WO2010007605A1

WO2010007605A1 - A method of assessing bacterial load of a sample

Info

Publication number: WO2010007605A1
Application number: PCT/IE2009/000047
Authority: WO
Inventors: Kaye Burgess; Geraldine Duffy; Anthony Dolan
Original assignee: Agriculture And Food Development Authority (Teagasc)
Priority date: 2008-07-16
Filing date: 2009-07-15
Publication date: 2010-01-21

Abstract

A method of determining a bacterial load of a food product, especially a meat carcass, comprises the steps of processing a sample of nucleic acid isolated from the food product to amplify and quantify a target region associated gram positive bacteria, and a target region associated with gram negative bacteria, and correlating the quantity of amplified target regions with a bacterial load value for the food product. The target region associated with gram positive bacteria is amplified using a primer set comprising of SEQUENCE ID NO. 3 (gram positive forward primer) or a functional fragment thereof, and SEQUENCE ID NO. 4 (gram positive reverse primer) or a functional fragment thereof. The target region associated with gram negative bacteria is amplified using a primer set comprising of SEQUENCE ID NO. 5 (gram negative forward primer) or a functional fragment thereof, and SEQUENCE ID NO. 6 (gram negative reverse primer) or a functional fragment thereof. A kit of parts for performing the method of the invention is also described.

Description

A METHOD QF ASSESSING BACTERIAL LOAD OF A SAMPLE

Introduction

The invention relates to a method of assessing the bacterial load of a sample. It is particularly useful in quantifying bacterial numbers of a sample, especially a meat, fish or poultry product.

The microbial quality and safety of a food is dependent on the microbial load or numbers present and on the diversity and composition of the microflora [I]. Criteria to assess the microbiology as outlined in EU legislation (2001/471 /EEC) include the total viable count (TVC), a cultural technique that can be used to evaluate the microbiological quality of a food product and predict shelf life. Culture dependent methods for enumerating bacterial numbers are known to be biased since bacteria can only be cultivated if their metabolic and physiological requirements can be reproduced in vitro [2]. Where a rapid result is required, culture dependent techniques may be unsuitable as they can take several days to provide a result. The gold standard method to assess microbial numbers remains the TVC₅ also referred to as the aerobic standard plate count (SPC or APC), which is routinely carried out according to the International Organisation for Standards procedure [3]. All alternative methods must generally be correlated or validated against this method.

While "gold standard" indicates the method is without flaw, there are in fact some drawbacks to the method. The term "total viable count" implies that "all" viable microorganisms will be incorporated in the results of the assay. However viable but non-culturable (VBNC) microorganisms may have growth requirements not met by the incubation conditions. The failure of the assay to account for these organisms may lead to an under-estimation of the "true" microbial load and produce erroneous results which leads to poor prediction of shelf life. From a practical perspective the method is also very slow, requiring three days for colonies to form and a result to be obtained. For products with a short shelf life this delay is impractical and a product may be in retail distribution before the microbiological status has been ascertained. It is an object of the present invention to overcome at least one of the above- mentioned problems.

Statement of Invention

The invention relates to a method of assessing bacterial load of a sample product such as, for example, a food product. It is particularly useful in quantifying bacterial load of a sample, for example, determining whether any bacteria are present in the sample, or quantifying the bacterial load. In particular, the invention relates to a method of determining the Total Viable Count of a sample product, especially a food product such as meat carcass. The method employs quantitative PCR, especially real time quantitative PCR, and employs the rnp gene as the PCR target. The product of the rnp gene, RNase P, is partly a RNA molecule and, as such, will only be present in viable cells. In the assay, total RNA is suitably isolated, and then converted to cDNA using reverse transcriptase for detection by PCR. Typically, two target regions from the rnp gene are detected using PCR, one of which may be correlated with gram positive bacteria, and one of which may be correlated with gram negative bacteria. The primers employed to amplify the gram positive target region correspond to flanking sections of this target region which have been found to be conserved amongst gram positive bacteria. Likewise, the primers employed to amplify the gram negative target region correspond to flanking sections of this target region which have been found to be conserved amongst gram negative bacteria. The method of the invention typically involves detecting and quantifying both of these regions using PCR, and then correlating the level of each region (i.e. copy number) with viable bacteria counts for each of gram positive, and gram negative, bacteria. The two counts provided are then typically added to provide a TVC.

According to the invention, there is provided a method of assessing the bacterial load of a sample comprising a step of providing a sample of the microflora from the sample, isolating nucleic acid from the sample, performing real time PCR on the nucleic acid to amplify and quantify a target region from the sample, and correlating the level of target region with bacterial load, wherein the target region is rnp or a fragment thereof.

