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WO2009036956A1 - Détection de staphilococcus aureus dans du lait de vaches souffrant de mastite - Google Patents

Détection de staphilococcus aureus dans du lait de vaches souffrant de mastite Download PDF

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Publication number
WO2009036956A1
WO2009036956A1 PCT/EP2008/007735 EP2008007735W WO2009036956A1 WO 2009036956 A1 WO2009036956 A1 WO 2009036956A1 EP 2008007735 W EP2008007735 W EP 2008007735W WO 2009036956 A1 WO2009036956 A1 WO 2009036956A1
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Prior art keywords
aureus
milk
gene
bacteria
qpcr
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Hans Ulrich Graber
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Universitaet Bern
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Universitaet Bern
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the invention relates to a method for detecting Staphylococcus aureus in milk and the diagnosis of bovine mastitis. It further relates to an improved method of extracting bacteria from any kind of biological fluid, e.g. body fluids such as milk.
  • Staphylococcus aureus (S. aureus) is causing mastitis in cows and represents one of the most important death causes apart from reproduction disorders. This causes substantial financial damage to milk farming, and is estimated to amount to 200 Mio. $ per year in Switzerland alone.
  • Diagnostics of S. aureus based on bacteriological testing of milk is unsatisfactory. For a single test under optimal condition diagnostic sensitivity reaches only 75% on average and may be as low as 41% (Sears, P.M. et al., J. Dairy Sci., 73:2785-2789, 1990). A reasonable sensitivity (98%) can only be reached by analysis of three consecutive samples. Reasons for the unsatisfactory sensitivity are the high limit of detection in bacterial testing and the intermittent excretion of S. aureus into milk. The low diagnostic sensitivity of existing S. aureus tests make systematic survey of herds difficult, time- consuming and expensive. There is a clear need for improved, reliable S. aureus testing in bovine milk.
  • the invention relates to a method of detection and quantification of S. aureus in milk comprising the steps of trypsinizi ⁇ g a milk sample, extracting bacteria and isolating nucleic acids, and quantifying DNA specific for S. aureus using primers for a gene or gene fragment specific for S. aureus in a polymerase chain reaction.
  • the method is superior to the bacterial assays of the state of the art both in specificity and sensitivity.
  • the method is used to diagnose mastitis caused by S. aureus in cows.
  • trypsination of milk samples is performed in the presence of Lactobacillus casei (L casei) or related bacteria.
  • the invention further relates to a method of improved extraction and isolation of bacterial nucleic acids from biological fluids, e.g. body fluids such as milk, or from dairy products, meat and meat products, in the presence of L casei or related bacteria.
  • biological fluids e.g. body fluids such as milk, or from dairy products, meat and meat products
  • This method is suitable for any kind of bacteria to be detected and optionally quantified, e.g. to pathogenic bacteria.
  • Body fluids considered are e.g. milk, urine, serum, plasma or liquor, swabs or lavages of mucosa, homogenates of body tissue, or homogenates of feces.
  • the invention relates to a method of detection and quantification of S. aureus in milk.
  • diagnosis of mastitis in cows is mainly based on an indirect test, e.g. the so- called California Mastitis Test, which indicates inflammatory problems based on the number of leukocytes found in milk.
  • California Mastitis Test which indicates inflammatory problems based on the number of leukocytes found in milk.
  • a more extensive analysis uses bacteriological identification of S. aureus and other mastitis-related organisms in milk by plating milk specimens on nutritients supporting growth of S. aureus and other pathogens, and detection based on morphology, biochemical properties and hemolysis.
  • the present invention describes and claims a much improved diagnostic method based on quantitative polymerase chain reaction (QPCR).
  • QPCR quantitative polymerase chain reaction
  • the invention relates to a method of detection and quantification of S. aureus in milk, preferably bovine milk, comprising the steps of trypsinizing a milk sample, extracting bacteria and isolating nucleic acids, and quantifying DNA of S. aureus using primers for a gene or gene fragment specific for S. aureus, for example a primer for the nuc gene, in a polymerase chain reaction.
  • Suitable primers for a gene or gene fragment specific for S. aureus are, for example, primers for the coa (Coagulase) gene, spa (Protein A) gene, elf A (Clumping factor A) gene, or for S. aureus specific fragments of 23S rRNA.
  • the method is superior to the bacterial assays of the state of the art both in the analytical specificity and sensitivity.
