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

Heterogeneous rpoS and rhlR mRNA Levels and 16S rRNA/rDNA (rRNA Gene) Ratios within Pseudomonas aeruginosa Biofilms, Sampled by Laser Capture Microdissection▿

The local environmental conditions in biofilms are dependent on the impinging aqueous solution, chemical diffusion, and the metabolic activities of cells within the biofilms. Chemical gradients established in biofilms lead to physiological heterogeneities in bacterial gene expression. Previously, we used laser capture microdissection (LCM) and quantitative reverse transcription (RT)-PCR to target defined biofilm subpopulations for gene expression studies. Here, we combined this approach with quantitative PCR of bacterial DNA to normalize the amount of gene expression per cell. By comparing the ratio of 16S rRNA to 16S rDNA (rRNA gene), we demonstrated that cells at the top of thick Pseudomonas aeruginosa biofilms have 16S rRNA/genome ratios similar to those of cells in a transition from the exponential phase to the stationary phase. Cells in the middle and bottom layers of these biofilms have ratios that are not significantly different from those of stationary-phase planktonic cultures. Since much of each biofilm appeared to be in a stationary-phase-like state, we analyzed the local amounts of the stationary-phase sigma factor rpoS gene and the quorum-sensing regulator rhlR gene per cell. Surprisingly, the amount of rpoS mRNA was largest at the top of the biofilms at the air-biofilm interface. Less than one rpoS mRNA transcript per cell was observed in the middle or base of the biofilms. The rhlR mRNA content was also greatest at the top of the biofilms, and there was little detectable rhlR expression at the middle or bottom of the biofilms. While the cell density was slightly greater at the bottom of the biofilms, expression of the quorum-sensing regulator occurred primarily at the top of the biofilms, where the cell metabolic activity was greatest, as indicated by local expression of the housekeeping gene acpP and by expression from a constitutive Ptrc promoter. The results indicate that in thick P. aeruginosa biofilms, cells in the 30 μm adjacent to the air-biofilm interface actively express genes associated with stationary phase, while cells in the interior portions do not express these genes and therefore are in a late-stationary-phase-like state and may be dormant

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

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

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

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

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

Surveillance of Vermont wildlife in 2021–2022 reveals no detected SARS-CoV-2 viral RNA

Abstract Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Surprisingly, we initially detected a number of N1 and/or N2 positive samples with high cycle threshold values, though after conducting environmental swabbing of the laboratory and verifying with a second independent primer set (WHO-E) and PCR without reverse transcriptase, we showed that these were false positives due to plasmid contamination from a construct expressing the N gene in the general laboratory environment. Our final results indicate that no sampled wildlife were positive for SARS-CoV-2 RNA, and highlight the importance of physically separate locations for the processing of samples for surveillance and experiments that require the use of plasmid DNA containing the target RNA sequence. These negative findings are surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American’s wildlife populations