Comparison of three DNA extraction methods for Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium

Aims:  To compare three methods for DNA extraction from Mycobacterium bovis , Mycobacterium tuberculosis and Mycobacterium avium subsp. avium .
Methods and Results:  The DNA was extracted from mycobacterial cultures using enzymatic extraction, combined bead beating and enzymatic extraction and cetyltrimethylammonium bromide (CTAB) extraction. The yield and quality of DNA were compared by spectrophotometry, agarose gel electrophoresis, restriction endonuclease analysis and PCR. The combined bead beating and enzymatic extraction method yielded more DNA. However, that method produced some sheared DNA, visible either by agarose gel electrophoresis or by restriction endonuclease analysis. All methods were appropriate for PCR amplification of a 123 bp fragment of IS 6110 in M. bovis and M. tuberculosis , and of a 1700 bp fragment of FR300 region in M. avium avium .
Conclusions:  Combined bead beating and enzymatic extraction method was the most efficient and easy method for extracting DNA from bacteria of the M. tuberculosis complex.
Significance and Impact of the Study:  The results reveal important differences among the DNA extraction methods for mycobacteria, which are relevant for the success of further downstream molecular analysis.     

Rapid Aeromonas hydrophila identification by TaqMan PCR assay: comparison with a phenotypic method

Aims:  Aeromonas hydrophila is recognized as a human pathogen following wound exposure or ingestion of contaminated water and food. For rapid identification of this bacterium, a TaqMan-based real-time PCR assay has been developed.
Methods and Results:  Primers and probes that target specific sequences of the 16S rRNA gene and cytolytic enterotoxin gene ( aerA ) were combined in a duplex assay. Presence and size of PCR products were confirmed with microchannel fluidics electrophoresis analysis. After validation, using type strain CIP7614T DNA, the PCR assay was tested on 12 positive and negative controls. Twenty-one Aeromonas strains were isolated from environmental samples and were identified with biochemical tests as Aer. sobria , Aer. caviae and Aer. hydrophila . Only Aer. hydrophila strains tested positive by PCR assay.
Conclusions:  The PCR developed here was successfully applied for the identification of Aer. hydrophila from reference, clinical and environmental samples and showed a high discrimination between Aer. hydrophila and other Aeromonas species.
Significance and Impact of the Study:  This molecular method is convenient, rapid (2·5 h vs 24 h), specific to identify Aer. hydrophila and usable for diagnosis in medical and veterinary laboratories.     

Mycobacterial Agglutination-Inhibition Test

The mycobacterial agglutination-inhibition test (MAIT) is described as a modification of the mycobacterial agglutination of Schaefer (MATS). It is an organized assemblage of absorption and agglutination tests which is especially useful in the identification of Mycobacterium avium isolates normally excluded from serological identification because of unstable cell suspension characteristics. The identity of 100 unstable M. avium and 3 unstable Mycobacterium intracellulare isolates was determined by the MAIT method and by animal pathogenicity tests. The comparability of the MAIT and MATS methods was demonstrated by testing 10 stable M. avium and 10 stable M. intracellulare isolates by both methods and obtaining the same serological identification for each culture. The reproducibility of the MAIT was confirmed when the same result was obtained in three consecutive tests on each of the 123 cultures used in this evaluation.     

Molecular identification and characterization of rifampicin-resistant Mycobacterium tuberculosis isolates by line probe assay: an approach for rapid diagnosis of multidrug-resistant tuberculosis

Aim:  Early identification and characterization of rifampicin-resistant (R^r) Mycobacterium tuberculosis isolates recovered from the samples of tuberculosis (TB) patients in the Aegean (West Anatolian) Region was intended.
Methods and Results:  Sixty isolates [47 (78·3%) multidrug-resistant (MDR)], which were identified as M. tuberculosis complex and phenotypically resistant to rifampicin by both BACTEC mycobacteria growth indicator tube (MGIT) 960 and 460 systems were analysed by a commercial line probe assay (INNO-LiPA Rif TB). The concordance of LiPA with the in vitro susceptibility test was found as 98·3%. Among the isolates, S531L (R5 pattern; 46·7%) and L511P/R, S512T, Q513L/K (ΔS1 pattern; 11·7%) were the most frequent mutation patterns. As compared with the BACTEC systems and conventional techniques for cultivation, identification and in vitro susceptibility testing, INNO-LiPA Rif TB after cultivation in BACTEC MGIT 960 system provided an average of 20 days early diagnosis of R^r M. tuberculosis isolates.
Conclusions:  Rapid molecular identification and characterization of R^r M. tuberculosis isolates after BACTEC MGIT 960 cultivation would be useful for faster diagnosis, infection control and planning of accurate treatment in MDR-TB patients.
Significance and Impact of the Study:  Patients with MDR-TB need a specified treatment and efficient follow-up strategies. Rapid and practical methodologies to diagnose and follow these patients should be applied in routine use.     