Suitably, the nucleic acid isolated from the sample is total RNA, which is then typically converted to cDNA which is detected by PCR. In an alternative embodiment, total DNA is extracted from the sample of microflora, which is detected using PCR. As RNA is generally only present in viable cells, it is preferable for the purpose of this assay to isolate total RNA from the sample of microflora, and then convert the mRNA to cDNA for PCR detection.

The method of the invention is suitably employed to quantify bacterial load of a sample. In a preferred embodiment of the invention, the method of the invention is suitably employed to quantify Total Viable Count (TVC) of bacteria of a sample. These terms may be used interchangeably. In this regard, two target regions from rnp are amplified and quantified, one of which is associated with gram positive bacteria, and one of which is associated with gram negative bacteria. The target region associated with gram positive bacteria has flanking sections that are conserved amongst typical gram positive bacteria associated with food spoilage, especially meat spoilage. Thus, the use of primers for nucleic acid amplification which correspond to these flanking regions will result in amplification of the target region only when those gram positive bacteria associated with food spoilage are present in the sample. Likewise, the target region associated with gram nagetive bacteria has flanking sections that are conserved amongst typical gram negative bacteria associated with food spoilage, especially meat spoilage. Thus, the use of primers for nucleic acid amplification which correspond to these flanking regions will result in amplification of the target region only when those gram negative bacteria associated with food spoilage are present in the sample.

In one embodiment, the target region associated with gram positive bacteria consists of the sequence provided in SEQUENCE ID NO: 1. Fig. IA shows the Enterococcus faecalis ribonuclease P RNA (rηpB) gene sequence, showing the forward and reverse primer binding sites that define the target region and which have been found to be conserved amongst gram positive bacteria. The target region may be amplified using a primer pair consisting of the oligonucleotides of SEQUENCE ID NO: 3 (forward primer) and 4 (reverse primer), or functional fragments of either. The binding sites for these primers are shown in Fig. IA. It should be noted that the reverse primer will not bind to the sequence indicated in Fig. 1, but will bind to a complementary sequence on the complementary strand (not shown). Ideally, the forward primer is a degenerate primer set of SEQUENCE ID NO: 3 (i.e. comprising each of the four possible primers of SEQUENCE ID NO: 3), typically in equal amounts.

In one embodiment, the target region conserved amongst gram negative bacteria consists of the sequence provided in SEQUENCE ID NO: 2. Fig. IB shows the

Escherichia coli RNA component of ribonuclease P sequence showing the primer binding sites. The target region may be amplified using a primer pair consisting of the oligonucleotides of SEQUENCE ID NO: 5 and 6, or functional fragments of either.

The binding sites for these primers are shown in Fig. IB. It should be noted that the reverse primer will not bind to the sequence indicated, but will bind to the complementary sequence on the complementary strand.

The term "functional fragment" should be understood as meaning a fragment of the recited primer which is capable of binding to, and initiating amplification of, the target region. A functional fragment will contain at least 10 contiguous nucleotides of the recited primer, and ideally at least 11, 12, 13, 14 or 15 contiguous nucleotides.

The method of the invention may be employed to assess bacterial load of a variety of samples, including food samples and clinical samples. Preferably, the food sample is a meat, fish or poulty product. In a preferred embodiment, the food sample is a carcass, especially a meat, fish, or poultry carcass. The sample may also be clinical sample such as a biological fluid such as blood, plasma, serum, saliva, cerebrospinal fluid, dental pulp, or the like.

The method of the invention may be also employed to detect bacterial contamination of a sample, for example a sample which is required to have a degree of sterility. An example of such a product would be a food product intended for consumption by a baby, or a surgical instrument or a medical device for use in the human body. In this regard, the two target regions of the rnp gene may be assayed according to the techniques described above, i.e. for both gram negative and gram positive bacteria, which will allow detection of any bacterial contamination of the sample.

The method of the invention may also be employed to quantify the loading of gram positive bacteria in a sample. In this regard, only the target region conserved in gram positive bacteria will be amplified and detected. The method of the invention may also be employed to quantify the loading of gram negative bacteria in a sample. In this regard, only the target region conserved in gram negative bacteria will be amplified and detected.

In one embodiment of the invention, the sample of microflora is obtained from the sample by taking a swab of the sample. Suitably, a plurality of swabs will be obtained from different parts of the one sample. Ideally, the swab is a sterile sponge. The sample of microflora obtained from the sample is then placed into a diluent, and then typically homogenised. In another embodiment, the sample may be obtained by stomaching a sample of food, for example, beef pieces (ISO 4833:2003 entitled: Microbiology of Food and Animal Feeding Stuffs - horizontal method for the enumeration of microorganisms — colony count technique at 3O⁰C).

