Quantitative detection of meat spoilage bacteria by using the polymerase chain reaction (PCR) and an enzyme linked immunosorbent assay (ELISA)

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw meat has been developed using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). Amplified PCR products generated using a digoxigenin-labelled primer were automatically hybridized to a biotinylated probe included in the PCR reaction. The hybridization was performed as part of the PCR programme. The biotin-digoxigenin hybrids were quantified by an enzyme-linked immunosorbent assay (ELISA). Streptavidin bound to the wells of a microtitre plate was used to capture the biotin-digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying meat samples containing bacteria in the range 10²–10⁷ cfu cm⁻². The detection threshold for the PCR-ELISA assay developed in this work is 10² cfu cm⁻².     

Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E. coli (STEC) strains, including enterohaemorrhagic E. coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII. Both ETEC and STEC are pathogenic to humans, pigs and cattle. As contamination of environmental water by any of these pathogenic E. coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E. coli was developed. The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories. The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E. coli cells as well as non- E. coli bacteria. Its detection limit was 10²–10³ cfu each of the target cells per assay. When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10⁰ cells 100 ml⁻¹ of the water sample could be detected. Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.     

Detection of Yersinia enterocolitica in food by PCR amplification

A polymerase chain reaction (PCR) assay was developed for detection of pathogenic, virulent strains of Yersinia enterocolitica . By using both virulence loci virF and ail as markers for pathogenicity, detection of species with a virulence factor present was possible. DNA preparation in the presence of hexadecyl trimethy ammonium bromide (CTAB) was followed by two 44 cycle amplification reactions, one for each of the markers. As few as 10² Y. enterocolitica cells were detected in ground pork in the presence of 10⁵–10⁶ bacteria of other species. The described PCR assay provides a sensitive robust assay for the detection of virulent Y. enterocolitica in food.     

Detection and quantification of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification

A species-specific 16S rRNA oligonucleotide probe (ASRB1) was developed for the detection of Desulforhabdus amnigenus in anaerobic granular sludge. The presence of nucleic acids from cells of D. amnigenus in granular sludge was determined using ASRB1 as a specific primer for polymerase chain reaction (PCR) amplification or as a probe for dot blot hybridizations. The detection threshold and the reproducibility of these two methods were determined with sludge amended with 10⁴–10¹⁰ D. amnigenus cells per gram of volatile suspended solids (VSS). For D. amnigenus cells with a ribosomal RNA content of 15 fg cell⁻¹, the lowest number of target cells detected by hybridization was 1 × 10⁸ cells g⁻¹ VSS. With the PCR amplification method the lowest number of target cells which could be detected was 1 × 10⁷ g⁻¹ VSS. This corresponds to a threshold level for hybridization of 0·1–0·001‰ of the total bacterial sludge population, while the threshold level obtained with the PCR approach amounted to 0·01–0·0001‰. The rRNA content of D. amnigenus was found to be affected by the growth rate and the growth phase, and it ranged from 19 fg cell⁻¹ in slow-growing cultures to 90 fg cell⁻¹ in fast-growing cultures. Therefore, the detection threshold of the dot blot hybridization method for fast-growing cells is lower than for slow-growing cells.     

Specific monitoring by PCR amplification and bioluminescence of firefly luciferase gene-tagged bacteria added to environmental samples

Abstract The firefly luciferase gene, luc , was demonstrated to hold promise as a specific marker for monitoring of genetically modified bacteria in the environment. PCR amplification and bioluminescence procedures were modified and compared for environmental monitoring of luc -tagged bacteria, using Escherichia coli as a model. The methods were used to track luc -tagged bacterial cells added to intact sediment core microcosms. Detection limits for the luc -tagged cells were the following, expressed as cells per 0.5 g of sediment: 10², by PCR amplification; 10³, by whole cell luminescence; and 10³−10⁴, by measurement of luminescence in cell extracts.     