Comparison of a multiplex-PCR assay with mycolic acids analysis and conventional methods for the identification of mycobacteria

A fast, sensitive and cost-effective multiplex-PCR assay for Mycobacterium tuberculosis complex (MTC) and Mycobacterium avium (M. avium) identification for routine diagnosis was evaluated. A total of 158 isolates of mycobacteria from 448 clinical specimens from patients with symptoms of mycobacterial disease were analyzed. By conventional biochemical methods 151 isolates were identified as M. tuberculosis, five as M. avium and two as Mycobacterium chelonae (M. chelonae). Mycolic acid patterns confirmed these results. Multiplex-PCR detected only IS6110 in isolates identified as MTC, and IS1245 was found only in the M. avium isolates. The method applied to isolates from two patients, identified by conventional methods and mycolic acid analysis, one as M. avium and other as M. chelonae, resulted positive for IS6110, suggesting co-infection with M. tuberculosis. These patients were successfully submitted to tuberculosis treatment. The multiplex-PCR method may offer expeditious identification of MTC and M. avium, which may minimize risks for active transmission of these organisms and provide useful treatment information.     

Relationships between Mycobacterium isolates from patients with pulmonary mycobacterial infection and potting soils

High numbers of mycobacteria, including known pathogenic species such as Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chelonae, were recovered from aerosols produced by pouring commercial potting soil products and potting soil samples provided by patients with pulmonary mycobacterial infections. The dominant mycobacteria in the soil samples corresponded to the dominant species implicated clinically. Profiles of large restriction fragments obtained by pulsed-field gel electrophoresis demonstrated a closely related pair of M. avium isolates recovered from a patient and from that patient's own potting soil. Thus, potting soils are potential sources of infection by environmental mycobacteria. Use of dust-excluding masks should be considered during potting or other activities that generate aerosol with soil.     

Development of a real-time PCR-based method for rapid differential identification of Mycobacterium species

AIMS: To develop a real-time PCR method for rapid differential identification of many clinically important mycobacteria to the species level. METHODS AND RESULTS: Eighteen Mycobacterium species that are considered clinically important were targeted for the identification. One primer pair and 21 pairs of hybridization probes (HybProbes) specific for the genus, species or complex were designed based on the rpoB gene sequences of mycobacteria. Twenty-five different Mycobacterium reference species were tested. In a single round of real-time PCR, all the nontuberculous mycobacteria (NTM) species tested were identified at the genus level and 16 of the 18 targeted species were differentially identified to the species or complex level during the amplification cycles; subsequent melting curve analysis allowed the specific identification of all the target species at the species or complex level without cross-reactivity with the other species. CONCLUSIONS: The developed real-time PCR assay rapidly identifies the NTM at the genus level and 18 clinically important Mycobacterium species at the species or complex level. SIGNIFICANCE AND IMPACT OF THE STUDY: This real-time PCR assay provides a useful tool for the rapid differentiation of most clinically important Mycobacterium species.     

Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in drinking water distribution systems

Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples). Numbers of mycobacteria ranged from 10 to 700,000 CFU liter(-1). The number of M. avium in raw waters was correlated with turbidity. Water treatment substantially reduced the number of mycobacteria in raw waters by 2 to 4 log units. Mycobacterial numbers were substantially higher in the distribution system samples (average, 25,000-fold) than in those collected immediately downstream from the treatment facilities, indicating that mycobacteria grow in the distribution system. The increase in mycobacterial numbers was correlated with assimilable organic carbon and biodegradable organic carbon levels (r(2) = 0.65, P = 0.03). Although M. intracellulare was seldom recovered from water samples, it was frequently recovered (six of eight systems) in high numbers from biofilms (average, 600 CFU/cm(2)). Evidently, the ecological niches of M. avium and M. intracellulare are distinct.     

Detection and identification of mycobacteria by amplification of mycobacterial DNA

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Taq polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 10(6) human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.     

Evaluation of ISO enrichment real-time PCR methods with internal amplification control for detection of Listeria monocytogenes and Salmonella enterica in fresh fruit and vegetables

