PCR Assay With Host Specific Internal Control for Staphylococcus Aureus From Bovine Milk Samples

Abstract Staphylococcus aureus is considered as one of the most important and common pathogens of bovine mastitis. Polymerase Chain Reaction is frequently proposed in the diagnosis of S. aureus directly from milk samples instead of classical culture. However, false-negative results may occur in the polymerase chain reaction analysis performed directly from clinical material. For the purpose of disclosing the false negative results, the use of internal amplification controls can be beneficial. Therefore, in this study a new polymerase chain reaction technique with host specific internal amplification control was developed by optimizing S. aureus-specific primers in combination with bovine specific primers. The effectiveness of the developed technique in this study was attempted in milk samples from bovine subclinical mastitis. This technique has the potential to detect S. aureus from bovine milk samples or dairy products.


INTRODUCTION
Staphylococcus aureus, one of the most prevalent pathogens of clinical and subclinical bovine mastitis, can spread rapidly throughout the herd if management precautions are not taken (1,2).S. aureus is one of the most important and common pathogens of bovine mastitis.It causes significant economic losses in the dairy industry (3). S. aureus damages milk-producing tissue and significantly decreases milk yield.In the early stages of infection, damage may be reversible, but when diagnosis is delayed, tissue damage is excessive and irreversible (4).Therefore, a rapid and reliable method of identifying the bacteria responsible for mastitis is important for disease management in the herd (5).Moreover, S. aureus infections are considered a serious problem that may affect public health because they can be transmitted by milk and milk products (6).
While different methods have been used to detect these agents, bacterial culture is considered the gold standard method for identifying mastitiscausing microorganisms.However, conventional culture is slow, requiring 24-48 h, and needs labour intensive study for definitive identification (7,8).Therefore, it is of commercial interest to accelerate this procedure by investigating alternative rapid DNA-based methods.PCR methods for detecting S. aureus in milk samples to identify cow mastitis have been proposed (9,10,11).However, PCR analyses from direct clinical material can result in false negative results caused by inhibitors in the clinical specimens (12,13).Therefore the use of internal amplification controls with different strategies is recommended to determine false negative results of PCR analyses from direct clinical material or food samples (14,15).
The aim of the present study was to develop PCR analyses with internal amplification control to detect S. aureus.A specific primer set for S. aureus was used and combined with bovinespecific primers.

Samples and Primers
Total of 50 milk samples from subclinical bovine mastitis were obtained from cattle farms in the Hatay region (The clinical samples were taken with permission with MKÜ Local Ethics committee; Meeting Date 17.06.2010:Meeting No: 2010/02: Decision No: 30) and nucleic acids were extracted from milk samples using the phenol-chloroform method.Extracted DNA was stored at -20°C (16).
Primers for S. aureus (Sau 327 and Sau 1645) and Bovine specific (12SM-FW and 12SBT-REV2) were used in this study.Simplex PCR protocols and procedures were carried out according to the recommendations in the literature for those primers.The properties of the primers used in this study are shown in Table 1.

Optimisation of internally controlled PCR mix
All procedures and protocols for multiplex PCR assays were optimized according to Henegariu et al., (18).The amplification mixture for multiplex PCR was carried out in a final volume of 25 μL.

Simplex PCR reaction results
In the PCR analyses of milk samples, 3 of 50 samples were found to be S. aureus bysimplex PCR and the specific amplification products (1318 bp).They are shown in Figure 1. S. aureus-specific bands were amplified very effectively with simplex PCR.

Multiplex PCR reaction results
In the PCR analysis results, specific bands for bovine gene as an internal control and specific bands for S. aureus were amplified effectively in the same PCR mixture; the amplification products are shown in Figure 2.

DISCUSSION
Staphylococcus aureus is considered an important and common pathogen causing bovine mastitis that can also be dangerous to human health.There are some studies including PCR detection of S. aureus from bovine milk samples (9,10,11).However, in the PCR analyses from clinical material false negative results can be encountered due to inhibitory substances.In order to avoid these false negative results, using internal control was recommended (12,13).Particularly, using of host specific internal control can be useful to determine false negative results due to mistakes in sampling, DNA extraction or preparing PCR mixtures and thermal cycler failures (14,15,19).Therefore, a PCR technique with host specific internal control was developed in this study by combining S. aureus specific primers and cow specific primers.
In this study a preliminary type, a specific primer set for S. aureus, was combined with specific bovine gene primers to develop an easy and rapid multiplex PCR system with high specificity.Using this optimised combination, a PCR analysis with an internal control specific for bovine genes was developed to detect S. aureus.The primer sets successfully amplified the target genes in the multiplex PCR without nonspecific or additional bands.There was no difficulty in discriminating between each band for the target strain (1318 bp) and the internal control (346 bp).Despite the low numbers of positive samples used in this study, the authors are confident that the developed technique will be reproducible in larger experiments.
At the end of the optimisation experiments, the best amplification results were obtained by decreasing 10x PCR buffer (2 μL) and host-specific primers 12SM-FW and 12SBT-REV2 (internal control, 5 pmol) and increasing to 20 pmol primers (Sau 327 and Sau 1645) specific for S. aureus.While low and high concentrations of MgCl 2 negatively affected the multiplex PCR, the best results were obtained with 3 mM of MgCl 2 concentration.The other important parameter was the annealing temperature: the best results were obtained at 54°C.The effects of these parameters were found to be in agreement with the recommendations of Henegariu et al. (18) and Phuektes et al. (20).
Despite the small number of samples analysed, the results of this preliminary study showed that this method can be used as a reliable and effective method for the diagnosis of S. aureus in the individual bovine milk or bulk milk samples.This technique should be tried for S. aureus detection from bovine milk products In conclusion, conventional PCR has already become widespread and is less expensive than other molecular techniques.The developed technique in this study might have the potential to detect S. aureus from bovine clinical samples.This method can be used for detecting S. aureus in the bovine milk or milk products.The technique can also be extended to detect other contagious mastitis pathogens, such as Streptococcus agalactiae and Trueperella pyogenes.

Table 1 .
Properties of primers used in the study