Cross-priming amplification targeting the coagulase gene for rapid detection of coagulase-positive Staphylococci.
OBJECTIVE
To develop and evaluate cross-priming amplification (CPA) combined with immuno-blotting for the detection of coagulase-positive Staphylococci including Staphylococcus aureus.


RESULTS
Twenty-four sets of cross and detection primers were designed according to four sequences of coagulase gene in Staph. aureus. The most specific primer pair was screened out for the next amplification and interaction. The specificity was evaluated in a total of 53 species of Staph. aureus and non-Staph. aureus. Two red lines indicating positive were always observed on the BioHelix Express strip for 12 subspecies of Staph. aureus. In contrast, only one signal line showing negative results was detected in all of non-Staph. aureus samples. The limit of detection (LOD) of CPA was 3·6 ± 2·7 fg for the genomic DNA, which is about 100 and 10 times sensitive than those of PCR and loop-mediated isothermal amplification respectively. For the pure culture of Staph. aureus and milk powders, the LODs of CPA were about 1·34 CFU per reaction and 5·2 ± 3·7 CFU per 100 g of milk powder respectively. The CPA method was also successfully applied to evaluate the contamination of Staph. aureus in 318 samples of daily food.


CONCLUSIONS
CPA is a very sensitive and rapid method to detect Staph. aureus by simple laboratory instrument.


CONCLUSIONS
It is the first report on the application of the CPA with immuno-blotting for detection of coagulase-positive Staphylococci including Staph. aureus.
School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
B
B
Qiao
School of Life Science, Shandong Province Key Laboratory of Applied Mycology, Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural University, Qingdao, Shandong Province, China.
J-Y
JY
Cui
Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China.
L
L
Sun
School of Life Science, Shandong Province Key Laboratory of Applied Mycology, Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural University, Qingdao, Shandong Province, China.|Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, China.
S
S
Yang
Department of Medical Microbiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China.
Y-L
YL
Zhao
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003030:Coagulase / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013203:Staphylococcal Infections / Q000382:microbiology*; D013210:Staphylococcus / Q000235:genetics; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D003030:Coagulase; D017931:DNA Primers
Staphylococcus aureus; coagulase gene; cross-priming amplification; immuno-blotting analysis; polymerase chain reaction