Ambient Stable Quantitative PCR Reagents for the Detection of Yersinia pestis

Background

Although assays for detecting Yersinia pestis using TaqMan probe-based real-time PCR have been developed for years, little is reported on room-temperature-stable PCR reagents, which will be invaluable for field epidemic surveillance, immediate response to public health emergencies, counter-bioterrorism investigation, etc. In this work, a set of real-time PCR reagents for rapid detection of Y. pestis was developed with extraordinary stability at 37°C.

Methods/Principal Findings

TaqMan-based real-time PCR assays were developed using the primers and probes targeting the 3a sequence in the chromosome and the F1 antigen gene caf1 in the plasmid pMT1of Y. pestis, respectively. Then, carbohydrate mixtures were added to the PCR reagents, which were later vacuum-dried for stability evaluation. The vacuum-dried reagents were stable at 37°C for at least 49 days for a lower concentration of template DNA (10 copies/µl), and up to 79 days for higher concentrations (≥10² copies/µl). The reagents were used subsequently to detect soil samples spiked with Y. pestis vaccine strain EV76, and 5×10⁴ CFU per gram of soil could be detected by both 3a- and caf1-based PCR reagents. In addition, a simple and efficient method for soil sample processing is presented here.

Conclusions/Significance

The vacuum-dried reagents for real-time PCR maintain accuracy and reproducibility for at least 49 days at 37°C, indicating that they can be easily transported at room temperature for field application if the machine for performing real-time PCR is available. This dry reagent is of great significance for routine plague surveillance.     

Diagnostic Performance of Schistosoma Real-Time PCR in Urine Samples from Kenyan Children Infected with Schistosoma haematobium: Day-to-day Variation and Follow-up after Praziquantel Treatment

Background

In an effort to enhance accuracy of diagnosis of Schistosoma haematobium, this study explores day-to-day variability and diagnostic performance of real-time PCR for detection and quantification of Schistosoma DNA compared to other diagnostic tools in an endemic area before and after treatment.

Methodology

Previously collected urine samples (N = 390) from 114 preselected proven parasitological and/or clinical S. haematobium positive Kenyan schoolchildren were analyzed by a Schistosoma internal transcribed spacer-based real-time PCR after 14 years of storage. Pre-treatment day-to-day fluctuations of PCR and microscopy over three consecutive days were measured for 24 children using intra-class correlation coefficient. A combined ‘gold standard’ (PCR and/or microscopy positive) was used to measure sensitivity and negative predictive value (NPV) of several diagnostic tools at baseline, two and 18 months post-treatment with praziquantel.

Principal Findings

All 24 repeatedly tested children were PCR-positive over three days with little daily variation in median Ct-values, while 83.3% were found to be egg-positive for S. haematobium at day 1 and 75.0% at day 2 and 3 pre-treatment, signifying daily fluctuations in microscopy diagnosis. Of all 114 preselected schoolchildren, repeated microscopic measurements were required to detect 96.5% versus 100% of positive pre-treatment cases by single PCR. At two months post-treatment, microscopy and PCR detected 22.8% versus 69.3% positive children, respectively. Based on the ‘gold standard’, PCR showed high sensitivity (>92%) as compared to >31% sensitivity for microscopy, both pre- and post-treatment.

Conclusions/Significance

Detection and quantification of Schistosoma DNA in urine by real-time PCR was shown to be a powerful and specific diagnostic tool for detection of S. haematobium infections, with less day-to-day variation and higher sensitivity compared to microscopy. The superior performance of PCR before, and two and 18 months post-treatment provides a compelling argument for PCR as an accurate and reproducible tool for monitoring treatment efficacy.     

Loop Mediated Isothermal Amplification (LAMP) Accurately Detects Malaria DNA from Filter Paper Blood Samples of Low Density Parasitaemias

Background

Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots.

Methods and Findings

Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6–782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94–99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0–4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1–98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2–99.9) and 76.9% (95%CI 46.2–95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1–100) in both study groups.

