The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification

This paper describes a novel approach, termed the 'phage amplification assay', for the rapid detection and identification of specific bacteria. The technique is based on the phage lytic cycle with plaque formation as the assay end-point. It is highly sensitive, quantitative and gives results typically within 4 h. The assay comprises four main stages : (1) phage infection of target bacterium ; (2) destruction of exogenous phage ; (3) amplification of phage within infected host and (4) plaque formation from infected host with the aid of helper bacteria. A key component of this assay is a potent virucidal agent derived from natural plant extracts, pomegranate rind extract (PRE). In combination with ferrous sulphate PRE can bring about an 11 log-cycle reduction in phage titre within 3 min. This is achieved without any injury to the infected target bacteria. Subsequently, any resulting plaques are derived only from infected target organisms. Data are presented for a range of bacterial hosts including Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus. The detection limit for Ps. aeruginosa was 40 bacteria ml⁻¹ in a time of 4 h and 600 bacteria m⁻¹ for Salm. typhimurium. Application of the principles of this technology to other bacterial genera is discussed.     

Viable Staphylococcus aureus quantitation using ¹⁵N metabolically labeled bacteriophage amplification coupled with a multiple reaction monitoring proteomic workflow

A multiple reaction monitoring liquid chromatography method with tandem mass spectrometric detection for quantitation of Staphylococcus aureus via phage amplification detection is described. This phage amplification detection method enables rapid and accurate quantitation of viable S. aureus by detecting an amplified capsid protein from a specific phage. A known amount of metabolically labeled (15)N reference bacteriophage, utilized as the input phage and as the internal standard for quantitation, was spiked into S. aureus samples. Following a 2-h incubation, the sample was subjected to a 3-min rapid trypsin digest and analyzed by high-throughput liquid chromatography tandem mass spectrometric detection targeting peptides unique to both the (15)N (input phage) and (14)N (progeny phage) capsid proteins. Quantitation was achieved by comparing peak areas of target peptides from the metabolically labeled (15)N bacteriophage peptide internal standard with that of the wild-type (14)N peptides that were produced by phage amplification and subsequent digestion when the host bacteria was present. This approach is based on the fact that a labeled species differs from the unlabeled one in terms of its mass but exhibits almost identical chemical properties such as ion yields and retention times. A 6-point calibration curve for S. aureus concentration was constructed with standards ranging from 5.0 × 10(4) colony forming units (CFU) ml(-1) to 2.0 × 10(6) CFU ml(-1), with the (15)N reference phage spiked at a concentration of 1.0 × 10(9) plaque forming units (PFU) ml(-1). Amplification with (15)N bacteriophage coupled with LC-MS/MS detection offers speed (3 h total analysis time), sensitivity (LOD: < 5.0 × 10(4) CFU ml(-1)), accuracy, and precision for quantitation of S. aureus.     

Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef

AIM: To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef. METHODS AND RESULTS: Fresh ground beef samples were experimentally inoculated with varying concentrations of E. coli O157:H7. PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation, and lysed using enzymatic digestion and successive freeze/thaw cycles. DNA was purified and concentrated via phenol/chloroform extraction and the Shiga toxin 1 gene (stx1) was amplified using PCR to evaluate the sample preparation method. Without prior enrichment of cells in broth media, the detection limit was 103 CFU g-1 beef. When a 6 h enrichment step was incorporated, the detection limit was 1 CFU g-1 beef. The total time required from beginning to end of the procedure was 12 h. CONCLUSIONS: The sample preparation method developed here enabled substantially improved sensitivity in the PCR-based detection of E. coli O157:H7 in ground beef, as compared to previous reports. SIGNIFICANCE AND IMPACT OF THE STUDY: Superb sensitivity, coupled with quick turn-around time, relative ease of use and cost-effectiveness, makes this a useful method for detecting E. coli O157:H7 in ground beef.     

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents

AIM: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. METHODS AND RESULTS: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. CONCLUSIONS: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. SIGNIFICANCE AND IMPACT OF STUDY: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.     