The invention also relates to a kit suitable for assessing the bacterial load of a sample comprising a primer set suitable for amplifying a target region in the rnp gene associated with gram positive bacteria and/or a primer set suitable for amplifying a target region in the rnp gene associated with gram negative bacteria. As above, term "associated with" as applied to a target region should be taken to mean that flanking sections of the region are conserved, which allows for the design of primer pairs that correspond to the flanking regions allowing the region to be amplified using the primer pairs and, ideally, PCR.

Typically, the primer pair suitable for detection of a target region associated with gram positive bacteria comprises the oligonucleotides of SEQUENCE ID NO:s 3 and 4, or a functional fragment of either. An example of a target region for gram positive bacteria is provided in SEQUENCE ID NO. 1. Real time quantitative PCR using this primer pair results in the target region being amplified, and the level of product may then be quantified and correlated with viable bacterial count for gram positive bacteria.

Typically, the primer pair suitable for detection of a target region associated with gram negative bacteria comprises the oligonucleotides of SEQUENCE ID NO:s 5 and 6, or a functional fragment of either. An example of a target region for gram negative bacteria is provided in SEQUENCE ID NO. 2. Real time quantitative PCR using this primer pair results in the target region being amplified, and the level of product may then be quantified and correlated with viable bacterial count for gram negaitive bacteria.

In one embodiment, in which the kit is suitable for determining the Total Viable Count of a sample, the kit comprises a primer set suitable for amplifying a target region associated with gram positive bacteria and a primer set suitable for amplifying a target region associated with gram negative bacteria. Suitably, the two primer pairs consist of the oligonucleotides of SEQUENCE ID NO: 3 (g+ve forward) and 4 (g+ve reverse), and the oligonucleotides of SEQUENCE ID NO: 5 (g-ve forward) and 6 (g- ve reverse). A functional fragment of one or more of these oligonucelotides may also employed. An example of the target region is shown in Figs. IA and IB. Ideally, the gram positive forward primer is a degenerate primer. In a preferred embodiment of the invention, the kit comprises means for correlating a level of amplified target with bacterial TVC. Various means may be provided for such a correlation, including standard curves, algorithms, or software, the details of which will be obvious to a person skilled in the art. For example, the quantity or level of the amplified target region associated with gram positive bacteria is correlated with bacterial count using a gram positive amplification standard curve, and the quantity of the amplified target region associated with gram negative bacteria is correlated with bacterial count using a gram negative amplification standard curve. Suitably, the gram positive amplification standard curve is obtained from a mixed broth culture of Enterococcus faecalis, Lactococcus lactis, and Staphylococcus saprophytics. Typically, the gram negative amplification standard curve is obtained from a mixed broth culture of Enterobacter aerogenes, Escherichia coli, and Pseudomonas putida. The kit of the invention may further include one or more of the following reagents: means for extracting RNA; reverse transcriptase enzyme; DNA polymerase; dye reagent suitable for binding to double stranded DNA; one or more swabs for collecting the sample of microflora from the product to be analysed; maximum recovery diluent; spc agar; and cDNA standards for generating one or more standard curve.

Suitably, the swabs are adapted for collection a sample of microflora from a food product, especially a meat, fish, or poultry carcass.

In a particularly preferred embodiment of the invention, there is provided a kit suitable for the rapid determination of TVC in a sample, especially a food product, the kit comprising: - a primer pair consisting of the oligonucleotides of SEQUENCE ID NO: 3 (g+ve forward) and 4 (g+ve reverse), or a functional fragments of either;

- a primer pair consisting of the oligonucleotides of SEQUENCE ID NO: 5 (g-ve forward) and 6 (g-ve reverse), or a functional fragment of either;

- optionally, an amount of reverse transcriptase enzyme; - optionally, an amount of DNA polymerase; optionally, an amount of dye reagent suitable for detecting double stranded DNA; optionally, one or more swabs suitable for collecting a sample of microflora from the sample; and

- optionally, cDNA standard(s) for the generation of one or more standard curves.

Ideally, the gram positive forward primer is a degenerate primer.

The invention also relates to the use of the rnp gene as a target region in a quantitative PCR assay for determining the bacterial load, and in particular, quantifying the viable bacteria, in a sample. Preferably, two target regions are employed, one of which is a region of rnp conserved amongst gram negative bacteria, and one of which is a region of rnp conserved amongst gram positive bacteria. Suitably, the target region conserved amongst gram positive bacteria is the sequence of SEQUENCE ID NO: 1. Ideally, the target region conserved amongst gram negative bacteria is the sequence of SEQUENCE ID NO: 2

The invention also relates to an isolated oligonucleotide consisting of a sequence selected from the group consisting of: SEQUENCE ID NO's: 3 to 6, or a functional fragment of any of the four oligobnucleotides.

The invention also relates to a primer pair consisting of the isolated oligonucelotides of SEQUENCE ID NO's 3 and 4, or a functional fragments of either

The invention also relates to a primer pair consisting of the isolated oligonucelotides of SEQUENCE ID NO's 5 and 6, or a functional fragment of either.