Conclusion

Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low-density malaria infections in pre-elimination settings.     

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

Background

Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics.

Methodology/Principal Findings

The objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10³ CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR.

Conclusions/Significance

Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.     

Impact of glucose levels on expression of hypha-associated secreted aspartyl proteinases in Candida albicans

Background

Ten secreted aspartyl proteinase (Sap) genes were identified in Candida albicans. The products of SAP genes are considered to be virulent factors of C. albicans that participated in causing mucocutaneous and systemic candidiasis in humans. Depending on environmental conditions, C. albicans may stay in yeast-form or convert into invasive hypha-form, and these issues may affect the expression of SAP genes. In this study we explored the component(s) of culture media that may affect the expression of hypha-associated SAP genes.

Results

We demonstrate that glucose levels modulate both the hyphae development and the expression strength of hypha-associated SAP genes (SAP4-6). In contrast to high glucose concentration (2%), lower glucose level (0.1%) is more potent to promote hyphae development and to promptly elicit the expression of hypha-associated Sap proteins during yeast-to-hypha transition of C. albicans. Both Cph1-mediated MAP kinase cascade and Efg1-mediated cAMP/PKA pathway, although the latter seemed dominant, participate in convey the glucose signaling to regulate the expression of hypha-associated SAP genes and this glucose level effect may perform at very early stage of yeast-to-hypha transition. In addition, when C. albicans was co-cultured with THP-1 human monocytes, the engulfed C. albicans was developing hypha efficiently within 1 hr and the expression of hypha-associated Sap proteins could be detected on the distal surface of hyphae.

Conclusion

We propose that the glucose level of bloodstream (approximately 0.1%) may be facilitated for stimulation of C. albicans to develop invasive hypha-form and to elicit promptly production of high-level hypha-associated Sap proteins.     

Optimized Pan-species and Speciation Duplex Real-time PCR Assays for Plasmodium Parasites Detection in Malaria Vectors

Background

An accurate method for detecting malaria parasites in the mosquito’s vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus.

Methods

Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin.

Results

The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was “excellent” (κ = 0.8, P<0.05). The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P = 0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed.

Conclusion

This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the utility of employing an accurate molecular diagnostic tool for detecting malaria parasites in field mosquito populations.     

Abbott RealTime HBV Assay Is More Sensitive in Detection of Low Viral Load and Little Impacted by Drug Resistant Mutation in Chronic Hepatitis B Patients under Nucleot(s)ide Analogues Therapy

Background

Selection of drug-resistant strains may lead to failure of HBV antiviral therapy. There is little information whether there is detection difference in drug resistant mutations between different viral load assays of HBV.

Objectives

This study is aimed to investigate whether there is drug-resistant strains related detection difference between Abbott RealTime HBV (RealTime) and CobasAmpliPrep/CobasTaqMan HBV assays 2.0 (TaqMan).

Study Design

One hundred and thirty-four CHB patients who received HBV anti-viral therapy were enrolled. HBV virological markers were tested 3 months apart regularly. Serum HBV DNA levels were determined using the TaqMan and RealTime. YMDD (rt180M and rt204V) mutation was checked in patients who experienced virologic breakthrough (VBT).

Results

The correlation of HBV DNA observed between the RealTime and TaqMan was good for all 571 samples (R² = 0.797; P<0.001). However, the correlation in the 434 samples with HBV DNA level <3 log₁₀ IU/ml was not as good as in all samples (R² = 0.457). Overall, 21.5% of samples had a detection difference of ≥1 log₁₀ IU/ml with 91.9% of these having HBV DNA level <3 log₁₀ IU/ml. Twenty-four patients experiencedVBT. Three of these patients had acquired the YMDD mutation and exhibited discordant viral load results between the two methods tested. In each case, persistent HBV DNA was detected by RealTime and undetectable with TaqMan. Of the patients who experienced a VBT and had acquired YMDD mutation, 4.7% had undetectable HBV DNA by TaqMan while all were detectable with RealTime.

