Development of PCR primers and a DNA macroarray for the simultaneous detection of major Staphylococcus species using groESL gene.
Staphylococcus spp., including S. aureus, S. intermedius, S. hyicus, S. epidermidis, S. saprophyticus, S. haemolyticus, S. xylosus, and S. carnosus, are major bacterial species associated with food poisoning, and human and veterinary clinics. Traditional methods for the identification of these staphylococci are time-consuming, laborious, or inaccurate. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and polymerase chain reaction (PCR) primers for the detection of the aforementioned Staphylococcus species. These primers were proved to be specific for the detection of their corresponding target strains. Furthermore, by using a consensus primer pair, we were able to co-amplify the intergenic region of groES-groEL for these staphylococci. Followed by a chromogenic macroarray system with the specific probes on the plastic chips, these staphylococci in milk products or clinical samples could be simultaneously detected. When the system was used for the inspection of milk or urine samples containing N × 10⁰ target cells per milliliter of the sample, all these staphylococcal species could be identified after an 8-h pre-enrichment step. This system also allowed the adequate diagnosis of bacteremia, since N × 10⁰ target cells per milliliter of the blood samples could be detected after a 12-h pre-enrichment. Compared to the multiplex PCR method, this approach has the additional advantage that it allowed the discrimination of more bacterial strains-even some bacterial strains that may generate PCR products with the same molecular sizes.
Department of Food Science and Technology, Hung Kuang University, Shalu, Taichung, Taiwan, ROC. honda224@sunrise.hk.edu.tw
Yu-Cheng
YC
Chiang
Hsi-Chi
HC
Lu
Sheng-Chih
SC
Li
Yu-Hsin
YH
Chang
Hsin-Yen
HY
Chen
Chia-Wei
CW
Lin
Hau-Yang
HY
Tsen
D000818:Animals; D016470:Bacteremia / Q000097:blood / Q000175:diagnosis / Q000382:microbiology / Q000652:urine; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism; D018833:Chaperonins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry*; D004269:DNA, Bacterial / Q000097:blood / Q000378:metabolism / Q000652:urine; D021901:DNA, Intergenic / Q000097:blood / Q000378:metabolism / Q000652:urine; D005513:Food Inspection / Q000379:methods; D005516:Food Microbiology; D015870:Gene Expression; D006801:Humans; D008892:Milk / Q000382:microbiology; D058889:Molecular Typing; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012703:Serotyping / Q000379:methods; D013202:Staphylococcal Food Poisoning / Q000097:blood / Q000175:diagnosis / Q000382:microbiology / Q000652:urine; D013203:Staphylococcal Infections / Q000097:blood / Q000175:diagnosis / Q000382:microbiology / Q000652:urine; D013210:Staphylococcus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D017931:DNA Primers; D004269:DNA, Bacterial; D021901:DNA, Intergenic; C097970:GroESL protein, Bacteria; D018833:Chaperonins