The invention also relates to a primer set consisting of the isolated oligonucelotides of SEQUENCE ID NO's 3, 4, 5, and 6, or a functional fragment of any of the four oligonucleotides.

Brief description of the Figures

Figure 1

(A) The Enterococcus faecalis ribonuclease P RNA (rnpB) gene sequence showing the primer binding sites. Available with accession number: U64887 (B) The Escherichia coli RNA component of ribonuclease P sequence showing the priming sites. Available with accession number: X00211.

Figure 2

A 2% agarose gel of the PCR products of gram positive organisms showing the 197 bp amplicon. Lanes: 1 and 15 100 bp ladder, 2 No Template Control, 3 Staphylococcus haemolyticus, 4 Staphylococcus saprophyticus, 5 Enterococcus faecalis, 6 Enterococcus faecium, 7 Lactococcus lactis, 8 Lactobacillus sakei, 9 Enterococcus gallinarum, 10 Staphylococcus epidermis, 11 Staphylococcus scuiri, 12 Enterococcus hirae, 13 Brevibacterium linens, 14 Staphylococcus arlettae. Figure 3

A 2% agarose gel of the PCR products of gram negative organisms showing the 299 bp amplicon. Lanes: 1 and 15 100 bp ladder, 2 No Template Control, 3 Escherichia coli, 4 Shewanella putrefaciens, 5 Pseudomonas aeruginosa, 6 Pseudomonas putida, 7 Pseudomonas fragi, 8 Acinetobacter calcoaceticus, 9 Citrobacter freundii, 10 Enter obacter aerogenes, 11 Aeromonas hydrophila, 12 Aeromonas sahnonicida, 13 Klebsiella oxytoca, 14 Enterobacter sakazaki.

Figure 4

Amplification curves for gram positive assay (left to right) duplicates of 6.4x10⁷ cfu/ml, 6.4xlO⁶ cfu/ml, 6.4xlO⁵ cfu/ml, 6.4xlO⁴ cfu/ml and 6.4xlO³ cfu/ml, No RT enzyme (contaminating DNA) control, no template reverse transcriptase control and PCR negative (water) control.

Figure 5

Comparison of RT-RTQ-PCR counts from the LightCycler 2.0 and total viable counts by Standard Plate Count Agar technique, (y = 0.539 + 0.9017x, R²=0.88).

Detailed Description of the Invention

Isolation of microflora fi'om beef carcasses

In a commercial beef abattoir, the carcasses of 30 slaughtered steers were sampled. Carcass swabs were taken using a sponge swab technique of four sites before chilling, as outlined in the EU 471 directive. The swabs were examined by plating a dilution series on to Standard Plate Count Agar A.P.H.A (SPC) (Oxoid Ltd. U.K.) at 30°C for 72 hours to establish the total viable count, and on to a range of selective agars in order to examine the swabs for particular groups of microflora; MRS (De Man, Rogosa, Sharpe) agar at 30⁰C for 48 hours and 22°C for an additional 24 hours for mesophilic-psychrotrophic lactic acid bacteria, Pseudomonas agar with cetrimide-fucidin-cephaloridine (C-F-C) selective supplement at 25°C for 48 hours for Pseudomonas spp., MacConkey agar at 37°C for 24 hours for Enterobacteriaceae, Violet Red Bile Glucose agar 42°C for 18 hours for thermotolerant coliforms and Baird Parker Egg Yolk-Tellurite medium at 37°C for 24 hours for Staphylococcus spp. (all Oxoid Ltd. U.K.),.

Identification of dominant microflora In order to identify as many different bacterial species as possible so that they could be used as a reference panel, 40 colonies were selected for identification by 16S rDNA sequencing. Colonies were selected on the basis of diverse colony morphology on different growth media and on the particular growth media on which they developed. A gram stain was performed and the colony was inoculated into either Tryptone Soya Broth or MRS broth and incubated over night at 30°C. 1 ml of the culture was transferred to a sterile 1.5 ml microcentrifuge tube and centrifuged for lOmin at 10000 rpm. The supernatant was discarded and DNA extraction was carried out using the DNeasy Blood and Tissue kit (QIAGEN Ltd., U.K) on the basis of the gram reaction and according to the manufacturers' instructions. The quality and concentration of DNA extracted from each isolate was assessed using the Nanodrop NDlOOO instrument (LabTech International, U.K.).

