Detection of Staphylococcus aureus in Dairy Products by Polymerase Chain Reaction Assay

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Agricultural Sciences in China 2007, 6(7): 857-862

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ScienceDirect

July 2007

Detection of Staphylococcus aureus in Dairy Products by Polymerase Chain Reaction Assay YANG Yangl, SU X u - d o n g l , YUAN Y a o - w u l , KANG Chun-yu1, LI Y m g - j u n l , ZHANG wei1 and ZHONG ximyingz 1

2

Laboratory for Microorganism Detection, College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, P.R. China Baoding Center for Disease Control and Prevention in Hebei Province, Baoding 071001, P.R.China

Abstract A polymerase chain reaction (PCR) assay was employed for direct detection of Staphylococcus aureus without enrichment in dairy products. A solvent extraction procedure was successfully modified for the extraction of Staphylococcus aureus DNA from artificially contaminated whole milk, skim milk, and cheese. A primer targeting the thermostable nuclease gene (nuc) was used in the PCR. A DNA fragment of 279 bp was amplified. The PCR product was confirmed by DNA sequencing. In this study, the PCR, GB- 4789.10-94,Perifilm RSA.Count Plate, and Baird-Parker + RPF Agar were compared. The sensitivity of the PCR was 10 CFU m L - 1 of whole milk, skim milk, and 55 CFU g-1 of cheese. The developed methodology allowed for detection of Staphylococcus aureus in dairy products in less than 6 h. The time taken for the development of this PCR assay was 12-24 h, less than the time taken by the general PCR assay using the enrichment method, and the coincidence rate of this developed PCR was 94.3%,the sensitivity was 100%. It was a rapid, sensitive, and effective method for PCR to detect Staphylococcus aureus in milk and milk products.

Key words: PCR, detection, dairy products, Staphylococcus aureus

INTRODUCTION Staphylococcus aureus (Staph. aureus) found on the skin and mucous membranes of mammals is an important human pathogen that also causes a diverse range of diseases in animals. It is a ubiquitous bacterium, and enterotoxins produced by Staph. aureus cause alimentary toxicosis. In accordance with the report of the Center for Disease Control and Prevention of the USA, the incidents of food poisoning caused by Staph. aureus among the foodborne pathogens were 33% in USA and 45% in Canada. In China it also represents the signal of great hazard to human health. The traditional meth-

ods of detecting Staph. aureus is loaded with trivial details which needed a long time, so it is an urgent task to establish procedures, which are rapid, sensitive, specific, and easy to handle. Specific primers for PCR detection of Staph. aureus have been directed to the nuc gene, encoding the thermostable nuclease (Wilson et al. 1991). In Wilson et al’s article, enterotoxigenic Staph. aureus cells could be detected in artificially contaminated dried skimmed milk samples at levels of lo5CFU mL-’within 8 h. The sensitivity of the PCR was low, the reason for which could be that the genomic DNA isolated from the dried skimmed milk contained some factors, which interfered with the reaction of PCR; Brakstad et al. (1992) em-

This paper is nanslated from its Chinese version in Scientia Agriculfura Sinica. Correspondence ZHANG Wei, Tel: c86-312-7528422. E-mail: [email protected]

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YANG Yang et al.

858

ployed the method of PCR to detect the Staph. aureus in both glucose broth and blood, the lower detection limit was < 10 CFU mL and lo3CFU mL-'. The reduced sensitivity of the nuc PCR for detection of Staph. aureus in blood could be because of the interfering components, such as, calcium ions, plasmin, and protein (Wilson 1997). Mclauchlin er al. (2000) reported that the detection of Sraph. aiireus was less successful in three types of cheese and in cream because of food matrice factors. In this article. a solvent extraction procedure was successfully modified for the extraction of Staph. aureus DNA, in which the interfering components including ions, protein, and fatty matter were eliminated. By using a PCR identification system, which detected thermostable nuclease gene (nuc),the samples of milk and milk products with Staph. aureus were identified sensitively, without amplifying the bacterial cells, and the results were rapidly obtained.

MATERIALS AND METHODS Bacterial strains and culture The bacterial strains used in this article are summatized in Table 1. The Staph. aureus strains were added to glucose broth, and the suspension was shaken and continually incubated at 37°C until a concentration of 10' CFU mL-' was reached.

Contaminated samples preparation The samples of milk and skim milk (purchased in the local supermarket), which had been proved to have no Staph. aureus were artificially contaminated by Staph. aureus. The decimal dilutions of Sraph. aureus in milk samples were selected (lo", 10'. lo2, lo3, lo4, lo5, lo6, lo', and loxCFU mL-') for DNA extraction, Total viable counts were determined by plating dilutions on plate count agar (glucose broth agar). A sample of 20 g cheese (purchased in the local supermarket) was triturated in mortar, and was then added to 90 mL sodium citratein solution (2%) in a conical flask. which had been heated to 75°C. The homogeneous suspension was then artificially contami-

Table 1 The bacterial strains used in experiment No.

