Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i>

<div><p>Background</p><p>The detection of <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> (MAP) infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure.</p><p>Methodology/Principal Findings</p><p>In this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA) assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%.</p><p>Conclusions/Significance</p><p>The RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP.</p></div

Probit analylsis results for the MAP RPA (black) and real-time PCR (orange) assays using STATISTICA.

<p>Data sets of eight RPA and real-time PCR assay runs as showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168733#pone.0168733.g001" target="_blank">Fig 1</a> was used. The limit of detection in RPA and real-time PCR at 95% probability were 16 and one DNA molecules of the molecule plasmid DNA standard, respectively.</p

Reproducibility of the RPA (A) and real-time PCR (B) assays using data set of eight runs of serial dilution of the MAP plasmid molecular standard in PRISM.

<p>RPA assay produced results between 2 to 10 minutes. In RPA 5x10⁶-10² DNA molecules were detected in 8 out of 8 runs; 50, 7/8 and 5, 1/8 by the RPA assay. The error bars represent the range. The real-time PCR produced more linear results, due to the regular cycle format of the PCR, while there is no strict separation of the amplification cycles in the isothermal RPA technology.</p

FMDV RT-RPA primers and exo-probe sequences aligned with the consensus sequence of 100 FMDV 3D genes downloaded from the Genbank.

<p>(Geneious® 6.1.5, Biomatters Limited, New Zealand). Mismatches are indicated in bold and underlined. The consensus sequence represents nt 7847–7961 of FMDV sequence JF749843. NNN are sites of the quencher and fluropohore in following order (BHQ1-dT) (Tetrahydrofuran) (FAM-dT). Y is C & T; R: A & G.</p

FMDV RT-RPA.

<p>Fluorescence development over time using a dilution range of 10⁷-10¹ molecules/µl of the FMDV RNA standard (Graph generated by ESEquant tubescanner software). F04+R20+P2 were employed and the analytical sensitivity was 10². 10⁷ represented by black line; 10⁶, gray; 10⁵, red; 10⁴, blue; 10³, green; 10², cyan; 10¹, dark khaki; negative control, orange.</p

Detection of FMDV in experimentally infected animals using real-time RT-PCR and RT-RPA.

<p>Dpi, days post infection;</p>1<p>infected with serotype A22 Iraq 24/64;</p>2<p>infected with O Bulgaria 2011; neg, negative.</p

Performance and analytical sensitivity of the FMDV RT-RPA assay.

<p>A: Semi-logarithmic regression of the data collected from eight FMDV RT-RPA test runs on the RNA standard using Prism Software. It yielded results between 4–10 minutes. B: Probit regression analysis using Statistica software on data of the eight runs. The limit of detection at 95% probability (1436 RNA molecules) is depicted by a triangle.</p

Additional file 1: of Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay

Figure S1. LD RPA primers and probe sequences aligned with the LD amplicon. One RPA exo probes (P), 8 forward primers (FPs) and 9 reverse primers (RPs) were tested to select combinations yielding the highest analytical LD RPA sensitivity. Figure S2. Field exercise in Mymensingh, Bangladesh. (A) The suitcase laboratory, where the nucleic acid extraction using the SpeedXtract kit was performed in 20 min. (B) The RPA assay was accomplished in another suitcase laboratory to avoid cross-contamination. (C) The team while screening blood samples. (D) The research team were able to operate the mobile laboratory during a power cut in the hospital because the laboratory was powered by the solar power pack. Figure S3. Mapping 100 sequences derived by BLAST nucleotide search to the LD RPA amplicon as well as RPA primers and probe. The Genbank accession number and the species of Leishmania were given. Grey represents the identical sequence. A, C, G, T were highlighted in red, violet, yellow, green, respectively, whenever a mismatch to the LD RPA amplicon was recorded. DNA sequence of L. donovani, L. infantum, L. major and L. chagasi were picked up by the BLAST search but not other leishmania species or nucleotide sequence of other pathogens. Table S1. Screening 48 samples with leishmania real-time PCR and RPA assays. Table S2. Testing clinical samples from patient hospitalized at the Surya Kanta Kala-azar Research Center in Mymensingh, Bangladesh. (DOCX 12654 kb