The 16S rDNA from each isolate was amplified by PCR with a universal primer set; forward primer 5'-AGTTTGATCCTGGCTCAG-S' and reverse primer 5'- TACCTTGTTACGACTT-3' [I]. PCR amplification was performed in a 50 μl reaction of 25 μl of 2X Taq PCR Master Mix (QIAGEN Ltd., U.K.), 0.6 μM each primer (MWG Biotech, Germany) and 50 ηg of template DNA. The amplified products were purified using the QIAQuick PCR purification kit (QIAGEN Ltd., U.K) according to the manufacturers' instructions and sequenced commercially. The National Center for Biotechnology Information BLAST service (http://www.ncbi.nlm.nih.gov/BLAST) was used to identify each isolate from the sequencing results [2].

Choice of target gene and primer design for rnp genes and sequencing Based on the identification and bioinformatic analysis of the predominant microflora present in the meat carcass samples, the ribonuclease P gene (rnp) was surprisingly found to be a suitable choice of target gene.

Sequences were obtained for 8 typical meat flora bacteria from the RNase P Database [3] and the GenBank database [4]. Oligonucleotide primers (Table 1) were designed using the DNAstar software to target the rnp genes (Figure. 1). Prospective primers were checked for hairpin structure, dimers, cross-dimers and repeats using the Primer Design program (Lasergene, USA). Initial trials showed that it was not possible to design one set of primers which would encompass total bacteria using the selected target. Instead, one set was designed for gram positive organisms and the second pair for gram negative organisms. Using DNA extracted from a number of strains, PCR was performed according to the origin of the DNA (i.e. gram positive or gram negative). All PCR was carried out in a DNA Engine Dyad™ (Bio-Rad Ltd., U.K.). Table 1 - RNP primer sequences

10 The gram negative PCR conditions consisted of; initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95⁰C for 30 s, annealing at 47⁰C for 40 s and primer extension at 72°C for 30 s, with a final extension step at 72°C for 5min and cooling to 4°C. All products were subjected to electrophoresis on a 2% agarose gel. The expected 299 bp product for each gram negative strain was observed.

15 The gram positive PCR conditions consisted of; initial denaturation at 95⁰C for 5 min, 40 cycles of denaturation at 95⁰C for 30 s, annealing at 54⁰C for 40 s and primer extension at 72°C for 30 s, with a final extension step at 72°C for 5min and cooling to 4°C. All products were subjected to electrophoresis on a 2% agarose gel. The expected 197 bp product for each gram positive strain was observed. 15 of the products were randomly

20 selected and extracted from the gel using the QIAQuick Gel Extraction kit (Qiagen, U.K.) according to the manufacturers' instructions. Purified PCR products were sequenced commercially and sequencing data was analysed as described above. To test the specificities of each primer set, conventional and real-time PCR assays were conducted against a range meat microflora (Table 2). In addition, the gram positive primer set was tested against a mix of cDNA (10⁶ cell equivalents) from gram negative bacteria and vice versa to determine if any cross reaction took place.

Table 2

Taxon Strain Reaction with the following primers

Gpos-mpF/ Gneg-rnpF/

Gram Positive bacteria Gpos-rnpR Gneg-rnpR

Bacillus cereus NCTC 07464 Enterococcus faecalis NCTC 12697 Enterococcus faecium ATCC 35667 Lactobacillus plantarum ATCC 8014 Lactobacillus sakei UC7012 Lactococcus lactis NCDO 509 Pediococcus acidilactici NCDO 521 Staphylococcus saprophyticus ATCC 15305 Staphylococcus haemolyticus ATCC 29970 Staphylococcus sciuri * Staphylococcus epidermis * Brevibacterium linens* Enterococcus hirae* Staphylococcus arlettae* Enterococcus gallinarum*

Gram negative bacteria

Aeromonas hydrophila ATCC 35654 +

Pseudomonas aeruginosa NCTC 12903 +

Pseudomonas putida ATCC 49128 +

Citrobacter freundii NCTC 09750 +

Enterobacter aerogenes NCTC 10006 +

Citrobacter diversus CCFRA 7119 +

Klebsiella oxytoca ATCC 43086 +

Pseudomonas fragi DSM 3456 +

Acinetobacter calcoaceticus ATCC 23055 +

Shewanella putrefaciens DSM 6067 +

E.CO// K12 ER 2925 +

Alcaligenes faecalis subsp. Parafaecalis DSM 13975 +

Enterobacter agglomerans NCTC 09381 +

Serratia liquefaciens DSM 4487 +

Enterobacter aerogenes ATCC 49469 +

Hafnia alvei DSM 30163 +

Enterobacter sakazaki NCTC 11467 +

Acinetobacter iwoffii DSM 2403 +

Citrobacter koseri NCTC 10768 +

Enterobacter clocae NCTC 10005 +

Aeromonas salmonicida* +

Salmonella Typhimurmim* +

"non-typed isolate Enzymatic pre-treatment of SYBR Green I mastermix

The contamination of reagents with bacterial DNA was a difficulty, which was observed while performing the above experiments. This was further exacerbated by the sensitive quantitative nature of the PCR. Investigations of a number of reagents used in the real- time PCR indicated the Taq polymerase SYBR Green I mix was the source of the contamination. Enzymatic pre-treatment of mixes has been previously described [5, 6]. In this study, a combination of AIu I (Roche, U.K.) and Amplification Grade DNase I (Invitrogen, U.S.A) were used at a concentration of 0.5U/ul of mastermix and 0.05U/ul of mastermix respectively. The conditions for the treatment were 37°C for 20 mins (mixed half way), 8O⁰C for 2 mins, and cooled. Primers, water and DNA or cDNA were added after incubation.

