title: Comparison between a chimeric lysin ClyH and other enzymes for extracting DNA to detect methicillin resistant Staphylococcus aureus by quantitative PCR.
abstract: Extracting DNA from Staphylococcus aureus cells is important for detecting MRSA by PCR. However, S. aureus cells are known to be difficult to disrupt due to their compact cell walls. Here, we systematically studied the efficiency of a highly active lysin ClyH for extracting DNA of S. aureus in comparison with commonly used enzymes, such as lysostaphin and achromopeptidase (ACP), and its compatibility in quantitative PCR (qPCR) detection of MRSA. qPCR analysis of S. aureus specific gene femB showed that ClyH was much faster than lysostaphin, ACP and lysozyme for releasing DNA. Five minutes disruption with ClyH at room temperature was enough to release all the DNA from S. aureus. Analysis of the spiked nasal swabs by a dual qPCR assay of the β-lactam resistance mecA gene and the staphylococcal cassette chromosome (SCCmec)-open reading frame X (orfX) junction (SCCmec-orfX) after ClyH lysis showed 100% sensitivity and specificity to the commercial BD GeneOhm™ MRSA test with ACP lysis, but the lysis time was reduced from 20 min by ACP to 5 min by ClyH. Our research shows that ClyH could be a better option than the currently used enzymes for DNA extraction from S. aureus, which can provide simpler and faster PCR detection of MRSA.
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.|University of Chinese Academy of Sciences, Beijing, 100039, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
authors.forename: Hang
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Wuhan Scithera Microbial Technologies Inc., Wuhan, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
authors.forename: Junping
authors.initials: J
authors.lastname: Yu
authors.affiliation: Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China. hpwei@wh.iov.cn.
authors.forename: Hongping
authors.initials: H
authors.lastname: Wei
mesh_terms: D002473:Cell Wall / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008248:Lysostaphin / Q000737:chemistry; D055624:Methicillin-Resistant Staphylococcus aureus / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D009088:Mucoproteins / Q000737:chemistry*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012697:Serine Endopeptidases / Q000737:chemistry; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial; D009088:Mucoproteins; C010393:lysin, gastropoda; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D008248:Lysostaphin
keywords: Bacteriophage lysin; DNA extraction; Quantitative PCR (qPCR); Staphylococcus aureus