Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR

Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.     

Polymerase chain reaction for Mycobacterium tuberculosis complex DNA. Use on negative archival Ziehl-Neelsen cytologic samples

OBJECTIVE: To evaluate the usefulness of a nested polymerase chain reaction (PCR) for Mycobacterium tuberculosis complex on routinely stained cytologic samples from patients with extrapulmonary tuberculosis. STUDY DESIGN: Nested PCR for the detection of a fragment of the IS6110 insertion sequence of M tuberculosis complex was applied to Ziehl-Neelsen-negative archival cytologic slides of serous effusions (pleural [n = 7], peritoneal [n = 1] and pericardial [n = 1]) and a lymph node fine needle aspirate (n = 1) from nine human immunodeficiency virus (HIV)-positive patients with autopsy-proven active extrapulmonary tuberculosis. Malignant effusions and aspirates from nine HIV-positive patients with non-Hodgkin's lymphoma and pleural effusions from seven HIV-negative patients with heart failure were used as controls. DNA was extracted after removing the coverslip and gently scraping the cytologic sample from the slides. RESULTS: In all cases, enough DNA was obtained for PCR without any significant loss of integrity, as demonstrated by PCR positive for HLA-Dq. PCR for M tuberculosis was positive in 8 of the 10 samples (80%) from patients with tuberculosis but also in three samples (30%) from HIV-positive patients in the control group. None of the samples from the HIV-negative patients was positive. CONCLUSION: PCR for M tuberculosis can be reliably performed on archival cytologic slides from extrapulmonary samples, but although it is highly sensitive, it may lead to positive results in immunocompromised patients without any sign of active tubercular disease.     

Diagnosis of Superficial Mycoses by a Rapid and Effective PCR Method from Samples of Scales,Nails and Hair

Superficial mycoses are the most frequently diagnosed affections of the stratum corneum of the skin, nails and hair. It is generally caused by the presence of yeasts and dermatophytes. Onychomycosis is the most common infection with an incidence of 80–90% in Europe generally produced by Trichophyton rubrum. The aim of this study is to compare the traditional diagnostic techniques of superficial mycoses with a homemade and wide-spectrum fungal polymerase chain reaction (PCR) technique that amplifies a specific region of the 18S ribosomal RNA (rRNA) directly from samples of scales, nails and hair. A total of 626 clinical samples (obtained in the Basurto University Hospital, Bilbao, Spain) were analysed by traditional culture, microscopy and PCR. DNA extraction was carried out by using an extraction buffer and bovine serum, and amplification of samples and performance of the PCR were checked by conventional agarose gel electrophoresis with subsequent sequencing of amplified samples. A total of 211 samples (34%) resulted in positive diagnosis with at least one of the two applied methods: culture (21%) and PCR (22%). Despite the low percentage of identification achieved by the sequencing technique (40%), the value contributed by the amplification of the 18S region of the rRNA was considered important in the identification as it showed a high predictive values for both positive and negative diagnoses (90.9% and 94.6%, respectively). The proposed PCR method has been confirmed as a complementary, rapid, and effective method in the diagnosis of superficial mycoses. Additionally, it reduces the time to obtain satisfactory results from 4 weeks to 7 h.     

Serological,culture and molecular survey of <Emphasis Type="Italic">Mycobacterium avium paratuberculosis</Emphasis> in a goat flock in Tuscany

Mycobacterium avium paratuberculosis (Map) is a pathogen which causes a chronic progressive granulomatous enteritis known as paratuberculosis or Johne’s disease and it primarily affects wild and domestic ruminants. The aim of this research was to examine a flock which consisted of 294 goats and was located in Garfagnana district (Tuscany, Italy) performing ELISA tests, culture and IS900 PCR assay; direct diagnostic methods were carried out not only on bulk tank milk and cheese samples but also on individual milk and tissue specimens collected from nine subjects positive to ELISA tests. Out of 294 animals, 20 goats (6.8%) were positive to ELISA surveys. Bulk tank milk samples were negative to culture and to PCR assay carried out on the DNA extracted directly from them, while, with respect to cheese, Map was detected by culture in 2/12 (16.66%) cheeses ripened for 3–7 days, and by PCR in 2/12 (16.66%) cheeses ripened for 3–7 days and in 3/12 (25%) cheeses ripened for 45 days. Regarding individual milk samples, Map was detected by culture in 2/9 (22.22%) specimens and by PCR in 5/9 (55.55%) samples. Furthermore, Map was isolated from the intestine in 9/9 (100%) animals, from the mesenteric lymph nodes in 8/9 (88.88%) subjects, from the liver in 4/9 (44.44%) goats, from the spleen in 5/9 (55.55%) animals, while Map DNA was found in all the tissue samples analyzed.The results demonstrated the presence of paratuberculosis in a goat flock located in Garfagnana district (Tuscany, Italy).     