RNA extraction from bacterial species and reverse transcription

A 1 ml aliquot of each standard or sample was transferred to a 1.5ml sterile microcentrifuge tube and centrifuged at 10000 rpm for 10 min. The supernatant was discarded and RNA extraction was carried out using the RiboPure Yeast RNA extraction kit (Ambion Europe, U.K.) with amendment to the elution volume to achieve a higher concentration and yield of RNA (2 x 26μl). In addition a second DNase I treatment was found to be necessary at an early stage of development, and was carried out using Amplification Grade DNase I (Invitrogen, USA) as described in the manufacturer's instructions. Reverse transcription was carried out using the ImProm-II Reverse Transcription system (Promega, USA) according to the manufacturer's instructions. Each 20μl reaction contained; 4μl of DNase treated RNA, 0.5μM reverse primer (gram positive or gram negative), 2.5 mM MgCl₂, 1 μl of dNTPs, 2 U of reverse transcriptase and 4 μl of RT 5X buffer. Two reverse transcriptase reactions were carried out for any mixed or unknown sample, one for gram negative species and one for gram positive species. The thermal conditions for the reverse transcription were: 25°C for 5 min, 42⁰C for 1 hour, 70°C for 15 mins followed by a cooling step.

Real time PCR amplification ofrnp cDNA

PCR was conducted using the LightCycler 2.0 instrument (Roche Diagnostics, GmbH Mannheim, Germany). Reaction mixtures contained a total volume of 20μl consisting of; 4.4μl of enyzmatically treated 5X SYBR Green I PCR Master Mix (Roche Diagnostics, Germany), 0.225μM of forward primer and 0.125μM of reverse primer (MWG Biotech, Germany) and 2 μl of cDNA. The conditions for the gram negative amplification were 1 cycle at 95°C for 10 min, followed by 45 cycles of 32 s at 95⁰C, annealing 16 s at 49°C, 14 s at 72⁰C and 84°C for 2 s for data acquisition. The conditions for the gram positive amplification were as above except for annealing for 16 s at 55°C, 11 s at 72°C and 76°C for 2 s for data acquisition.

Standard curve and melt curve analysis

In order to generate a gram negative standard curve a mixed broth culture of Escherichia coli Kl 2 (ER 2925), Enterobacter aerogenes (ATCC 49469) and Pseudomonas putida (ATCC 49128) was serial diluted. These species were chosen as representatives of the gram negative microflora typical to meat. To generate a gram positive standard curve a mixed broth culture of Enterococcus faecalis (NCTC 12697), Lactococcus lactis (NCDO 509) and Staphylococcus saprophytics (ATCC 15305) was serial diluted. These were chosen as representative species of the typical gram positive microflora typical to meat. Each dilution was then plated and grown on plate count agar. RNA was extracted from 1 ml of each dilution as described. Following calculation of the initial amount of bacteria in the broth culture, the quantity of bacteria in each dilution was established. Reverse transcription of standards was carried out as described above and each standard was amplified in duplicate using the real-time PCR conditions described above. The concentration of each standard in cfu ml^"1 was inputted into the software and using the threshold cycle value (C_T) for the corresponding standard in the absolute quantification analysis tools on the LightCycler 2.0 software, a standard curve was generated which was then used to interpolate the number of bacteria present in unknown or blind samples. Melt curve analysis was carried out after the amplification program to ensure a single peak was present and to identify any non-targeted PCR products. The melting curve was obtained by heating at temperatures from 65 to 95°C at a ramp rate of 0.2°C/s with continuous fluorescence monitoring. Subsequent agarose gel electrophoresis was used to visualise the amplification products to ensure a single product was formed.

Application of RT-RTQ-PCR to enumerate total viable counts

A random selection of bacteria from the panel (Table. 2) were grown over night. The bacteria were then mixed and diluted to create an assorted group of 20 cocktail samples of unknown composition and concentration. Each mixed sample was diluted in a 10-fold dilution series and 1 ml of each dilution was plated in duplicate. They were then overlaid with sterile molten Standard Plate Count Agar using the pour plate method. Separately, a 1 ml aliquot of each neat sample was taken for RNA extraction and subsequent reverse transcription. This was carried out independently of microbiological analysis. Cell numbers were then quantified by RT-RTQ-PCR using the standard curves generated on the LightCycler instrument. The threshold cycle values (C_T) in the linear range of the assay were applied to the standard curves generated previously to determine the number of total bacteria in the extracted sample and then converted to log₁₀ cfu ml^"1.

