Simon A. Weller

The Potential of TaqMan Array Cards for Detection of Multiple Biological Agents by Real-Time PCR

The TaqMan Array Card architecture, normally used for gene expression studies, was evaluated for its potential to detect multiple bacterial agents by real-time PCR. Ten PCR assays targeting five biological agents (Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis) were incorporated onto Array Cards. A comparison of PCR performance of each PCR in Array Card and singleplex format was conducted using DNA extracted from pure bacterial cultures. When 100 fg of agent DNA was added to Array Card channels the following levels of agent detection (where at least one agent PCR replicate returned a positive result) were observed: Y. pestis 100%, B. mallei & F. tularensis 93%; B. anthracis 71%; B. pseudomallei 43%. For B. mallei & pseudomallei detection the BPM2 PCR, which detects both species, outperformed PCR assays specific to each organism indicating identification of the respective species would not be reproducible at the 100 fg level. Near 100% levels of detection were observed when 100 fg of DNA was added to each PCR in singleplex format with singleplex PCRs also returning sporadic positives at the 10 fg per PCR level. Before evaluating the use of Array Cards for the testing of environmental and clinical sample types, with potential levels of background DNA and PCR inhibitors, users would therefore have to accept a 10-fold reduction in sensitivity of PCR assays on the Array Card format, in order to benefit for the capacity to test multiple samples for multiple agents. A two PCR per agent strategy would allow the testing of 7 samples for the presence of 11 biological agents or 3 samples for 23 biological agents per card (with negative control channels).

Buffer AVL alone does not inactivate Ebola virus in a representative clinical sample type

ABSTRACT Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 108 50% tissue culture infective dose per milliliter [TCID50 · ml−1]) and murine blood (EBOV concentration of 1 × 107 TCID50 · ml−1) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples.

Evaluation of two multiplex real-time PCR screening capabilities for the detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples generated from murine infection models.

Two multiplex PCR screening capabilities (TaqMan Array Cards and FilmArray) were evaluated for their ability to detect Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples obtained from respective murine infection models. Blood samples were obtained from infected mice at 24 h intervals after exposure. Multiplex PCR results were compared with standard blood culture and singleplex real-time PCR. Across all three models, 71 mice were tested in total, within which a subset of 43 samples was shown to contain an infecting agent by at least one of the detection technologies. Within this subset of positive samples, for each model studied, the detection rates of each technology were compared. The B. anthracis model blood culture (14 of 15 agent-containing samples tested) and FilmArray PCR (12 of 15) were shown to have equivalent detection rates, which were significantly higher (at the 95 % confidence level) than singleplex (five of 14) or Array Card (two of 14) PCRs. The F. tularensis model blood culture (12 of 12) was shown to have a significantly higher (at 95 % confidence level) detection rate than all PCR technologies, with FilmArray (seven of 11) and singleplex (seven of 12) PCRs shown to have significantly higher (at 95 % confidence level) detection rates than the Array Card PCR (two of 11). Within the Y. pestis model, there was no significant difference in detection rates between blood culture (10 of 16), singleplex PCR (14 of 16), Array Card PCR (10 of 16) and FilmArray PCR (10 of 13).

Comparison of eight methods for the extraction of Bacillus atrophaeus spore DNA from eleven common interferents and a common swab.

Eight DNA extraction products or methods (Applied Biosystems PrepFiler Forensic DNA Extraction Kit; Bio-Rad Instagene Only, Bio-Rad Instagene & Spin Column Purification; EpiCentre MasterPure DNA & RNA Kit; FujiFilm QuickGene Mini80; Idaho Technologies 1-2-3 Q-Flow Kit; MoBio UltraClean Microbial DNA Isolation Kit; Sigma Extract-N-Amp Plant and Seed Kit) were adapted to facilitate extraction of DNA under BSL3 containment conditions. DNA was extracted from 12 common interferents or sample types, spiked with spores of Bacillus atropheaus. Resulting extracts were tested by real-time PCR. No one method was the best, in terms of DNA extraction, across all sample types. Statistical analysis indicated that the PrepFiler method was the best method from six dry powders (baking, biological washing, milk, plain flour, filler and talcum) and one solid (Underarm deodorant), the UltraClean method was the best from four liquids (aftershave, cola, nutrient broth, vinegar), and the MasterPure method was the best from the swab sample type. The best overall method, in terms of DNA extraction, across all sample types evaluated was the UltraClean method.

Development of real-time PCR assays for the specific detection of Francisella tularensis ssp. tularensis, holarctica and mediaasiatica

Real-time polymerase chain reaction (PCR) assays were developed to detect Francisella tularensis (Ft), the causative agent of tularaemia in humans. Two real-time PCRs (FTT0376 and FTT0523) were designed in genetic sequences identified by the Insignia genome comparison tool (http://insignia.cbcb.umd.edu/) as being unique to pathogenic subspecies of F. tularensis. Both PCRs identified all pathogenic F. tularensis subspecies but did not cross react with avirulent Francisella philomiragia or F. tularensis ssp. novicida or other environmental bacteria. Limits of detection from DNA purified from pure culture (FTT0376 approximately 80 Ft genome equivalents (GEs) per PCR; FTT0523 approximately 20 Ft GEs per PCR;) and DNA purified from spiked blood samples (4 x 10(4) to 4 x 10(3) cfu ml(-1), both assays) were determined.