Conclusions

RealTime assay is more sensitive and is little impacted by the development of drug resistant mutation.     

Hepatitis B Virus Infection Does Not Significantly Influence Plasmodium Parasite Density in Asymptomatic Infections in Ghanaian Transfusion Recipients

Background

Areas endemic for malaria and Hepatitis B virus (HBV) infection largely overlap geographically. A recent study has suggested the existence of an interaction between the two pathogens in symptomatic co-infected individuals on the South-American continent. We examined this issue in a hyperendemic area for both pathogens in sub-Saharan Africa.

Methodology and Findings

Pre-transfusion samples from a retrospective cohort of 154 blood transfusion recipients were screened for both serological and molecular markers of HBV and Plasmodium genomes using species-specific nested PCR and quantitative real-time PCR. Thirty-seven individuals met exclusion criteria and were subsequently eliminated from further analysis. Of 117 participants, 90% of recipients exhibited evidence of exposure to HBV, 42% with HBsAg and/or HBV DNA and 48% anti-HBc reactive without detectable HBV DNA. Plasmodium genome prevalence by NAT was 50%. Parasitemic individuals were significantly younger than non-parasitemic individuals (P = 0.04). Parasitemia level was not significantly lower in individuals with HBV DNA positive infections compared to those with HBV DNA negative exposures. HBV DNA load was not significantly different in parasitemic and non-parasitemic individuals.

Conclusion

The data presented suggests that, in sub-Saharan Africa, asymptomatic co-infections with these two ubiquitous pathogens do not appear to significantly affect each other and evolve independently.     

High throughput, multiplexed pathogen detection authenticates plague waves in medieval Venice, Italy

Background

Historical records suggest that multiple burial sites from the 14th–16^th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics.

Methodology/Principal Findings

High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype.

Conclusions

These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.     

Prevalence of Bloodstream Pathogens Is Higher in Neonatal Encephalopathy Cases vs. Controls Using a Novel Panel of Real-Time PCR Assays

Background

In neonatal encephalopathy (NE), infectious co-morbidity is difficult to diagnose accurately, but may increase the vulnerability of the developing brain to hypoxia-ischemia. We developed a novel panel of species-specific real-time PCR assays to identify bloodstream pathogens amongst newborns with and without NE in Uganda.

Methodology

Multiplex real-time PCR assays for important neonatal bloodstream pathogens (gram positive and gram negative bacteria, cytomegalovirus (CMV), herpes simplex virus(HSV) and P. falciparum) were performed on whole blood taken from 202 encephalopathic and 101 control infants. Automated blood culture (BACTEC) was performed for all cases and unwell controls.

Principal Findings

Prevalence of pathogenic bacterial species amongst infants with NE was 3.6%, 6.9% and 8.9%, with culture, PCR and both tests in combination, respectively. More encephalopathic infants than controls had pathogenic bacterial species detected (8.9%vs2.0%, p = 0.028) using culture and PCR in combination. PCR detected bacteremia in 11 culture negative encephalopathic infants (3 Group B Streptococcus, 1 Group A Streptococcus, 1 Staphylococcus aureus and 6 Enterobacteriacae). Coagulase negative staphylococcus, frequently detected by PCR amongst case and control infants, was considered a contaminant. Prevalence of CMV, HSV and malaria amongst cases was low (1.5%, 0.5% and 0.5%, respectively).

Conclusion/Significance

This real-time PCR panel detected more bacteremia than culture alone and provides a novel tool for detection of neonatal bloodstream pathogens that may be applied across a range of clinical situations and settings. Significantly more encephalopathic infants than controls had pathogenic bacterial species detected suggesting that infection may be an important risk factor for NE in this setting.     

Candida albicans possesses Sap7 as a pepstatin A-insensitive secreted aspartic protease

Background

Candida albicans, a commensal organism, is a part of the normal flora of healthy individuals. However, once the host immunity is compromised, C. albicans opportunistically causes recurrent superficial or fatal systemic candidiasis. Secreted aspartic proteases (Sap), encoded by 10 types of SAP genes, have been suggested to contribute to various virulence processes. Thus, it is important to elucidate their biochemical properties for better understanding of the molecular mechanisms that how Sap isozymes damage host tissues.