Direct detection by polymerase chain reaction (PCR) of Escherichia coli in water and soft cheese and identification of enterotoxigenic strains

PCR was used to develop a method to detect Escherichia coli in surface water and soft cheese, which does not require cultivation of bacteria. DNA sequences from the ma /B operon of E. coli were amplified to specifically detect this bacterium. Samples of surface water and soft cheese naturally contaminated with E. coli from less than 100 cells per g up to several times 10⁵cells per g were analysed by both the classical culture method and the PCR assay. Comparable results were obtained with both methods. Soft cheese samples artificially contaminated with various levels of enterotoxigenic E. coli were analysed with a second PCR test specific for the heat-labile enterotoxin type I (LTI) of E. coli. The detection limit was about 1000 bacteria per g of soft cheese. In addition, two soft cheese samples naturally contaminated with 2 times 10⁵and 6 times 10⁵ E. coli per g as determined by the culture method were analysed by LTI-PCR and found to contain low levels of enterotoxigenic E. coli.     

Detection and counting of Nitrobacter populations in soil by PCR.

Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient.     

Improved PCR detection sensitivity of Erwinia carotovora subsp. atroseptica in potato tuber peel extract by prior enrichment on a selective medium

A simple and sensitive method was developed to replace the need for complex and laborious DNA extraction to remove inhibitory substances in potato tuber peel extract before detection of Erwinia carotovora subsp. atroseptica (Eca) by PCR. Eca was enriched by a factor of 10⁵ when peel extract was inoculated onto a selective medium, CVP, and incubated at 27°C for 24 h. Bacterial micro-colonies which developed were suspended in 500 μl of water and the bacteria diluted in water 100-fold, or 10-fold followed by washing by centrifugation, before PCR testing. The sensitivity of detection obtained with the former was ca 10¹–10² cells ml⁻¹ and with the latter ca 10¹ cells ml⁻¹, when different numbers of streptomycin-resistant Eca strain were added to peel extract from three Eca-free potato cultivars. The method was validated and the sensitivity confirmed relative to two different commonly used Eca detection methods using naturally contaminated tubers.     

Bacterivory by the ciliate Euplotes in different states of hunger

Abstract: The feeding of the marine ciliate Euplotes mutabilis was studied using bacteria ( Vibrio natriegens ) doubly labelled with ³H-thymidine and ¹⁴C-leucine. In the presence of abundant bacteria (30 × 10⁶ bacteria ml⁻¹), an average Euplotes cell (initially without food vacuoles) with a protein content of 12 ng consumed 16 × 10³ bacteria in the first hour and 27 × 10³ bacteria over four hours, accumulating about 60% of the bacterial protein into ciliate macromolecules. Euplotes which had been starved or under-fed to reduce cell protein biomass to 7 or 9 ng consumed significantly fewer bacteria, but the gross growth efficiency for protein did not change. The rate of consumption of bacteria by large Euplotes of protein content 15 ng was initially less than that of 12 ng cells, and it decreased markedly before the end of a 4-hour experiment. Recently divided cells ingested bacteria rapidly, but showed a reduced gross growth efficiency of about 40%. At low bacterial concentrations (6 × 10⁶ bacteria ml⁻¹) the rates of ingestion were markedly reduced to between     and     of maximal levels; the smallest cells could not sustain feeding activity at the low prey concentration and gross growth efficiency fell from 43 to 20% during a 4-hour experiment. The strategy adopted by Euplotes in response to local fluctuations in food supply involves rapid consumption with high growth efficiency in times of plenty, but slow shrinkage without cell division to survive in times of shortage.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw milk

We have developed a quantitative PCR-ELISA for the rapid enumeration of bacteria inrefrigerated raw milk using primers designed from conserved regions in the 16S ribosomal RNAgene (rRNA). The designed primers permitted the amplification of a 147 bp DNA fragment froma wide selection of bacteria which may grow in milk at refrigeration temperatures. Amplified PCRproducts generated using a digoxigenin-labelled primer were heat-denatured before beingquantified by an enzyme-linked immunosorbent assay (ELISA). A biotinylated probe immobilizedonto streptavidin-coated microplates was used to capture the digoxigenin-labelled fragments thatwere detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion ofsubstrate gave distinct absorbence differences when assaying milk samples containing bacteria inthe range 10³–10⁷ cfu ml⁻¹. The detection threshold for thePCR-ELISA assay developed in this work is 103 cfu ml⁻¹.     