Influence of phage population on the phage-mediated bioluminescent adenylate kinase (AK) assay for detection of bacteria

AIMS: The effect of phage concentration on the activity of adenylate kinase (AK) released from the cells lysed during infection was investigated in order to optimize a bioluminescent phage-mediated method for bacterial enumeration. METHODS AND RESULTS: The number of bacteria lysed by phages specific to Salmonella enteritidis and E. coli was determined using a bioluminescent method for the detection of AK released. In order to optimize the assay, the effect of phage concentration and time of infection on the amount of AK released was investigated. The release of AK was greatest at a multiplicity of infection (moi) of 10-100. CONCLUSION: The amount of AK released from Salmonella enteritidis and E. coli G2-2 cells by specific phages, SJ2 and AT20, respectively, depended on the type of bacteria, the stage of growth, the nature of phage, moi and time. SIGNIFICANCE AND IMPACT OF THE STUDY: An assay is described which allows detection of E. coli and Salmonella Enteritidis within 2 h at levels of 103 cfu ml-1.     

Rapid monitoring of microbial contamination on herbal medicines by fluorescent staining method

AIMS: To apply fluorescent staining method for fast assessment of microbial quality of herbal medicines. METHODS AND RESULTS: The number of total bacteria and esterase-active bacteria on powdered traditional Chinese medicines were enumerated by fluorescent staining method using 6-carboxyfluorescein diacetate (6CFDA) and 4',6-diamidino-2-phenylindole (DAPI), and they were compared with colony-forming units (CFU). The CFU was approximately 10(3) per gram in ginseng radix, and no bacterial colonies were detected from others. However, the total bacterial number (TDC) was more than 10(7) per gram, and number of bacteria possessing esterase activity ranged from 1 to 3% of TDC. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Many bacteria in each Chinese medicine had enzyme activity and most of them could not be detected by conventional plate counting technique. Enumeration of bacterial cells on traditional Chinese medicines by fluorescent staining method requires less than 1 h. The double staining method with 6CFDA and DAPI could be applicable to rapid microbial monitoring of crude drugs.     

Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction

Aims: A new real‐time polymerase chain reaction‐based method was developed for the detection of Salmonella enterica in food. Methods and Results: The method consisted of a novel two‐step enrichment involving overnight incubation in buffered peptone water and a 5‐h subculture in Rappaport–Vassiliadis medium, lysis of bacterial cells and a Salmonella‐specific 5′‐nuclease real‐time PCR with an exogenous internal amplification control. Because a two‐step enrichment was used, the detection limit for dead S. enterica cells in artificially contaminated ice cream and salami samples was high at 10⁷ CFU (25 g)^?1, eliminating potential false‐positive results. When the method was evaluated with a range of 100 naturally contaminated food samples, three positive samples were detected by both the real‐time PCR‐based method and by the standard microbiological method, according to EN ISO 6579. When the real‐time PCR‐based method was evaluated alongside the standard microbiological method according to EN ISO 6579 with 36 food samples artificially contaminated at a level of 10⁰ CFU (25 g)^?1, identical results were obtained from both methods. Conclusions: The real‐time PCR‐based method involving a two‐step enrichment produced equivalent results to EN ISO 6579 on the day after sample receipt. Significance and Impact of the Study: The developed method is suitable for rapid detection of S. enterica in food.     

Protein III-based single-chain antibody phage display using bacterial cells bearing an additional genome of a gene-III-lacking helper phage

We present herein a novel method of pIII-based antibody phage display using Hpd3cells--bacterial cells bearing the genome of a gene-III-lacking helper phage (VCSM13d3). A high level of single-chain variable fragments (scFvs) was displayed in the consequent phagemid particles using Hpd3cells to rescue the phagemid encoding scFv-pIII. Hpd3cells considerably improved the specific enrichment factor when used for constructing an immunized antibody library. In addition, using Hpd3cells could overcome pill resistance and can contribute to the efficient enrichment of specific binding antibodies from a phage display library, thereby increasing the chance of obtaining more diverse antibodies specific for target antigens.     

