Quantitative reverse transcription-PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus.
OBJECTIVE
To evaluate a new quantitative reverse transcription-PCR (qRT-PCR) assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA).


RESULTS
Primers for Staphylococcus-specific regions of 16S rRNA gene, spa gene and mecA gene were newly designed. RNAs extracted from broth-cultured strains were tested by qRT-PCR targeting each primer, and the bacterial counts obtained correlated well with those counted by the plating method with detection limits of 10(0), 10(1) and 10(2) CFU. The qRT-PCR assay targeting the 16S rRNA was 6430-fold or more sensitive than qPCR assay. All Staph. aureus strains tested were detected and none of the other Staphylococcus species and genus strains tested cross-reacted with the assay targeting the spa gene. All MRSAs tested were detected by the assay targeting the mecA gene. Clinical samples, faecal material and bronchial washout solutions were tested by our assay, and MRSAs were detected with a high sensitivity within 6 h.


CONCLUSIONS
Our qRT-PCR assay targeting three new primers to the target genes is a rapid and sensitive tool for the detection of MRSA directly from clinical samples.


CONCLUSIONS
Because of its sensitivity and rapidity, our qRT-PCR assay is considered to be a valuable tool for clinical management.
Wada
M
M
 Laboratory for Probiotics Research, Juntendo University School of Medicine, Tokyo, Japan.| Department of Pediatrics, Juntendo University School of Medicine, Tokyo, Japan.
Lkhagvadorj
E
E
 Laboratory for Probiotics Research, Juntendo University School of Medicine, Tokyo, Japan.
Bian
L
L
 Laboratory for Probiotics Research, Juntendo University School of Medicine, Tokyo, Japan.
Wang
C
C
 Laboratory for Probiotics Research, Juntendo University School of Medicine, Tokyo, Japan.
Chiba
Y
Y
 Laboratory for Probiotics Research, Juntendo University School of Medicine, Tokyo, Japan.| Department of Pediatrics, Juntendo University School of Medicine, Tokyo, Japan.
Nagata
S
S
 Laboratory for Probiotics Research, Juntendo University School of Medicine, Tokyo, Japan.| Department of Pediatrics, Juntendo University School of Medicine, Tokyo, Japan.
Shimizu
T
T
 Department of Pediatrics, Juntendo University School of Medicine, Tokyo, Japan.
Yamashiro
Y
Y
 Laboratory for Probiotics Research, Juntendo University School of Medicine, Tokyo, Japan.
Asahara
T
T
 Yakult Central Institute for Microbiological Research, Tokyo, Japan.
Nomoto
K
K
 Yakult Central Institute for Microbiological Research, Tokyo, Japan.
D058491:Bacterial Load; D001426:Bacterial Proteins / Q000235:genetics; D001769:Blood / Q000382:microbiology; D017931:DNA Primers; D005243:Feces / Q000382:microbiology; D006801:Humans; D057230:Limit of Detection; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*; D046915:Penicillin-Binding Proteins; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000382:microbiology
D001426:Bacterial Proteins; D017931:DNA Primers; D046915:Penicillin-Binding Proteins; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C487789:mecA protein, Staphylococcus aureus
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