Polymerase chain reaction for the rapid identification of Clostridium botulinum type A strains and detection in food samples

A polymerase chain reaction (PCR) was developed for the detection of Clostridium botulinum type A, a cause of human botulism. A two primer set and an oligonucleotide detection probe were used to specifically detect Cl. botulinum type A neurotoxin gene (BoNT/A). After 40 cycles of amplification, detection of a 798 bp amplified DNA fragment was carried out by agarose gel electrophoresis and Southern blot hybridization. This assay was able to detect 12.5 fg of purified target DNA or five bacteria per reaction. The sensitivity in artificially contaminated food samples after an 18 h enrichment step ranges from 10 to 10³ bacteria per g according to the type of food samples. No cross-reactions were observed with the other Cl. botulinum toxinotypes and other bacteria found routinely in food. This PCR method may provide a suitable and rapid alternative to standard techniques for detection of Cl. botulinum type A in food samples.     

Survival of κ-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g⁻¹ of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 10⁹ cfu g⁻¹ of dry soil to below the detection threshold of both selective (14 cfu g⁻¹ of dry soil) and nonselective (1 × 10³ cfu g⁻¹ of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 10⁶ to 2.9 × 10⁸ cfu g⁻¹ of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 10⁶ cfu g⁻¹ of dry soil after the freeze-thaw treatment.     

COMPARISON OF THREE POLYMERASE CHAIN REACTION-BASED METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN FOOD

Three PCR-based methods for the detection of Listeria monocytogenes in food (BAX for Screening, Probelia and a method according to Kaclíková et al. (2003) were compared on the basis of the determination of detection limits for 15 artificially contaminated food products. Detection limits of all methods for all samples were 10⁰ cfu per 10 g, with the exception of three cheese samples which did not produce valid results because of the inhibition of Probelia PCR. Detection limits for nonviable L. monocytogenes cells were sufficiently high ( 10⁹ cfu per 10 g) for BAX and the method according to Kaclíková et al. (2003), but considerably low ( 10⁶ cfu per 10 g) for Probelia. The results demonstrate that BAX for Screening as well as the Kaclíková et al. (2001) method fulfill the sensitivity requirements for a rapid alternative method for the detection of L. monocytogenes in food, which would be equivalent to the standard method EN ISO 11290–1.     

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA

Abstract Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1⁺/2⁺) and avirulent (pXO1⁺/2⁻, pXO1⁻/2⁺ and pXO1⁻/2⁻) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria.     

Design and evaluation of malolactic enzyme gene targeted primers for rapid identification and detection of Oenococcus oeni in wine

Rapid identification and detection of Oenococcus oeni was achieved by species-specific PCR. Two primers flanking a 1025 bp region of the O. oeni gene encoding the malolactic enzyme were designed. The expected DNA amplificate was obtained only when purified DNA from O. oeni was used. The identity of PCR product was confirmed by nested PCR and restriction analysis. Within 8 h, 10³ cfu ml⁻¹ of oenococci were detected in fermenting grape must containing 10⁷ yeast cells, whereas the detection limit in wine was 10⁴ cfu ml⁻¹. The rapidity and reliability of the PCR procedure established suggests that the method may be profitably applied in winery laboratories for quality control.     

Development of a PCR assay for identification of the Bacillus cereus group species

Aims:  A PCR technique was developed as a reliable and rapid identification method for the Bacillus cereus group species, based on a unique conserved sequence of the motB gene (encoding flagellar motor protein) from B. cereus , Bacillus thuringiensis and Bacillus anthracis .
Methods and Results:  Primer locations were identified against eight strains of the B. cereus group spp. from nucleotide sequences available in the National Centre for Biotechnology Information database. The PCR assay was applied for the identification of 117 strains of the B. cereus group spp. and 19 strains from other microbial species, with special emphasis on foodborne pathogens.
Conclusion:  The designed cross-species primers are group specific and did not react with DNA from other Bacillus and non- Bacillus species either motile or not. The primers system enabled us to detect 10³ CFU of B. cereus cells per millilitre of sample.
Significance and Impact of the Study:  Bacillus cereus group spp. belongs to one of the most prevalent foodborne pathogens. Bacterial growth results in production of different toxins; therefore, consumption of food containing >10⁶ bacteria per gram may result in emetic and diarrhoeal syndromes. A rapid and sensitive bacterial detection method is significant for food safety.     