Detection of Salmonella by indicator agar media and PCR as affected by alfalfa seed homogenates and native bacteria

AIMS: To investigate and prevent the undesirable effect of native bacteria and alfalfa seed homogenates on detection of Salmonella in alfalfa seeds by indicator agar media and polymerase chain reaction (PCR). METHODS AND RESULTS: The relative sensitivity of five indicator agar media, including modified semisolid RV (MSRV), xylose-lysine-Tergitol 4 (XLT4), Hektoen enteric agar (HEA), brilliant green agar (BGA) and bismuth sulphite agar (BSA), for detection of Salmonella in the presence of a large number of native bacteria from alfalfa seeds was examined. The detection limit as measured by the ratio between the numbers of native bacteria and Salmonella was estimated to be 10(6) to 1 for MSRV and 10(3) to 1 for XLT4, HEA, BGA or BSA. Presence of alfalfa seed homogenates markedly reduced the sensitivity of Salmonella detection by PCR. The minimal number of Salmonella detectable by PCR was determined to be 1-10 and 100-1000 CFU in the absence and presence of seed homogenate, respectively. Application of anti-Salmonella immunomagnetic beads permitted detection of 2-5 CFU of heat-injured cells in 25 g of seeds within 24 h by PCR. CONCLUSIONS: The MSRV medium is more sensitive than other indicator agars for detecting a small number of motile Salmonella in samples containing a large number of native bacteria. Application of immunomagnetic beads eliminates the PCR-inhibitory activity of seed homogenates and improves the detection of Salmonella in inoculated seeds. SIGNIFICANCE AND IMPACT: The results generated from this study will aid the seed distributors, sprout growers and public health officials to identify and recall the Salmonella-contaminated seed lots to be used for sprout production.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.     

PCR-BASED FLUORESCENT METHOD FOR RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN POULTRY SAMPLES

A DNA binding fluorescence method based on polymerase chain reaction (PCR) products was evaluated for rapid detection of Salmonella Typhimurium in poultry products. Wash water samples of chicken carcasses and ground turkey were inoculated with S. Typhimurium to obtain final concentrations of 10° - 10⁵ CFU/mL. One mL of each sample was used to get the DNA template and 5 μL of the sample template was added into 25 μL of SYBR Green PCR Master Mix and two specific Salmonella ompC gene primers. The negative control was the same except 5 μL of each wash solution was added instead of 5 μL sample template. The reaction was carried out in a thermocycler. Finally, the fluorescence signal of each PCR product was measured using a fluorometer. The PCR products were also confirmed by ethidium bromide agarose gel, and the DNA concentrations of the PCR products were measured by a filter fluorescence photometer. The results showed that when bacterial cells increased from 0 to 2 CFU/mL, the fluorescence signal increased significantly. The PCR-based fluorescence method could detect the target bacteria in minutes after PCR amplification compared to hours by gel electrophoresis and also could be done at an earlier time during PCR amplification. The detection limit of this method for S. Typhimurium in the poultry samples was 2 CFU/mL without any enrichment.     

PCR-Based Detection of Artificial Latent Infections of Geranium by Xanthomonas campestris pv. pelargonii

A procedure entailing biological enrichment and PCR amplification was developed to detect small populations of Xanthomonas campestris pv. pelargonii (X.c, pv. pelargonii ) in tissues of geranium. Known numbers of colony forming units (CFU) of X.c. pv. pelargonii were introduced into 'Red Elite' geraniums through wounding of petioles and stems. Immediately after inoculation, sections of the petioles and stems were harvested and incubated for 24 or 48 h in nutrient broth (biological enrichment). After enrichment, bacterial cells were collected by centrifugation, followed by rapid extraction of total nucleic acid from the cells with GeneReleaser™, PCR amplification of DNA with pathovar-specific primers, and ethidium bromide-stained agarose gel electrophoretic analysis of the PCR products. After 48 h biological enrichment, it was possible to detect as few as 1 CFU of Xc. pv. pelargonii in stems and petioles collected immediately after inoculation, with the detection limit ranging between 1 and 120 CFU during multiple experiments. It also was possible to detect systemic movement of the bacterium in intact plants sampled 24 h after inoculation with a minimal inoculum (4 CFU). This procedure may have application in geranium certification programs concerned with the detection of latent infections associated with low levels of X.c .pv. pelargonii.     

Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples

PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger. Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h. A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h. Low levels of microbial contamination were detected in all raw materials and products using PCR assays. Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.     

RAPID DETECTION OF MICROBIAL CONTAMINATION IN TRICLOSAN AND HIGH FLUORIDE DENTIFRICES USING AN ATP BIOLUMINESCENCE ASSAY

Abstract The Celsis ATP Bioluminescence method was optimized and validated to detect the presence of microbial contamination in High Fluoride and Triclosan dentifrice formulations. Several enrichment broths were evaluated by using a 24–27 h incubation period. The ATP concentrations of the enrichment broths were found to a range from 0.012 to 0.040 nM. None of the tested enrichment broths were found to exhibit any sample inhibition/enhancement effects on the ATP Bioluminescence reaction. Dentifrice suspensions were inoculated with bacteria, yeast, and mold. All test microorganisms (ca. 1–15 CFU/g) were detected within a 24–27 h incubation period by using TAT Broth Base enrichment broths containing different concentrations of the following ingredients: Tween 20, Neopeptone, Dextrose, Triton X-100, Thiosulfate, Sodium Dibasic Phosphate, and Glycine. Negative ATP response after 24–27 h of incubation at 35C indicates the absence of contamination from these products.     

SIMULTANEOUS PCR DETECTION OF BACTERIA AND MOLD DNA SEQUENCES IN PHARMACEUTICAL SAMPLES BY USING A GRADIENT THERMOCYCLER

A gradient thermocycler, the Stratagene RoboCycler 96-Gradient, was evaluated for the simultaneous PCR amplification of microbial genes which indicated the presence of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger in pharmaceutical samples. Suspensions of pharmaceutical products were inoculated with pure cultures of bacteria and mold. After a 24 h incubation, bacterial DNA was extracted from each enrichment broth using a mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling the samples in Tris-EDTA-SDS buffer for 1 h. A 10 μL aliquots of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50 μL aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. The individual samples were loaded into the RoboCycler 96-Gradient thermocycler. Simultaneous detection of all microbial genes was performed by using a gradient profile that allowed the use of DNA primers with different annealing temperatures. Standard methods for quality control evaluation of pharmaceutical products required 6–8 days while simultaneous PCR detection of bacteria and mold DNA sequences was completed within 27 h.     

Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease

A multiplex PCR (mPCR) method was designed for the simultaneous detection of 4 major fish pathogens, Flavobacterium psychrophilum, Lactococcus garvieae, Pseudomonas aeruginosa, and P. putida. Each of the 4 pairs of oligonucleotide primers exclusively amplified the 16S rDNA gene of their targeted microorganism. The average detection limits for each organism amplified by mPCR were 2 colony-forming units (CFU) of F. psychrophilum, 3 CFU of L. garvieae, 3 CFU of P. aeruginosa, and 5 CFU of P. putida in mixed cultures. Multiplex PCR did not produce any nonspecific amplification products when tested against 28 related species of bacteria. High amounts of DNA from 1 bacterial species had a significant effect on the amplification sensitivity of the other bacterial species when these were present in lower concentrations in the multiplex reaction. The mPCR assay proved useful for the detection of the bacteria in naturally infected fish. The assay is a sensitive, specific, and reproducible diagnostic tool for the simultaneous detection of 4 pathogenic bacteria that cause disease in fish and offers a potentially useful alternative to the conventional culture-based method.     

