Phosphorothioated Primers Lead to Loop-Mediated Isothermal Amplification at Low Temperatures

Loop-mediated isothermal amplification (LAMP) is an extremely powerful tool for the detection of nucleic acids with high sensitivity and specificity. However, LAMP shows optimal performance at around 65 °C, which limits applications in point-of-care-testing (POCT). Here, we have developed a version of LAMP that uses phosphorothioated primers (PS-LAMP) to enable more efficient hairpin formation and extension at the termini of growing concatamers, and that therefore works at much lower temperatures. By including additional factors such as chaotropes (urea) and single-stranded DNA binding protein (SSB), the sensitivities and selectivities for amplicon detection with PS-LAMP at 40 °C were comparable with a regular LAMP reaction at 65 °C

Exogenous MMTV RNA Loads Are Higher in the Presence of Endogenous <i>Mtv</i> Proviruses

<div><p>(A) RT-PCR indicates higher levels of C3H MMTV RNA in spleens derived from infected BALB/c mice compared to those from infected <i>Mtv</i>-null mice. The numbers above each lane represent separate BALB/c or <i>Mtv</i>-null animals as indicated. Mice injected with cloned HYB-MTV-producing cells are designated HM, whereas mice infected by foster-nursing are designated F. Organs were harvested between 6–15 mo post-infection in BALB/c mice at the time of tumor development, whereas organs from <i>Mtv</i>-null mice were harvested between 10–15 mo post-infection. MT, mammary tumor</p><p>(B) RT-PCR analysis shows lower TBLV levels in <i>Mtv</i>-null versus <i>Mtv</i>-positive strains. Organs were harvested between 4–10 mo post-infection in BALB/c mice at the time of tumor development; organs from <i>Mtv</i>-negative mice were obtained at 10 mo post-infection.</p></div

Bacterial Load Is Decreased in <i>Mtv</i>-Null Mice

<p>Three different doses of V. cholerae were given to 5- to 6-d-old pups (three to four animals/dose). After 24 h, the mice were sacrificed, and the small intestines were assayed for bacterial growth. The means (numbers shown inside bars) and standard deviations for the recovered CFUs of each set of animals are indicated. Two-tailed Student's <i>t</i> tests were used for pair-wise comparisons of BALB/c and <i>Mtv</i>-null animals infected at each dose; <i>p</i>-values of <0.05 are shown by an asterisk.</p

BALB/c X <i>Mtv</i>-Null F1 Hybrids and Mice Carrying Single <i>Mtv</i> Proviruses Exhibit C3H MMTV Sag-Specific T-Cell Deletion

<p>PBLs isolated from MMTV-infected (dark bars) and control non-infected (light bars) mice at different times were dually stained with mouse-specific CD4-PE and Vβ8 or Vβ14-FITC antibodies followed by FACS analysis. The percentages of C3H Sag-reactive CD4+Vβ14+ (A–E) and non-reactive CD4+Vβ8+ (F–J) T cells obtained from BALB/c X <i>Mtv</i>-null F1 (A and F), BALB/<i>Mtv6</i> (B and G), BALB/<i>Mtv8</i> (C and H), BALB/<i>Mtv9</i> (D and I), and BALB/<i>Mtv</i>-null (E and J) are depicted. Numbers inside the bars represent numbers of animals tested. The average percentages of T cells in the MMTV-infected <i>Mtv</i>-positive strains (including standard deviation) were significantly different from non-infected controls of the same strains by the two-tailed Student's <i>t</i> test (<i>p</i> < 0.05) (asterisks). In contrast, infected <i>Mtv</i>-null animals failed to demonstrate T-cell deletion. PE, phycoerythrin.</p

BALB/<i>Mtv</i>-Null Mice Infected by Milk-Borne MMTV Lack Virus-Specific Neutralizing Antibodies

<div><p>(A) Both BALB/c and <i>Mtv</i>-null animals sporadically have low levels of MMTV-specific antibodies. Sera from milk-borne C3H MMTV-infected (gray symbols) or age-matched non-infected (white symbols) BALB/c (squares) and <i>Mtv</i>-null (circles) mice were analyzed weekly by ELISA to detect virion-specific antibodies. Each symbol represents an individual mouse. The serum antibody titer was calculated as the reciprocal of the highest serum dilution that reacted with MMTV virions. Since one non-infected animal showed a titer of 1:80, values above 1:100 (the heavy line) were considered positive.</p><p>(B) Sporadic, low-titer antibodies in <i>Mtv</i>-null animals have reactivity to MMTV capsid antigen. Proteins from purified MMTV particles were separated on SDS-containing polyacrylamide gels, subjected to Western blotting, and incubated with a 20-fold dilution of serum from two MMTV-infected <i>Mtv</i>-null mice with the highest antibody titers (1:320) at 4–5 wk of age. MMTV capsid antigen (CA) or surface envelope (SU)-specific monoclonal antibodies were used as positive controls. Sera from two age-matched non-infected BALB/<i>Mtv</i>-null mice were used as negative controls.</p></div