Rapid detection and quantification of bisulfite reductase genes in oil field samples using real-time PCR

Sulfate-reducing bacteria (SRB) pose a serious problem to offshore oil industries by producing sulfide, which is highly reactive, corrosive and toxic. The dissimilatory sulfite reductase ( dsr ) gene encodes for enzyme dissimilatory sulfite reductase and catalyzes the conversion of sulfite to sulfide. Because this gene is required by all sulfate reducers, it is a potential candidate as a functional marker. Denaturing gradient gel electrophoresis fingerprints revealed the presence of considerable genetic diversity in the DNA extracts achieved from production water collected from various oil fields. A quantitative PCR (qPCR) assay was developed for rapid and accurate detection of dsrB in oil field samples. A standard curve was prepared based on a plasmid containing the appropriate dsrB fragment from Desulfomicrobium norvegicum . The quantification range of this assay was six orders of magnitude, from 4.5 × 10⁷ to 4.5 × 10² copies per reaction. The assay was not influenced by the presence of foreign DNA. This assay was tested against several DNA samples isolated from formation water samples collected from geographically diverse locations of India. The results indicate that this qPCR approach can provide valuable information related to the abundance of the bisulfite reductase gene in harsh environmental samples.     

The use of compound continuous flow diffusion chemostats to study the interaction between nitrifying and nitrate-reducing bacteria

Abstract The interactions occuring between populations of a nitrate-respiring Vibrio sp. and autotrophic nitrifying bacteria belonging to the genera Nitrosomonas and Nitrobacter have been investigated in a compound bi-directional flow diffusion chemostat at a dilution rate of 0.025 h⁻¹ and a temperature of 25°C. When grown under NO⁻₃ limitation, the Vibrio sp. produced NH⁺₄ as the principal end-product of nitrate respiration, and there was a corresponding significant increase in cell numbers of the Nitrosomonas sp. population, which derived energy by the oxidation of NH⁺₄ to NO⁻₂. Nitrite in turn was used by the Nitrobacter sp. population as an energy source with the concomitant regeneration of NO⁻₃. Under NO⁻₃ excess growth conditions the Vibrio sp. produced NO⁻₂ rather than NH⁺₄ as the major product of NO⁻₃ dissimilation, and growth of the Nitrobacter population was stimulated as increased quantities of NO⁻₂ became available. In contrast, the Nitrosomonas sp. population declined sharply as the energy source NH⁺₄ became limiting. These data demonstrate that defined mixed populations of obligately aerobic nitrifying bacteria and facultatively anaerobic nitrate respiring bacteria can co-exist for extended time periods and operate an internal nitrogen cycle which is energetically beneficial to both populations.     

Rapid detection of pathogenic Vibrio parahaemolyticus by a sensitive and specific duplex PCR-hybridization probes assay using LightCycler

Aims:  To develop a real-time polymerase chain reaction (PCR) hybridization probe assay for rapid and specific detection of thermostable direct haemolysin-producing Vibrio parahaemolyticus.
Methods and Results:  Primers and hybridization probes were designed to target the toxR and tdh2 genes. Mismatches were introduced in the tdh2 primers for specific amplification of the target. The 3' ends of donor probes for both genes were labelled with fluorescein. The 5' ends of recipient probes for tdh2 and toxR were labelled with LC Red 640 and LC Red 705, respectively. The real-time assay was evaluated against conventional biochemical tests and the KAP-RPLA kit (Kanagawa phenomenon detection kit by reverse passive latex agglutination). toxR and tdh2 were detected in 100% and 91% of clinical V. parahaemolyticus isolates ( n  = 118), respectively. Specificity and sensitivity of the real-time assay for toxR and tdh2 were 100%, respectively. Dynamic range of detection for toxR was 10⁷–10¹ CFU ml⁻¹ and that for tdh2 was 10⁷–10⁴ CFU ml⁻¹.
Conclusions:  The LightCycler assay described is sensitive and highly specific for detection of pathogenic V. parahaemolyticus in a single reaction tube within 80 min.
Significance and Impact of the Study:  The assay developed allows accurate detection of pathogenic V. parahaemolyticus , which is valuable for rapid tracing of infection source during outbreaks.