Real-time PCR detection of Staphylococcus aureus in milk and meat using new primers designed from the heat shock protein gene htrA sequence.
Staphylococcus aureus may cause foodborne disease outbreaks and staphylococcal infections and is one of the major causes of mastitis. Rapid and reliable methods for detection of this microorganism in milk and other foods are needed. In this study, we designed a primer set from the sequence of the heat shock protein gene htrA, a gene coding for high-temperature-requirement A (HtrA) protein, and used it for real-time PCR detection of S. aureus isolates: 16 reference strains and 40 strains isolated from food-poisoning cases. All strains tested generated positive results. Bacterial strains other than S. aureus, including strains of other Staphylococcus species, did not produce positive results. When this primer set was used for the real-time PCR detection of S. aureus in milk and meat samples without the preenrichment step, samples with target cell numbers greater than 10(3) CFU/ml or CFU/g could be detected, indicating the potential quantitative ability of this real-time PCR assay. With a 10-h preenrichment step, however, a detection limit of 1 CFU/ml or CFU/g could be obtained.
Chiang
Yu-Cheng
YC
Institute of Industrial Biotechnology, Hung Kuang University, 34 Chung-Chi Road, Shalu, Taichung County, 433 Taiwan, Republic of China.
Fan
Chih-Ming
CM
Liao
Wan-Wen
WW
Lin
Chien-Ku
CK
Tsen
Hau-Yang
HY
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D015169:Colony Count, Microbial; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D008460:Meat / Q000382:microbiology*; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013211:Staphylococcus aureus / Q000302:isolation & purification*; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006360:Heat-Shock Proteins; C113725:HspA protein, bacteria