BALB/<i>Mtv</i>-Null Mice Lack Endogenous <i>Mtv</i>s and Have Impaired Sag-Mediated T-Cell Deletion after Exogenous MMTV Infection

<div><p>(A) PCR analysis of mouse genomic DNA using endogenous <i>Mtv</i>-specific primers. Genomic DNA samples from mice with single endogenous <i>Mtv</i>s were used as positive controls for PCR amplification of individual proviruses (lanes 2–4). Genomic DNA samples from three <i>Mtv-</i>null mice were analyzed (lanes 5–7). PCR with no added DNA template was the negative control (lane 8).</p><p>(B) Flow cytometry of endogenous <i>Mtv</i> Sag-reactive T-cell subsets in PBLs. PBLs from 6-mo-old BALB/c and <i>Mtv</i>-null mice were dually stained with mouse specific CD4-phycoerythrin and mouse-specific TCR Vβ3, 5, 7, or 12-FITC antibodies followed by FACS and software analysis. Three individual animals of each strain were tested, and the average proportion of T-cell subsets (± standard deviation) is shown. Flow cytometry also was performed on PBLs isolated after MMTV infection of BALB/c J (C) or <i>Mtv</i>-null (D) mice. The numbers inside the bars indicate the numbers of animals tested. At 11 mo post-infection of BALB/c animals, only two animals were tested for T-cell deletion because the other animals had died from mammary tumors. Cells were stained with mouse-specific CD4-PE and mouse-specific Vβ8 or Vβ14-FITC antibodies. The percentages of C3H MMTV Sag-reactive (CD4+Vβ14+) (left panels) and non-reactive (CD4+Vβ8+) (right panels) T cells among the PBLs isolated from mice inoculated with C3H MMTV-infected rat cells (dark bars) were compared to non-injected control mice (light bars). PE, phycoerythrin.</p></div

Real-Time Detection of Isothermal Amplification Reactions with Thermostable Catalytic Hairpin Assembly

Catalytic hairpin assembly (CHA) is an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification reactions. Here, for the first time, we engineered CHA to be thermostable from 37 to 60 °C and in consequence have generalized its application to the real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high- and low-temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection but also improved the sensitivity by as much as 25- to 10000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise

Strand Displacement Probes Combined with Isothermal Nucleic Acid Amplification for Instrument-Free Detection from Complex Samples

Sensitive and specific detection of pathogens via nucleic acid amplification is currently constrained to laboratory settings and portable equipment with costly fluorescent detectors. Nucleic acid-detecting lateral flow immunoassay strips (LFIAs) offer a low-cost visual transduction strategy at points of need. Unfortunately, these LFIAs frequently detect amplification byproducts that can yield spurious results which can only be deciphered through statistical analysis. We integrated customizable strand displacement probes into standard loop mediated isothermal amplification (LAMP) assays to prevent byproduct capture on commercial LFIAs. We find that combining strand displacement with LAMP (SD-LAMP) yields LFIA test band intensities that can be unequivocally interpreted by human subjects without additional instrumentation, thereby alleviating the need for a portable reader’s analysis. Using SD-LAMP, we capture target amplicons on commercially available LFIAs from as few as 3.5 <i>Vibrio cholerae</i> and 2 750 <i>Escherichia coli</i> bacteria without false positive or false negative interpretation. Moreover, we demonstrate that LFIA capture of SD-LAMP products remain specific even in the presence of complex sample matrixes, providing a significant step toward reliable instrument-free pathogen detection outside of laboratories

Robust Strand Exchange Reactions for the Sequence-Specific, Real-Time Detection of Nucleic Acid Amplicons

Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10⁹ copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (<i>rpoB</i>) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of <i>rpoB</i> in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests

Determination of the technical LOD of one-pot asymmetric five-primer OSD-RT-LAMP assays designed for MERS-CoV detection.

<p>Technical LOD of the ORF1a.55, ORF1b.59 and UpE.9 asymmetric five-primer LAMP primer sets was determined by amplification of specific <i>in vitro</i> transcribed MERS-CoV RNA segments. Representative amplification curves of the ORF1a.55, ORF1b.59 and UpE.9 assays are depicted in panels A, C and E, respectively. Probit regression analysis plots with the calculated LOD of the ORF1a.55, ORF1b.59 and UpE.9 assays are depicted in panels B, D and F, respectively. Samples labeled ‘VERO’ consists of RNA extracted from uninfected Vero cell culture supernatants.</p