Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.

Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 10(4) copies of HIV-1 DNA in a background of 0-14 μg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 μg when 10(6) copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads.

[1]  R. Sutton,et al.  Characterization and detection of artificial replication-competent lentivirus of altered host range. , 2003, Molecular therapy : the journal of the American Society of Gene Therapy.

[2]  Paul Yager,et al.  Enhanced sensitivity of lateral flow tests using a two-dimensional paper network format. , 2011, Analytical chemistry.

[3]  J. Nurnberger,et al.  A non-organic and non-enzymatic extraction method gives higher yields of genomic DNA from whole-blood samples than do nine other methods tested. , 1992, Journal of biochemical and biophysical methods.

[4]  Felix von Stetten,et al.  Rapid Molecular Assays for the Detection of Yellow Fever Virus in Low-Resource Settings , 2014, PLoS neglected tropical diseases.

[5]  E. Abrams,et al.  Virus load as a marker of disease progression in HIV-infected children. , 1996, AIDS research and human retroviruses.

[6]  F. Bier,et al.  Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis , 2014, Malaria Journal.

[7]  Rebecca Richards-Kortum,et al.  Nucleic Acid Test to Diagnose Cryptosporidiosis: Lab Assessment in Animal and Patient Specimens , 2014, Analytical chemistry.

[8]  B. Hoffmann,et al.  A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-and-Mouth Disease Virus , 2013, PloS one.

[9]  Rochelle P Walensky,et al.  Early infant HIV-1 diagnosis programs in resource-limited settings: opportunities for improved outcomes and more cost-effective interventions , 2011, BMC medicine.

[10]  Olaf Piepenburg,et al.  Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification , 2013, mBio.

[11]  Torsten Seemann,et al.  Genomic Insights to Control the Emergence of Vancomycin-Resistant Enterococci , 2013, mBio.

[12]  Avettand‐Fènoël Véronique,et al.  LTR real‐time PCR for HIV‐1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01) , 2009 .

[13]  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.

[14]  Veronica Leautaud,et al.  A Lateral Flow Assay for Quantitative Detection of Amplified HIV-1 RNA , 2012, PloS one.

[15]  Sujit R. Jangam,et al.  Rapid, Point-of-Care Extraction of Human Immunodeficiency Virus Type 1 Proviral DNA from Whole Blood for Detection by Real-Time PCR , 2009, Journal of Clinical Microbiology.

[16]  Olaf Piepenburg,et al.  DNA Detection Using Recombination Proteins , 2006, PLoS biology.

[17]  I. Mäger,et al.  Sensitive and rapid detection of Chlamydia trachomatis by recombinase polymerase amplification directly from urine samples. , 2014, The Journal of molecular diagnostics : JMD.

[18]  Rebecca Richards-Kortum,et al.  Equipment-Free Incubation of Recombinase Polymerase Amplification Reactions Using Body Heat , 2014, PloS one.

[19]  S. Santiago-Felipe,et al.  Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis. , 2014, Analytica chimica acta.

[20]  F. Hufert,et al.  Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis , 2012, Journal of Clinical Microbiology.