tyrosine or tryptophan aromatic groups were tested as electroactive ‘labels’ for the electrochemical detection of double-stranded DNA amplicons obtained by recombinase polymerase amplification.

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30

Cai, Qiqi, Rui Wang, Zhaohui Qiao, and Wenge Yang. "Single-digit Salmonella detection with the naked eye using bio-barcode immunoassay coupled with recombinase polymerase amplification and a CRISPR-Cas12a system." Analyst 146, no. 17 (2021): 5271–79. http://dx.doi.org/10.1039/d1an00717c.

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An ultrasensitive, rapid, and visual detection platform for Salmonella Typhimurium based on the bio-barcode assay and recombinase polymerase amplification (RPA) coupled with a CRISPR-Cas12a cleavage system is presented.

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31

Abd El Wahed, Ahmed, Pranav Patel, Oumar Faye, Sasikanya Thaloengsok, Doris Heidenreich, Ponpan Matangkasombut, Khajohnpong Manopwisedjaroen, et al. "Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection." PLOS ONE 10, no. 6 (June 15, 2015): e0129682. http://dx.doi.org/10.1371/journal.pone.0129682.

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32

Euler, M., Y. Wang, P. Otto, H. Tomaso, R. Escudero, P. Anda, F. T. Hufert, and M. Weidmann. "Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis." Journal of Clinical Microbiology 50, no. 7 (April 18, 2012): 2234–38. http://dx.doi.org/10.1128/jcm.06504-11.

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33

Martorell, Sara, Sarai Palanca, Ángel Maquieira, and Luis A. Tortajada-Genaro. "Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene." Analytical Biochemistry 544 (March 2018): 49–56. http://dx.doi.org/10.1016/j.ab.2017.12.013.

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34

Davi, Saskia Dede, Jonas Kissenkötter, Martin Faye, Susanne Böhlken-Fascher, Christiane Stahl-Hennig, Oumar Faye, Ousmane Faye, et al. "Recombinase polymerase amplification assay for rapid detection of Monkeypox virus." Diagnostic Microbiology and Infectious Disease 95, no. 1 (September 2019): 41–45. http://dx.doi.org/10.1016/j.diagmicrobio.2019.03.015.

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35

Martorell, Sara, Luis A. Tortajada-Genaro, and Ángel Maquieira. "Magnetic concentration of allele-specific products from recombinase polymerase amplification." Analytica Chimica Acta 1092 (December 2019): 49–56. http://dx.doi.org/10.1016/j.aca.2019.10.006.

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36

Chi, Yuan-kai, Wei Zhao, Meng-di Ye, Farman Ali, Tao Wang, and Ren-de Qi. "Evaluation of Recombinase Polymerase Amplification Assay for Detecting Meloidogyne javanica." Plant Disease 104, no. 3 (March 2020): 801–7. http://dx.doi.org/10.1094/pdis-07-19-1473-re.

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Meloidogyne javanica is one of the most widespread and economically important nematodes in many countries, including China. In this study, a recombinase polymerase amplification (RPA) assay was evaluated for the detection of M. javanica based on the sequences of a sequence-characterized amplified regions marker gene segment. The RPA assay specifically detected M. javanica from individual juvenile or adult female, M. javanica-induced galls, and nematodes in the soil samples. The detection limit of M. javanica RPA assay was 1 pg of purified genomic DNA, 0.01 adult female, or 0.1 second-stage juvenile, which was 10 times more sensitive than conventional PCR assay. Furthermore, combined with lateral flow dipstick (LFD), a visual detection method of LFD-RPA assay was developed, which is suitable for onsite surveys and routine diagnostics. Results indicate that the RPA assay is rapid, sensitive, and reliable for detection and molecular identification of M. javanica.

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Chia, Catherine T., Andrew T. Bender, Lorraine Lillis, Benjamin P. Sullivan, Coleman D. Martin, Wynn Burke, Charles Landis, David S. Boyle, and Jonathan D. Posner. "Rapid detection of hepatitis C virus using recombinase polymerase amplification." PLOS ONE 17, no. 10 (October 25, 2022): e0276582. http://dx.doi.org/10.1371/journal.pone.0276582.