Development of multisample detection system using a tag insertion primer and an electrochemical DNA chip

We have developed a novel multisample detection system by employing a technology combining a tag insertion primer and an electrochemical DNA chip. In the first application, Helicobacter species-infected mouse samples were detected. The primers that insert a different tag sequence in each sample were prepared, and loop-mediated isothermal amplification (LAMP) reaction was carried out. Then amplification products in which a part of the sequence was different in each sample could be obtained. The target sample in which these amplification products were mixed was injected into a cassette that included the DNA chip with immobilized probes. After the cassette was set in the DNA detection system, Genelyzer, the processes of hybridization, washing, and detection were performed by the system automatically. The positive and negative concordance rates of the existing nested polymerase chain reaction (PCR) method and this method were 100% (40/40 samples) and 97.3% (117/120 samples), respectively. This is a simple high-throughput method. Moreover, the cost per sample can be drastically lowered. Therefore, it is expected to contribute to the diagnosis of infectious agents in humans and animals.     

PCR法快速检测临床标本中结核杆菌DNA

应用聚合酶链反应（ＰＣＲ）快速检测临床标本（脑脊液、胸水、腹水、血、痰液）中的结核杆菌ＤＮＡ,特异性扩增片段１２３ｂｐ,为结核杆菌的特异性重复序列ＩＳ６１１０部分基因。ＰＣＲ检测人型结核杆菌的敏感性达１０ｆｇＤＮＡ。临床标本的ＰＣＲ检测阳性率（２３．３％）明显高于抗酸染色涂片（２．９％）和细菌培养（５．７％）的阳性率（Ｐ〈０．０５）。通过设立对照系统及对扩增产物酶切分析,表明该法无假阴性结果（特异     

A Novel Method of Identifying Mycobacterium tuberculosis Beijing Strains by Detecting SNPs in Rv0444c and Rv2629

A particular genotype of tuberculosis, named Beijing strain, is strongly associated with drug resistance and high virulence. Therefore, rapid prospective identification of Mycobacterium tuberculosis Beijing strains is very important for identifying and controlling tuberculosis of Beijing genotype. In the present study, we found that the co-mutation, A191C in Rv2629 and G243C in Rv0444c, is closely related to Beijing genotype. Gene Rv2629 and Rv0444c of 139 clinical isolates of M. tuberculosis were analyzed by PCR amplification and sequencing. Among 99 Beijing strains, 86 % (n = 85) isolates had the mutation G243C in Rv0444c and 92.93 % (n = 92) isolates had the mutation A191C in Rv2629. Among 40 non-Beijing isolates, only six isolates carried the mutation G243C in Rv0444c and eight isolates carried the mutation A191C in Rv2629. The co-mutation existed in 84.85 % (n = 84) of 99 clinical genome samples of W-Beijing strains and in only 12.5 % (n = 5) of the 40 non-Beijing strains, and the positive predictive value of 94.38 %, obtained in our experiment with a designed ratio of Beijing isolates, is similar to that in China at present. This result suggested that the detection method of the co-mutation, A191C in Rv2629 and G243C in Rv0444c, proposed in this study was a rapid, reliable, and sensitive one for identifying tuberculosis with Beijing genotype.     

Comparison of Dermatophyte PCR Kit with Conventional Methods for Detection of Dermatophytes in Skin Specimens

The laboratory diagnosis of dermatophytosis is usually based on direct microscopic examination and culturing of clinical specimens. A commercial polymerase chain reaction kit (Dermatophyte PCR) has had favorable results when used for detection of dermatophytes and identification of Trichophyton rubrum in nail specimens. This study investigated the efficacy of the Dermatophyte PCR kit for detecting dermatophytosis in 191 hair or skin specimens from patients with suspected dermatophytosis. PCR was positive for 37 % of samples, whereas 31 and 39 % of the specimens were positive by culturing and direct microscopy, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for PCR analysis were 83, 84, 71, and 91 %, respectively. The sensitivity of the PCR test was higher in specimens obtained from skin (88 %) than in those obtained from hair (58 %), while the specificity remained almost the same (84 and 86 % for skin and hair, respectively). Our results show that the Dermatophyte PCR kit is a promising diagnostic tool for detection of dermatophytosis in skin samples, providing clinicians with a rapid diagnosis.     