  • the method of the invention shows a low intra-assay and inter-assay variability and is more sensitive than the standard bacterial assay by a factor of more than 25. Correlation of the inventive method with the state of the art bacterial assay is very high. Compared to state of the art phenotype bacteriology the method of the invention allows a safe decision with respect to presence and source of S. aureus in milk, and a reliable quantification without having to make use of dilution series.
  • Diagnosis of mammary infection in cows is based on the presence or absence of mastitis-related bacteria such as S. aureus in milk as determined with the method of the invention.
  • mastitis-related bacteria such as S. aureus in milk
  • To confirm mastitis it might be desirable to further test the cows for inflammation typical of mastitis, for example by measuring the content of leukocytes in milk. Such measurements may be based on machine or manual cell counting or the standard California Mastitis Test as described hereinabove.
  • the invention further relates to a method of diagnosis of mastitis caused by S. aureus in cows comprising the steps of drawing milk samples, quantifying S. aureus by trypsinizing the milk sample, extracting bacteria and isolating nucleic acids, and quantifying DNA of S. aureus using primers for a gene or gene fragment specific for S. aureus, for example a primer for the nuc gene, in a polymerase chain reaction, wherein mastitis is diagnosed when the count of S. aureus is higher than approx. 1400 staphylococcal cell equivalents (SCE) per ml of milk.
  • SCE staphylococcal cell equivalents
  • the invention further relates to a method of improved extraction and isolation of bacterial nucleic acids from biological fluids wherein the fluid is loaded with 10 6 to 2 x 10 10 colony forming units (CFU) of L casei or related bacteria per ml of fluid, treated with trypsin, incubated between 37°C and 60 0 C for 10 to 60 minutes, and distributed between an apolar solvent (optionally including chaotropic agents such as urea, guanidine or guanidine isothiocyanate) and an aqueous phase, followed by standard methods of bacterial extraction, nucleic acid isolation and optionally nucleic acid detection and/or quantification.
  • CFU colony forming units
  • a preferred concentration of L. casei is between 10 9 and 10 10 , e.g. around 5 x 10 9 CFU per ml of biological fluid.
  • Samples for extraction and isolation of bacterial nucleic acids are usually between 0.1 ml and 1000 ml, preferably around 0.4 ml. This means that around 1.5 x 10 9 CFU for a sample size of 0.4 ml fluid, e.g. a body fluid such as milk, are used.
  • Body fluids considered are e.g. milk, urine, serum, plasma, liquor cerebrospinalis, abdominal and thoracic fluid, lavages or swabs of mucosa, e.g.
  • the method of the invention for an improved extraction of bacterial nucleic acids using L casei or related bacteria for example Lactobacillus spp., Lactococcus spp., Streptococcus spp., Streptomyces spp., Propionibactehum spp., Bifidobacterium spp., or non pathogenic laboratory bacteria such as strains of Escherichia coli, which are used in molecular biology.
  • the method is not restricted to detection of nucleic acids from S. aureus, but may be used for any problem to be solved involving detection and quantification of small amounts of any particular (potentially pathogenic) bacteria in combination with suitable probes for such bacteria and QPCR. Addition of L.
  • casei or related bacteria any bacterium is suitable as long as the detection by QPCR of the specific nucleic acid from the particular bacteria to be isolated is not hindered
  • a sufficient amount of L. casei or related bacteria further improves the repeatability and reliability of quantification results of QPCR.
  • DNA from L casei or related bacteria supports the detection and analysis of the integrity of isolated DNA by gel electrophoresis.
  • Use of the method of the invention for an improved extraction of bacterial nucleic acids using L casei or related bacteria is not restricted to cases of extraction and nucleic acid isolation by centrifugation but is equally well suited for filtration methods or filtration steps within extraction and isolation methods.
  • Trypsination is performed according to standard procedures in the art. Trypsin of different sources may be used, although Trypsin 250TM of Becton Dickinson is preferred. Alternatively, proteinase K, pronase (Roche; a mixture of proteases) or other trypsin/protease products may be used. Detergents such as Triton-X100 or other non- ionic detergents (e.g. Tween derivatives) or ionic detergents (e.g. sodium dodecyl sulfate or bile acid salts), chelating agents such as EDTA or EGTA (to eliminate calcium in milk), and various buffers (e.g. phosphate buffer) are added.