Bacteria

Source')

1

Staphylococcus epidermrds-ATCC26069

CGMCC

2

Sraphvlococcus xylosus-ATCC29971

CGMCC

3

Sraphylococcus albus-l . I 84

CMCC

3

Sraphylococcus intermedius-I109

CMCC

5

Escherichia colr-ATCC25922

CGMCC

6

Escherrchra coli-ATCC35401

CGMCC

7

Salmonella ryphimurium-ATCC14028

CGMCC

8

Salmonella typhimurium-5007- 1

CMCC

9

Shigella dysenreriae- 1.1869

CMCC

10

Shigella flerneri- 1,1868

CMCC

11

Shigella boydii-ATCC9209

CGMCC

12

Shigella sonnei-ATCC9290

CGMCC

13

Srreprococcus hemolyfis-a-32205

CMCC

I3

Srreprococcus hemolytis-P32204

CMCC

IS

Lisrerra monocyrogenes-ATCC35152

CGMCC

16

Listeria monocyrogenes-ATCC35152

CGMCC

17 18

Bacillus cereus-ATCCI 1778 Bacillus subfilis-ATCC605 1

CGMCC CGMCC

19

Yersinia enterocolirica-52001

CMCC

20

Vibrio parahaemolyricus-2000 1

CMCC

21

Sraphylococcus aureus-ATCC6538

CGMCC

22

Sraphylococcus aureus-ATCC6538p

CGMCC

23

Sraphylococcus aureus- 1.800

CMCC

21

Sraphylococcus aureus- 1.1416

CMCC

2s

Sraphylococcus aureus-26003-21

CMCC

26

Sraphylococcus anreus-ATCC25923

CGMCC

21

Sraphylococcus aureus-ATCC13565

CGMCC

28

CGMCC

29

Sraphylococcus aureus-ATCC14458 Staphylococcus aureus-ATCC8095

30

52 strains of Sraphylococcus aureus

LMDAUH

'

CGMCC

CGMCC. China Microbiological Culture Collection Center; CMCC, China Medical Culture Collection Center; LMDAUH, Laboratory for Microorganism Detection of Agricultural University of Hebei.

nated by Staph. aureus. The different concentrations of Staph. aureus (loo, lo', lo2, lo3, lo4, lo5, lo6, lo7, and lox CFU mL-') were also obtained and used for DNA extraction.

DNA extraction The DNA extraction was carried out from 5 mL contaminated milk, skim milk or homogeneous suspension of cheese. One milliliter of NH,, 1 mL of ethanol, and 1 mL of petroleum ether were added. Then the system was mixed and harvested by centrifugation at 12 000 x g for 10 min, and nearly all of the bacterial cells were detected in the pellet. The final pellet was dissolved in 300 pL of TE (10 mM TRIS, 1 mM EDTA, pH 7.8)? followed by the addition of 5 pL lysostaphin (1.8 U pLY; Sigma, Deisenhofen, Germany). After incubation for

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Detection of Staphylococcus aureus in Dairy Products by Polymerase Chain Reaction Assay

1 h at 37°C with interval shakes, 50 yL SDS (10%) was added to the system and then it was continually heated (100°C) for 5 min. Chloroform of equivalence volume was mixed with the system for extracting. 0.1 total volume of ammonium acetate (2.5 M) and 2.5 volume of ethanol were added to the extraction, and then the system was centrifuged at 17 000 x g for 20 min at 4°C. Finally, the pellet was dried and resuspended in 100 pL ddH,O.

PCR amplification The thermostable nuclease gene (nuc) was amplified for the detection of enterotoxigenic Staph. aureus. One pair of primers was used (Pri-1: 5'-GCGATTGATGGT GATACGGTT-3'; and Pri-2: 5 '-AGCCAAGCCTT GACGAACTAAAGC-3'). For PCR amplification,the reaction mixture (50 pL), contained 0.5 pL of Pri- 1 (10 pm pL-'), 0.5 pL of Pri2 (10 pm pL-'), 4 pL of deoxynucleoside triphosphate (10 mm; MBI Fermentas, St Leon-Rot, Germany), 5 pL of 10x thermophilic buffer (Promega, Mannheim, Germany), 2 yL of MgC1, (25 mM; Promega), 0.5 pL of Taq DNA polymerase (5 U pL-'; Promega), and 35.5 pL of distilled water. Finally, 2.0 yL of the DNA preparation was added to the reaction tube. Amplification was carried out for 32 reaction cycles of denaturing at 94°C for 1 min, annealing at 52°C for 0.5 min, and extension at 72°C for 1.5 min, with an additional step of extension at 72°C for 10 min. The PCR products were sequenced by Shanghai Sangon Biological Engineering Technology Services Co., China. Moreover the sequence was blasted at the NCBI web site.