Construction and sequencing of DNA library

To ensure that the primers for either reaction were not biased towards the amplification of DNA from certain strains, a PCR clone library was set up. To test the gram negative primer set, PCR amplicons from a 1:1 :1 mixture of: Klebsiella pneumoniae, Pseudomonas fragi and Enterobacter aerogenes, were cloned into the plasmid. Similarly, for the gram positive set, PCR amplicons from a 1:1:1 mixture of Lactobacillus sakei, Enterococcus faecium and Staphylococcus saprophytics were taken. The cloned PCR products and subsequent transformation were carried out using the Qiagen PCR Cloning PLUS kit (QIAGEN Ltd, U.K.) as per the manufacturer's instructions. Breifly, white colonies/blue-white colonies were selected at random and grown overnight in LB broth containing 50μg/ml kanomycin. 2 x 1 ml of culture was then centrifuged in a 1.5ml microcentrifuge tube and plasmid purification was carried out. The plasmid was extracted using the QIAprep Miniprep kit (QIAGEN Ltd., U.K.). The purified plasmid was then digested using EcoRI (Promega, USA) and subjected to agarose gel electrophoresis to check for the appropriate PCR product insert. Any reaction showing the incorporation of the insert was sent for sequencing commercially using T7 promoter standard primer. In all, 17 reactions from the gram negative PCR and 13 samples from the gram positive PCR were analysed. Sequences were analysed using the BLAST service as described.

Statistical analysis

The relationship between the real-time PCR method and the traditional plate count method for determining total viable counts for mixed microflora in liquid media was examined using linear regression analysis (Genstat, VSN International Ltd., U.K.)- For the linear regression equation, the adjusted R² value (which measures the quality of fit of the equation) and the 95% confidence limits for a predicted value of plate count were obtained.

Results

Identification of microbial species

Nucleotide sequence analysis of 16S rDNA PCR fragments was used to investigate the bacterial diversity of beef carcasses. If a sequence was within 2 bases of matching a known sequence, the colony was placed into the corresponding taxon. It was thus assumed that a 2-base mismatch represented Taq polymerase error or strain variation [7]. In previous studies sequencing of the 16S rDNA gene has been shown to be successful in identifying bacteria when coupled with sequencing [1, 7]. Here the 40 bacterial strains identified belonged to 11 different taxonomic families. Among the species detected and identified at this stage of the beef slaughter process, Staphylococcus spp., Aeromonas spp. and Escherichia coli were predominant. Although previously proposed meat spoilers such as Pseudomonas spp. and Acinetobacter spp. were also detected here [8, 9].

rnp gene PCR By using two sets of primer pairs for the gram negative and positive rnp genes, DNA from the panel of bacteria was successfully amplified. Using the conditions described above it was possible to generate a single 197 bp product for a gram positive bacteria panel using the primer set (Figure 2) and a single 299 bp for all gram negative bacteria panel using primer set (Figure. 3). Sequencing confirmed the products to be rnp genes.

RT-RTQ-PCR

The melting curve analysis of each PCR showed a single peak. The gram positive peak appeared at ~85°C while the gram negative peak appeared at ~90°C. Sample peaks showed up between 80-86°C and 86-92⁰C for the gram positive and gram negative assay respectively. Agarose gel electrophoresis showed the presence of the expected amplicons. The gram positive and gram negative assays showed linearity over the range 8.9xl0⁷-lxl0³ CFU ml^"1 and 9.1xl0⁸-lχl0³ CFU ml^"1 respectively. In both assays, non- specific products were formed after 40 cycles in the no template reverse transcriptase control (no RNA), no reverse transcriptase enzyme control (contaminating DNA) and PCR control water (Figure 4). These products were analysed by agarose gel electrophoresis and found to be either primer dimer or a <100 bp artefact. Subsequently, any sample or standard that gave an amplification product after 40 cycles was discounted.

Statistical Comparison of RT-RTQ-PCR and traditional cultural method

To investigate the relationship between a total viable count by culture and a total count by RT-RTQ-PCR, the results from the gram negative assay and gram positive assay were added together. Correlation analysis was performed on both methods of detection and they were shown to be significantly correlated over the range LogAs shown in Figure. 5, the result from the PCR assay and the plate count technique were found to correlate well over the range from Log₁₀ 2.3 to Log₁₀ 6.8 CFU/ml (R² = 0.88).