A piezoelectric immunosensor for specific capture and enrichment of viable pathogens by quartz crystal microbalance sensor, followed by detection with antibody-functionalized gold nanoparticles

A sensitive bacteria enrichment and detection system for viable Escherichia coli O157:H7 was developed using a piezoelectric biosensor-quartz crystal microbalance (QCM) with antibody-functionalized gold nanoparticles (AuNPs) used as detection verifiers and amplifiers. In the circulating-flow QCM system, capture antibodies for E. coli O157:H7 were first immobilized onto the QCM chip. The sample containing E. coli O157:H7 was circulated through the system in the presence of 10ml of brain heart infusion (BHI) broth for 18h. The cells of E. coli O157:H7 specifically captured and enriched on the chip surface of the QCM were identified by QCM frequency changes. Listeria monocytogenes and Salmonella Typhimurium were used as negative controls. After bacterial enrichment, detection antibody-functionalized AuNPs were added to enhance the changes in detection signal. The use of BHI enrichment further enhanced the sensitivity of the developed system, achieving a detection limit of 0-1log CFU/ml or g. The real-time monitoring method for viable E. coli O157:H7 developed in this study can be used to enrich and detect viable cells simultaneously within 24h. The unique advantages of the system developed offer great potential in the microbial analysis of food samples in routine settings.     

A simple filtration technique to detect enterohemorrhagic Escherichia coli O157:H7 and its toxins in beef by multiplex PCR.

Primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, were coupled with oligonucleotides for the shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. A minimum of 10(2) CFU per PCR (10 microliters) was necessary to amplify E. coli O157:H7-specific bands by multiplex PCR. Food particles as well as various unknown metabolic by-products of bacteria inhibited the PCR, but a simple two-step filtration procedure eliminated this inhibition. To reliably generate PCR products, an E. coli inoculum of 10(3) CFU g of food slurry-1 in a nonspecific medium was required with 6 h of enrichment at 37 degrees C. However, when the food homogenate was incubated overnight, E. coli O157:H7 at an initial inoculum of even 1 CFU g-1 was detected.     

Detection of low levels of Listeria monocytogenes cells by using a fiber-optic immunosensor

Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 x 10(3) CFU/ml for a pure culture of L. monocytogenes grown at 37 degrees C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10 degrees C with 3.5% NaCl, the detection threshold was 4.1 x 10(4) or 2.8 x 10(7) CFU/ml, respectively. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth.     

Comparison of agar plate and real-time PCR on enumeration of Lactobacillus, Clostridium perfringens and total anaerobic bacteria in dog faeces

AIMS: To compare agar plate and real-time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces. METHODS AND RESULTS: Thirty-two faecal specimens from Labrador retriever dogs were used to compare agar plate and real-time PCR enumeration methods for Lactobacillus, C. perfringens and total anaerobic bacteria. Total anaerobic bacteria, C. perfringens and Lactobacillus of faeces were counted (as CFU g(-1) faeces) for 48-h incubation at 37 degrees C in an anaerobic gas chamber on genus-selective media. Total genomic DNA from samples was extracted by the QIAamp DNA stool mini kit. The quantification of DNA (as DNA copy per gram faeces) by real-time PCR was performed with a LightCycler system with the QuantiTect SYBR green PCR kit for PCR amplification. The results indicated that there was a significant correlation between CFU and DNA copy of Lactobacillus (R2 = 0.78, P < 0.01) and total anaerobic bacteria (R2 = 0.21, P < 0.05); but no correlation was found between CFU and DNA copy of C. perfringens. The regression equations for Lactobacillus and total anaerobic bacteria were log(DNA copy) = 0.83 x log(CFU) + 1.43 and log(DNA copy) = 1.62 x log(CFU) - 6.32 respectively. CONCLUSIONS: The real-time PCR method could be used to enumerate Lactobacillus within 2 days when compared with plating method which requires 5-6 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR method and the primer set for Lactobacillus spp. harboured in the dog intestine can be used for rapid enumeration of lactobacilli and monitoring of the faecal Lactobacillus community.