Comparisonbetween the uniplex PCR and other detection methods of Staph. aureus The methods of uniplex PCR, GB- 4789.10-94, Perifilm

859

RSA.Count Plate, and Baird-Parker + RPF Agar were used to detect the 35 dairy products together [including 20 products of whole milk, five products of sterile (UHT) milk, five products of milk powder, and five products of cheese], which were purchased in the local supermarket. According to the results, the sensitivity and accordance rate of each were counted. Sensitivity = The number of true-positive sample/ (The number of true-positive samples + The number of false-negative samples). Accordance rate = (The number of true-positive samples + The number of true-negative samples)/The total number of samples detected.

RESULTS Specific of primers Of the tested bacterial strains, only the DNA of Staph. aureus could be amplified by using the primers, and other strains produced no reaction product (Fig. 1). Therefore, it could be proved that the primers were highly specific.

Detectingof PCR on dairy productscontaminated by Staph. aureus In the whole milk, skim milk, and cheese, as low as 10' CFU mL-'of Staph. aureus could be detected by targeting the nuc gene (Fig.2). But it was found that cheeses had difficult matrices to be assayed by PCR because of the high fat content, which could affect the DNA extraction and PCR amplification. The products amplified with this pair of primers were relatively few (Fig. 1-C). The results of the PCR amplifications of the DNA directly extracted from the whole milk, skim milk, and cheese are summarized in Table 2. The detection limits of whole milk and skim milk artificially contaminated

Table 2 The lower detection limit of Staph. Aureus in dairy products after being artificially contaminated by PCR Samples Whole milk

skim milk Cheese

Concentration of Staph. Aureus (CFU mL-')

1OR

107

106

105

109

103

102

10'

+ +

+ + +

+

+

+

+

c

+

+ +

+ +

+

+

+

+ + +

+

+

+

~~

+, the detection result of PCR

was positive;

10"

-. the detection result of X R was negative 02007,CAAS. All rights resewed. Published by Elsevier Ltd.

YANG Yanrr et al.

860

by Staph. aureus were both LO CFU rnL-'. The sample of cheese was triturated as a homogeneous liquid state, according to the disposal method, and the detection limit of cheese could be calculated. It was 55 CFU g-'

(10CFUmL'x110mL20g1).

The production of PCR A DNA fragment of 279 bp was amplified by PCR pig. 1). The sequence identified by Shanghai Sangon Biological Engineering Technology Services Co., was

Fig. 1 The detection result of Staphylococcus aureus and Non-Staphylococcus aureus by PCR. A: 1, Staphylococcus epidennids; 2, Staphylococcus qlosus; 3. Staphylococcus albus; 4. Staphylococcus intermedius; 5 , Escherichia coli: 6 , Escherichia coli; I , Salmonella ty~himrtriutn;8, Salmonella qphirnuriurn: 9. Shigella dysenteriae; 10, negative control; 1 1, Staphylococcus aureus; 12, positive control; 13, marker DL2000; 14, Shigella flexneri; 15, Shigella boydii; 16, Shigella sonnei; 17, Streptococcus hemolytis-a; 18, Streptococcus hemolyiis- b; 19, Listeria monocytogenes: 20, Listeria monocytogenes: 2 1, Bacillus cereus; 22, Bacillus subtilis; 23, Yersinia enterocolitica; 24, Vibrio parahaemolwicus. B : 1. marker DL2000; 2-21, Staphylococcus aureus; 22, negative control; 23, positive control; 24, marker DL2000. C: 1. marker DL2000: 2-21, Staphylococcus aureus: 22, negative control; 23, positive control; 24, marker DL2000. D: 1-9, Staphylococcus aureus: 10, negative control; 1 1. positive control; 12, marker DL2000; 13-23, Staphylococcus aureus.

Fig. 2 The detected result of Siaphylococcus aureus by PCR in artificially contaminated whole milk (A), skim milk (B), and cheese ( C ) . 1 , marker DL2000: 2, positive control; 3 , negative control. The concentration of Staphylococcus aureus from lane 4 to lane 12 is 108 to loo CFU mL I respectively; the concentration of Siaphylococcrts aureus from lane 13 to lane 21 is lo8 to looCFU mL 1 respectively; the concentration of Sruphylococcus aureus from lane 22 to lane 30 is lo8 to loo CFU g-1, respectively.

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Detection of Sraphylococcus aureus in Dairy Products by Polymerase Chain Reaction Assay

blasted at the NCBI web site, and it had a sequence homology of 99.6% to the holotype gene.