  • Triton-X100 e.g. Tween derivatives
  • ionic detergents e.g. sodium dodecyl sulfate or bile acid salts
  • chelating agents such as EDTA or EGTA (to eliminate calcium in milk)
  • Trypsination is preferably accomplished for 10 to 20 minutes, e.g. 15 minutes, at temperatures around 55 0 C.
  • a preferred apolar solvent is pentane. If pentane and temperatures around 25°C are used, then the apolar phase will remain fluid on centrif ligation. After treatment of the samples with an aqueous base such as sodium hydroxide solution, they are cent ⁇ fuged at top speed (between 19'00Og and 30'00Og) at temperature near 0 c C, e.g. around 4 0 C, in order to ease separation of supernatant from damaged bacteria.
  • an aqueous base such as sodium hydroxide solution
  • Isolation of DNA is accomplished according to standard procedures, for example as described by Chavagnat, F. et al., FEMS Microbiol. Lett., 217:177-183, 2002, using lysozyme, mutanolysin and the "High Pure PCR Template Preparation Kit” of Roche Diagnostics, but is not restricted to those particular reaction conditions. It is preferable to use standardized analysis conditions in order to obtain repeatable recovery and repeatable analytical sensitivity.
  • CMT California Mastitis Test
  • the teat ends are thoroughly purified with gauze pledgets moistened with 70% ethanol, and individual quarter milk samples are collected aseptically in sterile vials.
  • the obtained probes are cooled to 4°C, transported into the laboratory at 4°C and stored at - 2O 0 C.
  • the agar plates are incubated aerobically at 37°C and are analyzed after 24 h and 48 h of incubation. Bacteria are identified according to the guidelines of the National Mastitis Council (NMC 1999) which include morphology, biochemical properties, and detection of hemolysis. On BA plates, colonies are considered typical for S. aureus if they are large, greyish-white to yellow showing ⁇ or double hemolysis. On BP plates, large black to grey colonies surrounded by an unambiguous, opaque precipitation halo are accepted as S. aureus. After 24h and 48h, colonies typical for S. aureus are counted on BP and BA plates. A total of 38 epidemiological ⁇ unrelated S. aureus and 36 CNS isolates were obtained from the diagnostic laboratory Dr.
  • NA nucleic acids
  • Standard PCR PCR for 1 sample is performed in 25 ⁇ l containing 1x HotStarTaq® Master Mix (QIAGEN AG, Hombrechtikon, Switzerland), 2.5 ⁇ l of NA, 300 nM (final concentration) of each of the spa, coa or clfA gene primers (Akineden, O. et al., Clin. Diagn. Lab. Immunol., 8:959- 964, 2001) and 1 ⁇ M for both nuc gene primers, respectively (see table 1). All the primers are obtained from Microsynth, Balgach, Switzerland.
  • a pre-PCR step is run at 95 0 C for 15 minutes followed by 35 cycles under following conditions: denaturation at 94°C for 1 minute, annealing at 6O 0 C for 1 minute and extension at 72 0 C for 1 minute.
  • the reaction is completed by a 10 minutes step at 72 0 C followed by cooling down to 4 0 C.
  • the PCR products are analyzed by agarose gel electrophoresis in 0.5x TBE including GelRed stain. Table 1.
  • the genes nuc, coa, spa and clfA are specific for Staphylococcus aureus (S. aureus).
  • oligonucleotide served as a QPCR probe. It was labeled with 6-carboxyfluorescein (FAM) at the 5' end and with 6-carboxytetramethylrhodamin (TAMRA) at the 3' end.
  • FAM 6-carboxyfluorescein
  • TAMRA 6-carboxytetramethylrhodamin
  • DNA sequencing nuc gene amplicons are purified using the "QIAquick PCR Purification Kit” (QIAGEN) according to the protocol of the manufacturer. The DNA concentration of the eluates is measured by spectroscopy using the Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies, Rockland, DE, USA). 100 ng are then added to a total volume of 10 ⁇ l containing 20 pmol of one of the sequencing primers (see table 1). The sequencing procedures are done at Microsynth. Each amplicon is sequenced once on both strands which is enough for reliable evaluation due to the very high quality of the obtained sequences.