Methodology/Principal Findings

The SAP7 gene was cloned from C. albicans SC5314 and heterogeneously produced by Pichia pastoris. Measurement of Sap7 proteolytic activity using the FRETS-25Ala library showed that Sap7 was a pepstatin A-insensitive protease. To understand why Sap7 was insensitive to pepstatin A, alanine substitution mutants of Sap7 were constructed. We found that M242A and T467A mutants had normal proteolytic activity and sensitivity to pepstatin A. M242 and T467 were located in close proximity to the entrance to an active site, and alanine substitution at these positions widened the entrance. Our results suggest that this alteration might allow increased accessibility of pepstatin A to the active site. This inference was supported by the observation that the T467A mutant has stronger proteolytic activity than the wild type.

Conclusions/Significance

We found that Sap7 was a pepstatin A-insensitive protease, and that M242 and T467 restricted the accessibility of pepstatin A to the active site. This finding will lead to the development of a novel protease inhibitor beyond pepstatin A. Such a novel inhibitor will be an important research tool as well as pharmaceutical agent for patients suffering from candidiasis.     

C. albicans colonization of human mucosal surfaces

Background

Candida albicans is a low level commensal organism in normal human populations with the continuous potential to expand and cause a spectrum of clinical conditions.

Methodology/Principal Findings

Using ex vivo human organ cultures and populations of primary human cells, we have developed several related experimental systems to examine early-stage interactions between C. albicans and mucosal surfaces. Experiments have been conducted both with exogenously added C. albicans and with overtly normal human mucosal surfaces supporting pre-existing infections with natural isolates of Candida. Under different culture conditions, we have demonstrated the formation of C. albicans colonies on human target cells and filament formation, equivalent to tissue invasion.

Conclusions/Significance

These organ culture systems provide a valuable new resource to examine the molecular and cellular basis for Candida colonization of human mucosal surfaces.     

Occult Hepatitis B Virus Infection in Anti-HBs-Positive Infants Born to HBsAg-Positive Mothers in China

Objective

To investigate the prevalence of occult HBV infection (OBI) among children and to characterize virology of occult HBV, we conducted an epidemiological survey.

Methods

186 HB-vaccinated infants born to HBsAg-positive mothers were included in the study. Serological tests for HBV markers were performed using commercial ELISA kits. Real-time quantitative PCR and nested PCR were used to detect HBV DNA. PCR products of the C and pre-S/S regions were sequenced and analyzed.

Results

1.61% (3/186) infants were HBsAg positive, and 4.92% (9/183) infants were considered as occult infection. The viral load of mothers was associated with occult infection (P = 0.020). Incomplete three-dose injections of HB vaccine was associated with HBV infection (P = 0.022). Six OBI infants were positive for anti-HBs, but their titers were not greater than 100 mIU/mL. Seven isolated HBV pre-S/S sequences were obtained from nine OBI infants. Three of the sequences were genotype C, and four of the sequences were genotype C/D. Escape mutation S143L was found in the four sequences of genotype C/D. All seven sequences lacked G145R and other escape mutation in S region.

Conclusions

Occult HBV infection was detected in anti-HBs positive infants born to HBsAg-positive mothers in China. Occult infection was associated with absent anti-HBs or with low anti-HBs level, high maternal viral loads and escape mutations in the S gene.     

Rapid qualitative urinary tract infection pathogen identification by SeptiFast real-time PCR

Background

Urinary tract infections (UTI) are frequent in outpatients. Fast pathogen identification is mandatory for shortening the time of discomfort and preventing serious complications. Urine culture needs up to 48 hours until pathogen identification. Consequently, the initial antibiotic regimen is empirical.

Aim

To evaluate the feasibility of qualitative urine pathogen identification by a commercially available real-time PCR blood pathogen test (SeptiFast^®) and to compare the results with dipslide and microbiological culture.