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Over 71 million people are infected with hepatitis C virus (HCV) worldwide, and approximately 400,000 global deaths result from complications of untreated chronic HCV. Pan-genomic direct-acting antivirals (DAAs) have recently become widely available and feature high cure rates in less than 12 weeks of treatment. The rollout of DAAs is reliant on diagnostic tests for HCV RNA to identify eligible patients with viremic HCV infections. Current PCR-based HCV RNA assays are restricted to well-resourced central laboratories, and there remains a prevailing clinical need for expanded access to decentralized HCV RNA testing to provide rapid chronic HCV diagnosis and linkage to DAAs in outpatient clinics. This paper reports a rapid, highly accurate, and minimally instrumented assay for HCV RNA detection using reverse transcription recombinase polymerase amplification (RT-RPA). The assay detects all HCV genotypes with a limit of detection of 25 copies per reaction for genotype 1, the most prevalent in the United States and worldwide. The clinical sensitivity and specificity of the RT-RPA assay were both 100% when evaluated using 78 diverse clinical serum specimens. The accuracy, short runtime, and low heating demands of RT-RPA may enable implementation in a point-of-care HCV test to expand global access to effective treatment via rapid chronic HCV diagnosis.

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Rathore, Himankshi, Radhika Biyani, Hirotomo Kato, Yuzuru Takamura, and Manish Biyani. "Palm-size and one-inch gel electrophoretic device for reliable and field-applicable analysis of recombinase polymerase amplification." Analytical Methods 11, no. 39 (2019): 4969–76. http://dx.doi.org/10.1039/c9ay01476d.

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A newly designed handheld one-inch gel electrophoresis-based detection system and recombinase polymerase amplification (RPA) can revolutionize nucleic acid-based molecular diagnostics for people in settings with poor healthcare infrastructure.

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Ivanov, A. V., A. V. Zherdev, and B. B. Dzantiev. "ANALYTICAL SYSTEMS BASED ON RECOMBINASE POLYMERASE AMPLIFICATION FOR RAPID DETECTION OF VIRAL, VIROID AND BACTERIAL PLANT PATHOGENS." http://eng.biomos.ru/conference/articles.htm 1, no. 19 (2021): 242–44. http://dx.doi.org/10.37747/2312-640x-2021-19-242-244.

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Test systems have been developed for the detection of phytopathogens, combining recombinase polymerase amplification and membrane test strips. Test systems provide detection of potato virus X, potato spindle tuber viroid, potato blackleg pathogen (Dickeya solani), as well as multi-analysis of three viruses. Amplification is carried out at 37 °C. The analysis time does n ot exceed 30 min.

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40

Crannell, Zachary Austin, Miguel Mauricio Cabada, Rebecca Richards-Kortum, Arthur Clinton White, Ayesha Irani, and Alejandro Castellanos-Gonzalez. "Recombinase Polymerase Amplification-Based Assay to Diagnose Giardia in Stool Samples." American Journal of Tropical Medicine and Hygiene 92, no. 3 (March 4, 2015): 583–87. http://dx.doi.org/10.4269/ajtmh.14-0593.

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Lai, Meng-Yee, Choo-Huck Ooi, and Yee-Ling Lau. "Rapid Detection of Plasmodium knowlesi by Isothermal Recombinase Polymerase Amplification Assay." American Journal of Tropical Medicine and Hygiene 97, no. 5 (November 8, 2017): 1597–99. http://dx.doi.org/10.4269/ajtmh.17-0427.

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42

Wang, Hua, Xinli Ding, Wenbo Sun, Zhi Chen, Linyi Bai, Hongkun Liang, Yujie Liu, et al. "Recombinase polymerase amplification assay for rapid detection of Seneca Valley Virus." Analytical Biochemistry 642 (April 2022): 114564. http://dx.doi.org/10.1016/j.ab.2022.114564.

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43

Kobialka, Rea Maja, Arianna Ceruti, Michelle Bergmann, Katrin Hartmann, Uwe Truyen, and Ahmed Abd El Wahed. "Molecular Detection of Feline Coronavirus Based on Recombinase Polymerase Amplification Assay." Pathogens 10, no. 10 (September 25, 2021): 1237. http://dx.doi.org/10.3390/pathogens10101237.

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Feline coronavirus (FCoV) is endemic in cat populations worldwide. Persistently, subclinically infected cats play a significant role in spreading the infection. Testing fecal samples of cats may facilitate efforts to decrease the viral burden within a population. Real-time RT-PCR is highly sensitive and specific for the detection of FCoV but must be performed in a fully equipped laboratory. A simple and accurate assay is needed to identify FCoV at the point-of-need. The aim of this study was to develop a rapid FCoV detection assay based on isothermal amplification technology, i.e., reverse transcription-recombinase polymerase amplification (RT-RPA). Primers were designed to target the highly conserved 3′ untranslated region of the 7b gene. Running on a constant temperature of 42 °C, reverse transcription as well as DNA amplification and detection was achieved in a maximum of 15 min. A probit analysis revealed a detection limit of 58.5 RNA copies/reaction. For cross-detection, nucleic acids from 19 viruses were tested. Both RT-RPA and real-time RT-PCR showed cross-detection with canine coronavirus and transmissible gastroenteritis virus, but not with other pathogens. To evaluate clinical performance, RNA was extracted from 39 fecal samples from cats. All samples were tested simultaneously with real-time RT-PCR resulting in a RT-RPA sensitivity and specificity of 90.9% and 100%, respectively. RT-RPA can be considered a promising simple method for rapid detection of FCoV.