Choosing the Best Plant for the Job: A Cost-Effective Assay to Prescreen Ancient Plant Remains Destined for Shotgun Sequencing

DNA extracted from ancient plant remains almost always contains a mixture of endogenous (that is, derived from the plant) and exogenous (derived from other sources) DNA. The exogenous ‘contaminant’ DNA, chiefly derived from microorganisms, presents significant problems for shotgun sequencing. In some samples, more than 90% of the recovered sequences are exogenous, providing limited data relevant to the sample. However, other samples have far less contamination and subsequently yield much more useful data via shotgun sequencing. Given the investment required for high-throughput sequencing, whenever multiple samples are available, it is most economical to sequence the least contaminated sample. We present an assay based on quantitative real-time PCR which estimates the relative amounts of fungal and bacterial DNA in a sample in comparison to the endogenous plant DNA. Given a collection of contextually-similar ancient plant samples, this low cost assay aids in selecting the best sample for shotgun sequencing.     

The use of the polymerase chain reaction in the diagnosis of pulmonary tuberculosis

The data on the use of the polymerase chain reaction (PCR) with primers INS1 and INS2 for the diagnosis of pulmonary tuberculosis are presented. All stages of PCR are described: from the treatment of biological material to the conditions necessary for carrying PCR and the registration of the results. Simultaneously with this reaction, PCR in a Cobas Amplicor apparatus was carried out and Mycobacterium tuberculosis culture was grown in a liquid medium in an MB/BacT apparatus. The study revealed that the method of PCR in pure M. tuberculosis culture made it possible to detect even the DNA of those cells which formed no colonies on L?wenstein--Jensen medium. The detection of M. tuberculosis in clinical samples (sputum, pleural exudate) taken from 31 patients with different pulmonary pathology showed that in 87.1% of cases diagnostics with the use of PCR carried out in a Cobas Amplicor apparatus and with primers INS1 and INS2 yielded similar results. In patients with pulmonary tuberculosis the results of PCR were positive, while the results of the analysis made with use of an MB/BacT apparatus were negative. The proposed primers INS1 and INS2, the conditions of amplification and detection make up the test system for the detection of mycobacteria of the tuberculosis complex.     

Statistical assessment of DNA extraction methodology for culture-independent analysis of microbial community associated with diverse environmental samples

Cost-effectiveness, quality, time-effectiveness and ease of the methodology are the most crucial factors in isolating quality DNA from wide variety of samples. Thus, research efforts focusing on the development of an efficient DNA extraction protocol is the need of the hour. The present study therefore, focuses on development of an efficient, rapid and free of inhibitory substances based methodology for extracting metagenomic DNA from diverse environmental samples viz. anaerobic biogas digesta, ruminant stomach, human feces, soil, and microbial starter cultures used for preparation of fermented food. PCR–DGGE based analysis and quality metagenomic library preparation, using DNA extraction methodology, validates the developed protocol. The developed protocol is cost effective, capable of isolating DNA from small sample size (100–1000 µl), time efficient (1.5–2.0 h protocol) and results in significantly higher DNA yield (4–8 times increased yield) when compared to previously available DNA extraction method and a commercial DNA extraction kit. The DNA extracted from the samples using different protocols was evaluated based on its ability to identify diverse microbial species using PCR–DGGE profiles targeting variable region within the 16S rRNA gene. The results of microbial community analysis revealed comparability of the developed protocol to commercial kits, in effectively identifying dominant representatives of the microbial community in different samples. Using the DNA extracted from the presented methodology, metagenomic libraries were prepared, which were found suitable for sequencing on Illumina platform.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA? kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

