An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases.

A simple, point of care, inexpensive, disposable cassette for the detection of nucleic acids extracted from pathogens was designed, constructed, and tested. The cassette utilizes a single reaction chamber for isothermal amplification of nucleic acids. The chamber is equipped with an integrated, flow-through, Flinders Technology Associates (Whatman FTA®) membrane for the isolation, concentration, and purification of DNA and/or RNA. The nucleic acids captured by the membrane are used directly as templates for amplification without elution, thus simplifying the cassette's flow control. The FTA membrane also serves another critical role-enabling the removal of inhibitors that dramatically reduce detection sensitivity. Thermal control is provided with a thin film heater external to the cassette. The amplification process was monitored in real time with a portable, compact fluorescent reader. The utility of the integrated, single-chamber cassette was demonstrated by detecting the presence of HIV-1 in oral fluids. The HIV RNA was reverse transcribed and subjected to loop-mediated, isothermal amplification (LAMP). A detection limit of less than 10 HIV particles was demonstrated. The cassette is particularly suitable for resource poor regions, where funds and trained personnel are in short supply. The cassette can be readily modified to detect nucleic acids associated with other pathogens borne in saliva, urine, and other body fluids as well as in water and food.

[1]  Mehmet Toner,et al.  Enhancing the performance of a point-of-care CD4+ T-cell counting microchip through monocyte depletion for HIV/AIDS diagnostics. , 2009, Lab on a chip.

[2]  H. Moon,et al.  Multiplex PCR for the simultaneous detection of pseudorabies virus, porcine cytomegalovirus, and porcine circovirus in pigs. , 2007, Journal of virological methods.

[3]  Michael G. Roper,et al.  A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability , 2006, Proceedings of the National Academy of Sciences.

[4]  R. P. Bucy,et al.  Quantitation of HIV-1 by real-time PCR with a unique fluorogenic probe. , 2001, Journal of virological methods.

[5]  D. Rudolph,et al.  Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP). , 2008, Journal of virological methods.

[6]  Christopher D Pilcher,et al.  HIV Infection: Point-of-Care Testing , 2004, The Annals of pharmacotherapy.

[7]  M. Kalish,et al.  Alternative Algorithms for Human Immunodeficiency Virus Infection Diagnosis Using Tests That Are Licensed in the United States , 2008, Journal of Clinical Microbiology.

[8]  A. Foulkes,et al.  Low-Cost HIV-1 Diagnosis and Quantification in Dried Blood Spots by Real Time PCR , 2009, PloS one.

[9]  V. Gant,et al.  Multiplex real-time PCR and blood culture for identification of bloodstream pathogens in patients with suspected sepsis. , 2009, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[10]  Ji Yoon Kang,et al.  A polymer lab-on-a-chip for reverse transcription (RT)-PCR based point-of-care clinical diagnostics. , 2008, Lab on a chip.

[11]  Amy E Herr,et al.  Microfluidic immunoassays as rapid saliva-based clinical diagnostics , 2007, Proceedings of the National Academy of Sciences.

[12]  Daniel Malamud,et al.  An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids , 2010, Biomedical microdevices.

[13]  Doyoung Byun,et al.  A disposable, self-contained PCR chip. , 2009, Lab on a chip.

[14]  Yasuyoshi Mori,et al.  Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products , 2008, Nature Protocols.

[15]  Bryan Lincoln,et al.  Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics. , 2008, Lab on a chip.

[16]  D. Malamud.  Oral diagnostic testing for detecting human immunodeficiency virus-1 antibodies: a technology whose time has come. , 1997, The American journal of medicine.

[17]  B. Branson.  Point‐of‐Care Rapid Tests for HIV Antibodies , 2003 .

[18]  T. Rutigliano,et al.  A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load , 2008, Nature Protocols.

[19]  Jerome P Ferrance,et al.  A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples. , 2006, Analytical chemistry.

[20]  Helen H. Lee,et al.  Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings. , 2007, The Analyst.

[21]  Lincoln Young,et al.  Weak solvent based chip lamination and characterization of on-chip valve and pump , 2010, Biomedical microdevices.

[22]  D. Malamud,et al.  Point Detection of Pathogens in Oral Samples , 2005, Advances in dental research.

[23]  Rustem F Ismagilov,et al.  Nanoliter multiplex PCR arrays on a SlipChip. , 2010, Analytical chemistry.

[24]  R. Mathies,et al.  Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis. , 2001, Lab on a chip.

[25]  Robin H. Liu,et al.  Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. , 2004, Analytical chemistry.

[26]  N. Johnson,et al.  Inhibitory effect of salivary fluids on PCR: potency and removal. , 1994, PCR methods and applications.

[27]  J. Kong,et al.  Loop-mediated isothermal amplification integrated on microfluidic chips for point-of-care quantitative detection of pathogens. , 2010, Analytical chemistry.

[28]  A. Ziober,et al.  Lab‐on‐a‐chip for oral cancer screening and diagnosis , 2008, Head & neck.

[29]  Suhyeon Kim,et al.  Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification. , 2006, Lab on a chip.

[30]  Xianbo Qiu,et al.  A timer-actuated immunoassay cassette for detecting molecular markers in oral fluids. , 2009, Lab on a chip.

[31]  Kristen L. Helton,et al.  Microfluidic Overview of Global Health Issues Microfluidic Diagnostic Technologies for Global Public Health , 2006 .

[32]  M. Re,et al.  Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by SYBR green real-time RT-PCR technique in HIV-1 seropositive patients. , 2004, Journal of virological methods.

[33]  D. Rudolph,et al.  Sequence‐specific detection method for reverse transcription, loop‐mediated isothermal amplification of HIV‐1 , 2009, Journal of medical virology.

[34]  Tsugunori Notomi,et al.  Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus , 2004, Journal of Clinical Microbiology.

[35]  Frederic D. Bushman,et al.  A quantitative assay for HIV DNA integration in vivo , 2001, Nature Medicine.

[36]  Phillip I. Tarr,et al.  Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Common Strains of Escherichia coli , 2008, Journal of Clinical Microbiology.

[37]  T. Notomi,et al.  Loop-mediated isothermal amplification of DNA. , 2000, Nucleic acids research.

[38]  Xavier Anglaret,et al.  Transfer and Evaluation of an Automated, Low-Cost Real-Time Reverse Transcription-PCR Test for Diagnosis and Monitoring of Human Immunodeficiency Virus Type 1 Infection in a West African Resource-Limited Setting , 2005, Journal of Clinical Microbiology.

[39]  Zongyuan Chen,et al.  A disposable microfluidic cassette for DNA amplification and detection. , 2006, Lab on a chip.

[40]  Jesse V Jokerst,et al.  Integration of semiconductor quantum dots into nano-bio-chip systems for enumeration of CD4+ T cell counts at the point-of-need. , 2008, Lab on a chip.

[41]  Daniel Malamud,et al.  A Microfluidic System for Saliva‐Based Detection of Infectious Diseases , 2007, Annals of the New York Academy of Sciences.

[42]  John W. Mellors,et al.  New Real-Time Reverse Transcriptase-Initiated PCR Assay with Single-Copy Sensitivity for Human Immunodeficiency Virus Type 1 RNA in Plasma , 2003, Journal of Clinical Microbiology.

[43]  D. Wong,et al.  Salivary diagnostics: enhancing disease detection and making medicine better. , 2008, European journal of dental education : official journal of the Association for Dental Education in Europe.

[44]  Jitae Kim,et al.  A PCR reactor with an integrated alumina membrane for nucleic acid isolation. , 2010, The Analyst.