Aim:  To find a quick and reliable diagnostic technique for penicillin nonsusceptible Streptococcus pneumoniae (PNSSP) from Nanjing, China.
Methods and Results:  A total of 130 S. pneumoniae clinical isolates and 50 healthy carriers' isolates were collected from seven teaching hospitals in Nanjing between March 2005 and June 2007. Three different methods, PCR, seminested-PCR, and PCR-RFLP, were applied in detecting PNSSP. The prevalence of PNSSP was 73·9%, the penicillin-resistance rate of children's group and adults' group were 87·8% and 31·2%, respectively. The PCR method had difficulty in identifying intermediate-resistant and resistant isolates, the PCR-RFLP method could only be used for susceptible isolates and had poor sensitivity. Seminested-PCR was fast, sensitive for detection, and successfully differentiated between moderately and highly resistant S. pnemoniae ; 96·1% of the PCR results were in agreement with the minimum inhibitory concentration (MIC) data.
Conclusions:  The penicillin resistance rate in isolates from children was much higher than of isolates from adults in China. Seminested-PCR for pbp1A was the best diagnostic method.
Significance and Impact of the Study:  We compared three different strategies for the first time in identification of PNSSP in China. In conclusion, seminested-PCR can be applied in clinical microbiology laboratories for detection of serious infections caused by S. pneumoniae .     

Development of a real-time PCR assay with an internal amplification control for the screening of Shiga toxin-producing Escherichia coli in foods

Aims:  To develop and evaluate a real-time PCR assay incorporating an internal amplification control (IAC) suitable for the screening of Shiga toxin (Stx)-producing Escherichia coli (STEC) in foods.
Methods and Results:  A competitive IAC was constructed and included in an stx -specific real-time PCR assay. Coupled to 18-h enrichment and automated DNA extraction, the assay could reliably detect the presence of STEC in minced meats inoculated at 10 CFU per 25 g. Its performance was evaluated on 415 minced beef and 112 raw milk cheese samples and compared with that of a PCR-ELISA method. Fifty-three minced meats and 31 cheeses were found stx -positive, giving 98·3% and 93·75% concordance, respectively, with the PCR-ELISA reference method.
Conclusions:  A highly sensitive stx -specific real-time PCR method including an IAC was developed, facilitating monitoring of false-negative results due to PCR inhibitors.
Significance and Impact of the Study:  Combined with automated DNA extraction, the stx -IAC real-time PCR assay represents a suitable method for rapid screening of STEC in foods.     

Distribution of non-tuberculosis mycobacteria strains from suspected tuberculosis patients by heat shock protein 65 PCR–RFLP

The genus Mycobacterium contains more than 150 species. Non-tuberculosis mycobacteria (NTM) often cause extrapulmonary and pulmonary disease. Mycobacteria detection at species level is necessary and provides useful information on epidemiology and facilitates successful treatment of patients. This retrospective study aimed to determine the incidence of the NTM isolates and Mycobacterium tuberculosis (Mtb) in clinical specimens collected from Iranian patients during February 2011–December 2013, by PCR–restriction fragment length polymorphism analysis (PRA) of the hsp65 gene. We applied conventional biochemical test and hsp65–PRA identification assay to identify species of mycobacteria in specimens from patients suspected of having mycobacterial isolates. This method was a sensitive, specific and effective assay for detecting mycobacterial species and had a 100% sensitivity and specificity for Mtb and Mycobacterium avium complex (MAC) species. Using PRA for 380 mycobacterial selected isolates, including 317 Mtb, four Mycobacterium bovis and of the 59 clinical isolates, the most commonly identified organism was Mycobacterium kansasii (35.6%), followed by Mycobacterium simiae (16.9%), Mycobacterium gordonae (16.9%), Mycobacterium fortuitum (5.1%), Mycobacterium intracellulare (5.1%), Mycobacterium avium (5.1%), Mycobacterium scrofulaceum (3.4%), Mycobacterium gastri (3.4%), Mycobacterium flavescens (3.4%), Mycobacterium chelonae (3.4%) and Mycobacterium nonchromogenicum (1.7%). PRA method, in comparison with classical methods, is rapid, useful and sensitive for the phylogenetic analysis and species detection of mycobacterial strains. Mycobacterium kansasii is the most common cause of infection by NTM in patients with non-HIV and HIV which demonstrated a high outbreak and diversity of NTM strains in our laboratory.     

An alternative real-time PCR method for the detection of thermotolerant Bacillus sensu lato contaminants in naturally-contaminated gelatine

Aims:  Comparison of an internally-controlled real-time PCR assay with the current plate-based assay for the detection of Bacillus sensu lato contaminants in gelatine.
Methods and Results:  A comprehensive TaqMan^® probe was designed allowing the real-time PCR assay to be fully inclusive for the gelatine-contaminating Bacillus s.l. species. An internal amplification control was implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (Kappa value = 0·94) was observed between the performance of the real-time PCR and the current plate-based method on naturally contaminated gelatines ( n  = 162). Relative accuracy, relative sensitivity and relative specificity were 97·5%.
Conclusions:  The real-time PCR assay is an adequate alternative of the current plate-based assay.
Significance and Impact of the Study:  The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h. The gelatine-producing industry can ensure gelatine quality in a much faster way.     