Using Surface-Enhanced Raman Spectroscopy and Electrochemically Driven Melting to Discriminate Yersinia pestis from Y. pseudotuberculosis Based on Single Nucleotide Polymorphisms within Unpurified Polymerase Chain Reaction Amplicons

The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

Influence of the length of target DNA overhang proximal to the array surface on discrimination of single-base mismatches on a 25-mer oligonucleotide array

BackgroundThe performance of probes on an oligonucleotide microarray can be characterised in terms of hybridisation signal strength and the ability to discriminate sequence mismatches between the probe and the hybridising target strand, such as those resulting from SNPs. Various properties of the probe affect mismatch discrimination, such as probe length and the position of mismatched bases, and the effects of these factors have been well characterised in a variety of array formats.ResultsA low-density microarray was developed to systematically investigate the effect of a probe’s position within hybridised target PCR products on the tolerance and discrimination of single-nucleotide mismatches between the probe and target. In line with previous reports, hybridisation signals were attenuated by different degrees depending on the identity of the mismatch, the position of the mismatch within the probe, and the length of the PCR product. However, the same mismatch caused different degrees of attenuation depending on the position of the probe within the hybridising product, such that improved mismatch discrimination was observed for PCR products where a greater proportion of the total length was proximal to the array surface.ConclusionsThese results suggest that the degree of mismatch discrimination can be influenced by the choice of PCR primers, providing a means by which array performance could be fine-tuned in addition to manipulation of the properties of the probes themselves.

Cross-institute evaluations of inhibitor-resistant PCR reagents for direct testing of aerosol and blood samples containing biological warfare agent DNA.

ABSTRACT Rapid pathogen detection is crucial for the timely introduction of therapeutics. Two groups (one in the United Kingdom and one in the United States) independently evaluated inhibitor-resistant PCR reagents for the direct testing of substrates. In the United Kingdom, a multiplexed Bacillus anthracis (target) and Bacillus subtilis (internal-control) PCR was used to evaluate 4 reagents against 5 PCR inhibitors and down-selected the TaqMan Fast Virus 1-Step master mix (Life Technologies Inc.). In the United States, four real-time PCR assays (targeting B. anthracis, Brucella melitensis, Venezuelan equine encephalitis virus [VEEV], and Orthopoxvirus spp.) were used to evaluate 5 reagents (plus the Fast Virus master mix) against buffer, blood, and soil samples and down-selected the KAPA Blood Direct master mix (KAPA Biosystems Inc.) with added Platinum Taq (Life Technologies). The down-selected reagents underwent further testing. In the United Kingdom experiments, both reagents were tested against seven contrived aerosol collector samples containing B. anthracis Ames DNA and B. subtilis spores from a commercial formulation (BioBall). In PCR assays with reaction mixtures containing 40% crude sample, an airfield-collected sample induced inhibition of the B. subtilis PCR with the KAPA reagent and complete failure of both PCRs with the Fast Virus reagent. However, both reagents allowed successful PCR for all other samples—which inhibited PCRs with a non-inhibitor-resistant reagent. In the United States, a cross-assay limit-of-detection (LoD) study in blood was conducted. The KAPA Blood Direct reagent allowed the detection of agent DNA (by four PCRs) at higher concentrations of blood in the reaction mixture (2.5%) than the Fast Virus reagent (0.5%), although LoDs differed between assays and reagent combinations. Across both groups, the KAPA Blood Direct reagent was determined to be the optimal reagent for inhibition relief in PCR.

Development of Three Real-Time PCR assays to Detect Bacillus anthracis and Assessment of Diagnostic Utility

Three real-time PCR assays to detect Bacillus anthracis genetic targets (pXO1; pXO2 and chromosome) were developed. Two of the PCR assays (pXO1-MGB and Ba chr-MGB) were tested against DNA extracts produced from whole blood samples obtained from a replicated B. anthracis murine infection model. Across all three models 45 samples were tested in total, within which a subset of 41 samples were shown to contain B. anthracis by either PCR or microbiological culture. Using microbiological culture as an analogue of conventional blood culture (as used in clinical settings) the detection rates of PCR and blood culture were compared. In two of the murine models blood culture had a significantly higher detection rate than PCR (BA1, p=0.004; BA3, p=0.013). In the BA2 model there was no significant difference between the detection rates of PCR and blood culture.

Development and Operation of Ebola Diagnostic Laboratories Led By Public Health England in Sierra Leone during the West African Ebola Outbreak 2013-2015

The Ebola outbreak 2013-2015 created an urgent need for humanitarian response. Public Health England (PHE), in partnership with the Ministry of Defence (MoD) and Defence Science and Technology Laboratory (DSTL), were tasked by the UK Government (through the Department for International Development (DfID)) to provide Ebola Virus Disease (EVD) diagnostic laboratories. These diagnostic laboratories supported the Ebola Treatment Units (ETU) being established in Sierra Leone. PHE operated arguably the largest diagnostic facilities in Sierra Leone by one unilateral donor: operating 3 laboratories (co-located with ETUs), each processing up to 200 samples a day. During the time of operation (October 2014 to December 2015) over 400 civilian UK staff on rotation were deployed in these laboratories, and between them processed greater than 40,000 samples (~6% positivity rate). Here we summarise the laboratory set-up, design rationale, scope and processes deployed. This information can inform planning in response to future outbreaks. Correspondence to: Daniel Bailey, Public Health England, Porton Down, Salisbury SP4 0JG, UK, Tel: +44 (0)1980 619913; E-mail: Daniel.Bailey@phe.gov.uk