Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings

Oligonucleotide primers and probes.

<p>The oligonucleotides chosen for amplification and detection of IS<i>6110</i> and IS<i>1081</i> are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103091#pone-0103091-t002" target="_blank">Table 2</a>. F = dT-FAM, H = tetra hydrofuran and Q = dT-Black Hole Quencher 1.</p

Limit of detection of IS<i>6110</i> RPA and IS<i>1081</i> RPA.

<p>Results shown are for purified DNA extracted from <i>M. bovis</i> BCG. DNA concentrations were measured prior to the preparation of serial dilutions for testing by RPA. The numbers of positive test results and the total number of tests run at each concentration of DNA are recorded.</p

Tuberculosis detection by indirect smear microscopy vs RPA IS<i>6110</i>.

<p>Testing pulmonary specimens (n = 90) by indirect smear microscopy and RPA IS<i>6110</i> to detect tuberculosis, with comparison to liquid culture based test data. RPA <i>IS6110</i> was more sensitive than indirect smear microscopy (87.5% (95% CI: 81.7, 93.2) vs 70.8% (95% CI: 62.91, 78.75)) and also more specific (95.4 (95% CI: 92.3, 98.1) vs 88% (95% CI: 83.6, 92.4)).</p

DNA amplification by Recombinase Polymerase Amplification.

<p>The three core proteins, recombinase, single-strand DNA binding protein (SSB) and strand-displacing polymerase enable PCR-like DNA amplification without the need for thermal cycling or an initial chemical or thermal melting step. This diagram was created by TwistDx Ltd (<a href="http://www.twistdx.co.uk/our_technology/" target="_blank">http://www.twistdx.co.uk/our_technology/</a>) and is licensed under a Creative Commons Attribution 3.0 United States License.</p

Strains used for specificity testing.

<p>Non-tuberculous mycobacterial (NTM) strains and other bacterial species used in the determination of the specificity of the IS<i>6110</i> and IS<i>1081</i> RPA assays. All tested negative by both RPA assays. Isolates marked * were supplied by the Mycobacteriology Unit, Institute of Tropical Medicine, Antwerp, Belgium. All other isolates were from the Washington State Public Health Laboratory strain collection.</p

Real-time RPA amplification of IS<i>1081</i> and IS<i>6110</i>.

<p>Figure <b>2a</b> shows the real time detection of IS<i>1081</i> amplification from a dilution series of quantitated <i>M. bovis</i> BCG DNA with a sensitivity as low as 0.04 pg of DNA per reaction. Figure <b>2b</b> shows the real time detection of IS<i>6110</i> amplification from a dilution series of quantitated <i>M. bovis</i> BCG DNA BCG DNA with a sensitivity as low as 5.0 fg of DNA per reaction.</p