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44

Crannell, Zachary Austin, Brittany Rohrman, and Rebecca Richards-Kortum. "Equipment-Free Incubation of Recombinase Polymerase Amplification Reactions Using Body Heat." PLoS ONE 9, no. 11 (November 5, 2014): e112146. http://dx.doi.org/10.1371/journal.pone.0112146.

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45

Frimpong, Michael, Hubert Ahor, Francisca Sarpong, Ken Laing, Mark Wansbrough-Jones, and Richard Phillips. "OC 8173 RAPID DETECTION OF MYCOBACTERIUM ULCERANS BY RECOMBINASE POLYMERASE AMPLIFICATION." BMJ Global Health 4, Suppl 3 (April 2019): A2.1—A2. http://dx.doi.org/10.1136/bmjgh-2019-edc.2.

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BackgroundThere are no primary measures to prevent people from contracting Buruli ulcer, mainly due to poor understanding of its epidemiology. The current control strategy emphasises early diagnosis and prompt treatment, with the goal of avoiding the complications associated with advanced stages of the disease. There is no diagnostic test for the disease appropriate for use at the primary health care level where most cases are detected and treated. Diagnosis based on clinical signs is unreliable in inexperienced hands and complicated by infections that have similar presentations. This study was to develop and evaluate the use of recombinase polymerase amplification (RPA) assay for the detection of Mycobacterium ulcerans at the point of patient care.MethodsA specific fragment of IS2404 of M. ulcerans was amplified in 15 min at a constant temperature of 42°C, using the RPA assay and analysed on a portable fluorometre. Themethod was tested for sensitivity and specificity with molecular standard of IS2404 DNA fragment, various M.ulcerans strains, other mycobacteria and environmentally associated bacteria. Additionally, the assay performance as a diagnostic tool was tested with archived DNA from symptomatic patients. All results were compared with that of a highly sensitive IS2404 PCR.ResultsThe detection limit was 50 copies of IS2404 in 15 min using plasmid standard and 125 fg with genomic Mu DNA equivalent 25 genomic copies. The assay was highly specific in detecting all strains of M. ulcerans with no observed cross reactivity with other mycobacteria and common skin colonising bacteria. The clinical sensitivity and specificity of the BU-RPA assay using clinical samples was 86% and 100% respectively.ConclusionWe have developed a real-time isothermal RPA assay for the detection of M. ulcerans as a cheaper alternative to PCR. Combining this assay with a simple extraction protocol will maximise its use as point-of-care test for Buruli ulcer.

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46

Wang, Jianchang, Yongning Zhang, Ruoxi Zhang, Qingan Han, Jinfeng Wang, Libing Liu, Ruiwen Li, and Wanzhe Yuan. "Recombinase polymerase amplification assay for rapid detection of porcine circovirus 3." Molecular and Cellular Probes 36 (December 2017): 58–61. http://dx.doi.org/10.1016/j.mcp.2017.09.001.

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Chen, Zhengwei, Jun Huang, Fang Zhang, Yang Zhou, and Huijie Huang. "Detection of shrimp hemocyte iridescent virus by recombinase polymerase amplification assay." Molecular and Cellular Probes 49 (February 2020): 101475. http://dx.doi.org/10.1016/j.mcp.2019.101475.

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48

Crannell, Zachary Austin, Brittany Rohrman, and Rebecca Richards-Kortum. "Quantification of HIV-1 DNA Using Real-Time Recombinase Polymerase Amplification." Analytical Chemistry 86, no. 12 (May 29, 2014): 5615–19. http://dx.doi.org/10.1021/ac5011298.

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49

Ge, Junwei, Yunjia Shi, Xingyang Cui, Shanshan Gu, Lili Zhao, and Hongyan Chen. "Rapid and sensitive detection of mink circovirus by recombinase polymerase amplification." Journal of Virological Methods 256 (June 2018): 1–5. http://dx.doi.org/10.1016/j.jviromet.2018.02.022.

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50

Nybond, Susanna, Pedro Réu, Samuel Rhedin, Gustav Svedberg, Tobias Alfvén, Jesper Gantelius, and Helene Andersson Svahn. "Adenoviral detection by recombinase polymerase amplification and vertical flow paper microarray." Analytical and Bioanalytical Chemistry 411, no. 4 (November 29, 2018): 813–22. http://dx.doi.org/10.1007/s00216-018-1503-y.

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