Usefulness of the BACTEC MYCO/F lytic system for detection of mycobacteremia in a clinical microbiology laboratory

278 BACTEC MYCO/F lytic system blood cultures for mycobacteria were evaluated between 1997 and 1999. Sixty of them were read as positive by the system, being considered 15 of them as false positives. Twenty-seven yielded mycobacterial growth (13 Mycobacterium avium from 3 patients and 14 Mycobacterium tuberculosis from 8 patients). Other bacteria isolated were coagulase-negative Staphylococcus (13 samples), Corynebacterium sp. (5 samples), Salmonella enteritidis (2 samples) and Klebsiella pneumoniae (1 sample). Five of these isolates were considered as true episodes of bacteremia. The average time for detection of mycobacteria was 12.6 days for M. avium and 26.4 days for M. tuberculosis. BACTEC MYCO/F lytic system is useful for detection of mycobacteremia in clinical microbiology laboratories.     

Sequence-based differentiation of strains in the Mycobacterium avium complex.

The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.     

Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.     

Genetic diversity in clinical isolates of Mycobacterium avium complex from Guinea-Bissau, West Africa

Isolates of Mycobacterium avium complex (MAC) were cultured from sputum samples obtained from patients in Guinea-Bissau, West Africa. Twenty-eight isolates hybridising with MAC probe (AccuProbe) were further characterised by different molecular techniques: hybridisation with species-specific probes (AccuProbe) for M. avium and M. intracellulare, partial sequencing of 16S rRNA gene and PCR detection of the DT1-DT6 sequences and the macrophage-induced gene (mig). Only one of the 28 isolates reacted with the M. avium probe and four with the M. intracellulare probe. Two isolates expressed the DT1 sequence, and three the DT6. The mig was detected in 18 (64%) of the isolates. Sequencing of 16S rRNA had the greatest discriminative power of the typing methods applied, without strong correlation with any other technique. Clinical MAC isolates from Guinea-Bissau demonstrated a wide genetic diversity among the members of M. avium complex that might reflect on biotope variation.     

Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards

From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n=961), Mycobacterium a. avium (n=677), Mycobacterium a. silvaticum (n=5), and Mycobacterium a. hominissuis (n=1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n=2), Mycobacterium bovis (n=13), M. bovis BCG (n=4), and Mycobacterium caprae (n=10) were examined. From other mycobacterial species Mycobacterium intracellulare (n=60) and atypical mycobacteria (n=256) including Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium scrofulaceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900, IS901, IS1245, and flanking region (FR300) of IS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 10(1), 10(3), 10(2), and 10(2) molecules for ISIS900, ISIS901, ISIS1245, and ISFR300 were used in the PCR, respectively, when no bacterial DNA was added to the reaction. The system was tested to define the amount of internal standards that could be used in the PCR without affecting the amplification of the specific segment. Non-specific amplifications were observed in M. fortuitum with IS1245 PCR and mixed infections with M. a. avium and M. a. hominissuis from pigs and cattle were found. PCR results of typing were compared with serotyping and Accu-Probes analyses in selected field isolates.     

Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa. The Ziehl-Neelsen acid-fast stain could not be used to enumerate intracellular mycobacteria because uninfected protozoa contained acid-fast, bacterium-like particles. Starved, 7-day-old cultures of T. pyriformis transferred into fresh medium readily phagocytized M. avium, M. intracellulare, and M. scrofulaceum. Phagocytosis was rapid and reached a maximum in 30 min. M. avium, M. intracellulare, and M. scrofulaceum grew within T. pyriformis, increasing by factors of 4- to 40-fold after 5 days at 30 degrees C. Intracellular M. avium numbers remained constant over a 25-day period of growth (by transfer) of T. pyriformis. Intracellular M. avium cells also survived protozoan encystment and germination. The growth and viability of T. pyriformis were not affected by mycobacterial infection. The results suggest that free-living phagocytic protozoa may be natural hosts and reservoirs for M. avium, M. intracellulare, and M. scrofulaceum.     

An alternative real-time PCR method to detect the Bacillus cereus group in naturally contaminated food gelatine: a comparison study

Aims:  Comparison of an internally controlled real-time PCR assay with the standard plate-based assay (ISO 21871) for the detection of Bacillus cereus group cells in gelatine.
Methods and Results:  A comprehensive TaqMan probe was designed allowing the real-time PCR assay to be fully inclusive and exclusive. An internal amplification control was designed and implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (κ = 0·99) was observed between the performance of the real-time PCR and the standard plate-based method on naturally contaminated gelatines ( n  = 197). Relative accuracy, relative sensitivity and relative specificity were ≥99%.
Conclusions:  The real-time PCR assay is a valid alternative of the standard plate-based assay.
Significance and Impact of the Study:  The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h, while analysis cost did not increase. The gelatine-producing industry can ensure gelatine safety and quality in a much faster way.