Comparison between the uniplex PCR and other detection methods of Staph. aureus The methods of GB- 4789.10-94, Perifilm RSA.Count Plate, and Baird-Parker + RPF Agar were also employed in the experiment, with parallel detection of 35 dairy products. The sensitivity of the uniplex PCR was loo%, and accordance rate was 94.3%. The methods of Perifilm RSACount Plate and Baird-Parker + RPF Agar both had 100% sensitivity and 97.3% accordance rate (Table 3). As a result, the PCR method owned a higher detection rate (62.9%) than others. It could be that PCR could detect the live cells and dead cells at the

861

same time. Among these methods, it took 28-30 h to get the detection result with Perifilm RSA.Count Plate and Baird-Parker + RPF Agar. For example, routine detection of Staph. aureus in food is usually carried out by traditional methods based on the use of selective media (Baird-Parker + RPF) for direct enumeration or for the recovery of isolates after enrichment in selective broth for 24-30 h at 37°C. The conventional methodology (Baird-Parker agar) takes from five to six days, because besides enrichment the suspected colonies that are positive for DNase and coagulase production should be identified by biochemical tests. The PCR amplifying detection needed less than 6 h, thus, it was rapid, easy to handle, sensitive, and specific, and constituted a very valuable tool for microbiological applications.

Table 3 The sensitivity, specificity, and coincidence rates of PCR Samples

Total

Milk UHT milk Powdered milk Cheese Total Detection rate (%) Sensitivity (%) Accordance rate (%)

Perifilm RSA.Count Plate

FCR

+

+

5

20 1

0 4

20 0

5

0

5

0

1 22 62.9 100 94.3

13 60 100 97.1

20

5 35

4

Baird-Parker + RPF Agar

+ 0

5 5

GB- 4789.10-94

+

20 0

0 5

20

0

0

5

0

5 5

4 14

0 20

5 15

1

4

0 I

21 60

14

21

57.1

100 97.1

DISCUSSION Food matrices can reduce the sensitivity of PCR. Fat content in the food is reported to be one of the major factors reducing the sensitivityof the PCR assay (Wilson 1997). Cheeses are often regarded as difficult matrices to be assayed by PCR, because of the high fat content, which can affect the DNA extraction and PCR amplification (Wilson 1997). The method of DNA extraction used in this study has been studied previously and shown to be effective in extracting microbial DNA of complex bacterial populations from cheeses and other dairy matrices. Enrichment procedures were performed to increase the level of detection. However, the sensitivity could be usually increased after 24 h and further. Lantz et aZ. (1998) established a multiplex PCR assay to detect viable pathogenic strains of Yersinia enterocolitica in the samples and resulted in a detection level of 1 O2 CFU

d-'. This method used enrichment media to amplify cells. Jinneman et al. (1995) also employed multiplex PCR to identify E.coZi 0,57:H7,but before detection, a process of enriched strains was designed. They all needed 18-30 h to accomplish their experiments . In this article, the developed methodology of extracting DNA allows detection of Staphylococcus aureus in dairy products in less than 6 h without strain enrichment. The time for this developed PCR assay is 12-24 h less than the time for the general PCR assay, using enrichment method. The sensitivity of the PCR of whole milk, skim milk, and 55 CFU is 10 CFU d-' g-l of cheese. It is a sensitive and specific tool for microbiological applications. This method can not only be used in detecting the contaminated dairy food by Staph. aureus, but can also be used to trace the Staph. aureus, before and after dairy production formation, from raw milk, such as monitoring the critical control points during food pro-

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862

cessing in the factory. However. publications on the application of PCR detection for Staph. aureus in food are very scarce. In fact. PCR detection is not yet currently used in food microbiological analysis, probably because there is still a lack of standardized criteria for validation of PCR sample preparation methods, reaction components and assembly, as well as amplification conditions for pathogens and various food matrices. Following the development of PCR technology, the advantage will appear in practice.

CONCLUSION A solvent extraction procedure was successfully modi-

fied for extraction of Stuphylococcus aureus DNA from artificially contaminated whole milk, skim milk, and cheese. Fat content, which was the factor reducing the sensitivity of the PCR in the food was eliminated. The developed methodology of extracting DNA allows detection of Stuphjlococcus aureus in dairy products in less than 6 h, without strain enrichment. The sensitivity of the PCR is 10 CFU mL-*of whole milk, skim milk, and 55 CFU g of cheese. The sensitivity of the uniplex PCR was 100’36, and the accordance rate was 94.3%.

Acknowledgements The study was supported by the Scientific Research Program of the Agricultural University of Hebei, China (2005-01 ).

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Microbiology, 30, 1654-1660.

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Wilson I G. 1997. Inhibition and facilitation of nucleic acid Microbiology, 63, amplification. AppZied and Env~r~in~e?ztul

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