DNA persistence after treatment of Lyme borreliosis

One hundred twenty-four patients—53 with neuroborreliosis, 48 with erythema migrans, and 23 with Lyme arthritis—were tested in a prospective study for the presence of the DNA of Borrelia burgdorferi sensu lato in plasma, cerebrospinal fluid (CSF), urine, and synovial fluid by nested polymerase chain reaction (PCR). Specific DNA was detected using five amplification systems simultaneously: three targeted chromosomal genes encoding 16S rDNA, flagellin, and p66; and two plasmid sequences of OspA and OspC. Patients were examined clinically and by PCR before and after treatment and again after 3 and 6 months. Before treatment, the specific DNA was detected in 78 patients (62.9 %). Forty-one neuroborreliosis patients were DNA-positive (77.4 %), with CSF positivity in 26 patients, urine in 25, and plasma in 16. Twenty-six erythema migrans patients were DNA-positive (54.2 %), with plasma positivity in 18 cases and urine in 14. Eleven Lyme arthritis cases (47.8 %) were DNA positive (six in urine, five in plasma, and four in synovial fluid). The frequency of PCR positives was comparable in CSF and urine, and it was lower by approximately 50 % in plasma. Specific DNA was also found in a significant number of patients in later testing periods: 48 patients after treatment, 29 patients after 3 months, and 6 patients after 6 months. The prolonged PCR positivity was not explainable by persistent infection according to the clinical manifestations of the disease. Possible explanations of the problem are discussed.     

Development of a Rapid Method for Identifying Carryover Contamination of Positive Control DNA,Using a Chimeric Positive Control and Restriction Enzyme for the Diagnosis of White Spot Syndrome Virus by Nested PCR

Chimeric positive plasmids have been developed to minimize false-positive reactions caused by polymerase chain reaction (PCR) contamination. Here, we developed a rapid method for identifying false-positive results while detecting white spot syndrome virus (WSSV) by nested PCR, using chimeric positive plasmids. The results of PCRs using WSSV diagnostic primer sets showed PCR products of a similar size (WSSV 1st PCR product, 1,447 bp; WSSV 2nd PCR product, 941 bp) using WSSV chimeric plasmids or DNA from shrimp infected with WSSV. The PCR products were digested with DraI for 1 h at 37 °C. The digested chimeric DNA separated into two DNA bands; however, the WSSV-infected shrimp DNA did not separate. Thus, chimeric plasmid DNA may be used as positive control DNA instead of DNA from WSSV-infected shrimp, in order to prevent PCR contamination. Thus, the use of restriction enzyme digestion allowed us to rapidly distinguish between WSSV DNA and WSSV chimeric plasmid DNA.     

Use of Mycological, nested PCR, and Real-time PCR Methods on BAL Fluids for Detection of Aspergillus fumigatus and A. flavus in Solid Organ Transplant Recipients

Invasive aspergillosis continues to be a significant cause of morbidity and mortality in solid organ transplant (SOT) recipients. A reliable and early diagnostic method is needed to improve survival. In this study, four methods direct microscopy, culture, nested PCR on internal transcribed spacer region, and TaqMan real-time PCR targeted β-tubulin gene were examined for the detection of Aspergillus fumigatus and A. flavus in sixty-four bronchoalveolar lavage (BAL) fluids that were obtained from SOT recipients. Direct examination with 20 % KOH (potassium hydroxide) and culture on mycological media were also performed. Of the 64 samples, seven (10.9 %) were positive in direct examination (five with septate hyphae and two with aseptate hyphae), and 15 (23 %) had positive culture including five A. flavus, four A. niger, two Penicillium spp., two Rhizopus spp., one Fusarium spp. and one mixed A. flavus/A. niger. Twenty five (39 %) samples had positive nested PCR with A. flavus and 6 (9.4 %) with A. fumigatus-specific primers. Only eight (12.5 %) had positive real-time PCR for A. flavus and nine (14 %) for A. fumigatus. The incidence of aspergillosis in these patients included proven invasive pulmonary aspergillosis (IPA) in two (3 %), probable IPA in 14 (22 %), possible IPA in 38 (59 %), and not IPA in 10 (16 %). A. flavus was the most common cause of pulmonary aspergillosis (PA) in the study. The results suggest that because nested PCR is too sensitive it may increase the number of false-positive results and is not recommended for BAL samples for diagnosis of PA. Although further studies with significant number of proved positive/negative standard BAL samples are necessary for better evaluation, the novel multiplex real-time PCR developed in the study could be promising as a valid diagnostic method for IPA.     

Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure.

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their sputum samples were collected and treated with a DNA extraction procedure using silica-guanidiniumthiocyanate. This procedure has been described to be highly efficient for extraction of different kind of nucleic acids from bacteria and clinical samples. Upon comparing PCR results with the number of acid-fast bacilli, no direct relation was observed between the number of bacilli present in the sample and PCR positivity. Part of the processed samples was therefore spiked with pure DNA of M. tuberculosis and inhibition of the PCR reaction was verified in 22 out of 36 (61%) of the samples, demonstrating that the extraction procedure as originally described should not be used for PCR analysis of sputum samples.     

人结核分枝杆菌特异性核酸探针制备及结核病PCR检测技术的初步临床应用

采用人结核分枝杆菌(Mycobacterium tuberculosis TB)染色体DNA为模板,选择位于插入片段IS6110中884～865和568～588碱基对处的两个片段为引物,扩增出317bp的特异性片段．将其克隆进pUCl9载体。酶切图谱分析和DNA序列测定证实为目的片段。该片段经DIG标记,分别与11种分枝杆菌DNA进行Southern杂交,结果证明只与人型复合分枝杆菌发生杂交反应。利用该对引物建立的PcR检测拄术对74份结核病痰液标本进行检测,并与临床细菌快速培养结果相比较,发现48份临床阳性均为PcR阳性,在26份临床阴性标本中亦发现11份PCR检测阳性。将标本PCR产物与克隆探针进行杂交,显示两者结果完全一致。说明PCR检测体系结果可靠,其灵敏度明显高于目前临床所采用的方法,可作为一种常规技术用于结核病的临床检测。     

Use of propidium monoazide and increased amplicon length reduce false-positive signals in quantitative PCR for bioburden analysis

Rapid microbiological methods (RMMs) as an alternative to conventional cultivation-based bioburden analysis are receiving increasing attention although no single technology is currently able to satisfy the needs of the health care industry. Among the RMMs, quantitative PCR (qPCR) seems particularly suited. Its implementation is, however, hampered by false-positive signals originating from free DNA in PCR reagents or from dead cells in the samples to be analysed. In this study, we assessed the capability of propidium monoazide (PMA) to inactivate exogenous DNA in PCR reagents and thus to minimise its impact in bioburden analysis. PMA is a membrane-impermeant dye that intercalates into DNA and covalently binds to it upon photoactivation leading to strong inhibition of PCR amplification. PMA is currently used mainly for treatment of microbiological samples to exclude signals from membrane-compromised cells, but is also very useful for suppression of exogenous DNA signals. In addition to testing the effect of different PMA concentrations on non-template controls and target DNA, we demonstrate the effect of amplicon length on the exclusion of background amplification. Targeting a 1,108-bp 16S rRNA gene fragment using universal bacterial primers and PCR reagents treated with 5 μM PMA resulted in complete suppression of signals from exogenous DNA within 50 cycles of amplification, while a limit of detection of 10 copies of Escherichia coli genomic DNA per PCR reaction was achieved. A combined PMA treatment of sample and PCR reagents furthermore improved the selective detection of live cells making this method appear a highly attractive RMM.     

Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to detect ultra low population of Ralstonia solanacearum (Smith 1896) Yabuchi et al. (1996)

Aims:  To develop a reliable and sensitive protocol for detection of Ralstonia solanacearum using MDA-PCR (Multiple displacement amplification–PCR amplification).
Methods and Results:  MDA-PCR technique was performed on pure cell lysates as well as soil samples. Pure cell lysate as well as that of soil DNA was used as template in MDA reaction. MDA of template DNA was carried out in the presence of sample buffer, reaction buffer and enzyme mix (Φ 29 DNA polymerase and random hexamers). The MDA amplified DNA was used for PCR amplification using R. solanacearum -specific PCR primers. MDA-PCR could detect as low as 1 colony forming unit (CFU ml⁻¹) of bacteria within 8 h including DNA isolation.
Conclusion:  MDA followed by standard PCR facilitated the detection of pathogen from very low count samples. The method is of great importance in managing the brown rot disease of potato.
Significance and Impact of study:  The ultrasensitive detection technique developed in the present study is sensitive and speedy enough to be included into integrated wilt disease control programmes.     

An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.