  • NA nucleic acids
  • the thawed milk samples used to determine the S. aureus plate counts are simultaneously processed to extract bacterial NA. For that reason, 400 ⁇ l milk of each sample is pipetted to a solution containing 250 ⁇ l Triton-X100 2% (w/v; Merck), 150 ⁇ l L case/ (1.5x10 9 CFU) and 125 ⁇ l 1% trypsin solution (for details see below). After incubating at 55 0 C for 15 minutes, 925 ⁇ l pentane (Merck) is added to the suspension followed by vortexing the samples three times for 10 seconds. Afterwards, the specimens are centrifuged at 18'00Og for 10 minutes (25°C).
  • the supernatant is discarded and the cell wall of the pel letted bacteria is lysed according to Chavagnat et al. (loc. cit.) with a minor modification: after the NaOH treatment centrifugation is performed at 20'80Og for 10 minutes (4 0 C). NA is then extracted with the "High Pure PCR Template Preparation Kit” (Roche Diagnostics). The standard protocol as listed below is preferably followed. The eluates (200 ⁇ l) are checked for DNA integrity and quantity by agarose gel electrophoresis. The extracts are stored at -20 0 C until further use.
  • L casei strain 18121 , was obtained from D. Isolini, Swiss Federal Research Station for Animal Production and Dairy Products, Liebefeld-Bern, Switzerland.
  • MRS De Man Rogosa Sharp
  • casei is done spectroscopically using a wave length of 650 nm and an own standard curve obtained from 3 independent dilution experiments including plating on MRS agar plates, incubation at 3O 0 C for 48 h and counting the grown colonies.
  • the bacteria are stored in MRS medium (10 9 CFU/100 ⁇ l) at 4 0 C for up to 3 weeks.
  • a pCI plasmid vector was obtained from Prof. Dr. A. Zurbriggen, Department of Clinical Veterinary Medicine, Vetsuisse-Faculty of Berne, Berne, Switzerland containing the full length N gene of the canine distemper virus (CDV- N gene). The insert is verified by sequencing the sense strand using the T7 primer (Microsynth). Afterwards, it serves as a template for a standard PCR to produce enough spiking DNA.
  • plasmid DNA is added to a final volume of 25 ⁇ l containing 1x HotStarTaq® Master Mix (QIAGEN), and 300 nM (final concentration) of the CDV-N-S and CDV-N-AS primer (see table 1).
  • Amplification is performed by a pre- PCR step at 95°C for 15 minutes followed by 33 cycles under following conditions: 94 0 C for 1 minute, 63 0 C for 1 minute and 72°C for 1 minute. The reaction is finished by a 10 minutes step at 72 0 C followed by cooling down to 4°C.
  • the PCR product ((788 bp) is analyzed by agarose gel electrophoresis in 0.5x TBE including Gel Red stain and is purified with the "QIAquick PCR Purification Kit” (QIAGEN) according to the protocol of the manufacturer. Quantification of the eluate DNA is done with the Nanodrop spectrometer.
  • DNA specific for S. aureus is quantified by QPCR. For that reason, 2 primers and a probe for the nuc target gene are designed (see table 1) amplifying a 166 bp fragment.
  • the probe is labeled at the 5 1 end with 6-carboxyfluorescein (FAM) and at the 3' end with 6-carboxytetramethylrhodamin (TAMRA; Microsynth).
  • FAM 6-carboxyfluorescein
  • TAMRA 6-carboxytetramethylrhodamin
  • Each 25 ⁇ l of QPCR mixture contains 1x TaqMan Universal PCR master mix (Applied Biosystems, Rotnch, Switzerland), 900 nM sense and antisense primer, 300 nM probe and 3.5 ⁇ l NA extracted from bacteria in milk.
  • the QPCR conditions are 5O 0 C for 2 minutes and 95°C for 10 minutes followed by 45 cycles of 95 0 C for 15 seconds and 60° for 1 minute. Cycling is performed in the GeneAmp 5700 Sequence Detection System (Applied Biosystems). To ensure that negative results are not due to nonspecific inhibition of the QPCR assay, the same NA is used in a second QPCR in the presence of an internal control DNA
  • CDV-N amplicon amplified with a CDV-N specific QPCR (amplified fragment: 130 bp).
  • each reaction 25 ⁇ l contains 1x TaqMan Universal PCR master mix (Applied Biosystems), 300 nM sense and antisense primer, 300 nM FAM/TAMRA labeled probe (see table 1 ; Microsynth), 3.5 ⁇ l NA and 10 5 copies of the CDV-N amplicon.