Cloned PCR products

BLAST analysis of the sequenced PCR products gave the following results for the 17 samples from the gram positive PCR: 10 Klebsiella spp., 4 Pseudomonas spp. and 3 unidentified. Similarly, analysis of the 13 samples from the gram positive reaction gave the following results as the closest match: 5 Staphylococcus saprophyticus, 3 Enter ΌCOCCUS faecalis, 4 Enterococcus faecium and 1 no significant similar match. Once again, strain variation was considered to be the cause of lack of significant matches or appearace of related species.

A linear curve suitable for quantification was achieved for both the gram positive and gram negative standard curves (R²=0.99 and 0.98 respectively), despite the use of a degenerate primer. Cloning the PCR products from a PCR with DNA from known species allowed the identification of any biased binding of the primers to one specific DNA source. Here none was seen, as 30 sequences were tested and no bias was observed in either the gram negative or gram positive section of the assay. Although sequencing did identify Enterococcus faecalis sequences in a mix that did not contain the addition of DNA from this source, Enterococcus faecalis and Enterococcus faecium, which was in the reaction mix, are closely related. Thus, by employing the methods of the invention, it is possible to quantify total bacteria with a high degree of accuracy, independent of culturability, which provides a genuine representation of the numbers and species present on a variety of different matrices.

Statistical analysis showed a good correlation between the cultural and the developed RT-RTQ-PCR method (R² = 0.88, residual standard deviation = 0.466).

The method and kits of the invention allow an accurate quantification of total bacteria with high sensitivity and accuracy. Furthermore, the real-time PCR results could be obtained in 8-12 h in comparison to 72 h for the colony count method. The novel method has the potential to be applied directly to enumerate total viable counts in food samples.

The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.

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Claims

1. A method of determining a bacterial load value of a food product comprising the steps of processing a sample of nucleic acid isolated from the food product to amplify and quantify a target region associated gram positive bacteria, and a target region associated with gram negative bacteria, and correlating the quantity of amplified target regions with a bacterial load value for the food product, wherein the target region associated with gram positive bacteria is amplified using a primer set comprising of SEQUENCE ID NO. 3 (gram positive forward primer) or a functional fragment thereof, and SEQUENCE ID NO. 4 (gram positive reverse primer) or a functional fragment thereof, and the target region associated with gram negative bacteria is amplified using a primer set comprising of SEQUENCE ID NO. 5 (gram negative forward primer) or a functional fragment thereof, and SEQUENCE ID NO. 6 (gram negative reverse primer) or a functional fragment thereof.

2. A method as claimed in Claim 1 in which the level of target region associated with gram positive bacteria is correlated with a viable count of gram positive bacteria, and the level of target region associated with gram negative bacteria is correlated with a viable count of gram negative bacteria, and wherein the bacterial load value is calculated by summation of the viable counts obtained.

3. A method as claimed in Claim 1 or 2 in which the food product is a meat product.

4. A method as claimed in Claim 3 in which the meat product is a meat carcass.

5. A method as claimed in any preceding Claim in which the gram positive forward primer comprises is a degenerate primer set of SEQUENCE ID NO: 3.

6. A method as claimed in any preceding Claim in which the quantity of amplified target regions is correlated with bacterial count using an amplification standard curve.

7. A method as claimed in Claim 6 in which the quantity of the amplified target region associated with gram positive bacteria is correlated with bacterial count using a gram positive amplification standard curve, and the quantity of the amplified target region

1 associated with gram negative bacteria is correlated with bacterial count using a gram negative amplification standard curve.

8. A method as claimed in Claim 7 in which the gram positive amplification standard curve is obtained from a mixed broth culture of Enterococcm faecalis, Lactococcus lactis, and Staphylococcus saprophyticus.

9. A method as claimed in Claim 7 in which the gram negative amplification standard curve is obtained from a mixed broth culture of Enterobacter aerogenes, Escherichia coli, and Pseudomonas putida.

10. A kit of parts suitable for the determination of a bacterial load value of a food product, the kit comprising the oligonucleotides of SEQUENCE ID NO: 3 (gram positive forward primer) or a functional fragment thereof, SEQUENCE ID NO: 4 (gram positive reverse primer) or a functional fragment, SEQUENCE ID NO: 5

(gram negative forward primer) or a functional fragment thereof, and SEQUENCE ID NO: 6 (gram negative reverse primer), or a functional fragment thereof.

11. A kit of parts as claimed in Claim 10 and further including one or more reagents selected from the group consisting of: reverse transcriptase enzyme; DNA polymerase; dye reagent suitable for detecting double stranded DNA; one or more cDNA standards for generating amplification standard curves; gram positive and gram negative amplification standard curves, and one or more swabs suitable for collecting a sample of microflora from the sample.

12. A kit of parts as claimed in Claim 10 or 11 in which the gram positive forward primer is a degenerate primer set of SEQUENCE ID NO: 3.