| Summary: | 碩士 === 弘光科技大學 === 生物產業研究所 === 97 === Abstract
Staphylococcus spp. including S. aureus, S. saprophyticus, S. epidermidis, S. haemolyticus and S. xylosus, etc. are important pathogenic bacteria, Diagnosis of these staphylococcus species isolated from food poisoning cases and clinical samples are important. Recently, studies revealed that heat shock protein gene, hsp60, hsp10 and hsp10-hsp60 (IGR) sequences can be used as a target gene for the identification of Staphylococcus spp. Therefore, the purpose of this study is to design of species specific PCR primers, i.e., S.aurF/R, S.sap-F/R, S.epi-F/R, and S.hae-F/R, respectively, these on the heat shock protein gene (hsp) sequences.
For Staphylococcus genus, universal primers, were also developed to amplify the hsp genes of Staphylococcus genus. Using S.aurF/R, S.sap-F/R, S.epi-F/R, and S.hae-F/R primer sets, bacterial species other than S. aureus, S. saprophyticus, S. epidermidis, S. haemolyticus, including other Staphylococcus. Spp. would not generate any false positive reaction. The detection limit were N × 103 cfu/ml. Such methods can be used for bacteria identification, rapid diagnosis, and these primers may be used on probes into biochip system.
Staphylococcus aureus, Streptococcus agalactiae, Strept. uberis, and Strept. bovis, etc., are common pathogens which may cause Mastitis in dairy herds. Conventional methods for the detection of these bacterial species are time consuming and laborious.
Thus, the development of rapid and reliable methods for the detection of these bacterial species is also important. In this study, we also designed the PCR primer sets, SAU3/SAU4, SAG3/SAG4, SUB3/SUB4 and SBO3/SBO4 from the heat shock protein gene, i.e., hsp70, hsp40, and hsp10, for the specific detection of Strep. agalactiae、Strep. uberis、Strep. bovis and S. aureus, respectively.
Using SAU3/SAU4, SAG3/SAG4, SUB3/SUB4 and SBO3/SBO4 primer sets, bacterial species other than S. aureus、Strep. agalactiae、Strep. uberis、Strep. bovis, including other Strept. spp. would not generate any false positive reaction.
As these PCR primer sets were used for multiplex PCR detection of target cells in pure culture, the detection limit was N × 103 cfu/ml, the detection limit were N × 104 and N × 100 cfu/ml milk depending on whereas the target cells were pre-enriched 10 hr, or not. Thus, the PCR and the multiplex PCR system so established offer a rapid method for detection of bovine mastitis pathogenic bacteria. Such method may be useful for the veterinary inspection agency and the dairy industry.
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