  • CDV-N QPCR is also used to check for PCR inhibitors contained in the NAs. If the slope (gradient?) of the amplification curve (log-transformed fluorescence intensities) is obviously flatter or the CT value (CT: cycle to reach the threshold) is more than 2 cycles larger than the mean, the NAs are diluted 1 :10 with H 2 O and re-analyzed by both the nuc and CDV-N gene QPCRs. In case of doubt, the NAs are diluted.
  • Data is expressed either as non-transformed or as log-transformed (logarithm base 10) values.
  • Counts of S. aureus on BA and BP plates are compared by the Wilcoxon signed- rank test for paired samples. Comparison of the QPCR and bacteriological results is done using log-transformed values together with the paired t test.
  • linear least square regression analysis is performed with the QPCR results (log SCE/ml) forming the dependent, the bacteriological results (log CFU/ml) the independent variable. Normality of the difference is demonstrated by quantile plot followed by the Lilliefors test.
  • the McNemar test is applied. For this purpose, the exact P value is calculated manually. For all the other computations, the Systat 10 software package (Systat Software Inc., CA, USA) is used. P values ⁇ 0.05 are considered to be significant.
  • the nuc gene is used as a target for the novel assay.
  • This gene coding for the thermonuclease is highly specific for S. aureus and is not found in other staphylococcal species or in further mastitis-relevant bacteria. Published nuc sequences are from human S. aureus isolates and might differ from the bovine cases.
  • the nuc gene of 20 epidemiological ⁇ unrelated S. aureus isolates from bovine mastitis milk is amplified by conventional PCR. The resulting full length PCR products are then sequenced on both strands.
  • the mutations are present in 2 alternative patterns of combination and each of the 19 isolates can be attributed to one of the 2 patterns.
  • the 20th isolate is very closely related to the human N315 strain with only 2 mutations observed in the 649 nucleotides analyzed. Based on these results, two primers and a fluorescent probe for QPCR are constructed which are expected to detect the nuc gene in all the bovine and human S. aureus isolates.
  • the assay is shown to be linear between 10 1 and 10 7 SCE/assay. It is even possible to measure down to 6 SCE/assay, but it is difficult to reproduce the results consistently. As a QPCR standard, therefore, concentrations between 10 1 and 3.8 x 10 4 SCE per reaction are chosen. For this range, a coefficient of correlation of 0.998 is obtained. Moreover, the lowest value (10 SCE/assay) could always be reproduced very well. The amplification efficiency is 0.96 (determined from the gradient).
  • the sensitivity of the assay procedure is evaluated by serially diluting counted liquid cultures of S. aureus in raw milk. These spiking experiments show an analytical sensitivity as low as 460 CFU/assay which translates to 1.8 CFU/10 ⁇ l of milk.
  • the QPCR method is 8.9 timers more sensitive than the bacteriological method (corresponds to 0.11 CFU/10 ⁇ l milk), when the analytical sensitivity of the bacteriological method is set to 1 CFU/10 ⁇ l milk. Since 1 CFU may, however, be the result of contamination, only more than 3 CFU of the same pathogen per plate as proof of pathogen originating from the udder. Under these preconditions the method of the invention represent an analytical sensitivity which is 27.1 times (corresponds to 0.037 CFU/10 ⁇ l) higher than the state of the art bacteriological method. For these calculations one has to consider that the change in analytical sensitivity of the QPCR method is not proportional since the correlation between SCE and CFU values is log linear.

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Abstract

L'invention concerne un procédé de détection et de quantification de S. aureus dans du lait, comprenant les étapes consistant à trypsiniser un échantillon de lait, à en extraire les bactéries et à en isoler les acides nucléiques, puis à quantifier l'ADN spécifique de S. aureus au moyen d'amorces pour le gène nuc dans le cadre d'une réaction de polymérisation en chaîne. Ce procédé se révèle supérieur aux dosages bactériens de l'état de l'art tant en termes de sensibilité que de spécificité analytique. Ce procédé est utilisé pour diagnostiquer une mastite chez les vaches. L'invention concerne, encore, un procédé amélioré d'extraction et d'isolement d'acides nucléiques bactériens présents dans du lait et d'autres liquides biologiques en présence de L. casei.
PCT/EP2008/007735 2007-09-19 2008-09-16 Détection de staphilococcus aureus dans du lait de vaches souffrant de mastite Ceased WO2009036956A1 (fr)

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US12023666B2 (en) 2017-09-15 2024-07-02 Biomeme, Inc. Methods and systems for automated sample processing
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