Design of study

Pilot study with prospectively collected urine samples.

Setting

University hospital.

Methods

82 prospectively collected urine samples from 81 patients with suspected UTI were included. Dipslide urine culture was followed by microbiological pathogen identification in dipslide positive samples. In parallel, qualitative DNA based pathogen identification (SeptiFast^®) was performed in all samples.

Results

61 samples were SeptiFast^® positive, whereas 67 samples were dipslide culture positive. The inter-methodological concordance of positive and negative findings in the gram+, gram- and fungi sector was 371/410 (90%), 477/492 (97%) and 238/246 (97%), respectively. Sensitivity and specificity of the SeptiFast^® test for the detection of an infection was 0.82 and 0.60, respectively. SeptiFast^® pathogen identifications were available at least 43 hours prior to culture results.

Conclusion

The SeptiFast^® platform identified bacterial DNA in urine specimens considerably faster compared to conventional culture. For UTI diagnosis sensitivity and specificity is limited by its present qualitative setup which does not allow pathogen quantification. Future quantitative assays may hold promise for PCR based UTI pathogen identification as a supplementation of conventional culture methods.     

Pre-Analytical Conditions in Non-Invasive Prenatal Testing of Cell-Free Fetal RHD

Background

Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the administration of antenatal anti-D prophylaxis only to women who carry an RhD positive fetus. Prophylaxis reduces the risk of immunization that may lead to hemolytic disease of the fetus and the newborn. The reliability of predicting the fetal RhD type depends on pre-analytical factors and assay sensitivity. We evaluated the testing setup in the Capital Region of Denmark, based on data from routine antenatal RHD screening.

Methods

Blood samples were drawn at gestational age 25 weeks. DNA extracted from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon 7/10. We investigated the effect of blood sample transportation time (n = 110) and ambient outdoor temperatures (n = 1539) on the levels of cffDNA and total DNA. We compared two different quantification methods, the delta Ct method and a universal standard curve. PCR pipetting was compared on two systems (n = 104).

Results

The cffDNA level was unaffected by blood sample transportation for up to 9 days and by ambient outdoor temperatures ranging from -10°C to 28°C during transport. The universal standard curve was applicable for cffDNA quantification. Identical levels of cffDNA were observed using the two automated PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median gestational age of 25 weeks (range 10–39, n = 1317).

Conclusion

The setup for real-time PCR-based, non-invasive prenatal testing of cffDNA in the Capital Region of Denmark is very robust. Our findings regarding the transportation of blood samples demonstrate the high stability of cffDNA. The applicability of a universal standard curve facilitates easy cffDNA quantification.     

Assessment of Tracer 99mTc(V)-DMSA Uptake as a Measure of Tumor Cell Proliferation In Vitro

Purpose

To examine whether ^99mTc(V)-DMSA could be used as a non-invasive measure of cancer cell proliferation.

Methods

Human breast cancer MCF-7, MDA-MB-231 and pII, and prostate cancer PC-3 cell lines were grown to 30, 50 and 100% confluency and pulsed with ^99mTc(V)-DMSA in media for 60 min at 37°C. DNA synthesis was analysed by quantification of the S phase using flow cytometry, [methyl-³H]thymidine incorporation and expression of proliferation markers PCNA and Ki-67 using realtime PCR. One way ANOVA was used to compare groups.

Results

In all cell lines rates of ^99mTc(V)-DMSA uptake were inversely related to cell density. This was paralleled by similar trends in S phase proportions, [methyl-³H]thymidine incorporation and expression of PCNA and Ki-67.

Conclusion

Rates of ^99mTc(V)-DMSA uptake into different types of tumour cells correlate well with cell density that is useful as a non-invasive measure of tumour cellular proliferation in vivo.     

Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition

Background

Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.

Methodology/Principal Findings

We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities.

Conclusions

M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×10⁵ cells g⁻¹ using Griffiths and 4.25×10⁶ cells g⁻¹ using FastDNA® Spin kit.